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Sample records for abundant plasma protein

  1. High-throughput fractionation of human plasma for fast enrichment of low- and high-abundance proteins.

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    Breen, Lucas; Cao, Lulu; Eom, Kirsten; Srajer Gajdosik, Martina; Camara, Lila; Giacometti, Jasminka; Dupuy, Damian E; Josic, Djuro

    2012-05-01

    Fast, cost-effective and reproducible isolation of IgM from plasma is invaluable to the study of IgM and subsequent understanding of the human immune system. Additionally, vast amounts of information regarding human physiology and disease can be derived from analysis of the low abundance proteome of the plasma. In this study, methods were optimized for both the high-throughput isolation of IgM from human plasma, and the high-throughput isolation and fractionation of low abundance plasma proteins. To optimize the chromatographic isolation of IgM from human plasma, many variables were examined including chromatography resin, mobile phases, and order of chromatographic separations. Purification of IgM was achieved most successfully through isolation of immunoglobulin from human plasma using Protein A chromatography with a specific resin followed by subsequent fractionation using QA strong anion exchange chromatography. Through these optimization experiments, an additional method was established to prepare plasma for analysis of low abundance proteins. This method involved chromatographic depletion of high-abundance plasma proteins and reduction of plasma proteome complexity through further chromatographic fractionation. Purification of IgM was achieved with high purity as confirmed by SDS-PAGE and IgM-specific immunoblot. Isolation and fractionation of low abundance protein was also performed successfully, as confirmed by SDS-PAGE and mass spectrometry analysis followed by label-free quantitative spectral analysis. The level of purity of the isolated IgM allows for further IgM-specific analysis of plasma samples. The developed fractionation scheme can be used for high throughput screening of human plasma in order to identify low and high abundance proteins as potential prognostic and diagnostic disease biomarkers.

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

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

    2014-01-01

    the application of pH-sensitive poly(N-isopropylacrylamide-acrylic acid) hydrogel particles for removal of abundant plasma proteins, prior to proteome analysis by MS. Protein depletion occurs by two separate mechanisms: (1) hydrogel particles incubated with low concentrations of plasma capture abundant proteins...... proteins are released and recovered in the eluate. We developed a series of distinct depletion protocols that proved useful for sample depletion and fractionation and facilitated targeted analysis of putative biomarkers such as IGF1-2, IBP2-7, ALS, KLK6-7, ISK5, and PLF4 by selected reaction monitoring...

  3. [Removal of high-abundance proteins in plasma of the obese by improved TCA/acetone precipitation method].

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    Wang, Jun; Feng, Liru; Yu, Wei; Xu, Jian; Yang, Hui; Liu, Xiaoli

    2013-09-01

    To develop an improved trichloroacetic acid (TCA)/acetone precipitation method for removal of high-abundance proteins in plasma of the obese. Volumes of TCA/acetone solution (1, 3, 4, 5, 6, 8, 10 and 20 times of the sample) and concentrations of TCA (10%, 30%, 50%, 60%, 70% TCA/acetone solution) have been investigated to optimize the conditions of sample preparation. SDS-PAGE were used to separate and tested proteins in the supernatant and sediment. The best concentration of the TCA/acetone solution was first determined by SDS-PAGE. The protein in precipitation from 10% TCA/acetone solution processing and the new determined concentration TCA/acetone solution processing were verified by 2-D-SDS-PAGE. And then the digested products of the protein in precipitation and supernatant by trypsin were analyzed by nano HPLC-Chip-MS/MS to verify which is the best concentration to process the plasma. The best volume of TCA/acetone is four times to sample, which less or more TCA/acetone would reduce the removal efficiency of high-abundance proteins. The concentration of TCA in acetone solution should be 60%, which may remove more high-abundance proteins in plasma than 10%, 30%, 50% TCA in acetone solution. If the TCA concentration is more than 60%, the reproducibility will be much poorer due to fast precipitation of proteins. The results of mass identification showed that human plasma prepared with 60% TCA/acetone (4 times sample volume) could be verified more low-abundance proteins than 10%. The most desirable conditions for removal of high-abundance proteins in plasma is 60% TCA/acetone (4 times sample volume), especially for the plasma of obesity.

  4. Relative Abundance of Proteins in Blood Plasma Samples from Patients with Chronic Cerebral Ischemia.

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    Kaysheva, Anna L; Kopylov, Artur T; Ponomarenko, Elena A; Kiseleva, Olga I; Teryaeva, Nadezhda B; Potapov, Alexander A; Izotov, Alexander А; Morozov, Sergei G; Kudryavtseva, Valeria Yu; Archakov, Alexander I

    2018-03-01

    A comparative protein profile analysis of 17 blood plasma samples from patients with ischemia and 20 samples from healthy volunteers was carried out using ultra-high resolution mass spectrometry. The analysis of measurements was performed using the proteomics search engine OMSSA. Normalized spectrum abundance factor (NSAF) in the biological samples was assessed using SearchGUI. The findings of mass spectrometry analysis of the protein composition of blood plasma samples demonstrate that the depleted samples are quite similar in protein composition and relative abundance of proteins. By comparing them with the control samples, we have found a small group of 44 proteins characteristic of the blood plasma samples from patients with chronic cerebral ischemia. These proteins contribute to the processes of homeostasis maintenance, including innate immune response unfolding, the response of a body to stress, and contribution to the blood clotting cascade.

  5. Hunting for low abundant redox proteins in plant plasma membranes.

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    Lüthje, Sabine; Hopff, David; Schmitt, Anna; Meisrimler, Claudia-Nicole; Menckhoff, Ljiljana

    2009-04-13

    Nowadays electron transport (redox) systems in plasma membranes appear well established. Members of the flavocytochrome b family have been identified by their nucleotide acid sequences and characterized on the transcriptional level. For their gene products functions have been demonstrated in iron uptake and oxidative stress including biotic interactions, abiotic stress factors and plant development. In addition, NAD(P)H-dependent oxidoreductases and b-type cytochromes have been purified and characterized from plasma membranes. Several of these proteins seem to belong to the group of hypothetical or unknown proteins. Low abundance and the lack of amino acid sequence data for these proteins still hamper their functional analysis. Consequently, little is known about the physiological function and regulation of these enzymes. In recent years evidence has been presented for the existence of microdomains (so-called lipid rafts) in plasma membranes and their interaction with specific membrane proteins. The identification of redox systems in detergent insoluble membranes supports the idea that redox systems may have important functions in signal transduction, stress responses, cell wall metabolism, and transport processes. This review summarizes our present knowledge on plasma membrane redox proteins and discusses alternative strategies to investigate the function and regulation of these enzymes.

  6. LDL receptor-related protein 1 regulates the abundance of diverse cell-signaling proteins in the plasma membrane proteome.

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    Gaultier, Alban; Simon, Gabriel; Niessen, Sherry; Dix, Melissa; Takimoto, Shinako; Cravatt, Benjamin F; Gonias, Steven L

    2010-12-03

    LDL receptor-related protein 1 (LRP1) is an endocytic receptor, reported to regulate the abundance of other receptors in the plasma membrane, including uPAR and tissue factor. The goal of this study was to identify novel plasma membrane proteins, involved in cell-signaling, that are regulated by LRP1. Membrane protein ectodomains were prepared from RAW 264.7 cells in which LRP1 was silenced and control cells using protease K. Peptides were identified by LC-MS/MS. By analysis of spectral counts, 31 transmembrane and secreted proteins were regulated in abundance at least 2-fold when LRP1 was silenced. Validation studies confirmed that semaphorin4D (Sema4D), plexin domain-containing protein-1 (Plxdc1), and neuropilin-1 were more abundant in the membranes of LRP1 gene-silenced cells. Regulation of Plxdc1 by LRP1 was confirmed in CHO cells, as a second model system. Plxdc1 coimmunoprecipitated with LRP1 from extracts of RAW 264.7 cells and mouse liver. Although Sema4D did not coimmunoprecipitate with LRP1, the cell-surface level of Sema4D was increased by RAP, which binds to LRP1 and inhibits binding of other ligands. These studies identify Plxdc1, Sema4D, and neuropilin-1 as novel LRP1-regulated cell-signaling proteins. Overall, LRP1 emerges as a generalized regulator of the plasma membrane proteome.

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

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    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.

  8. Detection and quantitation of twenty-seven cytokines, chemokines and growth factors pre- and post-high abundance protein depletion in human plasma

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    Seong-Beom Ahn

    2014-06-01

    Full Text Available Cytokines, chemokines and growth factors (CCGFs in human plasma are analyzed for identification of biomarkers. However concentrations of CCGFs are very low; it is difficult to identify and quantify low abundance proteins in the presence of the high abundance proteins (HAPs unless HAPs are removed prior to analysis. However, there is a concern that the low abundance proteins such as CCGFs may also be removed during the HAP depletion process. In this study, we have examined whether or not depletion of the HAPs enhances detection of the CCGFs by immuno-assays. Top 14 HAPs were depleted from 10 healthy volunteers’ plasma using MARS-14 immuno-depletion column and a total of 27 CCGFs were analyzed by bead-based multiplexed immuno-assay. All 27 CCGFs were detected in neat plasma (NP, 25 were detected in flow through fraction (FT and 21 were detected in bound protein (BP fraction. Concentrations of 22 CCGFs were significantly higher in NP compared to FT and BP. Only one CCGF had higher concentration in FT compared to NP. The remaining 2 CCGFs were not different between NP and FT. It was counter-productive for the detection of 24 CCGFs after HAP removal, primarily due to post-depletion protein precipitation and/or re-suspension of pellets.

  9. Relative Abundance of Integral Plasma Membrane Proteins in Arabidopsis Leaf and Root Tissue Determined by Metabolic Labeling and Mass Spectrometry

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    Bernfur, Katja; Larsson, Olaf; Larsson, Christer; Gustavsson, Niklas

    2013-01-01

    Metabolic labeling of proteins with a stable isotope (15N) in intact Arabidopsis plants was used for accurate determination by mass spectrometry of differences in protein abundance between plasma membranes isolated from leaves and roots. In total, 703 proteins were identified, of which 188 were predicted to be integral membrane proteins. Major classes were transporters, receptors, proteins involved in membrane trafficking and cell wall-related proteins. Forty-one of the integral proteins, including nine of the 13 isoforms of the PIP (plasma membrane intrinsic protein) aquaporin subfamily, could be identified by peptides unique to these proteins, which made it possible to determine their relative abundance in leaf and root tissue. In addition, peptides shared between isoforms gave information on the proportions of these isoforms. A comparison between our data for protein levels and corresponding data for mRNA levels in the widely used database Genevestigator showed an agreement for only about two thirds of the proteins. By contrast, localization data available in the literature for 21 of the 41 proteins show a much better agreement with our data, in particular data based on immunostaining of proteins and GUS-staining of promoter activity. Thus, although mRNA levels may provide a useful approximation for protein levels, detection and quantification of isoform-specific peptides by proteomics should generate the most reliable data for the proteome. PMID:23990937

  10. Evaluation of three high abundance protein depletion kits for umbilical cord serum proteomics

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    Nie Jing

    2011-05-01

    Full Text Available Abstract Background High abundance protein depletion is a major challenge in the study of serum/plasma proteomics. Prior to this study, most commercially available kits for depletion of highly abundant proteins had only been tested and evaluated in adult serum/plasma, while the depletion efficiency on umbilical cord serum/plasma had not been clarified. Structural differences between some adult and fetal proteins (such as albumin make it likely that depletion approaches for adult and umbilical cord serum/plasma will be variable. Therefore, the primary purposes of the present study are to investigate the efficiencies of several commonly-used commercial kits during high abundance protein depletion from umbilical cord serum and to determine which kit yields the most effective and reproducible results for further proteomics research on umbilical cord serum. Results The immunoaffinity based kits (PROTIA-Sigma and 5185-Agilent displayed higher depletion efficiency than the immobilized dye based kit (PROTBA-Sigma in umbilical cord serum samples. Both the PROTIA-Sigma and 5185-Agilent kit maintained high depletion efficiency when used three consecutive times. Depletion by the PROTIA-Sigma Kit improved 2DE gel quality by reducing smeared bands produced by the presence of high abundance proteins and increasing the intensity of other protein spots. During image analysis using the identical detection parameters, 411 ± 18 spots were detected in crude serum gels, while 757 ± 43 spots were detected in depleted serum gels. Eight spots unique to depleted serum gels were identified by MALDI- TOF/TOF MS, seven of which were low abundance proteins. Conclusions The immunoaffinity based kits exceeded the immobilized dye based kit in high abundance protein depletion of umbilical cord serum samples and dramatically improved 2DE gel quality for detection of trace biomarkers.

  11. A combined blood based gene expression and plasma protein abundance signature for diagnosis of epithelial ovarian cancer - a study of the OVCAD consortium

    International Nuclear Information System (INIS)

    Pils, Dietmar; Sehouli, Jalid; Braicu, Ioana; Vergote, Ignace; Van Gorp, Toon; Mahner, Sven; Concin, Nicole; Speiser, Paul; Zeillinger, Robert; Tong, Dan; Hager, Gudrun; Obermayr, Eva; Aust, Stefanie; Heinze, Georg; Kohl, Maria; Schuster, Eva; Wolf, Andrea

    2013-01-01

    The immune system is a key player in fighting cancer. Thus, we sought to identify a molecular ‘immune response signature’ indicating the presence of epithelial ovarian cancer (EOC) and to combine this with a serum protein biomarker panel to increase the specificity and sensitivity for earlier detection of EOC. Comparing the expression of 32,000 genes in a leukocytes fraction from 44 EOC patients and 19 controls, three uncorrelated shrunken centroid models were selected, comprised of 7, 14, and 6 genes. A second selection step using RT-qPCR data and significance analysis of microarrays yielded 13 genes (AP2A1, B4GALT1, C1orf63, CCR2, CFP, DIS3, NEAT1, NOXA1, OSM, PAPOLG, PRIC285, ZNF419, and BC037918) which were finally used in 343 samples (90 healthy, six cystadenoma, eight low malignant potential tumor, 19 FIGO I/II, and 220 FIGO III/IV EOC patients). Using new 65 controls and 224 EOC patients (thereof 14 FIGO I/II) the abundances of six plasma proteins (MIF, prolactin, CA125, leptin, osteopondin, and IGF2) was determined and used in combination with the expression values from the 13 genes for diagnosis of EOC. Combined diagnostic models using either each five gene expression and plasma protein abundance values or 13 gene expression and six plasma protein abundance values can discriminate controls from patients with EOC with Receiver Operator Characteristics Area Under the Curve values of 0.998 and bootstrap .632+ validated classification errors of 3.1% and 2.8%, respectively. The sensitivities were 97.8% and 95.6%, respectively, at a set specificity of 99.6%. The combination of gene expression and plasma protein based blood derived biomarkers in one diagnostic model increases the sensitivity and the specificity significantly. Such a diagnostic test may allow earlier diagnosis of epithelial ovarian cancer

  12. 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.

  13. Clinical relevance of drug binding to plasma proteins

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    Ascenzi, Paolo; Fanali, Gabriella; Fasano, Mauro; Pallottini, Valentina; Trezza, Viviana

    2014-12-01

    Binding to plasma proteins highly influences drug efficacy, distribution, and disposition. Serum albumin, the most abundant protein in plasma, is a monomeric multi-domain macromolecule that displays an extraordinary ligand binding capacity, providing a depot and carrier for many endogenous and exogenous compounds, such as fatty acids and most acidic drugs. α-1-Acid glycoprotein, the second main plasma protein, is a glycoprotein physiologically involved in the acute phase reaction and is the main carrier for basic and neutral drugs. High- and low-density lipoproteins play a limited role in drug binding and are natural drug delivery system only for few lipophilic drugs or lipid-based formulations. Several factors influence drug binding to plasma proteins, such as pathological conditions, concurrent administration of drugs, sex, and age. Any of these factors, in turn, influences drug efficacy and toxicity. Here, biochemical, biomedical, and biotechnological aspects of drug binding to plasma proteins are reviewed.

  14. Microscale depletion of high abundance proteins in human biofluids using IgY14 immunoaffinity resin: analysis of human plasma and cerebrospinal fluid.

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    Hyung, Seok-Won; Piehowski, Paul D; Moore, Ronald J; Orton, Daniel J; Schepmoes, Athena A; Clauss, Therese R; Chu, Rosalie K; Fillmore, Thomas L; Brewer, Heather; Liu, Tao; Zhao, Rui; Smith, Richard D

    2014-11-01

    Removal of highly abundant proteins in plasma is often carried out using immunoaffinity depletion to extend the dynamic range of measurements to lower abundance species. While commercial depletion columns are available for this purpose, they generally are not applicable to limited sample quantities (depletion media can be prohibitive for larger-scale studies. Modern LC-MS instrumentation provides the sensitivity necessary to scale-down depletion methods with minimal sacrifice to proteome coverage, which makes smaller volume depletion columns desirable for maximizing sample recovery when samples are limited, as well as for reducing the expense of large-scale studies. We characterized the performance of a 346 μL column volume microscale depletion system, using four different flow rates to determine the most effective depletion conditions for ∼6-μL injections of human plasma proteins and then evaluated depletion reproducibility at the optimum flow rate condition. Depletion of plasma using a commercial 10-mL depletion column served as the control. Results showed depletion efficiency of the microscale column increased as flow rate decreased, and that our microdepletion was reproducible. In an initial application, a 600-μL sample of human cerebrospinal fluid (CSF) pooled from multiple sclerosis patients was depleted and then analyzed using reversed phase liquid chromatography-mass spectrometry to demonstrate the utility of the system for this important biofluid where sample quantities are more commonly limited.

  15. Phospholipase D specific for the phosphatidylinositol anchor of cell-surface proteins is abundant in plasma

    International Nuclear Information System (INIS)

    Low, M.G.; Prasad, A.R.S.

    1988-01-01

    An enzyme activity capable of degrading the glycosyl-phosphatidylinositol membrane anchor of cell-surface proteins has previously been reported in a number of mammalian tissues. The experiments reported here demonstrate that this anchor-degrading activity is also abundant in mammalian plasma. The activity was inhibited by EGTA or 1,10-phenanthroline. It was capable of removing the anchor from alkaline phosphatase, 5'-nucleotidase, and variant surface glycoprotein but had little or no activity toward phosphatidylinositol or phosphatidylcholine. Phosphatidic acid was the only 3 H-labeled product when this enzyme hydrolyzed [ 3 H]myristate-labeled variant surface glycoprotein. It could be distinguished from the Ca 2 =-dependent inositol phospholipid-specific phospholipase C activity in several rat tissues on the basis of its molecular size and its sensitivity to 1,10-phenanthroline. The data therefore suggest that this activity is due to a phospholipase D with specificity for glycosylphosphatidylinositol structures. Although the precise physiological function of this anchor-specific phospholipase D remains to be determined, these findings indicate that it could play an important role in regulating the expression and release of cell-surface proteins in vivo

  16. Increased uncoupling protein-2 mRNA abundance and glucocorticoid action in adipose tissue in the sheep fetus during late gestation is dependent on plasma cortisol and triiodothyronine

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    Gnanalingham, MG; Mostyn, A; Forhead, AJ; Fowden, AL; Symonds, ME; Stephenson, T

    2005-01-01

    The endocrine regulation of uncoupling protein-2 (UCP2), an inner mitochondrial protein, in fetal adipose tissue remains unclear. The present study aimed to determine if fetal plasma cortisol and triiodothyronine (T3) influenced the mRNA abundance of UCP2, glucocorticoid receptor (GR) and 11β-hydroxysteroid dehydrogenase type 1 (11βHSD1) and 2 (11βHSD2) in fetal adipose tissue in the sheep during late gestation. Perirenal–abdominal adipose tissue was sampled from ovine fetuses to which either cortisol (2–3 mg kg−1 day−1) or saline was infused for 5 days up to 127–130 days gestation, or near term fetuses (i.e. 142–145 days gestation) that were either adrenalectomised (AX) or remained intact. Fetal plasma cortisol and T3 concentrations were higher in the cortisol infused animals and lower in AX fetuses compared with their corresponding control group, and increased with gestational age. UCP2 and GR mRNA abundance were significantly lower in AX fetuses compared with age-matched controls, and increased with gestational age and by cortisol infusion. Glucocorticoid action in fetal adipose tissue was augmented by AX and suppressed by cortisol infusion, the latter also preventing the gestational increase in 11βHSD1 mRNA and decrease in 11βHSD2 mRNA. When all treatment groups were combined, both fetal plasma cortisol and T3 concentrations were positively correlated with UCP2, GR and 11βHSD2 mRNA abundance, but negatively correlated with 11βHSD1 mRNA abundance. In conclusion, plasma cortisol and T3 are both required for the late gestation rise in UCP2 mRNA and differentially regulate glucocorticoid action in fetal adipose tissue in the sheep during late gestation. PMID:15961419

  17. ESCRT-dependent degradation of ubiquitylated plasma membrane proteins in plants.

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    Isono, Erika; Kalinowska, Kamila

    2017-12-01

    To control the abundance of plasma membrane receptors and transporters is crucial for proper perception and response to extracellular signals from surrounding cells and the environment. Posttranslational modification of plasma membrane proteins, especially ubiquitin conjugation or ubiquitylation, is key for the determination of stability for many transmembrane proteins localized on the cell surface. The targeted degradation is ensured by a complex network of proteins among which the endosomal sorting complex required for transport (ESCRT) plays a central role. This review focuses on progresses made in recent years on the understanding of the function of the ESCRT machinery in the degradation of ubiquitylated plasma membrane proteins in plants. Copyright © 2017 Elsevier Ltd. All rights reserved.

  18. Comparison of Depletion Strategies for the Enrichment of Low-Abundance Proteins in Urine.

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    Filip, Szymon; Vougas, Konstantinos; Zoidakis, Jerome; Latosinska, Agnieszka; Mullen, William; Spasovski, Goce; Mischak, Harald; Vlahou, Antonia; Jankowski, Joachim

    2015-01-01

    Proteome analysis of complex biological samples for biomarker identification remains challenging, among others due to the extended range of protein concentrations. High-abundance proteins like albumin or IgG of plasma and urine, may interfere with the detection of potential disease biomarkers. Currently, several options are available for the depletion of abundant proteins in plasma. However, the applicability of these methods in urine has not been thoroughly investigated. In this study, we compared different, commercially available immunodepletion and ion-exchange based approaches on urine samples from both healthy subjects and CKD patients, for their reproducibility and efficiency in protein depletion. A starting urine volume of 500 μL was used to simulate conditions of a multi-institutional biomarker discovery study. All depletion approaches showed satisfactory reproducibility (n=5) in protein identification as well as protein abundance. Comparison of the depletion efficiency between the unfractionated and fractionated samples and the different depletion strategies, showed efficient depletion in all cases, with the exception of the ion-exchange kit. The depletion efficiency was found slightly higher in normal than in CKD samples and normal samples yielded more protein identifications than CKD samples when using both initial as well as corresponding depleted fractions. Along these lines, decrease in the amount of albumin and other targets as applicable, following depletion, was observed. Nevertheless, these depletion strategies did not yield a higher number of identifications in neither the urine from normal nor CKD patients. Collectively, when analyzing urine in the context of CKD biomarker identification, no added value of depletion strategies can be observed and analysis of unfractionated starting urine appears to be preferable.

  19. Plasma Protein Turnover Rates in Rats Using Stable Isotope Labeling, Global Proteomics, and Activity-Based Protein Profiling

    Energy Technology Data Exchange (ETDEWEB)

    Smith, Jordan N.; Tyrrell, Kimberly J.; Hansen, Joshua R.; Thomas, Dennis G.; Murphree, Taylor A.; Shukla, Anil K.; Luders, Teresa; Madden, James M.; Li, Yunying; Wright, Aaron T.; Piehowski, Paul D.

    2017-12-06

    Protein turnover is important for general health on cellular and organism scales providing a strategy to replace old, damaged, or dysfunctional proteins. Protein turnover also informs of biomarker kinetics, as a better understanding of synthesis and degradation of proteins increases the clinical utility of biomarkers. Here, turnover rates of plasma proteins in rats were measured in vivo using a pulse-chase stable isotope labeling experiment. During the pulse, rats (n=5) were fed 13C6-labeled lysine (“heavy”) feed for 23 days to label proteins. During the chase, feed was changed to an unlabeled equivalent feed (“light”), and blood was repeatedly sampled from rats over 10 time points for 28 days. Plasma samples were digested with trypsin, and analyzed with liquid chromatography-tandem mass spectrometry (LC-MS/MS). MaxQuant was used to identify peptides and proteins, and quantify heavy:light lysine ratios. A system of ordinary differential equations was used to calculate protein turnover rates. Using this approach, 273 proteins were identified, and turnover rates were quantified for 157 plasma proteins with half-lives ranging 0.3-103 days. For the ~70 most abundant proteins, variability in turnover rates among rats was low (median coefficient of variation: 0.09). Activity-based protein profiling was applied to pooled plasma samples to enrich serine hydrolases using a fluorophosphonate (FP2) activity-based probe. This enrichment resulted in turnover rates for an additional 17 proteins. This study is the first to measure global plasma protein turnover rates in rats in vivo, measure variability of protein turnover rates in any animal model, and utilize activity-based protein profiling for enhancing measurements of targeted, low-abundant proteins, such as those commonly used as biomarkers. Measured protein turnover rates will be important for understanding of the role of protein turnover in cellular and organism health as well as increasing the utility of protein

  20. Evidence for radical-oxidation of plasma proteins in humans

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    Wang, D.; Davies, M.; Dean, R.; Fu, S.; Taurins, A.; Sullivans, D.

    1998-01-01

    Oxidation of proteins by radicals has been implicated in many pathological processes. The hydroxyl radical is known to generate protein-bound hydroxylated derivatives of amino acids, for example hydroxyvaline (from Val), hydroxyleucine (from Leu), o-tyrosine (from Phe), and DOPA (from Tyr). In this study, we have investigated the occurrence of these oxidised amino acids in human plasma proteins from both normal subjects and dialysis patients. By employing previously established HPLC methods [Fu et al. Biochemical Journal, 330, 233-239, 1998], we have found that oxidised amino acids exist in normal human plasma proteins (n=32). The level of these oxidised amino acids is not correlated to age. Similar levels of oxidised amino acids are found in the plasma proteins of the dialysis patients (n=6), but a more detailed survey is underway. The relative abundance of the oxidised amino acids is similar to that resulting from oxidation of BSA by hydroxy radicals or Fenton systems [Fu et al. Biochemical Journal, 333, 519-525, 1998]. The results suggest that metal-ion catalysed oxyl-radical chemistry may be a key contributor to the oxidative damage in plasma proteins in vivo in humans

  1. Protein abundance profiling of the Escherichia coli cytosol

    DEFF Research Database (Denmark)

    Ishihama, Y.; Schmidt, T.; Rappsilber, J.

    2008-01-01

    sample. Using a combination of LC-MS/MS approaches with protein and peptide fractionation steps we identified 1103 proteins from the cytosolic fraction of the Escherichia coli strain MC4100. A measure of abundance is presented for each of the identified proteins, based on the recently developed emPAI...... approach which takes into account the number of sequenced peptides per protein. The values of abundance are within a broad range and accurately reflect independently measured copy numbers per cell. As expected, the most abundant proteins were those involved in protein synthesis, most notably ribosomal...

  2. The effect of colostrum intake on blood plasma proteome profile in newborn lambs: low abundance proteins

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    Hernandez Castellano, Lorenzo E; de Almeida, André Martinho; Ventosa, Miguel

    2014-01-01

    Colostrum intake by newborn lambs plays a fundamental role in the perinatal period, ensuring lamb survival. In this study, blood plasma samples from two groups of newborn lambs (Colostrum group and Delayed Colostrum group) at 2 and 14 h after birth were treated to reduce the content of high abund...

  3. Identification of clam plasma proteins that bind its pathogen Quahog Parasite Unknown.

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    Hartman, Rachel; Pales Espinosa, Emmanuelle; Allam, Bassem

    2018-06-01

    The hard clam (Mercenaria mercenaria) is among the most economically-important marine species along the east coast of the United States, representing the first marine resource in several Northeastern states. The species is rather resilient to infections and the only important disease of hard clams results from an infection caused by Quahog Parasite Unknown (QPX), a protistan parasite that can lead to significant mortality events in wild and aquacultured clam stocks. Though the presence of QPX disease has been documented since the 1960s, little information is available on cellular and molecular interactions between the parasite and the host. This study examined the interactions between the clam immune system and QPX cells. First, the effect of clam plasma on the binding of hemocytes to parasite cells was evaluated. Second, clam plasma proteins that bind QPX cells were identified through proteomic (LC-MS/MS) analyses. Finally, the effect of prior clam exposure to QPX on the abundance of QPX-reactive proteins in the plasma was evaluated. Results showed that plasma factors enhance the attachment of hemocytes to QPX. Among the proteins that specifically bind to QPX cells, several lectins were identified, as well as complement component proteins and proteolytic enzymes. Furthermore, results showed that some of these lectins and complement-related proteins are inducible as their abundance significantly increased following QPX challenge. These results shed light on plasma proteins involved in the recognition and binding of parasite cells and provide molecular targets for future investigations of factors involved in clam resistance to the disease, and ultimately for the selection of resistant clam stocks. Copyright © 2018 Elsevier Ltd. All rights reserved.

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

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    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.

  5. PLASMA PROTEIN AND HEMOGLOBIN PRODUCTION

    Science.gov (United States)

    Robscheit-Robbins, F. S.; Miller, L. L.; Whipple, G. H.

    1947-01-01

    Given healthy dogs fed abundant iron and protein-free or low protein diets with sustained anemia and hypoproteinemia, we can study the capacity of these animals to produce simultaneously new hemoglobin and plasma protein. Reserve stores of blood protein-building materials are measurably depleted and levels of 6 to 8 gm. per cent for hemoglobin and 4 to 5 gm. per cent for plasma protein can be maintained for weeks or months depending upon the intake of food proteins or amino acid mixtures. These dogs are very susceptible to infection and various poisons. Dogs tire of these diets and loss of appetite terminates many experiments. Under these conditions (double depletion) standard growth mixtures of essential amino acids are tested to show the response in blood protein output and urinary nitrogen balance. As a part of each tabulated experiment one of the essential amino acids is deleted from the complete growth mixture to compare such response with that of the whole mixture. Methionine, threonine, phenylalanine, and tryptophane when singly eliminated from the complete amino acid mixture do effect a sharp rise in urinary nitrogen. This loss of urinary nitrogen is corrected when the individual amino acid is replaced in the mixture. Histidine, lysine, and valine have a moderate influence upon urinary nitrogen balance toward nitrogen conservation. Leucine, isoleucine, and arginine have minimal or no effect upon urinary nitrogen balance when these individual amino acids are deleted from the complete growth mixture of amino acids during 3 to 4 week periods. Tryptophane and to a less extent phenylalanine and threonine when returned to the amino acid mixture are associated with a conspicuous preponderance of plasma protein output over the hemoglobin output (Table 4). Arginine, lysine, and histidine when returned to the amino acid mixture are associated with a large preponderance of hemoglobin output. Various amino acid mixtures under these conditions may give a positive

  6. Plasma Membrane Protein Profiling in Beta-Amyloid-Treated Microglia Cell Line.

    Science.gov (United States)

    Correani, Virginia; Di Francesco, Laura; Mignogna, Giuseppina; Fabrizi, Cinzia; Leone, Stefano; Giorgi, Alessandra; Passeri, Alessia; Casata, Roberto; Fumagalli, Lorenzo; Maras, Bruno; Schininà, M Eugenia

    2017-09-01

    In the responsiveness of microglia to toxic stimuli, plasma membrane proteins play a key role. In this study we treated with a synthetic beta amyloid peptide murine microglial cells metabolically differently labelled with stable isotope amino acids (SILAC). The plasma membrane was selectively enriched by a multi-stage aqueous two-phase partition system. We were able to identify by 1D-LC-MS/MS analyses 1577 proteins, most of them are plasma membrane proteins according to the Gene Ontology annotation. An unchanged level of amyloid receptors in this data set suggests that microglia preserve their responsiveness capability to the environment even after 24-h challenge with amyloid peptides. On the other hand, 14 proteins were observed to change their plasma membrane abundance to a statistically significant extent. Among these, we proposed as reliable biomarkers of the inflammatory microglia phenotype in AD damaged tissues MAP/microtubule affinity-regulating kinase 3 (MARK3), Interferon-induced transmembrane protein 3 (IFITM3), Annexins A5 and A7 (ANXA5, ANXA7) and Neuropilin-1 (NRP1), all proteins known to be involved in the inflammation processes and in microtubule network assembly rate. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  7. Proteomics of plasma membranes from poplar trees reveals tissue distribution of transporters, receptors, and proteins in cell wall formation.

    Science.gov (United States)

    Nilsson, Robert; Bernfur, Katja; Gustavsson, Niklas; Bygdell, Joakim; Wingsle, Gunnar; Larsson, Christer

    2010-02-01

    By exploiting the abundant tissues available from Populus trees, 3-4 m high, we have been able to isolate plasma membranes of high purity from leaves, xylem, and cambium/phloem at a time (4 weeks after bud break) when photosynthesis in the leaves and wood formation in the xylem should have reached a steady state. More than 40% of the 956 proteins identified were found in the plasma membranes of all three tissues and may be classified as "housekeeping" proteins, a typical example being P-type H(+)-ATPases. Among the 213 proteins predicted to be integral membrane proteins, transporters constitute the largest class (41%) followed by receptors (14%) and proteins involved in cell wall and carbohydrate metabolism (8%) and membrane trafficking (8%). ATP-binding cassette transporters (all members of subfamilies B, C, and G) and receptor-like kinases (four subfamilies) were two of the largest protein families found, and the members of these two families showed pronounced tissue distribution. Leaf plasma membranes were characterized by a very high proportion of transporters, constituting almost half of the integral proteins. Proteins involved in cell wall synthesis (such as cellulose and sucrose synthases) and membrane trafficking were most abundant in xylem plasma membranes in agreement with the role of the xylem in wood formation. Twenty-five integral proteins and 83 soluble proteins were exclusively found in xylem plasma membranes, which identifies new candidates associated with cell wall synthesis and wood formation. Among the proteins uniquely found in xylem plasma membranes were most of the enzymes involved in lignin biosynthesis, which suggests that they may exist as a complex linked to the plasma membrane.

  8. Identification of Differentially Abundant Proteins of Edwardsiella ictaluri during Iron Restriction.

    Directory of Open Access Journals (Sweden)

    Pradeep R Dumpala

    Full Text Available Edwardsiella ictaluri is a Gram-negative facultative anaerobe intracellular bacterium that causes enteric septicemia in channel catfish. Iron is an essential inorganic nutrient of bacteria and is crucial for bacterial invasion. Reduced availability of iron by the host may cause significant stress for bacterial pathogens and is considered a signal that leads to significant alteration in virulence gene expression. However, the precise effect of iron-restriction on E. ictaluri protein abundance is unknown. The purpose of this study was to identify differentially abundant proteins of E. ictaluri during in vitro iron-restricted conditions. We applied two-dimensional difference in gel electrophoresis (2D-DIGE for determining differentially abundant proteins and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF/TOF MS for protein identification. Gene ontology and pathway-based functional modeling of differentially abundant proteins was also conducted. A total of 50 unique differentially abundant proteins at a minimum of 2-fold (p ≤ 0.05 difference in abundance due to iron-restriction were detected. The numbers of up- and down-regulated proteins were 37 and 13, respectively. We noted several proteins, including EsrB, LamB, MalM, MalE, FdaA, and TonB-dependent heme/hemoglobin receptor family proteins responded to iron restriction in E. ictaluri.

  9. Distinctive serum protein profiles involving abundant proteins in lung cancer patients based upon antibody microarray analysis

    Directory of Open Access Journals (Sweden)

    Rom William N

    2005-08-01

    Full Text Available Abstract Background Cancer serum protein profiling by mass spectrometry has uncovered mass profiles that are potentially diagnostic for several common types of cancer. However, direct mass spectrometric profiling has a limited dynamic range and difficulties in providing the identification of the distinctive proteins. We hypothesized that distinctive profiles may result from the differential expression of relatively abundant serum proteins associated with the host response. Methods Eighty-four antibodies, targeting a wide range of serum proteins, were spotted onto nitrocellulose-coated microscope slides. The abundances of the corresponding proteins were measured in 80 serum samples, from 24 newly diagnosed subjects with lung cancer, 24 healthy controls, and 32 subjects with chronic obstructive pulmonary disease (COPD. Two-color rolling-circle amplification was used to measure protein abundance. Results Seven of the 84 antibodies gave a significant difference (p Conclusion Our results suggest that a distinctive serum protein profile involving abundant proteins may be observed in lung cancer patients relative to healthy subjects or patients with chronic disease and may have utility as part of strategies for detecting lung cancer.

  10. Differential abundances of four forms of Binder of SPerm 1 in the seminal plasma of Bos taurus indicus bulls with different patterns of semen freezability.

    Science.gov (United States)

    Magalhães, Marcos Jorge; Martins, Leonardo Franco; Senra, Renato Lima; Santos, Thaís Ferreira Dos; Okano, Denise Silva; Pereira, Paulo Roberto Gomes; Faria-Campos, Alessandra; Campos, Sérgio Vale Aguiar; Guimarães, José Domingos; Baracat-Pereira, Maria Cristina

    2016-08-01

    The Binder of SPerm 1 (BSP1) protein is involved in the fertilization and semen cryopreservation processes and is described to be both beneficial and detrimental to sperm. Previously, the relationship of BSP1 with freezability events has not been completely understood. The objective of this work was to determine the differential abundance of the forms of the BSP1 protein in cryopreserved seminal plasma of Bos taurus indicus bulls with different patterns of semen freezability using proteomics. A wide cohort of adult bulls with high genetic value from an artificial insemination center was used as donors of high quality, fresh semen. Nine bulls presenting different patterns of semen freezability were selected. Two-dimensional gel electrophoresis showed differential abundance in a group of seven protein spots in the frozen/thawed seminal plasma from the bulls, ranging from 15 to 17 kDa, with pI values from 4.6 to 5.8. Four of these spots were confirmed to be BSP1 using mass spectrometry, proteomics, biochemical, and computational analysis (Tukey's test at P semen freezability and its absence in bulls presenting high semen freezability. This is the first report showing that more than two forms of BSP1 are found in the seminal plasma of Nelore adult bulls and not all animals have a similar abundance of each BSP1 form. Different BSP1 forms may be involved in different events of fertilization and the cryopreservation process. Copyright © 2016 Elsevier Inc. All rights reserved.

  11. Simplified Enrichment of Plasma Membrane Proteins from Arabidopsis thaliana Seedlings Using Differential Centrifugation and Brij-58 Treatment.

    Science.gov (United States)

    Collins, Carina A; Leslie, Michelle E; Peck, Scott C; Heese, Antje

    2017-01-01

    The plasma membrane (PM) forms a barrier between a plant cell and its environment. Proteins at this subcellular location play diverse and complex roles, including perception of extracellular signals to coordinate cellular changes. Analyses of PM proteins, however, are often limited by the relatively low abundance of these proteins in the total cellular protein pool. Techniques traditionally used for enrichment of PM proteins are time consuming, tedious, and require extensive optimization. Here, we provide a simple and reproducible enrichment procedure for PM proteins from Arabidopsis thaliana seedlings starting from total microsomal membranes isolated by differential centrifugation. To enrich for PM proteins, total microsomes are treated with the nonionic detergent Brij-58 to decrease the abundance of contaminating organellar proteins. This protocol combined with the genetic resources available in Arabidopsis provides a powerful tool that will enhance our understanding of proteins at the PM.

  12. Highly efficient enrichment of low-abundance intact proteins by core-shell structured Fe3O4-chitosan@graphene composites.

    Science.gov (United States)

    Zhang, Peng; Fang, Xiaoni; Yan, Guoquan; Gao, Mingxia; Zhang, Xiangmin

    2017-11-01

    In proteomics research, the screening and monitoring of disease biomarkers is still a major challenge, mainly due to their low concentration in biological samples. However, the universal enrichment of intact proteins has not been further studied. In this work, we developed a Fe 3 O 4 -chitosan@graphene (Fe 3 O 4 -CS@G) core-shell composite to enrich low-abundance proteins from biological samples. Fe 3 O 4 -CS@G composite holds chitosan layer decorated Fe 3 O 4 core, which improves the hydrophilicity of materials greatly. Meanwhile, the graphene nanosheets shell formed via electrostatic assembly endows the composite with huge surface area (178m 2 /g). The good water dispersibility ensures the sufficient contact opportunities between graphene composites and proteins, and the large surface area provides enough adsorption sites for the enrichment of proteins. Using Fe 3 O 4 -CS@G, four standard proteins Cyt-c, BSA, Myo and OVA were enriched with better adsorption capacity and recovery rate, compared with previously reported magnetic graphene composites. Additionally, the mechanism of compared to" is corrected into "compared with". proteins adsorption on Fe 3 O 4 -CS@G was further studied, which indicates that hydrophobic and electrostatic interaction work together to facilitate the universal and efficient enrichment of proteins. Human plasma sample was employed to further evaluate the enrichment performance of Fe 3 O 4 -CS@G. Eventually, 123 proteins were identified from one of SAX fractions of human plasma, which is much better than commercial Sep-pak C18 enrichment column (39 proteins). All these outstanding performances suggest that Fe 3 O 4 -CS@G is an ideal platform for the enrichment of low-abundance intact proteins and thus holds great potential to facilitate the identification of biomarkers from biological samples in proteomics research. Copyright © 2017 Elsevier B.V. All rights reserved.

  13. Distinctive serum protein profiles involving abundant proteins in lung cancer patients based upon antibody microarray analysis

    International Nuclear Information System (INIS)

    Gao, Wei-Min; Haab, Brian B; Hanash, Samir M; Kuick, Rork; Orchekowski, Randal P; Misek, David E; Qiu, Ji; Greenberg, Alissa K; Rom, William N; Brenner, Dean E; Omenn, Gilbert S

    2005-01-01

    Cancer serum protein profiling by mass spectrometry has uncovered mass profiles that are potentially diagnostic for several common types of cancer. However, direct mass spectrometric profiling has a limited dynamic range and difficulties in providing the identification of the distinctive proteins. We hypothesized that distinctive profiles may result from the differential expression of relatively abundant serum proteins associated with the host response. Eighty-four antibodies, targeting a wide range of serum proteins, were spotted onto nitrocellulose-coated microscope slides. The abundances of the corresponding proteins were measured in 80 serum samples, from 24 newly diagnosed subjects with lung cancer, 24 healthy controls, and 32 subjects with chronic obstructive pulmonary disease (COPD). Two-color rolling-circle amplification was used to measure protein abundance. Seven of the 84 antibodies gave a significant difference (p < 0.01) for the lung cancer patients as compared to healthy controls, as well as compared to COPD patients. Proteins that exhibited higher abundances in the lung cancer samples relative to the control samples included C-reactive protein (CRP; a 13.3 fold increase), serum amyloid A (SAA; a 2.0 fold increase), mucin 1 and α-1-antitrypsin (1.4 fold increases). The increased expression levels of CRP and SAA were validated by Western blot analysis. Leave-one-out cross-validation was used to construct Diagonal Linear Discriminant Analysis (DLDA) classifiers. At a cutoff where all 56 of the non-tumor samples were correctly classified, 15/24 lung tumor patient sera were correctly classified. Our results suggest that a distinctive serum protein profile involving abundant proteins may be observed in lung cancer patients relative to healthy subjects or patients with chronic disease and may have utility as part of strategies for detecting lung cancer

  14. Detecting significant changes in protein abundance

    Directory of Open Access Journals (Sweden)

    Kai Kammers

    2015-06-01

    Full Text Available We review and demonstrate how an empirical Bayes method, shrinking a protein's sample variance towards a pooled estimate, leads to far more powerful and stable inference to detect significant changes in protein abundance compared to ordinary t-tests. Using examples from isobaric mass labelled proteomic experiments we show how to analyze data from multiple experiments simultaneously, and discuss the effects of missing data on the inference. We also present easy to use open source software for normalization of mass spectrometry data and inference based on moderated test statistics.

  15. Deep-Dive Targeted Quantification for Ultrasensitive Analysis of Proteins in Nondepleted Human Blood Plasma/Serum and Tissues

    Energy Technology Data Exchange (ETDEWEB)

    Nie, Song [Biological Sciences Division; Shi, Tujin [Biological Sciences Division; Fillmore, Thomas L. [Biological Sciences Division; Schepmoes, Athena A. [Biological Sciences Division; Brewer, Heather [Biological Sciences Division; Gao, Yuqian [Biological Sciences Division; Song, Ehwang [Biological Sciences Division; Wang, Hui [Biological Sciences Division; Rodland, Karin D. [Biological Sciences Division; Qian, Wei-Jun [Biological Sciences Division; Smith, Richard D. [Biological Sciences Division; Liu, Tao [Biological Sciences Division

    2017-08-11

    Mass spectrometry-based targeted proteomics (e.g., selected reaction monitoring, SRM) is emerging as an attractive alternative to immunoassays for protein quantification. Recently we have made significant progress in SRM sensitivity for enabling quantification of low ng/mL to sub-ng/mL level proteins in nondepleted human blood plasma/serum without affinity enrichment. However, precise quantification of extremely low abundant but biologically important proteins (e.g., ≤100 pg/mL in blood plasma/serum) using targeted proteomics approaches still remains challenging. To address this need, we have developed an antibody-independent Deep-Dive SRM (DD-SRM) approach that capitalizes on multidimensional high-resolution reversed-phase liquid chromatography (LC) separation for target peptide enrichment combined with precise selection of target peptide fractions of interest, significantly improving SRM sensitivity by ~5 orders of magnitude when compared to conventional LC-SRM. Application of DD-SRM to human serum and tissue has been demonstrated to enable precise quantification of endogenous proteins at ~10 pg/mL level in nondepleted serum and at <10 copies per cell level in tissue. Thus, DD-SRM holds great promise for precisely measuring extremely low abundance proteins or protein modifications, especially when high-quality antibody is not available.

  16. Overexpression of BAX INHIBITOR-1 Links Plasma Membrane Microdomain Proteins to Stress.

    Science.gov (United States)

    Ishikawa, Toshiki; Aki, Toshihiko; Yanagisawa, Shuichi; Uchimiya, Hirofumi; Kawai-Yamada, Maki

    2015-10-01

    BAX INHIBITOR-1 (BI-1) is a cell death suppressor widely conserved in plants and animals. Overexpression of BI-1 enhances tolerance to stress-induced cell death in plant cells, although the molecular mechanism behind this enhancement is unclear. We recently found that Arabidopsis (Arabidopsis thaliana) BI-1 is involved in the metabolism of sphingolipids, such as the synthesis of 2-hydroxy fatty acids, suggesting the involvement of sphingolipids in the cell death regulatory mechanism downstream of BI-1. Here, we show that BI-1 affects cell death-associated components localized in sphingolipid-enriched microdomains of the plasma membrane in rice (Oryza sativa) cells. The amount of 2-hydroxy fatty acid-containing glucosylceramide increased in the detergent-resistant membrane (DRM; a biochemical counterpart of plasma membrane microdomains) fraction obtained from BI-1-overexpressing rice cells. Comparative proteomics analysis showed quantitative changes of DRM proteins in BI-1-overexpressing cells. In particular, the protein abundance of FLOTILLIN HOMOLOG (FLOT) and HYPERSENSITIVE-INDUCED REACTION PROTEIN3 (HIR3) markedly decreased in DRM of BI-1-overexpressing cells. Loss-of-function analysis demonstrated that FLOT and HIR3 are required for cell death by oxidative stress and salicylic acid, suggesting that the decreased levels of these proteins directly contribute to the stress-tolerant phenotypes in BI-1-overexpressing rice cells. These findings provide a novel biological implication of plant membrane microdomains in stress-induced cell death, which is negatively modulated by BI-1 overexpression via decreasing the abundance of a set of key proteins involved in cell death. © 2015 American Society of Plant Biologists. All Rights Reserved.

  17. Plasma levels of selenium-containing proteins in Inuit adults from Nunavik.

    Science.gov (United States)

    Achouba, Adel; Dumas, Pierre; Ouellet, Nathalie; Lemire, Mélanie; Ayotte, Pierre

    2016-11-01

    Selenium (Se) is highly abundant in marine foods traditionally consumed by Inuit of Nunavik (Northern Quebec, Canada) and accordingly, their Se intake is among the highest in the world. However, little is known regarding the biological implications of this high Se status in this Arctic indigenous population. We used a method combining affinity chromatography and inductively coupled plasma-mass spectrometry with quantification by post-column isotope dilution to determine total Se levels and concentrations of Se-containing proteins in archived plasma samples of Inuit adults who participated to the 2004 Nunavik Inuit Health Survey (N = 852). Amounts of mercury (Hg) associated with Se-containing proteins were also quantified. Results show that glutathione peroxidase 3 (GPx3), selenoprotein P (SelP) and selenoalbumin (SeAlb) represented respectively 25%, 52% and 23% of total plasma Se concentrations. In addition, small amounts of Hg co-eluted with each Se-containing protein and up to 50% of plasma Hg was associated to SelP. Total plasma Se concentrations (median = 139 μg L− 1; interquartile range (IQR) = 22.7 μg L− 1) were markedly lower and less variable than whole blood Se concentration (median = 261 μg L− 1, IQR = 166 μg L− 1). A non linear relation was observed between whole blood Se and plasma Se levels, with plasma Se concentrations leveling off at approximately 200 μg L− 1, whereas 16% and 3% of individuals exhibited whole blood concentrations higher than 500 μg L− 1 and 1000 μg L− 1, respectively. In contrast, a linear relationship was previously reported in communities consuming Brazil nuts which are rich Se, mainly present as selenomethionine. This suggests that a different selenocompound, possibly selenoneine, is present in the Arctic marine food chain and accumulates in the blood cellular fraction of Inuit.

  18. Population-specific plasma proteomes of marine and freshwater three-spined sticklebacks (Gasterosteus aculeatus).

    Science.gov (United States)

    Kültz, Dietmar; Li, Johnathon; Zhang, Xuezhen; Villarreal, Fernando; Pham, Tuan; Paguio, Darlene

    2015-12-01

    Molecular phenotypes that distinguish resident marine (Bodega Harbor) from landlocked freshwater (FW, Lake Solano) three-spined sticklebacks were revealed by label-free quantitative proteomics. Secreted plasma proteins involved in lipid transport, blood coagulation, proteolysis, plasminogen-activating cascades, extracellular stimulus responses, and immunity are most abundant in this species. Globulins and albumins are much less abundant than in mammalian plasma. Unbiased quantitative proteome profiling identified 45 highly population-specific plasma proteins. Population-specific abundance differences were validated by targeted proteomics based on data-independent acquisition. Gene ontology enrichment analyses and known functions of population-specific plasma proteins indicate enrichment of processes controlling cell adhesion, tissue remodeling, proteolytic processing, and defense signaling in marine sticklebacks. Moreover, fetuin B and leukocyte cell derived chemotaxin 2 are much more abundant in marine fish. These proteins promote bone morphogenesis and likely contribute to population-specific body armor differences. Plasma proteins enriched in FW fish promote translation, heme biosynthesis, and lipid transport, suggesting a greater presence of plasma microparticles. Many prominent population-specific plasma proteins (e.g. apoptosis-associated speck-like protein containing a CARD) lack any homolog of known function or adequate functional characterization. Their functional characterization and the identification of population-specific environmental contexts and selective pressures that cause plasma proteome diversification are future directions emerging from this study. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. Two novel heat-soluble protein families abundantly expressed in an anhydrobiotic tardigrade.

    Directory of Open Access Journals (Sweden)

    Ayami Yamaguchi

    Full Text Available Tardigrades are able to tolerate almost complete dehydration by reversibly switching to an ametabolic state. This ability is called anhydrobiosis. In the anhydrobiotic state, tardigrades can withstand various extreme environments including space, but their molecular basis remains largely unknown. Late embryogenesis abundant (LEA proteins are heat-soluble proteins and can prevent protein-aggregation in dehydrated conditions in other anhydrobiotic organisms, but their relevance to tardigrade anhydrobiosis is not clarified. In this study, we focused on the heat-soluble property characteristic of LEA proteins and conducted heat-soluble proteomics using an anhydrobiotic tardigrade. Our heat-soluble proteomics identified five abundant heat-soluble proteins. All of them showed no sequence similarity with LEA proteins and formed two novel protein families with distinct subcellular localizations. We named them Cytoplasmic Abundant Heat Soluble (CAHS and Secretory Abundant Heat Soluble (SAHS protein families, according to their localization. Both protein families were conserved among tardigrades, but not found in other phyla. Although CAHS protein was intrinsically unstructured and SAHS protein was rich in β-structure in the hydrated condition, proteins in both families changed their conformation to an α-helical structure in water-deficient conditions as LEA proteins do. Two conserved repeats of 19-mer motifs in CAHS proteins were capable to form amphiphilic stripes in α-helices, suggesting their roles as molecular shield in water-deficient condition, though charge distribution pattern in α-helices were different between CAHS and LEA proteins. Tardigrades might have evolved novel protein families with a heat-soluble property and this study revealed a novel repertoire of major heat-soluble proteins in these anhydrobiotic animals.

  20. 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...... more efficient. The reaction of fresh diluted plasma with HOCl also gives rise to protein-derived nitrogen-centred radicals in a time- and HOCl-concentration-dependent manner; these have been detected by EPR spin trapping. Identical radicals have been detected with isolated HOCl-treated plasma proteins...

  1. The Origin of Solar Filament Plasma Inferred from In Situ Observations of Elemental Abundances

    Energy Technology Data Exchange (ETDEWEB)

    Song, H. Q.; Chen, Y.; Li, B. [Shandong Provincial Key Laboratory of Optical Astronomy and Solar-Terrestrial Environment, and Institute of Space Sciences, Shandong University, Weihai, Shandong 264209 (China); Li, L. P. [Key Laboratory of Solar Activity, National Astronomical Observatories, Chinese Academy of Sciences, Beijing 100012 (China); Zhao, L. [Department of Climate and Space sciences and Engineering, University of Michigan, Ann Arbor, MI 48105 (United States); He, J. S.; Duan, D. [School of Earth and Space Sciences, Peking University, Beijing 100871 (China); Cheng, X. [School of Astronomy and Space Science, Nanjing University, Nanjing, Jiangsu 210093 (China); Zhang, J., E-mail: hqsong@sdu.edu.cn [Department of Physics and Astronomy, George Mason University, Fairfax, VA 22030 (United States)

    2017-02-10

    Solar filaments/prominences are one of the most common features in the corona, which may lead to energetic coronal mass ejections (CMEs) and flares when they erupt. Filaments are about 100 times cooler and denser than the coronal material, and physical understanding of their material origin remains controversial. Two types of scenarios have been proposed: one argues that the filament plasma is brought into the corona from photosphere or chromosphere through a siphon or evaporation/injection process, while the other suggests that the material condenses from the surrounding coronal plasma due to thermal instability. The elemental abundance analysis is a reasonable clue to constrain the models, as the siphon or evaporation/injection model would predict that the filament material abundances are close to the photospheric or chromospheric ones, while the condensation model should have coronal abundances. In this Letter, we analyze the elemental abundances of a magnetic cloud that contains the ejected filament material. The corresponding filament eruption occurred on 1998 April 29, accompanying an M6.8 class soft X-ray flare located at the heliographic coordinates S18E20 (NOAA 08210) and a fast halo CME with the linear velocity of 1374 km s{sup −1} near the Sun. We find that the abundance ratios of elements with low and high first ionization potential such as Fe/O, Mg/O, and Si/O are 0.150, 0.050, and 0.070, respectively, approaching their corresponding photospheric values 0.065, 0.081, and 0.066, which does not support the coronal origin of the filament plasma.

  2. Dextrose-mediated aggregation of therapeutic monoclonal antibodies in human plasma: Implication of isoelectric precipitation of complement proteins.

    Science.gov (United States)

    Luo, Shen; Zhang, Baolin

    2015-01-01

    Many therapeutic monoclonal antibodies (mAbs) are clinically administered through intravenous infusion after mixing with a diluent, e.g., saline, 5% dextrose. Such a clinical setting increases the likelihood of interactions among mAb molecules, diluent, and plasma components, which may adversely affect product safety and efficacy. Avastin® (bevacizumab) and Herceptin® (trastuzumab), but not Remicade® (infliximab), were shown to undergo rapid aggregation upon dilution into 5% dextrose when mixed with human plasma in vitro; however, the biochemical pathways leading to the aggregation were not clearly defined. Here, we show that dextrose-mediated aggregation of Avastin or Herceptin in plasma involves isoelectric precipitation of complement proteins. Using mass spectrometry, we found that dextrose-induced insoluble aggregates were composed of mAb itself and multiple abundant plasma proteins, namely complement proteins C3, C4, factor H, fibronectin, and apolipoprotein. These plasma proteins, which are characterized by an isoelectronic point of 5.5-6.7, lost solubility at the resulting pH in the mixture with formulated Avastin (pH 6.2) and Herceptin (pH 6.0). Notably, switching formulation buffers for Avastin (pH 6.2) and Remicade (pH 7.2) reversed their aggregation profiles. Avastin formed little, if any, insoluble aggregates in dextrose-plasma upon raising the buffer pH to 7.2 or above. Furthermore, dextrose induced pH-dependent precipitation of plasma proteins, with massive insoluble aggregates being detected at pH 6.5-6.8. These data show that isoelectric precipitation of complement proteins is a prerequisite of dextrose-induced aggregation of mAb in human plasma. This finding highlights the importance of assessing the compatibility of a therapeutic mAb with diluent and human plasma during product development.

  3. Differential abundance of egg white proteins in laying hens treated with corticosterone.

    Science.gov (United States)

    Kim, Jimin; Choi, Yang-Ho

    2014-12-24

    Stressful environments can affect not only egg production and quality but also gene and protein abundance in the ovary and oviduct in laying hens. The oviductal magnum of laying hens is the organ responsible for the synthesis and secretion of egg white proteins. The objective of this study was to investigate the effects of dietary corticosterone as a stress model on the abundance of proteins in the egg white and of mRNA and proteins in the magnum in laying hens. After a 14-day acclimation, 40 laying hens were divided into two groups which were provided for the next 14 days with either control (Control) or corticosterone (Stress) diet containing at 30 mg/kg. Corticosterone treatment resulted in increased feed intake (P ≤ 0.05) and decreased egg production. Two-dimensional electrophoresis (2DE) with MALDI-TOF/TOF MS/MS using eggs obtained on days 0 and 5 revealed differential abundance of egg white proteins by Stress: transiently expressed in neural precursors (TENP), hemopexin (HPX), IgY-Fcυ3-4, and extracellular fatty acid-binding protein (Ex-FABP) were decreased while ovoinhibitor and ovalbumin-related protein X (OVAX) were increased on days 5 vs 0 (P ≤ 0.05). Expression of mRNAs and proteins was also significantly modulated in the magnum of hens in Stress on day 14 (P ≤ 0.05). In conclusion, the current study provides the first evidence showing that dietary corticosterone modulates protein abundance in the egg white in laying hens, and it suggests that environmental stress can differentially modify expression of egg white proteins in laying hens.

  4. Identification of stress responsive genes by studying specific relationships between mRNA and protein abundance.

    Science.gov (United States)

    Morimoto, Shimpei; Yahara, Koji

    2018-03-01

    Protein expression is regulated by the production and degradation of mRNAs and proteins but the specifics of their relationship are controversial. Although technological advances have enabled genome-wide and time-series surveys of mRNA and protein abundance, recent studies have shown paradoxical results, with most statistical analyses being limited to linear correlation, or analysis of variance applied separately to mRNA and protein datasets. Here, using recently analyzed genome-wide time-series data, we have developed a statistical analysis framework for identifying which types of genes or biological gene groups have significant correlation between mRNA and protein abundance after accounting for potential time delays. Our framework stratifies all genes in terms of the extent of time delay, conducts gene clustering in each stratum, and performs a non-parametric statistical test of the correlation between mRNA and protein abundance in a gene cluster. Consequently, we revealed stronger correlations than previously reported between mRNA and protein abundance in two metabolic pathways. Moreover, we identified a pair of stress responsive genes ( ADC17 and KIN1 ) that showed a highly similar time series of mRNA and protein abundance. Furthermore, we confirmed robustness of the analysis framework by applying it to another genome-wide time-series data and identifying a cytoskeleton-related gene cluster (keratin 18, keratin 17, and mitotic spindle positioning) that shows similar correlation. The significant correlation and highly similar changes of mRNA and protein abundance suggests a concerted role of these genes in cellular stress response, which we consider provides an answer to the question of the specific relationships between mRNA and protein in a cell. In addition, our framework for studying the relationship between mRNAs and proteins in a cell will provide a basis for studying specific relationships between mRNA and protein abundance after accounting for potential

  5. Athoropometric measurements and plasma proteins in protein ...

    African Journals Online (AJOL)

    Athoropometric measurements and plasma proteins in protein energy malnutrition. MH Etukudo, EO Agbedana, OO Akinyinka, BOA Osifo. Abstract. No Abstract. Global Journal of Medical Sciences Vol. 5(1) 2006: 7-11. Full Text: EMAIL FREE FULL TEXT EMAIL FREE FULL TEXT · DOWNLOAD FULL TEXT DOWNLOAD ...

  6. Identification of the hemoglobin scavenger receptor/CD163 as a natural soluble protein in plasma

    DEFF Research Database (Denmark)

    Møller, Holger Jon; Peterslund, Niels Anker; Graversen, Jonas Heilskov

    2002-01-01

    enabled identification of a soluble plasma form of HbSR (sHbSR) having an electrophoretic mobility equal to that of recombinant HbSR consisting of the extracellular domain (scavenger receptor cysteine-rich 1-9). A sandwich enzyme-linked immunosorbent assay was established and used to measure the s...... a level of sHbSR above the range of healthy persons. Patients with myelomonocytic leukemias and pneumonia/sepsis exhibited the highest levels (up to 67.3 mg/L). In conclusion, sHbSR is an abundant plasma protein potentially valuable in monitoring patients with infections and myelomonocytic leukemia....

  7. Snake venoms are integrated systems, but abundant venom proteins evolve more rapidly.

    Science.gov (United States)

    Aird, Steven D; Aggarwal, Shikha; Villar-Briones, Alejandro; Tin, Mandy Man-Ying; Terada, Kouki; Mikheyev, Alexander S

    2015-08-28

    While many studies have shown that extracellular proteins evolve rapidly, how selection acts on them remains poorly understood. We used snake venoms to understand the interaction between ecology, expression level, and evolutionary rate in secreted protein systems. Venomous snakes employ well-integrated systems of proteins and organic constituents to immobilize prey. Venoms are generally optimized to subdue preferred prey more effectively than non-prey, and many venom protein families manifest positive selection and rapid gene family diversification. Although previous studies have illuminated how individual venom protein families evolve, how selection acts on venoms as integrated systems, is unknown. Using next-generation transcriptome sequencing and mass spectrometry, we examined microevolution in two pitvipers, allopatrically separated for at least 1.6 million years, and their hybrids. Transcriptomes of parental species had generally similar compositions in regard to protein families, but for a given protein family, the homologs present and concentrations thereof sometimes differed dramatically. For instance, a phospholipase A2 transcript comprising 73.4 % of the Protobothrops elegans transcriptome, was barely present in the P. flavoviridis transcriptome (king cobra genome, suggesting that rapid evolution of abundant proteins may be generally true for snake venoms. Looking more broadly at Protobothrops, we show that rapid evolution of the most abundant components is due to positive selection, suggesting an interplay between abundance and adaptation. Given log-scale differences in toxin abundance, which are likely correlated with biosynthetic costs, we hypothesize that as a result of natural selection, snakes optimize return on energetic investment by producing more of venom proteins that increase their fitness. Natural selection then acts on the additive genetic variance of these components, in proportion to their contributions to overall fitness. Adaptive

  8. Identification of stress responsive genes by studying specific relationships between mRNA and protein abundance

    Directory of Open Access Journals (Sweden)

    Shimpei Morimoto

    2018-03-01

    Full Text Available Protein expression is regulated by the production and degradation of mRNAs and proteins but the specifics of their relationship are controversial. Although technological advances have enabled genome-wide and time-series surveys of mRNA and protein abundance, recent studies have shown paradoxical results, with most statistical analyses being limited to linear correlation, or analysis of variance applied separately to mRNA and protein datasets. Here, using recently analyzed genome-wide time-series data, we have developed a statistical analysis framework for identifying which types of genes or biological gene groups have significant correlation between mRNA and protein abundance after accounting for potential time delays. Our framework stratifies all genes in terms of the extent of time delay, conducts gene clustering in each stratum, and performs a non-parametric statistical test of the correlation between mRNA and protein abundance in a gene cluster. Consequently, we revealed stronger correlations than previously reported between mRNA and protein abundance in two metabolic pathways. Moreover, we identified a pair of stress responsive genes (ADC17 and KIN1 that showed a highly similar time series of mRNA and protein abundance. Furthermore, we confirmed robustness of the analysis framework by applying it to another genome-wide time-series data and identifying a cytoskeleton-related gene cluster (keratin 18, keratin 17, and mitotic spindle positioning that shows similar correlation. The significant correlation and highly similar changes of mRNA and protein abundance suggests a concerted role of these genes in cellular stress response, which we consider provides an answer to the question of the specific relationships between mRNA and protein in a cell. In addition, our framework for studying the relationship between mRNAs and proteins in a cell will provide a basis for studying specific relationships between mRNA and protein abundance after

  9. Effects of Long-Term Storage Time and Original Sampling Month on Biobank Plasma Protein Concentrations

    Directory of Open Access Journals (Sweden)

    Stefan Enroth

    2016-10-01

    Full Text Available The quality of clinical biobank samples is crucial to their value for life sciences research. A number of factors related to the collection and storage of samples may affect the biomolecular composition. We have studied the effect of long-time freezer storage, chronological age at sampling, season and month of the year and on the abundance levels of 108 proteins in 380 plasma samples collected from 106 Swedish women. Storage time affected 18 proteins and explained 4.8–34.9% of the observed variance. Chronological age at sample collection after adjustment for storage-time affected 70 proteins and explained 1.1–33.5% of the variance. Seasonal variation had an effect on 15 proteins and month (number of sun hours affected 36 proteins and explained up to 4.5% of the variance after adjustment for storage-time and age. The results show that freezer storage time and collection date (month and season exerted similar effect sizes as age on the protein abundance levels. This implies that information on the sample handling history, in particular storage time, should be regarded as equally prominent covariates as age or gender and need to be included in epidemiological studies involving protein levels.

  10. Plasma proteins and proteinuria in gestational malaria

    OpenAIRE

    Fisayo, Asaolu Modupe

    2007-01-01

    The plasma concentrations of total protein, albumin, immunoglobulins IgG, IgA and IgM and urinary protein were assayed in 250 pregnant Nigerian women with malaria and compared with 250 healthy pregnant women which served as controls. The mean values of plasma total proteins, albumin, IgG and IgA were significantly lowered (P

  11. Plasma proteomic study in patients with high altitude pulmonary edema (HAPE

    Directory of Open Access Journals (Sweden)

    Yong-jun LUO

    2012-01-01

    Full Text Available Objective  To investigate the differential expressions of protein in the plasma proteome in patients suffering from high altitude pulmonary edema (HAPE and their implications. Methods  The plasmas of six HAPE patients and six healthy controls were studied. The high-abundant proteins in the plasma were removed. The low-abundant proteins in the plasma/serum were segregated by 2-DE. MALDI-TOF/MS was adopted to measure the peptide fingerprints after the differential protein spots were digested by enzymes. Comparison and analysis were made in the GenBank. Results  The immunoglobulin K1 light chain, serum transferrin protein precursor, and α-trypsin inhibitor heavy chain-related protein expressions were upregulated in HAPE patients compared with the control group. However the human fibrin glue coagulation protein 3 was down-regulated. Conclusion  The differential expression of the above four proteins in the plasma of HAPE patients may be related to the occurrence of HAPE and can be used as the target point for the prediction of HAPE.

  12. Differential plasma protein binding to metal oxide nanoparticles

    International Nuclear Information System (INIS)

    Deng, Zhou J; Mortimer, Gysell; Minchin, Rodney F; Schiller, Tara; Musumeci, Anthony; Martin, Darren

    2009-01-01

    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 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 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.

  13. 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.

  14. Liver plasma membranes: an effective method to analyze membrane proteome.

    Science.gov (United States)

    Cao, Rui; Liang, Songping

    2012-01-01

    Plasma membrane proteins are critical for the maintenance of biological systems and represent important targets for the treatment of disease. The hydrophobicity and low abundance of plasma membrane proteins make them difficult to analyze. The protocols given here are the efficient isolation/digestion procedures for liver plasma membrane proteomic analysis. Both protocol for the isolation of plasma membranes and protocol for the in-gel digestion of gel-embedded plasma membrane proteins are presented. The later method allows the use of a high detergent concentration to achieve efficient solubilization of hydrophobic plasma membrane proteins while avoiding interference with the subsequent LC-MS/MS analysis.

  15. Zinc can increase the activity of protein kinase C and contributes to its binding to plasma membranes in T lymphocytes

    International Nuclear Information System (INIS)

    Csermely, P.; Szamel, M.; Resch, K.; Somogyi, J.

    1988-01-01

    In the primary structure of protein kinase C, the presence of a putative metal-binding site has been suggested. In the present report, the authors demonstrate that the most abundant intracellular heavy metal, zinc, can increase the activity of cytosolic protein kinase C. Zinc reversibly binds the enzyme to plasma membranes,and it may contribute to the calcium-induced binding as well. The intracellular heavy metal chelator N,N,N',N'-tetrakis(2-pyridylmethyl) ethylenediamine prevents the phorbol ester- and antigen-induced translocation of protein kinase C. This effect can be totally reversed by the concomitant addition of Zn 2+ , while Fe 2+ and Mn 2+ are only partially counteractive. The results suggest that zinc can activate protein kinase C and contributes to its binding to plasma membranes in T lymphocytes induced by Ca 2+ , phorbol ester, or antigen

  16. [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.

  17. Visualization and Dissemination of Multidimensional Proteomics Data Comparing Protein Abundance During Caenorhabditis elegans Development

    Science.gov (United States)

    Riffle, Michael; Merrihew, Gennifer E.; Jaschob, Daniel; Sharma, Vagisha; Davis, Trisha N.; Noble, William S.; MacCoss, Michael J.

    2015-11-01

    Regulation of protein abundance is a critical aspect of cellular function, organism development, and aging. Alternative splicing may give rise to multiple possible proteoforms of gene products where the abundance of each proteoform is independently regulated. Understanding how the abundances of these distinct gene products change is essential to understanding the underlying mechanisms of many biological processes. Bottom-up proteomics mass spectrometry techniques may be used to estimate protein abundance indirectly by sequencing and quantifying peptides that are later mapped to proteins based on sequence. However, quantifying the abundance of distinct gene products is routinely confounded by peptides that map to multiple possible proteoforms. In this work, we describe a technique that may be used to help mitigate the effects of confounding ambiguous peptides and multiple proteoforms when quantifying proteins. We have applied this technique to visualize the distribution of distinct gene products for the whole proteome across 11 developmental stages of the model organism Caenorhabditis elegans. The result is a large multidimensional dataset for which web-based tools were developed for visualizing how translated gene products change during development and identifying possible proteoforms. The underlying instrument raw files and tandem mass spectra may also be downloaded. The data resource is freely available on the web at http://www.yeastrc.org/wormpes/.

  18. The Human Plasma Proteome Draft of 2017: Building on the Human Plasma PeptideAtlas from Mass Spectrometry and Complementary Assays.

    Science.gov (United States)

    Schwenk, Jochen M; Omenn, Gilbert S; Sun, Zhi; Campbell, David S; Baker, Mark S; Overall, Christopher M; Aebersold, Ruedi; Moritz, Robert L; Deutsch, Eric W

    2017-12-01

    Human blood plasma provides a highly accessible window to the proteome of any individual in health and disease. Since its inception in 2002, the Human Proteome Organization's Human Plasma Proteome Project (HPPP) has been promoting advances in the study and understanding of the full protein complement of human plasma and on determining the abundance and modifications of its components. In 2017, we review the history of the HPPP and the advances of human plasma proteomics in general, including several recent achievements. We then present the latest 2017-04 build of Human Plasma PeptideAtlas, which yields ∼43 million peptide-spectrum matches and 122,730 distinct peptide sequences from 178 individual experiments at a 1% protein-level FDR globally across all experiments. Applying the latest Human Proteome Project Data Interpretation Guidelines, we catalog 3509 proteins that have at least two non-nested uniquely mapping peptides of nine amino acids or more and >1300 additional proteins with ambiguous evidence. We apply the same two-peptide guideline to historical PeptideAtlas builds going back to 2006 and examine the progress made in the past ten years in plasma proteome coverage. We also compare the distribution of proteins in historical PeptideAtlas builds in various RNA abundance and cellular localization categories. We then discuss advances in plasma proteomics based on targeted mass spectrometry as well as affinity assays, which during early 2017 target ∼2000 proteins. Finally, we describe considerations about sample handling and study design, concluding with an outlook for future advances in deciphering the human plasma proteome.

  19. Temporal course of pregnancy-associated plasma protein-A in angioplasty-treated ST-elevation myocardial infarction patients and potential significance of concomitant heparin administration

    DEFF Research Database (Denmark)

    Terkelsen, Christian J; Oxvig, Claus; Nørgaard, Bjarne L

    2009-01-01

    Pregnancy-associated plasma protein-A (PAPP-A) is a putative plaque instability marker. In acute coronary syndromes, the disrupted culprit plaque contains abundant PAPP-A, and circulating PAPP-A levels predict clinical outcomes. Determinants of circulating PAPP-A levels, however, are not fully...

  20. Bovine plasma protein fractionation by ion exchange chromatography.

    Science.gov (United States)

    Moure, F; Rendueles, M; Díaz, M

    2004-12-01

    An ion exchange chromatography process was developed to separate the main protein fractions of bovine blood plasma using a composite material, Q-HyperD resin, and a gel material, DEAE-Sepharose. The experiments were carried out at semipreparative scale. It was necessary to establish analytical methods of electrophoresis and HPLC to identify the fractionated proteins. Results show that these materials are able to adequately fractionate different protein groups from the raw blood plasma. This method may be used to avoid chemical fractionation using agents such as ethanol or PEG and, thus, decrease protein denaturation of the different fractions to be used for research or pharmaceutical purposes. The Q-HyperD resin presents a better retention capacity for plasma protein than DEAE-Sepharose under the experimental conditions employed.

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

    DEFF Research Database (Denmark)

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

    2015-01-01

    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 signature (P ....001) was associated with observed increased next-day C-reactive protein. The 82-plex protein signature outperformed routine clinical procedures. The category-free net reclassification index improved with 82-plex plasma protein signature (total net reclassification index, 88.3%). Using the 82-plex plasma protein...

  2. A Proteomic Approach for Plasma Biomarker Discovery with iTRAQ Labelling and OFFGEL Fractionation

    Directory of Open Access Journals (Sweden)

    Emilie Ernoult

    2010-01-01

    Full Text Available Human blood plasma contains a plethora of proteins, encompassing not only proteins that have plasma-based functionalities, but also possibly every other form of low concentrated human proteins. As it circulates through the tissues, the plasma picks up proteins that are released from their origin due to physiological events such as tissue remodeling and cell death. Specific disease processes or tumors are often characterized by plasma “signatures,” which may become obvious via changes in the plasma proteome profile, for example, through over expression of proteins. However, the wide dynamic range of proteins present in plasma makes their analysis very challenging, because high-abundance proteins tend to mask those of lower abundance. In the present study, we used a strategy combining iTRAQ as a reagent which improved the peptide ionization and peptide OFFGEL fractionation that has already been shown, in our previous research, to improve the proteome coverage of cellular extracts. Two prefractioning methods were compared: immunodepletion and a bead-based library of combinatorial hexapeptide technology. Our data suggested that both methods were complementary, with regard to the number of identified proteins. iTRAQ labelling, in association with OFFGEL fractionation, allowed more than 300 different proteins to be characterized from 400 μg of plasma proteins.

  3. A proteomic approach for plasma biomarker discovery with iTRAQ labelling and OFFGEL fractionation.

    Science.gov (United States)

    Ernoult, Emilie; Bourreau, Anthony; Gamelin, Erick; Guette, Catherine

    2010-01-01

    Human blood plasma contains a plethora of proteins, encompassing not only proteins that have plasma-based functionalities, but also possibly every other form of low concentrated human proteins. As it circulates through the tissues, the plasma picks up proteins that are released from their origin due to physiological events such as tissue remodeling and cell death. Specific disease processes or tumors are often characterized by plasma "signatures," which may become obvious via changes in the plasma proteome profile, for example, through over expression of proteins. However, the wide dynamic range of proteins present in plasma makes their analysis very challenging, because high-abundance proteins tend to mask those of lower abundance. In the present study, we used a strategy combining iTRAQ as a reagent which improved the peptide ionization and peptide OFFGEL fractionation that has already been shown, in our previous research, to improve the proteome coverage of cellular extracts. Two prefractioning methods were compared: immunodepletion and a bead-based library of combinatorial hexapeptide technology. Our data suggested that both methods were complementary, with regard to the number of identified proteins. iTRAQ labelling, in association with OFFGEL fractionation, allowed more than 300 different proteins to be characterized from 400 microg of plasma proteins.

  4. Zinc can increase the activity of protein kinase C and contributes to its binding to plasma membranes in T lymphocytes

    Energy Technology Data Exchange (ETDEWEB)

    Csermely, P.; Szamel, M.; Resch, K.; Somogyi, J.

    1988-05-15

    In the primary structure of protein kinase C, the presence of a putative metal-binding site has been suggested. In the present report, the authors demonstrate that the most abundant intracellular heavy metal, zinc, can increase the activity of cytosolic protein kinase C. Zinc reversibly binds the enzyme to plasma membranes,and it may contribute to the calcium-induced binding as well. The intracellular heavy metal chelator N,N,N',N'-tetrakis(2-pyridylmethyl) ethylenediamine prevents the phorbol ester- and antigen-induced translocation of protein kinase C. This effect can be totally reversed by the concomitant addition of Zn/sup 2 +/, while Fe/sup 2 +/ and Mn/sup 2 +/ are only partially counteractive. The results suggest that zinc can activate protein kinase C and contributes to its binding to plasma membranes in T lymphocytes induced by Ca/sup 2 +/, phorbol ester, or antigen.

  5. 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...... distribution of hemoglobin in haptoglobin-deficient mice resulted in abnormal iron deposits in proximal tubules during aging. Moreover, iron also accumulated in proximal tubules after renal ischemia-reperfusion injury or after an acute plasma heme-protein overload caused by muscle injury, without affecting...... morphological and functional parameters of renal damage. These data demonstrate that haptoglobin crucially prevents glomerular filtration of hemoglobin and, consequently, renal iron loading during aging and following acute plasma heme-protein overload....

  6. Depleting high-abundant and enriching low-abundant proteins in human serum: An evaluation of sample preparation methods using magnetic nanoparticle, chemical depletion and immunoaffinity techniques.

    Science.gov (United States)

    de Jesus, Jemmyson Romário; da Silva Fernandes, Rafael; de Souza Pessôa, Gustavo; Raimundo, Ivo Milton; Arruda, Marco Aurélio Zezzi

    2017-08-01

    The efficiency of three different depletion methods to remove the most abundant proteins, enriching those human serum proteins with low abundance is checked to make more efficient the search and discovery of biomarkers. These methods utilize magnetic nanoparticles (MNPs), chemical reagents (sequential application of dithiothreitol and acetonitrile, DTT/ACN), and commercial apparatus based on immunoaffinity (ProteoMiner, PM). The comparison between methods shows significant removal of abundant protein, remaining in the supernatant at concentrations of 4.6±0.2, 3.6±0.1, and 3.3±0.2µgµL -1 (n=3) for MNPs, DTT/ACN and PM respectively, from a total protein content of 54µgµL -1 . Using GeLC-MS/MS analysis, MNPs depletion shows good efficiency in removing high molecular weight proteins (>80kDa). Due to the synergic effect between the reagents DTT and ACN, DTT/ACN-based depletion offers good performance in the depletion of thiol-rich proteins, such as albumin and transferrin (DTT action), as well as of high molecular weight proteins (ACN action). Furthermore, PM equalization confirms its efficiency in concentrating low-abundant proteins, decreasing the dynamic range of protein levels in human serum. Direct comparison between the treatments reveals 72 proteins identified when using MNP depletion (43 of them exclusively by this method), but only 20 proteins using DTT/ACN (seven exclusively by this method). Additionally, after PM treatment 30 proteins were identified, seven exclusively by this method. Thus, MNPs and DTT/ACN depletion can be simple, quick, cheap, and robust alternatives for immunochemistry-based protein depletion, providing a potential strategy in the search for disease biomarkers. Copyright © 2017 Elsevier B.V. All rights reserved.

  7. Nitrogen 15 abundance in protein fractions of beans fertilized with (15NH4)2SO4

    International Nuclear Information System (INIS)

    Chaud, Saula Goulart; Oliveira, Admar Costa de; Trivelin, Paulo Cesar Ocheuze

    2002-01-01

    Studies evaluating the protein nutritive value of beans labelled with 15 N, using nitrogen balance and the quantitation of faecal and urinary endogenous nitrogen, determined by isotopic dilution, have been extensively used. The objective of this research was to verify if the isotopic labelling of raw, freeze dried beans (Phaseolus vulgaris L., cultivar Pirata 1) with 1.394 atoms % 15 N, resulted in the same abundance of the whole flour and of the protein fractions extracted from the beans with 0.5 mol L -1 NaCl. The isotopic abundance found in the whole bean flour, in the protein extract, in the globulin and albumin fractions were respectively: 1.394 +- 0.011; 1.403 +- 0.012; 1.399 +- 0.007 and 1.399 +- 0.028 atoms % of 15 N, presenting no difference (P > 0.05). However, a difference was found (P < 0.05) between the above mentioned abundances and the isotopic abundance found in the nitrogen of the proteins in the extraction residue, which was 0.969 +- 0.084. Since the abundances did not differ, the protein nutritive indexes, such as digestibility and biological value, determined from the nitrogen balance and corrected for isotopic dilution, would not be affected by extracting the proteins from the beans with 0.5 mol L 1 NaCl. If working with the nitrogen balance of the residual proteins after extraction and even with the whole flours, these indexes could present incorrect values, since the isotopic labelling of the residual proteins was less than that of the protein fractions. (author)

  8. Analysis of human blood plasma proteome from ten healthy volunteers from Indian population.

    Directory of Open Access Journals (Sweden)

    Poonam Gautam

    Full Text Available Analysis of any mammalian plasma proteome is a challenge, particularly by mass spectrometry, due to the presence of albumin and other abundant proteins which can mask the detection of low abundant proteins. As detection of human plasma proteins is valuable in diagnostics, exploring various workflows with minimal fractionation prior to mass spectral analysis, is required in order to study population diversity involving analysis in a large cohort of samples. Here, we used 'reference plasma sample', a pool of plasma from 10 healthy individuals from Indian population in the age group of 25-60 yrs including 5 males and 5 females. The 14 abundant proteins were immunodepleted from plasma and then evaluated by three different workflows for proteome analysis using a nanoflow reverse phase liquid chromatography system coupled to a LTQ Orbitrap Velos mass spectrometer. The analysis of reference plasma sample a without prefractionation, b after prefractionation at peptide level by strong cation exchange chromatography and c after prefractionation at protein level by sodium dodecyl sulfate polyacrylamide gel electrophoresis, led to the identification of 194, 251 and 342 proteins respectively. Together, a comprehensive dataset of 517 unique proteins was achieved from all the three workflows, including 271 proteins with high confidence identified by ≥ 2 unique peptides in any of the workflows or identified by single peptide in any of the two workflows. A total of 70 proteins were common in all the three workflows. Some of the proteins were unique to our study and could be specific to Indian population. The high-confidence dataset obtained from our study may be useful for studying the population diversity, in discovery and validation process for biomarker identification.

  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. Chloroform-assisted phenol extraction improving proteome profiling of maize embryos through selective depletion of high-abundance storage proteins.

    Directory of Open Access Journals (Sweden)

    Erhui Xiong

    Full Text Available The presence of abundant storage proteins in plant embryos greatly impedes seed proteomics analysis. Vicilin (or globulin-1 is the most abundant storage protein in maize embryo. There is a need to deplete the vicilins from maize embryo extracts for enhanced proteomics analysis. We here reported a chloroform-assisted phenol extraction (CAPE method for vicilin depletion. By CAPE, maize embryo proteins were first extracted in an aqueous buffer, denatured by chloroform and then subjected to phenol extraction. We found that CAPE can effectively deplete the vicilins from maize embryo extract, allowing the detection of low-abundance proteins that were masked by vicilins in 2-DE gel. The novelty of CAPE is that it selectively depletes abundant storage proteins from embryo extracts of both monocot (maize and dicot (soybean and pea seeds, whereas other embryo proteins were not depleted. CAPE can significantly improve proteome profiling of embryos and extends the application of chloroform and phenol extraction in plant proteomics. In addition, the rationale behind CAPE depletion of abundant storage proteins was explored.

  11. 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...... 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...... 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...

  12. Ion-Exchange Sample Displacement Chromatography as a Method for Fast and Simple Isolation of Low- and High-Abundance Proteins from Complex Biological Mixtures

    Directory of Open Access Journals (Sweden)

    Martina Srajer Gajdosik

    2014-01-01

    Full Text Available Sample displacement chromatography (SDC in reversed phase and ion-exchange modes was introduced at the end of 1980s. This chromatographic method was first used for preparative purification of synthetic peptides, and subsequently adapted for protein fractionation, mainly in anion-exchange mode. In the past few years, SDC has been successfully used for enrichment of low- and medium-abundance proteins from complex biological fluids on both monolithic and bulk chromatographic supports. If aqueous mobile phase is used with the application of mild chromatographic conditions, isolated proteins are not denatured and can also keep their biological activity. In this paper, the use of SDC in anion-exchange mode on a high-capacity chromatographic resin for separation of proteins from complex biological mixtures such as human plasma is demonstrated. By use of three and more columns coupled in series during sample application, and subsequent parallel elution of detached columns, additional separation of bound proteins was achieved. Highly enriched human serum albumin fraction and a number of physiologically active medium- and low-abundance proteins could be fractionated and detected by electrospray ionization tandem mass spectrometry (ESI-MS/MS and matrix assisted laser desorption/ionization time-of-flight tandem mass spectrometry (MALDI-TOF/TOF-MS. The use of the aforementioned columns that can be sanitized with 1 M sodium hydroxide for further application of SDC in biotechnology and food technology was discussed.

  13. Total plasma N-glycome changes during pregnancy.

    NARCIS (Netherlands)

    Ruhaak, L.R.; Uh, H.W.; Deelder, A.M.; Dolhain, R.E.; Wuhrer, M.

    2014-01-01

    During pregnancy, the mother faces a major immunological challenge. Most of the major plasma proteins have important immunological functions, and altered levels of these major proteins have been reported during pregnancy, potentially providing immunosuppression. A large number of the high abundance

  14. Functional Regulation of the Plasma Protein Histidine-Rich Glycoprotein by Zn2+ in Settings of Tissue Injury

    Directory of Open Access Journals (Sweden)

    Kristin M. Priebatsch

    2017-03-01

    Full Text Available Divalent metal ions are essential nutrients for all living organisms and are commonly protein-bound where they perform important roles in protein structure and function. This regulatory control from metals is observed in the relatively abundant plasma protein histidine-rich glycoprotein (HRG, which displays preferential binding to the second most abundant transition element in human systems, Zinc (Zn2+. HRG has been proposed to interact with a large number of protein ligands and has been implicated in the regulation of various physiological and pathological processes including the formation of immune complexes, apoptotic/necrotic and pathogen clearance, cell adhesion, antimicrobial activity, angiogenesis, coagulation and fibrinolysis. Interestingly, these processes are often associated with sites of tissue injury or tumour growth, where the concentration and distribution of Zn2+ is known to vary. Changes in Zn2+ levels have been shown to modify HRG function by altering its affinity for certain ligands and/or providing protection against proteolytic disassembly by serine proteases. This review focuses on the molecular interplay between HRG and Zn2+, and how Zn2+ binding modifies HRG-ligand interactions to regulate function in different settings of tissue injury.

  15. 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. © 2014. Published by The Company of Biologists Ltd.

  16. Zinc can increase the activity of protein kinase C and contributes to its binding to plasma membranes in T lymphocytes.

    Science.gov (United States)

    Csermely, P; Szamel, M; Resch, K; Somogyi, J

    1988-05-15

    In the primary structure of protein kinase C, the presence of a putative metal-binding site has been suggested (Parker, P.J., Coussens, L., Totty, N., Rhee, L., Young, S., Chen, E., Stabel, S., Waterfield, M.D., and Ullrich, A. (1986) Science 233, 853-859). In the present report, we demonstrate that the most abundant intracellular heavy metal, zinc, can increase the activity of cytosolic protein kinase C. Zinc reversibly binds the enzyme to plasma membranes, and it may contribute to the calcium-induced binding as well. The intracellular heavy metal chelator N,N,N',N'-tetrakis(2-pyridylmethyl) ethylenediamine prevents the phorbol ester- and antigen-induced translocation of protein kinase C. This effect can be totally reversed by the concomitant addition of Zn2+, while Fe2+ and Mn2+ are only partially counteractive. Our results suggest that zinc can activate protein kinase C and contributes to its binding to plasma membranes in T lymphocytes induced by Ca2+, phorbol ester, or antigen.

  17. Effect of dietary proteins on the incorporation of amino acids in plasma proteins of ruminants

    International Nuclear Information System (INIS)

    Mehra, Usha R.; Singh, U.B.; Kumar, S.

    1979-01-01

    Experiments were conducted on nine male calves (Hariana x Holstein) of about one and a half years of age and fed different amounts of crude protein. 14 C-DL-leucine was injected into the blood of the animals and specific radioactivity of plasma protein measured. There was linear correlation between nitrogen ingested, digested and retained by the animals and the specific radioactivity of total plasma proteins. The experiments suggest the possible use of the incorporation of amino acids into plasma proteins as an index of nutritional status of the animals. (auth.)

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

    International Nuclear Information System (INIS)

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

    1987-01-01

    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

  19. Differential profiling of breast cancer plasma proteome by isotope-coded affinity tagging method reveals biotinidase as a breast cancer biomarker

    International Nuclear Information System (INIS)

    Kang, Un-Beom; Ahn, Younghee; Lee, Jong Won; Kim, Yong-Hak; Kim, Joon; Yu, Myeong-Hee; Noh, Dong-Young; Lee, Cheolju

    2010-01-01

    Breast cancer is one of the leading causes of women's death worldwide. It is important to discover a reliable biomarker for the detection of breast cancer. Plasma is the most ideal source for cancer biomarker discovery since many cells cross-communicate through the secretion of soluble proteins into blood. Plasma proteomes obtained from 6 breast cancer patients and 6 normal healthy women were analyzed by using the isotope-coded affinity tag (ICAT) labeling approach and tandem mass spectrometry. All the plasma samples used were depleted of highly abundant 6 plasma proteins by immune-affinity column chromatography before ICAT labeling. Several proteins showing differential abundance level were selected based on literature searches and their specificity to the commercially available antibodies, and then verified by immunoblot assays. A total of 155 proteins were identified and quantified by ICAT method. Among them, 33 proteins showed abundance changes by more than 1.5-fold between the plasmas of breast cancer patients and healthy women. We chose 5 proteins for the follow-up confirmation in the individual plasma samples using immunoblot assay. Four proteins, α1-acid glycoprotein 2, monocyte differentiation antigen CD14, biotinidase (BTD), and glutathione peroxidase 3, showed similar abundance ratio to ICAT result. Using a blind set of plasmas obtained from 21 breast cancer patients and 21 normal healthy controls, we confirmed that BTD was significantly down-regulated in breast cancer plasma (Wilcoxon rank-sum test, p = 0.002). BTD levels were lowered in all cancer grades (I-IV) except cancer grade zero. The area under the receiver operating characteristic curve of BTD was 0.78. Estrogen receptor status (p = 0.940) and progesterone receptor status (p = 0.440) were not associated with the plasma BTD levels. Our study suggests that BTD is a potential serological biomarker for the detection of breast cancer

  20. Protein diffusion in plant cell plasma membranes: the cell-wall corral.

    Science.gov (United States)

    Martinière, Alexandre; Runions, John

    2013-01-01

    Studying protein diffusion informs us about how proteins interact with their environment. Work on protein diffusion over the last several decades has illustrated the complex nature of biological lipid bilayers. The plasma membrane contains an array of membrane-spanning proteins or proteins with peripheral membrane associations. Maintenance of plasma membrane microstructure can be via physical features that provide intrinsic ordering such as lipid microdomains, or from membrane-associated structures such as the cytoskeleton. Recent evidence indicates, that in the case of plant cells, the cell wall seems to be a major player in maintaining plasma membrane microstructure. This interconnection / interaction between cell-wall and plasma membrane proteins most likely plays an important role in signal transduction, cell growth, and cell physiological responses to the environment.

  1. 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.

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

    International Nuclear Information System (INIS)

    Song, Y.R.; Wu, B.; Yang, Y.T.; Chen, J.; Zhang, L.J.; Zhang, Z.W.; Shi, H.Y.; Huang, C.L.; Pan, J.X.; Xie, P.

    2015-01-01

    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

  3. Identification of Proteins of Altered Abundance in Oil Palm Infected with Ganoderma boninense

    Science.gov (United States)

    Al-Obaidi, Jameel R.; Mohd-Yusuf, Yusmin; Razali, Nurhanani; Jayapalan, Jaime Jacqueline; Tey, Chin-Chong; Md-Noh, Normahnani; Junit, Sarni Mat; Othman, Rofina Yasmin; Hashim, Onn Haji

    2014-01-01

    Basal stem rot is a common disease that affects oil palm, causing loss of yield and finally killing the trees. The disease, caused by fungus Ganoderma boninense, devastates thousands of hectares of oil palm plantings in Southeast Asia every year. In the present study, root proteins of healthy oil palm seedlings, and those infected with G. boninense, were analyzed by 2-dimensional gel electrophoresis (2-DE). When the 2-DE profiles were analyzed for proteins, which exhibit consistent significant change of abundance upon infection with G. boninense, 21 passed our screening criteria. Subsequent analyses by mass spectrometry and database search identified caffeoyl-CoA O-methyltransferase, caffeic acid O-methyltransferase, enolase, fructokinase, cysteine synthase, malate dehydrogenase, and ATP synthase as among proteins of which abundances were markedly altered. PMID:24663087

  4. Identification of Proteins of Altered Abundance in Oil Palm Infected with Ganoderma boninense

    Directory of Open Access Journals (Sweden)

    Jameel R. Al-Obaidi

    2014-03-01

    Full Text Available Basal stem rot is a common disease that affects oil palm, causing loss of yield and finally killing the trees. The disease, caused by fungus Ganoderma boninense, devastates thousands of hectares of oil palm plantings in Southeast Asia every year. In the present study, root proteins of healthy oil palm seedlings, and those infected with G. boninense, were analyzed by 2-dimensional gel electrophoresis (2-DE. When the 2-DE profiles were analyzed for proteins, which exhibit consistent significant change of abundance upon infection with G. boninense, 21 passed our screening criteria. Subsequent analyses by mass spectrometry and database search identified caffeoyl-CoA O-methyltransferase, caffeic acid O-methyltransferase, enolase, fructokinase, cysteine synthase, malate dehydrogenase, and ATP synthase as among proteins of which abundances were markedly altered.

  5. Identification of proteins of altered abundance in oil palm infected with Ganoderma boninense.

    Science.gov (United States)

    Al-Obaidi, Jameel R; Mohd-Yusuf, Yusmin; Razali, Nurhanani; Jayapalan, Jaime Jacqueline; Tey, Chin-Chong; Md-Noh, Normahnani; Junit, Sarni Mat; Othman, Rofina Yasmin; Hashim, Onn Haji

    2014-03-24

    Basal stem rot is a common disease that affects oil palm, causing loss of yield and finally killing the trees. The disease, caused by fungus Ganoderma boninense, devastates thousands of hectares of oil palm plantings in Southeast Asia every year. In the present study, root proteins of healthy oil palm seedlings, and those infected with G. boninense, were analyzed by 2-dimensional gel electrophoresis (2-DE). When the 2-DE profiles were analyzed for proteins, which exhibit consistent significant change of abundance upon infection with G. boninense, 21 passed our screening criteria. Subsequent analyses by mass spectrometry and database search identified caffeoyl-CoA O-methyltransferase, caffeic acid O-methyltransferase, enolase, fructokinase, cysteine synthase, malate dehydrogenase, and ATP synthase as among proteins of which abundances were markedly altered.

  6. Simulating the influence of plasma protein on measured receptor affinity in biochemical assays reveals the utility of Schild analysis for estimating compound affinity for plasma proteins.

    Science.gov (United States)

    Blakeley, D; Sykes, D A; Ensor, P; Bertran, E; Aston, P J; Charlton, S J

    2015-11-01

    Plasma protein binding (PPB) influences the free fraction of drug available to bind to its target and is therefore an important consideration in drug discovery. While traditional methods for assessing PPB (e.g. rapid equilibrium dialysis) are suitable for comparing compounds with relatively weak PPB, they are not able to accurately discriminate between highly bound compounds (typically >99.5%). The aim of the present work was to use mathematical modelling to explore the potential utility of receptor binding and cellular functional assays to estimate the affinity of compounds for plasma proteins. Plasma proteins are routinely added to in vitro assays, so a secondary goal was to investigate the effect of plasma proteins on observed ligand-receptor interactions. Using the principle of conservation of mass and the law of mass action, a cubic equation was derived describing the ligand-receptor complex [LR] in the presence of plasma protein at equilibrium. The model demonstrates the profound influence of PPB on in vitro assays and identifies the utility of Schild analysis, which is usually applied to determine receptor-antagonist affinities, for calculating affinity at plasma proteins (termed KP ). We have also extended this analysis to functional effects using operational modelling and demonstrate that these approaches can also be applied to cell-based assay systems. These mathematical models can potentially be used in conjunction with experimental data to estimate drug-plasma protein affinities in the earliest phases of drug discovery programmes. © 2015 The British Pharmacological Society.

  7. The plasma membrane proteome of germinating barley embryos

    DEFF Research Database (Denmark)

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

    2009-01-01

    Cereal seed germination involves a complex coordination between different seed tissues. Plasma membranes must play crucial roles in coordination and execution of germination; however, very little is known about seed plasma membrane proteomes due to limited tissue amounts combined...... with amphiphilicity and low abundance of membrane proteins. A fraction enriched in plasma membranes was prepared from embryos dissected from 18 h germinated barley seeds using aqueous two-phase partitioning. Reversed-phase chromatography on C-4 resin performed in micro-spin columns with stepwise elution by 2-propanol...... was used to reduce soluble protein contamination and enrich for hydrophobic proteins. Sixty-one proteins in 14 SDS-PAGE bands were identified by LC-MS/MS and database searches. The identifications provide new insight into the plasma membrane functions in seed germination....

  8. Influence of ionizing radiation on the plasma membrane proteins

    International Nuclear Information System (INIS)

    Dreval', V.I.

    1992-01-01

    The effect of ionizing radiation on the meat cattle thymocytes plasma membranes was studied. Using fluorescence quenching technique the effect of irradiation of proteins conformation was investigated. The influence of ionizing radiation on the plasma membranes was shown to be followed by changes of the protein structure-dynamic organization

  9. Protein diffusion in plant cell plasma membranes: The cell-wall corral

    Directory of Open Access Journals (Sweden)

    Alexandre eMartinière

    2013-12-01

    Full Text Available Studying protein diffusion informs us about how proteins interact with their environment. Work on protein diffusion over the last several decades has illustrated the complex nature of biological lipid bilayers. The plasma membrane contains an array of membrane-spanning proteins or proteins with peripheral membrane associations. Maintenance of plasma membrane microstructure can be via physical features that provide intrinsic ordering such as lipid microdomains, or from membrane-associated structures such as the cytoskeleton. Recent evidence indicates, that in the case of plant cells, the cell wall seems to be a major player in maintaining plasma membrane microstructure. This interconnection / interaction between cell-wall and plasma membrane proteins most likely plays an important role in signal transduction, cell growth, and cell physiological responses to the environment.

  10. Separomics applied to the proteomics and peptidomics of low-abundance proteins: Choice of methods and challenges - A review.

    Science.gov (United States)

    Baracat-Pereira, Maria Cristina; de Oliveira Barbosa, Meire; Magalhães, Marcos Jorge; Carrijo, Lanna Clicia; Games, Patrícia Dias; Almeida, Hebréia Oliveira; Sena Netto, José Fabiano; Pereira, Matheus Rodrigues; de Barros, Everaldo Gonçalves

    2012-06-01

    The enrichment and isolation of proteins are considered limiting steps in proteomic studies. Identification of proteins whose expression is transient, those that are of low-abundance, and of natural peptides not described in databases, is still a great challenge. Plant extracts are in general complex, and contaminants interfere with the identification of proteins involved in important physiological processes, such as plant defense against pathogens. This review discusses the challenges and strategies of separomics applied to the identification of low-abundance proteins and peptides in plants, especially in plants challenged by pathogens. Separomics is described as a group of methodological strategies for the separation of protein molecules for proteomics. Several tools have been used to remove highly abundant proteins from samples and also non-protein contaminants. The use of chromatographic techniques, the partition of the proteome into subproteomes, and an effort to isolate proteins in their native form have allowed the isolation and identification of rare proteins involved in different processes.

  11. Separomics applied to the proteomics and peptidomics of low-abundance proteins: choice of methods and challenges - a review

    Directory of Open Access Journals (Sweden)

    Maria Cristina Baracat-Pereira

    2012-01-01

    Full Text Available The enrichment and isolation of proteins are considered limiting steps in proteomic studies. Identification of proteins whose expression is transient, those that are of low-abundance, and of natural peptides not described in databases, is still a great challenge. Plant extracts are in general complex, and contaminants interfere with the identification of proteins involved in important physiological processes, such as plant defense against pathogens. This review discusses the challenges and strategies of separomics applied to the identification of low-abundance proteins and peptides in plants, especially in plants challenged by pathogens. Separomics is described as a group of methodological strategies for the separation of protein molecules for proteomics. Several tools have been used to remove highly abundant proteins from samples and also non-protein contaminants. The use of chromatographic techniques, the partition of the proteome into subproteomes, and an effort to isolate proteins in their native form have allowed the isolation and identification of rare proteins involved in different processes.

  12. Overexpression Analysis of emv2 gene coding for Late Embryogenesis Abundant Protein from Vigna radiata (Wilczek

    Directory of Open Access Journals (Sweden)

    Rajesh S.

    2008-10-01

    Full Text Available Late embryogenesis abundant (LEA proteins are speculated to protect against water stress deficit in plants. An over expression system for mungbean late embryogenesis abundant protein, emv2 was constructed in a pET29a vector, designated pET-emv2 which is responsible for higher expression under the transcriptional/translational control of T7/lac promoter incorporated in the Escherichia coli BL21 (DE3.Induction protocol was optimized for pET recombinants harboring the target gene. Overexpressed EMV2 protein was purified to homogeneity and the protein profile monitored by SDS-PAGE.

  13. 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.

  14. Interaction of monomeric Ebola VP40 protein with a plasma membrane: A coarse-grained molecular dynamics (CGMD) simulation study.

    Science.gov (United States)

    Mohamad Yusoff, Mohamad Ariff; Abdul Hamid, Azzmer Azzar; Mohammad Bunori, Noraslinda; Abd Halim, Khairul Bariyyah

    2018-06-01

    Ebola virus is a lipid-enveloped filamentous virus that affects human and non-human primates and consists of several types of protein: nucleoprotein, VP30, VP35, L protein, VP40, VP24, and transmembrane glycoprotein. Among the Ebola virus proteins, its matrix protein VP40 is abundantly expressed during infection and plays a number of critical roles in oligomerization, budding and egress from the host cell. VP40 exists predominantly as a monomer at the inner leaflet of the plasma membrane, and has been suggested to interact with negatively charged lipids such as phosphatidylinositol 4,5-bisphosphate (PIP 2 ) and phosphatidylserine (PS) via its cationic patch. The hydrophobic loop at the C-terminal domain has also been shown to be important in the interaction between the VP40 and the membrane. However, details of the molecular mechanisms underpinning their interactions are not fully understood. This study aimed at investigating the effects of mutation in the cationic patch and hydrophobic loop on the interaction between the VP40 monomer and the plasma membrane using coarse-grained molecular dynamics simulation (CGMD). Our simulations revealed that the interaction between VP40 and the plasma membrane is mediated by the cationic patch residues. This led to the clustering of PIP 2 around the protein in the inner leaflet as a result of interactions between some cationic residues including R52, K127, K221, K224, K225, K256, K270, K274, K275 and K279 and PIP 2 lipids via electrostatic interactions. Mutation of the cationic patch or hydrophobic loop amino acids caused the protein to bind at the inner leaflet of the plasma membrane in a different orientation, where no significant clustering of PIP 2 was observed around the mutated protein. This study provides basic understanding of the interaction of the VP40 monomer and its mutants with the plasma membrane. Copyright © 2018 Elsevier Inc. All rights reserved.

  15. 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 plasma membrane and intracellular membranes. We show that consistent with such shuttling, G proteins constitutively reside in endomembranes. Furthermore, we show that shuttling is inhibited by 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.

  16. TiO2-Based Phosphoproteomic Analysis of the Plasma Membrane and the Effects of Phosphatase Inhibitor Treatment

    DEFF Research Database (Denmark)

    Thingholm, Tine; Larsen, Martin Røssel; Ingrell, Christian

    2008-01-01

    Phosphorylation of plasma membrane proteins frequently initiates signal transduction pathways or attenuate plasma membrane transport processes. Because of the low abundance and hydrophobic features of many plasma membrane proteins and the low stoichiometry of protein phosphorylation, studies...... of the plasma membrane phosphoproteome are challenging. We present an optimized analytical strategy for plasma membrane phosphoproteomics that combines efficient plasma membrane protein preparation with TiO 2-based phosphopeptide enrichment and high-performance mass spectrometry for phosphopeptide sequencing....... We used sucrose centrifugation in combination with sodium carbonate extraction to achieve efficient and reproducible purification of low microgram levels of plasma membrane proteins from human mesenchymal stem cells (hMSCs, 10 (7) cells), achieving more than 70% yield of membrane proteins...

  17. The Magnaporthe oryzae Alt A 1-like protein MoHrip1 binds to the plant plasma membrane.

    Science.gov (United States)

    Zhang, Yi; Liang, Yingbo; Dong, Yijie; Gao, Yuhan; Yang, Xiufen; Yuan, Jingjing; Qiu, Dewen

    2017-10-07

    MoHrip1, a protein isolated from Magnaporthe oryzae, belongs to the Alt A 1 (AA1) family. mohrip1 mRNA levels showed inducible expression throughout the infection process in rice. To determine the location of MoHrip1 in M. oryzae, a mohrip1-gfp mutant was generated. Fluorescence microscopy observations and western blotting analysis showed that MoHrip1 was both present in the secretome and abundant in the fungal cell wall. To obtain MoHrip1 protein, we carried out high-yield expression of MoHrip1 in Pichia pastoris. Treatment of tobacco plants with MoHrip1 induced the formation of necrosis, accumulation of reactive oxygen species and expression of several defense-related genes, as well as conferred disease resistance. By fusion to green fluorescent protein, we showed that MoHrip1 was able to bind to the tobacco and rice plant plasma membrane, causing rapid morphological changes at the cellular level, such as cell shrinkage and chloroplast disorganization. These findings indicate that MoHrip1 is a microbe-associated molecular pattern that is perceived by the plant immune system. This is the first study on an AA1 family protein that can bind to the plant plasma membrane. Copyright © 2017 Elsevier Inc. All rights reserved.

  18. Effects of ethanol on plasma protein shedding in the human stomach

    International Nuclear Information System (INIS)

    Brassinne, A.

    1979-01-01

    Plasma protein shedding in the stomach was measured in 23 normal individuals before and after intragastric administration of a 30% solution of ethyl alcohol. Two different methods were used to assess plasma protein shedding. The first technique utilizes [ 131 I] albumin and requires neutralization of the gastric juice. It was used in 12 subjects and failed to demonstrate any increase of plasma protein shedding under the influence of ethanol. The second technique which utilizes [ 51 Cr] chloride was used in 11 subjects. It demonstrated a significant increase of the gastric clearance of plasma protein which reached 2.5 times the control values. The [ 51 Cr] chloride technique does not require prior neutralization of gastric acidity. It is concluded that, in normal man, ethanol administration increases plasma protein shedding in the stomach when it is given in the presence of an acid gastric juice. The effect is not observed when the gastric acidity is neutralized

  19. 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

  20. Function of plasma membrane microdomain-associated proteins during legume nodulation.

    Science.gov (United States)

    Qiao, Zhenzhen; Libault, Marc

    2017-10-03

    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.

  1. 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...

  2. Conservation of protein abundance patterns reveals the regulatory architecture of the EGFR-MAPK pathway

    Energy Technology Data Exchange (ETDEWEB)

    Shi, T.; Niepel, M.; McDermott, J. E.; Gao, Y.; Nicora, C. D.; Chrisler, W. B.; Markillie, L. M.; Petyuk, V. A.; Smith, R. D.; Rodland, K. D.; Sorger, P. K.; Qian, W. -J.; Wiley, H. S.

    2016-07-12

    It is not known whether cancer cells generally show quantitative differences in the expression of signaling pathway proteins that could dysregulate signal transduction. To explore this issue, we first defined the primary components of the EGF-MAPK pathway in normal human mammary epithelial cells, identifying 16 core proteins and 10 feedback regulators. We then quantified their absolute abundance across a panel of normal and cancer cell lines. We found that core pathway proteins were expressed at very similar levels across all cell types. In contrast, the EGFR and transcriptionally controlled feedback regulators were expressed at highly variable levels. The absolute abundance of most core pathway proteins was between 50,000- 70,000 copies per cell, but the adaptors SOS1, SOS2, and GAB1 were found at far lower levels (2,000-5,000 per cell). MAPK signaling showed saturation in all cells between 3,000-10,000 occupied EGFR, consistent with the idea that low adaptor levels limit signaling. Our results suggest that the core MAPK pathway is essentially invariant across different cell types, with cell- specific differences in signaling likely due to variable levels of feedback regulators. The low abundance of adaptors relative to the EGFR could be responsible for previous observation of saturable signaling, endocytosis, and high affinity EGFR.

  3. Multiple marker abundance profiling

    DEFF Research Database (Denmark)

    Hooper, Cornelia M.; Stevens, Tim J.; Saukkonen, Anna

    2017-01-01

    proteins and the scoring accuracy of lower-abundance proteins in Arabidopsis. NPAS was combined with subcellular protein localization data, facilitating quantitative estimations of organelle abundance during routine experimental procedures. A suite of targeted proteomics markers for subcellular compartment...

  4. Synaptic vesicle proteins under conditions of rest and activation: analysis by 2-D difference gel electrophoresis.

    Science.gov (United States)

    Burré, Jacqueline; Beckhaus, Tobias; Corvey, Carsten; Karas, Michael; Zimmermann, Herbert; Volknandt, Walter

    2006-09-01

    Synaptic vesicles are organelles of the nerve terminal that secrete neurotransmitters by fusion with the presynaptic plasma membrane. Vesicle fusion is tightly controlled by depolarization of the plasma membrane and a set of proteins that may undergo post-translational modifications such as phosphorylation. In order to identify proteins that undergo modifications as a result of synaptic activation, we induced massive exocytosis and analysed the synaptic vesicle compartment by benzyldimethyl-n-hexadecylammonium chloride (BAC)/SDS-PAGE and difference gel electrophoresis (DIGE) followed by MALDI-TOF-MS. We identified eight proteins that revealed significant changes in abundance following nerve terminal depolarization. Of these, six were increased and two were decreased in abundance. Three of these proteins were phosphorylated as detected by Western blot analysis. In addition, we identified an unknown synaptic vesicle protein whose abundance increased on synaptic activation. Our results demonstrate that depolarization of the presynaptic compartment induces changes in the abundance of synaptic vesicle proteins and post-translational protein modification.

  5. Proteomic analysis of plasma proteins in diabetic retinopathy patients by two dimensional electrophoresis and MALDI-Tof-MS.

    Science.gov (United States)

    Gopalakrishnan, Vidhya; Purushothaman, Parthiban; Bhaskar, Anusha

    2015-01-01

    Diabetic retinopathy is a highly specific vascular complication of diabetes mellitus and progresses from mild non-proliferative abnormalities characterized by increased vascular permeability to moderate and severe proliferative diabetic retinopathy characterized by the growth of blood vessels on the retina. The aim of the study was to identify the differentially expressed proteins in diabetic retinopathy using two-dimensional electrophoresis. Blood sample was drawn from subjects with diabetes mellitus (without retinopathy) who served as controls and patients with diabetic retinopathy in tubes containing EDTA as anticoagulant. Albumin and immunoglobulin IgG collectively removed to enrich proteins of lower abundance. 2de was carried out to see if there are any differentially expressed proteins. Approximately 48 and 61 spots were identified in control and diabetic retinopathy respectively, of which three protein spots RBP1 (retinol-binding protein 1), NUD10 (Diphosphoinositol polyphosphohydrolase 3 alpha), NGB (neuroglobin) were down regulated and HBG2 (hemoglobin) and BY55 (CD 160 antigen) were upregulated in diabetic retinopathy. These five protein spots were excised and were subjected to in-gel tryptic digestion, and their identities were determined by ultraflex MALDI-TOF-MS. We report a comprehensive patient-based plasma proteomic approach to the identification of potential biomarkers for diabetic retinopathy screening and detection. We identified 5 different proteins that were differentially expressed in the plasma of control diabetic patients (without retinopathy). Among these five proteins the expression of neuroglobin (NGB) protein varied significantly and may be a potential biomarker in diabetic retinopathy. Copyright © 2015 Elsevier Inc. All rights reserved.

  6. Abundances in planetary nebulae near the galactic centre .1. Abundance determinations

    NARCIS (Netherlands)

    Ratag, MA; Pottasch, [No Value; Dennefeld, M; Menzies, J

    1997-01-01

    Abundance determinations of about 110 planetary nebulae, which are likely to be in the Galactic Bulge are presented. Plasma diagnostics have been performed by making use of the available forbidden line ratios combined with radio continuum measurements. Chemical abundances of He, O, N, Ne, S, Ar, and

  7. Hepatic nuclear sterol regulatory binding element protein 2 abundance is decreased and that of ABCG5 increased in male hamsters fed plant sterols.

    Science.gov (United States)

    Harding, Scott V; Rideout, Todd C; Jones, Peter J H

    2010-07-01

    The effect of dietary plant sterols on cholesterol homeostasis has been well characterized in the intestine, but how plant sterols affect lipid metabolism in other lipid-rich tissues is not known. Changes in hepatic cholesterol homeostasis in response to high dietary intakes of plant sterols were determined in male golden Syrian hamsters fed hypercholesterolemia-inducing diets with and without 2% plant sterols (wt:wt; Reducol, Forbes Meditech) for 28 d. Plasma and hepatic cholesterol concentrations, cholesterol biosynthesis and absorption, and changes in the expression of sterol response element binding protein 2 (SREBP2) and liver X receptor-beta (LXRbeta) and their target genes were measured. Plant sterol feeding reduced plasma total cholesterol, non-HDL cholesterol, and HDL cholesterol concentrations 43% (P 6-fold (P = 0.029) and >2-fold (P sterol-fed hamsters compared with controls. Plant sterol feeding also increased fractional cholesterol synthesis >2-fold (P sterol feeding increased hepatic protein expression of cytosolic (inactive) SREBP2, decreased nuclear (active) SREBP2, and tended to increase LXRbeta (P = 0.06) and ATP binding cassette transporter G5, indicating a differential modulation of the expression of proteins central to cholesterol metabolism. In conclusion, high-dose plant sterol feeding of hamsters changes hepatic protein abundance in favor of cholesterol excretion despite lower hepatic cholesterol concentrations and higher cholesterol fractional synthesis.

  8. Rupturing Giant Plasma Membrane Vesicles to Form Micron-sized Supported Cell Plasma Membranes with Native Transmembrane Proteins.

    Science.gov (United States)

    Chiang, Po-Chieh; Tanady, Kevin; Huang, Ling-Ting; Chao, Ling

    2017-11-09

    Being able to directly obtain micron-sized cell blebs, giant plasma membrane vesicles (GPMVs), with native membrane proteins and deposit them on a planar support to form supported plasma membranes could allow the membrane proteins to be studied by various surface analytical tools in native-like bilayer environments. However, GPMVs do not easily rupture on conventional supports because of their high protein and cholesterol contents. Here, we demonstrate the possibility of using compression generated by the air-water interface to efficiently rupture GPMVs to form micron-sized supported membranes with native plasma membrane proteins. We demonstrated that not only lipid but also a native transmembrane protein in HeLa cells, Aquaporin 3 (AQP3), is mobile in the supported membrane platform. This convenient method for generating micron-sized supported membrane patches with mobile native transmembrane proteins could not only facilitate the study of membrane proteins by surface analytical tools, but could also enable us to use native membrane proteins for bio-sensing applications.

  9. 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.

  10. Separomics applied to the proteomics and peptidomics of low-abundance proteins: Choice of methods and challenges – A review

    Science.gov (United States)

    Baracat-Pereira, Maria Cristina; de Oliveira Barbosa, Meire; Magalhães, Marcos Jorge; Carrijo, Lanna Clicia; Games, Patrícia Dias; Almeida, Hebréia Oliveira; Sena Netto, José Fabiano; Pereira, Matheus Rodrigues; de Barros, Everaldo Gonçalves

    2012-01-01

    The enrichment and isolation of proteins are considered limiting steps in proteomic studies. Identification of proteins whose expression is transient, those that are of low-abundance, and of natural peptides not described in databases, is still a great challenge. Plant extracts are in general complex, and contaminants interfere with the identification of proteins involved in important physiological processes, such as plant defense against pathogens. This review discusses the challenges and strategies of separomics applied to the identification of low-abundance proteins and peptides in plants, especially in plants challenged by pathogens. Separomics is described as a group of methodological strategies for the separation of protein molecules for proteomics. Several tools have been used to remove highly abundant proteins from samples and also non-protein contaminants. The use of chromatographic techniques, the partition of the proteome into subproteomes, and an effort to isolate proteins in their native form have allowed the isolation and identification of rare proteins involved in different processes. PMID:22802713

  11. Spatial Mapping of Protein Abundances in the Mouse Brain by Voxelation Integrated with High-Throughput Liquid Chromatography ? Mass Spectrometry

    International Nuclear Information System (INIS)

    Petyuk, Vladislav A.; Qian, Weijun; Chin, Mark H.; Wang, Haixing H.; Livesay, Eric A.; Monroe, Matthew E.; Adkins, Joshua N.; Jaitly, Navdeep; Anderson, David J.; Camp, David G.; Smith, Desmond J.; Smith, Richard D.

    2007-01-01

    Temporally and spatially resolved mapping of protein abundance patterns within the mammalian brain is of significant interest for understanding brain function and molecular etiologies of neurodegenerative diseases; however, such imaging efforts have been greatly challenged by complexity of the proteome, throughput and sensitivity of applied analytical methodologies, and accurate quantitation of protein abundances across the brain. Here, we describe a methodology for comprehensive spatial proteome mapping that addresses these challenges by employing voxelation integrated with automated microscale sample processing, high-throughput LC system coupled with high resolution Fourier transform ion cyclotron mass spectrometer and a ''universal'' stable isotope labeled reference sample approach for robust quantitation. We applied this methodology as a proof-of-concept trial for the analysis of protein distribution within a single coronal slice of a C57BL/6J mouse brain. For relative quantitation of the protein abundances across the slice, an 18O-isotopically labeled reference sample, derived from a whole control coronal slice from another mouse, was spiked into each voxel sample and stable isotopic intensity ratios were used to obtain measures of relative protein abundances. In total, we generated maps of protein abundance patterns for 1,028 proteins. The significant agreement of the protein distributions with previously reported data supports the validity of this methodology, which opens new opportunities for studying the spatial brain proteome and its dynamics during the course of disease progression and other important biological and associated health aspects in a discovery-driven fashion

  12. Plasma cholesteryl ester transfer protein mass and phospholipid transfer protein activity are associated with leptin in type 2 diabetes mellitus

    NARCIS (Netherlands)

    Dullaart, R. P. F.; de Vries, R.; Dallinga-Thie, G. M.; van Tol, A.; Sluiter, W. J.

    Adipose tissue contributes to plasma levels of lipid transfer proteins and is also the major source of plasma adipokines. We hypothesized that plasma cholesteryl ester transfer protein (CETP) mass, phospholipid transfer protein (PLTP) activity and cholesteryl ester transfer (CET, a measure of CETP

  13. MAMP (microbe-associated molecular pattern)-induced changes in plasma membrane-associated proteins.

    Science.gov (United States)

    Uhlíková, Hana; Solanský, Martin; Hrdinová, Vendula; Šedo, Ondrej; Kašparovský, Tomáš; Hejátko, Jan; Lochman, Jan

    2017-03-01

    Plant plasma membrane associated proteins play significant roles in Microbe-Associated Molecular Pattern (MAMP) mediated defence responses including signal transduction, membrane transport or energetic metabolism. To elucidate the dynamics of proteins associated with plasma membrane in response to cryptogein, a well-known MAMP of defence reaction secreted by the oomycete Phytophthora cryptogea, 2D-Blue Native/SDS gel electrophoresis of plasma membrane fractions was employed. This approach revealed 21 up- or down-regulated protein spots of which 15 were successfully identified as proteins related to transport through plasma membrane, vesicle trafficking, and metabolic enzymes including cytosolic NADP-malic enzyme and glutamine synthetase. Observed changes in proteins were also confirmed on transcriptional level by qRT-PCR analysis. In addition, a significantly decreased accumulation of transcripts observed after employment of a mutant variant of cryptogein Leu41Phe, exhibiting a conspicuous defect in induction of resistance, sustains the contribution of identified proteins in cryptogein-triggered cellular responses. Our data provide further evidence for dynamic MAMP-induced changes in plasma membrane associated proteins. Copyright © 2016 Elsevier GmbH. All rights reserved.

  14. Effects of copper and tributyltin on stress protein abundance in the rotifer Brachionus plicatilis.

    Science.gov (United States)

    Cochrane, B J; Irby, R B; Snell, T W

    1991-01-01

    1. Exposure of the rotifer Brachionus plicatilis to elevated temperature resulted in the synthesis of a number of proteins, including a prominent one of 58,000 Da (SP58). 2. This protein is immunologically crossreactive with the 65,000 Da heat shock protein of the moth Heliothis virescens, which is a member of a highly conserved family of mitochondrial proteins. 3. Exposure of rotifers to sublethal doses of CuSO4 leads to a 4-5-fold increase in abundance of SP58, with maximum increase occurring at a dose that is approximately 5% of the LC50 for that compound. 4. A similar response was seen with tributyl tin (TBT). Kinetics of induction were sigmoidal, with induction occurring in the range of 20-30 micrograms/l. 5. No response was observed when rotifers were exposed to aluminum chloride, mercury chloride, pentachlorophenol, sodium arsenite, sodium azide, sodium dodecyl sulfate, or zinc chloride. 6. These results indicate that changes in stress protein abundance may prove useful as a biomarker of exposure to particular toxicants.

  15. Differences in abundances of cell-signalling proteins in blood reveal novel biomarkers for early detection of clinical Alzheimer's disease.

    Directory of Open Access Journals (Sweden)

    Mateus Rocha de Paula

    Full Text Available BACKGROUND: In November 2007 a study published in Nature Medicine proposed a simple test based on the abundance of 18 proteins in blood to predict the onset of clinical symptoms of Alzheimer's Disease (AD two to six years before these symptoms manifest. Later, another study, published in PLoS ONE, showed that only five proteins (IL-1, IL-3, EGF, TNF- and G-CSF have overall better prediction accuracy. These classifiers are based on the abundance of 120 proteins. Such values were standardised by a Z-score transformation, which means that their values are relative to the average of all others. METHODOLOGY: The original datasets from the Nature Medicine paper are further studied using methods from combinatorial optimisation and Information Theory. We expand the original dataset by also including all pair-wise differences of z-score values of the original dataset ("metafeatures". Using an exact algorithm to solve the resulting Feature Set problem, used to tackle the feature selection problem, we found signatures that contain either only features, metafeatures or both, and evaluated their predictive performance on the independent test set. CONCLUSIONS: It was possible to show that a specific pattern of cell signalling imbalance in blood plasma has valuable information to distinguish between NDC and AD samples. The obtained signatures were able to predict AD in patients that already had a Mild Cognitive Impairment (MCI with up to 84% of sensitivity, while maintaining also a strong prediction accuracy of 90% on a independent dataset with Non Demented Controls (NDC and AD samples. The novel biomarkers uncovered with this method now confirms ANG-2, IL-11, PDGF-BB, CCL15/MIP-1; and supports the joint measurement of other signalling proteins not previously discussed: GM-CSF, NT-3, IGFBP-2 and VEGF-B.

  16. Ready-made chromatography columns for extracellular vesicle isolation from plasma

    Directory of Open Access Journals (Sweden)

    Joanne Louise Welton

    2015-03-01

    Full Text Available Proteomic studies of circulating vesicles are hampered by difficulties in purifying vesicles from plasma and serum. Isolations are contaminated with high-abundance blood proteins that may mask genuine vesicular-associated proteins and/or simply provide misleading data. In this brief report, we explored the potential utility of a commercially available size exclusion chromatography column for rapid vesicle purification. We evaluated the performance of the column, with cancer cell line conditioned medium or healthy donor plasma, in terms of removing non-vesicular protein and enriching for vesicles exhibiting exosome characteristics. Serial fractions revealed a peak for typical exosomal proteins (CD9, CD81 etc. that preceded the peak for highly abundant proteins, including albumin, for either sample type, and harvesting only this peak would represent elimination of >95% of protein from the sample. The columns showed good reproducibility, and streamlining the workflow would allow the exosome-relevant material to be collected in less than 10 minutes. Surprisingly, however, subsequent post-column vesicle concentration steps whilst resulting in some protein loss also lead to low vesicle recoveries, with a net effect of reducing sample purity (assessed by the particle-to-protein ratio. The columns provide a convenient, reproducible and highly effective means of eliminating >95% of non-vesicular protein from biological fluid samples such as plasma.

  17. Protein degradation rate is the dominant mechanism accounting for the differences in protein abundance of basal p53 in a human breast and colorectal cancer cell line.

    Directory of Open Access Journals (Sweden)

    Eszter Lakatos

    Full Text Available We determine p53 protein abundances and cell to cell variation in two human cancer cell lines with single cell resolution, and show that the fractional width of the distributions is the same in both cases despite a large difference in average protein copy number. We developed a computational framework to identify dominant mechanisms controlling the variation of protein abundance in a simple model of gene expression from the summary statistics of single cell steady state protein expression distributions. Our results, based on single cell data analysed in a Bayesian framework, lends strong support to a model in which variation in the basal p53 protein abundance may be best explained by variations in the rate of p53 protein degradation. This is supported by measurements of the relative average levels of mRNA which are very similar despite large variation in the level of protein.

  18. Protein degradation rate is the dominant mechanism accounting for the differences in protein abundance of basal p53 in a human breast and colorectal cancer cell line.

    Science.gov (United States)

    Lakatos, Eszter; Salehi-Reyhani, Ali; Barclay, Michael; Stumpf, Michael P H; Klug, David R

    2017-01-01

    We determine p53 protein abundances and cell to cell variation in two human cancer cell lines with single cell resolution, and show that the fractional width of the distributions is the same in both cases despite a large difference in average protein copy number. We developed a computational framework to identify dominant mechanisms controlling the variation of protein abundance in a simple model of gene expression from the summary statistics of single cell steady state protein expression distributions. Our results, based on single cell data analysed in a Bayesian framework, lends strong support to a model in which variation in the basal p53 protein abundance may be best explained by variations in the rate of p53 protein degradation. This is supported by measurements of the relative average levels of mRNA which are very similar despite large variation in the level of protein.

  19. Preliminary study on plasma proteins in pregnant and non-pregnant female dogs.

    Science.gov (United States)

    Szczubiał, Marek; Wawrzykowski, Jacek; Dąbrowski, Roman; Krawczyk, Magdalena; Kankofer, Marta

    2017-07-15

    In this study, we used a combined approach based on 2-dimensional electrophoresis (2-DE), difference in gel electrophoresis (DIGE), and mass spectrometry (MS) to identify the plasma protein composition in pregnant female dogs and compared it with non-pregnant female dogs. We used the plasma samples obtained from four female dogs during I, II, and III thirds of pregnancy, three days after parturition, as well as from four non-pregnant female dogs in diestrus phase. Analysis of 2-DE gel image exhibited of 249 protein spots. The intensity of staining of 35 spots differed significantly (P dogs. We used matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI TOF MS) to identify 47 spots corresponding to 52 different proteins. Five identified protein spots, including zinc finger BED domain-containing protein 5, hemoglobin subunit beta-2, integrator complex subunit 7, apolipoprotein A-I, and glutamyl aminopeptidase were differentially presented in the plasma of pregnant and non-pregnant female dogs. To the best of our knowledge, this is the first report on the plasma protein profile of pregnant and non-pregnant female dogs. In this study, we identified proteins that have not been previously identified in dogs. Our findings showed that numerous protein spots were differentially presented in the plasma of female dogs during normal pregnancy. Although we identified only a limited number of differentially presented proteins, our study demonstrated that the plasma protein profile changed during pregnancy in female dogs, which suggests its importance in maintaining pregnancy. Further studies are necessary to define complete plasma protein profile of pregnant female dogs and to identify all proteins that are differentially presented in the pregnant animals compared with the non-pregnant ones. In addition, studies are warranted to explain the role of those proteins in maintaining the pregnancy and their usefulness in detection of early pregnancy

  20. Shotgun proteomics of plant plasma membrane and microdomain proteins using nano-LC-MS/MS.

    Science.gov (United States)

    Takahashi, Daisuke; Li, Bin; Nakayama, Takato; Kawamura, Yukio; Uemura, Matsuo

    2014-01-01

    Shotgun proteomics allows the comprehensive analysis of proteins extracted from plant cells, subcellular organelles, and membranes. Previously, two-dimensional gel electrophoresis-based proteomics was used for mass spectrometric analysis of plasma membrane proteins. In order to get comprehensive proteome profiles of the plasma membrane including highly hydrophobic proteins with a number of transmembrane domains, a mass spectrometry-based shotgun proteomics method using nano-LC-MS/MS for proteins from the plasma membrane proteins and plasma membrane microdomain fraction is described. The results obtained are easily applicable to label-free protein semiquantification.

  1. Rapid Upregulation of Orai1 Abundance in the Plasma Membrane of Platelets Following Activation with Thrombin and Collagen Related Peptide

    Directory of Open Access Journals (Sweden)

    Guilai Liu

    2015-11-01

    Full Text Available Background: Blood platelets accomplish primary hemostasis following vascular injury and contribute to the orchestration of occlusive vascular disease. Platelets are activated by an increase of cytosolic Ca2+-activity ([Ca2+]i, which is accomplished by Ca2+-release from intracellular stores and subsequent store operated Ca2+ entry (SOCE through Ca2+ release activated Ca2+ channel moiety Orai1. Powerful activators of platelets include thrombin and collagen related peptide (CRP, which are in part effective by activation of small G- protein Rac1. The present study explored the influence of thrombin and CRP on Orai1 protein abundance and cytosolic Ca2+-activity ([Ca2+]i in platelets drawn from wild type mice. Methods: Orai1 protein surface abundance was quantified utilizing CF™488A conjugated antibodies, and [Ca2+]i was determined with Fluo3-fluorescence. Results: In resting platelets, Orai1 protein abundance and [Ca2+]i were low. Thrombin (0.02 U/ml and CRP (5ug/ml within 2 min increased [Ca2+]i and Orai1 protein abundance at the platelet surface. [Ca2+]i was further increased by Ca2+ ionophore ionomycin (1 µM and by store depletion with the sarcoendoplasmatic Ca2+ ATPase inhibitor thapsigargin (1 µM. However, Orai1 protein abundance at the platelet surface was not significantly affected by ionomycin and only slightly increased by thapsigargin. The effect of thrombin and CRP on Orai1 abundance and [Ca2+]i was significantly blunted by Rac1 inhibitor NSC23766 (50 µM. Conclusion: The increase of [Ca2+]i following stimulation of platelets with thrombin and collagen related peptide is potentiated by ultrarapid Rac1 sensitive translocation of Orai1 into the cell membrane.

  2. 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...... in plasma, and 2) determine whether the plasma proteins are likely to be causally implicated in disease. We report here the results of genome-wide association (GWA) studies of 83 proteins considered relevant to cardiovascular disease (CVD), measured in 3,394 individuals with multiple CVD risk factors. We...... on coronary artery disease, and highlight several potentially causal associations. Overall, a majority of the plasma proteins studied showed evidence of regulation at the genetic level. Our results enable future studies of the causal architecture of human disease, which in turn should aid discovery of new...

  3. Evolutionary plasticity of plasma membrane interaction in DREPP family proteins.

    Science.gov (United States)

    Vosolsobě, Stanislav; Petrášek, Jan; Schwarzerová, Kateřina

    2017-05-01

    The plant-specific DREPP protein family comprises proteins that were shown to regulate the actin and microtubular cytoskeleton in a calcium-dependent manner. Our phylogenetic analysis showed that DREPPs first appeared in ferns and that DREPPs have a rapid and plastic evolutionary history in plants. Arabidopsis DREPP paralogues called AtMDP25/PCaP1 and AtMAP18/PCaP2 are N-myristoylated, which has been reported as a key factor in plasma membrane localization. Here we show that N-myristoylation is neither conserved nor ancestral for the DREPP family. Instead, by using confocal microscopy and a new method for quantitative evaluation of protein membrane localization, we show that DREPPs rely on two mechanisms ensuring their plasma membrane localization. These include N-myristoylation and electrostatic interaction of a polybasic amino acid cluster. We propose that various plasma membrane association mechanisms resulting from the evolutionary plasticity of DREPPs are important for refining plasma membrane interaction of these signalling proteins under various conditions and in various cells. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. Modulating Protein Adsorption on Oxygen Plasma Modified Polysiloxane Surfaces

    International Nuclear Information System (INIS)

    Marletta, G.

    2006-01-01

    In the present paper we report the study on the adsorption behaviour of three model globular proteins, Human Serum Albumin, Lactoferrin and Egg Chicken Lysozyme onto both unmodified surfaces of a silicon-based polymer and the corresponding plasma treated surfaces. In particular, thin films of hydrophobic polysiloxane (about 90 degree of static water contact angle, WCA) were converted by oxygen plasma treatment at reduced pressure into very hydrophilic phases of SiOx (WCA less than 5 degree). The kinetics of protein adsorption processes were investigated by QCM-D technique, while the chemical structure and topography of the protein adlayer have been studied by Angular resolved-XPS and AFM respectively. It turned out that Albumin and Lysozyme exhibited the opposite preferential adsorption respectively onto the hydrophobic and hydrophilic surfaces, while Lactoferrin did not exhibit significant differences. The observed protein behaviour are discussed both in terms of surface-dependent parameters, including surface free energy and chemical structure, and in terms of protein-dependent parameters, including charge as well as the average molecular orientation in the adlayers. Finally, some examples of differential adsorption behaviour of the investigated proteins are reported onto nanopatterned polysiloxane surfaces consisting of hydrophobic nanopores surrounded by hydrophilic (plasma-treated) matrix and the reverse

  5. Plasma protein loss associated with gastrointestinal parasitism in grazing sheep.

    Science.gov (United States)

    Yakoob, A Y; Holmes, P H; Parkins, J J; Armour, J

    1983-01-01

    Some pathophysiological effects of parasitic gastroenteritis in two groups of lambs grazing paddocks either heavily or lightly contaminated with trichostrongyle larvae were investigated between July and October 1980. The leak of plasma protein was measured on three occasions at pasture using 51chromic chloride. Total faecal output was measured indirectly using chromic oxide. Losses of 51chromic chloride-labelled plasma protein into the gastrointestinal tract were significantly higher in the lambs grazing the heavily contaminated pasture than in those grazing lightly infected ground in both July and August. The increased plasma losses were associated with high faecal egg counts, hypoalbuminaemia and elevated levels of plasma pepsinogen.

  6. [Purification of arsenic-binding proteins in hamster plasma after oral administration of arsenite].

    Science.gov (United States)

    Wang, Wenwen; Zhang, Min; Li, Chunhui; Qin, Yingjie; Hua, Naranmandura

    2013-01-01

    To purify the arsenic-binding proteins (As-BP) in hamster plasma after a single oral administration of arsenite (iAs(III)). Arsenite was given to hamsters in a single dose. Three types of HPLC columns, size exclusion, gel filtration and anion exchange columns, combined with an inductively coupled argon plasma mass spectrometer (ICP MS) were used to purify the As-BP in hamster plasma. SDS-PAGE was used to confirm the arsenic-binding proteins at each purification step. The three-step purification process successfully separated As-BP from other proteins (ie, arsenic unbound proteins) in hamster plasma. The molecular mass of purified As-BP in plasma was approximately 40-50 kD on SDS-PAGE. The three-step purification method is a simple and fast approach to purify the As-BP in plasma samples.

  7. Nitrogen 15 abundance in protein fractions of beans fertilized with ({sup 15}NH{sub 4}){sub 2}SO{sub 4}

    Energy Technology Data Exchange (ETDEWEB)

    Chaud, Saula Goulart; Oliveira, Admar Costa de [Universidade Estadual de Campinas, SP (Brazil). Faculdade de Estudos Agricolas. Dept. de Planejamento Alimentar e Nutricao; Trivelin, Paulo Cesar Ocheuze [Centro de Energia Nuclear na Agricultura (CENA), Piracicaba, SP (Brazil). Lab. de Isotopos Estaveis]. E-mail: admarco@fea.unicamp.br

    2002-12-01

    Studies evaluating the protein nutritive value of beans labelled with 15 N, using nitrogen balance and the quantitation of faecal and urinary endogenous nitrogen, determined by isotopic dilution, have been extensively used. The objective of this research was to verify if the isotopic labelling of raw, freeze dried beans (Phaseolus vulgaris L., cultivar Pirata 1) with 1.394 atoms % 15 N, resulted in the same abundance of the whole flour and of the protein fractions extracted from the beans with 0.5 mol L{sup -1} NaCl. The isotopic abundance found in the whole bean flour, in the protein extract, in the globulin and albumin fractions were respectively: 1.394 +- 0.011; 1.403 +- 0.012; 1.399 +- 0.007 and 1.399 +- 0.028 atoms % of 15 N, presenting no difference (P > 0.05). However, a difference was found (P < 0.05) between the above mentioned abundances and the isotopic abundance found in the nitrogen of the proteins in the extraction residue, which was 0.969 +- 0.084. Since the abundances did not differ, the protein nutritive indexes, such as digestibility and biological value, determined from the nitrogen balance and corrected for isotopic dilution, would not be affected by extracting the proteins from the beans with 0.5 mol L 1 NaCl. If working with the nitrogen balance of the residual proteins after extraction and even with the whole flours, these indexes could present incorrect values, since the isotopic labelling of the residual proteins was less than that of the protein fractions. (author)

  8. Low plasma adiponectin concentrations do not predict weight gain in humans

    DEFF Research Database (Denmark)

    Vozarova, Barbora; Stefan, Norbert; Lindsay, Robert S

    2002-01-01

    Low concentrations of plasma adiponectin, the most abundant adipose-specific protein, are observed in obese individuals and predict the development of type 2 diabetes. Administration of adiponectin to rodents prevented diet-induced weight gain, suggesting a potential etiologic role of hypoadipone......Low concentrations of plasma adiponectin, the most abundant adipose-specific protein, are observed in obese individuals and predict the development of type 2 diabetes. Administration of adiponectin to rodents prevented diet-induced weight gain, suggesting a potential etiologic role...... of hypoadiponectinemia in the development of obesity. Our aim was to prospectively examine whether low plasma adiponectin concentrations predict future weight gain in Pima Indians, explaining the predictive effect of adiponectin on the development of type 2 diabetes. We measured plasma adiponectin concentrations in 219...... nondiabetic Pima Indians (112 M/107 F, age 31 +/- 9 years, body weight 96 +/- 20 kg [mean +/- SD]) in whom body weight and height were measured and BMI calculated at baseline and follow-up. Cross-sectionally, plasma adiponectin concentrations were negatively associated with body weight (r = -0.28, P = 0...

  9. Mass spectrometric identification of diagnostic markers for chronic prostatitis in seminal plasma by analysis of seminal plasma protein clinical samples.

    Science.gov (United States)

    Rokka, A; Mehik, A; Tonttila, P; Vaarala, M

    2017-08-15

    There are few specific diagnostic markers for chronic prostatitis. Therefore, we used mass spectrometry to evaluate differences in seminal plasma protein expression among patients with prostatitis and young and middle-aged healthy controls. We analysed pooled seminal plasma protein samples from four prostatitis patients (two pools), three young controls (one pool), and three middle-aged controls (one pool). The samples were analysed by liquid chromatography-tandem mass spectrometry. Of the 349 proteins identified, 16 were differentially expressed between the two control pools. Five proteins were up- or down-regulated in both of the prostatitis pools compared to middle-aged controls but not between young and middle-aged pools. Progestagen-associated endometrial protein (PAEP) was over-expressed in prostatitis samples compared to young and middle-aged controls. Our findings and those of previous studies indicate that PAEP is a potential seminal plasma marker for chronic prostatitis. In conclusion, we found age-related changes in seminal plasma protein expression. PAEP expression in seminal plasma should be investigated further to evaluate its potential as a diagnostic marker for chronic prostatitis.

  10. Inhibition of platelet [3H]- imipramine binding by human plasma protein fractions

    International Nuclear Information System (INIS)

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

    1988-01-01

    Inhibition of high-affinity [ 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 1 acid glycoprotein, high density and low density lipoprotein, IgG and α 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 [ 3 H]-imipramine binding site

  11. Solubilization of rat kidney plasma membrane proteins associated with 3H-aldosterone

    International Nuclear Information System (INIS)

    Ozegovic, B.; Dobrovic-Jenik, D.; Milkovic, S.

    1988-01-01

    The treatment of rat kidney plasma membranes with sodium dodecyl sulphate (SDS) did not essentially affect the ability of the membranes for 3 H-aldosterone binding as compared with the intact plasma membranes (Ozegovic et al., 1977). A gel filtration of 3 H-aldosterone - kidney plasma membranes complex on Sepharose 6B yielded 2 protein and 2 3 H-aldosterone peaks. The proteins which were eluted in the first peak were associated with the first 3 H-aldosterone peak while the second 3 H-aldosterone peak was eluted with Ve corresponding to Ve of free 3 H-aldosterone. Spironolactone, a competitive antagonist of aldosterone, prevented the binding of 3 H-aldosterone to the membrane proteins. The results demonstrated a high affinity of the kidney plasma membranes solubilized with SDS and a specificity of aldosterone binding to the plasma membrane proteins of higher molecular mass. (author)

  12. Enrichment and identification of the most abundant zinc binding proteins in developing barley grains by Zinc-IMAC capture and nano LC-MS/MS

    DEFF Research Database (Denmark)

    Dionisio, Giuseppe; Uddin, Mohammad Nasir; Vincze, Eva

    2018-01-01

    exhibited zinc binding. In the aleurone/subaleurone, zinc affinity captured proteins were late abundant embryogenesis proteins, dehydrins, many isoforms of non-specific lipid transfer proteins, and alpha amylase trypsin inhibitor. Conclusions: We have shown evidence that abundant barley grain proteins have......Background: Zinc accumulates in the embryo, aleurone, and subaleurone layers at different amounts in cereal grains. Our hypothesis is that zinc could be stored bound, not only to low MW metabolites/proteins, but also to high MW proteins as well. Methods: In order to identify the most abundant zinc...

  13. 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

  14. Irradiation of porcine plasma protein powder, 1

    International Nuclear Information System (INIS)

    Hayashi, Toru; Saito, Masayoshi; Todoroki, Setsuko; Tajima, Makoto; Biagio, R.

    1987-01-01

    Recently interest in the use of animal blood protein as a food ingradient has been increasing. A study was conducted on the decontamination effect of gamma rays and electrons beam on plasma protein powder prepared from slaughtered porcine blood. Non irradiated sample was mainly contaminated with heat-resistant becterial spores (B. subtilis) and the total mocrobial count was 9.6 x 10 3 per 1 g of dried powder. The D 10 values of total microbial count for gamma rays and electrons beam were 0.82 kGy and 1.06 kGy, respectively. For B. subtilis, the D 10 values obtained under aerobic condition were 1.40 kGy for gamma rays and 1.45 kGy for electrons beam, with the survival curve for electrons beam showing a shoulder until 0.1 kGy. From these results, both types of irradiation were effective for the decotamination of plasma proteins. (author)

  15. Looking deep inside: detection of low-abundance proteins in leaf extracts of Arabidopsis and phloem exudates of pumpkin.

    Science.gov (United States)

    Fröhlich, Andreas; Gaupels, Frank; Sarioglu, Hakan; Holzmeister, Christian; Spannagl, Manuel; Durner, Jörg; Lindermayr, Christian

    2012-07-01

    The field of proteomics suffers from the immense complexity of even small proteomes and the enormous dynamic range of protein concentrations within a given sample. Most protein samples contain a few major proteins, which hamper in-depth proteomic analysis. In the human field, combinatorial hexapeptide ligand libraries (CPLL; such as ProteoMiner) have been used for reduction of the dynamic range of protein concentrations; however, this technique is not established in plant research. In this work, we present the application of CPLL to Arabidopsis (Arabidopsis thaliana) leaf proteins. One- and two-dimensional gel electrophoresis showed a decrease in high-abundance proteins and an enrichment of less abundant proteins in CPLL-treated samples. After optimization of the CPLL protocol, mass spectrometric analyses of leaf extracts led to the identification of 1,192 proteins in control samples and an additional 512 proteins after the application of CPLL. Upon leaf infection with virulent Pseudomonas syringae DC3000, CPLL beads were also used for investigating the bacterial infectome. In total, 312 bacterial proteins could be identified in infected Arabidopsis leaves. Furthermore, phloem exudates of pumpkin (Cucurbita maxima) were analyzed. CPLL prefractionation caused depletion of the major phloem proteins 1 and 2 and improved phloem proteomics, because 67 of 320 identified proteins were detectable only after CPLL treatment. In sum, our results demonstrate that CPLL beads are a time- and cost-effective tool for reducing major proteins, which often interfere with downstream analyses. The concomitant enrichment of less abundant proteins may facilitate a deeper insight into the plant proteome.

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

    African Journals Online (AJOL)

    Dr Olaleye Samuel

    macroalbuminuric type II diabetes subjects compared with type II diabetes patients with microalbuminuria and healthy subjects , showing an upregulation of hepatic secretory proteins ... order to reduce the effect of diet on plasma proteins.

  17. 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)

    Fagundes, B.

    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.

  18. 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

    The effect of experimentally increasing the postpartum protein supply on plasma protein synthesis, rumen tissue proliferation, and immune homeostasis was studied using 8 periparturient Holstein cows in a complete randomized design. At calving, cows were assigned to abomasal infusion of water (CTRL......) or casein (CAS) in addition to a lactation diet. Casein infusion was gradually decreased from 696 ± 1 g/d at +2 d relative to calving (DRTC) to 212 ± 10 g/d at +29 DRTC to avoid excessive supply. Synthesis rate of plasma proteins was measured at –14, +4, +15, and +29 DRTC by measuring [13C]Phe isotopic...... 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...

  19. Aldosterone-mineralocorticoid receptor promotes urine prostasin through glomerular barrier injury and not tissue abundance

    DEFF Research Database (Denmark)

    Stolzenburg Oxlund, Christina; Kurt, B.; Schwarzensteiner, I.

    2015-01-01

    with placebo or the mineralocorticoid antagonist spironolactone. Western immunoblotting of creatinine-normalized urine samples was performed from placebo and spironolactone treated patients with and without albuminuria. Tissue prostasin was measured in membranes from human nephrectomy recieving either ACE......-i/ANGII or no antihypertensive treatment prior to operation. Urine and tissue prostasin was measured in puromycin-induced nephrotic syndrome rats. Results: Plasma prostasin concentration increased significantly with spironolactone but was not changed with placebo. Urine prostasin concentration was below detection limit....... Puromycin-induced nephrotic syndrome in rats was associated with significant increase in u-prostasin while kidney tissue prostasin protein abundance was not changed. Prostasin protein abundance was similar in membranes from human nephrectomy homogenate from patients treated preoperatively with ACE...

  20. 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...

  1. 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.

  2. Profiling of kidney vascular endothelial cell plasma membrane proteins by liquid chromatography-tandem mass spectrometry.

    Science.gov (United States)

    Liu, Zan; Xu, Bo; Nameta, Masaaki; Zhang, Ying; Magdeldin, Sameh; Yoshida, Yutaka; Yamamoto, Keiko; Fujinaka, Hidehiko; Yaoita, Eishin; Tasaki, Masayuki; Nakagawa, Yuki; Saito, Kazuhide; Takahashi, Kota; Yamamoto, Tadashi

    2013-06-01

    Vascular endothelial cells (VECs) play crucial roles in physiological and pathologic conditions in tissues and organs. Most of these roles are related to VEC plasma membrane proteins. In the kidney, VECs are closely associated with structures and functions; however, plasma membrane proteins in kidney VECs remain to be fully elucidated. Rat kidneys were perfused with cationic colloidal silica nanoparticles (CCSN) to label the VEC plasma membrane. The CCSN-labeled plasma membrane fraction was collected by gradient ultracentrifugation. The VEC plasma membrane or whole-kidney lysate proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and digested with trypsin in gels for liquid chromatography-tandem mass spectrometry. Enrichment analysis was then performed. The VEC plasma membrane proteins were purified by the CCSN method with high yield (approximately 20 μg from 1 g of rat kidney). By Mascot search, 582 proteins were identified in the VEC plasma membrane fraction, and 1,205 proteins were identified in the kidney lysate. In addition to 16 VEC marker proteins such as integrin beta-1 and intercellular adhesion molecule-2 (ICAM-2), 8 novel proteins such as Deltex 3-like protein and phosphatidylinositol binding clathrin assembly protein (PICALM) were identified. As expected, many key functions of plasma membranes in general and of endothelial cells in particular (i.e., leukocyte adhesion) were significantly overrepresented in the proteome of CCSN-labeled kidney VEC fraction. The CCSN method is a reliable technique for isolation of VEC plasma membrane from the kidney, and proteomic analysis followed by bioinformatics revealed the characteristics of in vivo VECs in the kidney.

  3. Spectral counting assessment of protein dynamic range in cerebrospinal fluid following depletion with plasma-designed immunoaffinity columns

    Directory of Open Access Journals (Sweden)

    Borg Jacques

    2011-06-01

    Full Text Available Abstract Background In cerebrospinal fluid (CSF, which is a rich source of biomarkers for neurological diseases, identification of biomarkers requires methods that allow reproducible detection of low abundance proteins. It is therefore crucial to decrease dynamic range and improve assessment of protein abundance. Results We applied LC-MS/MS to compare the performance of two CSF enrichment techniques that immunodeplete either albumin alone (IgYHSA or 14 high-abundance proteins (IgY14. In order to estimate dynamic range of proteins identified, we measured protein abundance with APEX spectral counting method. Both immunodepletion methods improved the number of low-abundance proteins detected (3-fold for IgYHSA, 4-fold for IgY14. The 10 most abundant proteins following immunodepletion accounted for 41% (IgY14 and 46% (IgYHSA of CSF protein content, whereas they accounted for 64% in non-depleted samples, thus demonstrating significant enrichment of low-abundance proteins. Defined proteomics experiment metrics showed overall good reproducibility of the two immunodepletion methods and MS analysis. Moreover, offline peptide fractionation in IgYHSA sample allowed a 4-fold increase of proteins identified (520 vs. 131 without fractionation, without hindering reproducibility. Conclusions The novelty of this study was to show the advantages and drawbacks of these methods side-to-side. Taking into account the improved detection and potential loss of non-target proteins following extensive immunodepletion, it is concluded that both depletion methods combined with spectral counting may be of interest before further fractionation, when searching for CSF biomarkers. According to the reliable identification and quantitation obtained with APEX algorithm, it may be considered as a cheap and quick alternative to study sample proteomic content.

  4. Molecular Characterization of Aquaporin 1 and Aquaporin 3 from the Gills of the African Lungfish, Protopterus annectens, and Changes in Their Branchial mRNA Expression Levels and Protein Abundance during Three Phases of Aestivation.

    Science.gov (United States)

    Chng, You R; Ong, Jasmine L Y; Ching, Biyun; Chen, Xiu L; Hiong, Kum C; Wong, Wai P; Chew, Shit F; Lam, Siew H; Ip, Yuen K

    2016-01-01

    African lungfishes can undergo long periods of aestivation on land during drought. During aestivation, lungfishes are confronted with desiccation and dehydration, and their gills become non-functional and covered with a thick layer of dried mucus. Aquaporins (Aqps) are a superfamily of integral membrane proteins which generally facilitate the permeation of water through plasma membranes. This study aimed to obtain the complete cDNA coding sequences of aqp1 and aqp3 from the gills of Protopterus annectens , and to determine their branchial mRNA and protein expression levels during the induction, maintenance and arousal phases of aestivation. Dendrogramic analyses of the deduced Aqp1 and Aqp3 amino acid sequences of P. annectens revealed their close relationships with those of Latimeria chalumnae and tetrapods. During the induction phase, there were significant decreases in the transcript levels of aqp1 and aqp3 in the gills of P. annectens , but the branchial Aqp1 and Aqp3 protein abundance remained unchanged. As changes in transcription might precede changes in translation, this could be regarded as an adaptive response to decrease the protein abundance of Aqp1 and Aqp3 in the subsequent maintenance phase of aestivation. As expected, the branchial transcript levels and protein abundance of aqp1 /Aqp1 and aqp3 /Aqp3 were significantly down-regulated during the maintenance phase, probably attributable to the shutdown of branchial functions and the cessation of volume regulation of branchial epithelial cells. Additionally, these changes could reduce the loss of water through branchial epithelial surfaces, supplementing the anti-desiccating property of the dried mucus. Upon arousal, it was essential for the lungfish to restore branchial functions. Indeed, the protein abundance of Aqp1 recovered partially, with complete recovery of mRNA expression level and protein abundance of Aqp3, in the gills of P. annectens after 3 days of arousal. These results provide insights

  5. In vivo versus in vitro protein abundance analysis of Shigella dysenteriae type 1 reveals changes in the expression of proteins involved in virulence, stress and energy metabolism

    Directory of Open Access Journals (Sweden)

    Donohue-Rolfe Arthur

    2011-06-01

    Full Text Available Abstract Background Shigella dysenteriae serotype 1 (SD1 causes the most severe form of epidemic bacillary dysentery. Quantitative proteome profiling of Shigella dysenteriae serotype 1 (SD1 in vitro (derived from LB cell cultures and in vivo (derived from gnotobiotic piglets was performed by 2D-LC-MS/MS and APEX, a label-free computationally modified spectral counting methodology. Results Overall, 1761 proteins were quantitated at a 5% FDR (false discovery rate, including 1480 and 1505 from in vitro and in vivo samples, respectively. Identification of 350 cytoplasmic membrane and outer membrane (OM proteins (38% of in silico predicted SD1 membrane proteome contributed to the most extensive survey of the Shigella membrane proteome reported so far. Differential protein abundance analysis using statistical tests revealed that SD1 cells switched to an anaerobic energy metabolism under in vivo conditions, resulting in an increase in fermentative, propanoate, butanoate and nitrate metabolism. Abundance increases of transcription activators FNR and Nar supported the notion of a switch from aerobic to anaerobic respiration in the host gut environment. High in vivo abundances of proteins involved in acid resistance (GadB, AdiA and mixed acid fermentation (PflA/PflB indicated bacterial survival responses to acid stress, while increased abundance of oxidative stress proteins (YfiD/YfiF/SodB implied that defense mechanisms against oxygen radicals were mobilized. Proteins involved in peptidoglycan turnover (MurB were increased, while β-barrel OM proteins (OmpA, OM lipoproteins (NlpD, chaperones involved in OM protein folding pathways (YraP, NlpB and lipopolysaccharide biosynthesis (Imp were decreased, suggesting unexpected modulations of the outer membrane/peptidoglycan layers in vivo. Several virulence proteins of the Mxi-Spa type III secretion system and invasion plasmid antigens (Ipa proteins required for invasion of colonic epithelial cells, and release

  6. 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

  7. Radioimmunoassay for pregnancy-associated plasma protein A

    International Nuclear Information System (INIS)

    Sinosich, M.J.; 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 μg/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

  8. 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.

  9. Effects of electroacupuncture on leukocytes and plasma protein in the X-irradiated rats

    International Nuclear Information System (INIS)

    Hau, D.M.

    1984-01-01

    The effects of electroacupuncture on leukocytes and plasma protein on the X ray-irradiated rats were investigated in the present study. The results showed that X-irradiation had an evident inhibitory effect on the counts of total leukocytes, lymphocytes and neutrocytes, and the concentration of the total plasma protein, plasma albumin, globulin and alpha- and beta-globulin in X-irradiated rats. The electroacupuncture was able to help the X-irradiated rats to recover the counts of the total leukocyte, lymphocyte and neutrocyte. The electroacupuncture had a helpful tendency to recover the concentration of the total plasma protein, albumin, globulin, and alpha- and beta-globulin in the irradiated rats

  10. Effects of electroacupuncture on leukocytes and plasma protein in the X-irradiated rats

    Energy Technology Data Exchange (ETDEWEB)

    Hau, D.M.

    The effects of electroacupuncture on leukocytes and plasma protein on the X ray-irradiated rats were investigated in the present study. The results showed that X-irradiation had an evident inhibitory effect on the counts of total leukocytes, lymphocytes and neutrocytes, and the concentration of the total plasma protein, plasma albumin, globulin and alpha- and beta-globulin in X-irradiated rats. The electroacupuncture was able to help the X-irradiated rats to recover the counts of the total leukocyte, lymphocyte and neutrocyte. The electroacupuncture had a helpful tendency to recover the concentration of the total plasma protein, albumin, globulin, and alpha- and beta-globulin in the irradiated rats.

  11. Radioimmunoassay of inactive creatine kinase B protein in human plasma

    Energy Technology Data Exchange (ETDEWEB)

    Burnam, M H; Shell, W E [California Univ., Los Angeles (USA). School of Medicine

    1981-08-27

    The authors describe a rapid, sensitive radioimmunoassay for enzymatically inactive creatine kinase B protein (CK-Bi) in plasma. /sup 125/I-CK-Bi of high specific activity and good stability was prepared by oxidant-based iodination. A 12-minute first antibody incubation was used. Bound and free antigen were separated by a second antibody system. Large excesses of purified CK-MM from human skeletal muscle did not react in the assay. Cross reactivity to CK-MB purified from the plasma of patients with acute myocardial infarction was negligible. The 95th percentile of plasma CK-Bi in 150 adults was 145 ..mu..g equivalents/ml. Within-assay and between-assay precision ranged from 5% to 9% and 6% to 10%, respectively. Evidence is presented indicating that the assay measures inactive creatine kinase B protein, a protein not measured by current assay systems dependent on biological activity.

  12. Radioimmunoassay of inactive creatine kinase B protein in human plasma

    International Nuclear Information System (INIS)

    Burnam, M.H.; Shell, W.E.

    1981-01-01

    The authors describe a rapid, sensitive radioimmunoassay for enzymatically inactive creatine kinase B protein (CK-Bi) in plasma. 125 I-CK-Bi of high specific activity and good stability was prepared by oxidant-based iodination. A 12-minute first antibody incubation was used. Bound and free antigen were separated by a second antibody system. Large excesses of purified CK-MM from human skeletal muscle did not react in the assay. Cross reactivity to CK-MB purified from the plasma of patients with acute myocardial infarction was negligible. The 95th percentile of plasma CK-Bi in 150 adults was 145 μg equivalents/ml. Within-assay and between-assay precision ranged from 5% to 9% and 6% to 10%, respectively. Evidence is presented indicating that the assay measures inactive creatine kinase B protein, a protein not measured by current assay systems dependent on biological activity. (Auth.)

  13. Plasma proteins predict conversion to dementia from prodromal disease.

    Science.gov (United States)

    Hye, Abdul; Riddoch-Contreras, Joanna; Baird, Alison L; Ashton, Nicholas J; Bazenet, Chantal; Leung, Rufina; Westman, Eric; Simmons, Andrew; Dobson, Richard; Sattlecker, Martina; Lupton, Michelle; Lunnon, Katie; Keohane, Aoife; Ward, Malcolm; Pike, Ian; Zucht, Hans Dieter; Pepin, Danielle; Zheng, Wei; Tunnicliffe, Alan; Richardson, Jill; Gauthier, Serge; Soininen, Hilkka; Kłoszewska, Iwona; Mecocci, Patrizia; Tsolaki, Magda; Vellas, Bruno; Lovestone, Simon

    2014-11-01

    The study aimed to validate previously discovered plasma biomarkers associated with AD, using a design based on imaging measures as surrogate for disease severity and assess their prognostic value in predicting conversion to dementia. Three multicenter cohorts of cognitively healthy elderly, mild cognitive impairment (MCI), and AD participants with standardized clinical assessments and structural neuroimaging measures were used. Twenty-six candidate proteins were quantified in 1148 subjects using multiplex (xMAP) assays. Sixteen proteins correlated with disease severity and cognitive decline. Strongest associations were in the MCI group with a panel of 10 proteins predicting progression to AD (accuracy 87%, sensitivity 85%, and specificity 88%). We have identified 10 plasma proteins strongly associated with disease severity and disease progression. Such markers may be useful for patient selection for clinical trials and assessment of patients with predisease subjective memory complaints. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

  14. 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.

  15. Plasma protein electrophoresis of Trachemys scripta and Iguana iguana.

    Science.gov (United States)

    Giménez, Mercè; Saco, Yolanda; Pato, Raquel; Busquets, Alex; Martorell, Jaime M; Bassols, Anna

    2010-06-01

    Protein electrophoresis is widely applied in veterinary medicine, but is not used often in reptiles, in part because of lack of reference values. The goals of this study were to compare plasma protein profiles obtained by cellulose acetate electrophoresis (CAE) and agarose gel electrophoresis (AGE), measure precision and examine interference by sample hemolysis, and establish preliminary reference intervals for 2 reptile species. Heparinized plasma samples from healthy and diseased adult female Iguana iguana (n=40) and Trachemys scripta (n=60) were analyzed by CAE and AGE. Total protein concentration was measured by the biuret method. Electrophoresis results were compared using Bland-Altman plots and Passing-Bablok regression analysis. Precision and the effects of sample hemolysis were determined. Results from clinically healthy animals were used to determine reference intervals. Five protein fractions were identified in both species, with bisalbuminemia observed in 23/40 iguanas. High correlation was observed between the 2 methods for all fractions, with few proportional and systematic errors. Coefficients of variation were lower using AGE vs CAE and for I. iguana vs T. scripta. Two additional bands were observed in hemolyzed samples from T. scripta; 1 additional band was observed for I. iguana. Minimum and maximum values were reported for healthy I. iguana (n=14) and T. scripta (n=22). Although both methods are acceptable, the performance of AGE was slightly better than that of CAE for analysis of plasma from reptiles. Furthermore, reptile electrophoretic patterns should be interpreted based on the method used, the species analyzed, and the quality of the plasma sample.

  16. An attempt to understand kidney's protein handling function by comparing plasma and urine proteomes.

    Directory of Open Access Journals (Sweden)

    Lulu Jia

    Full Text Available BACKGROUND: With the help of proteomics technology, the human plasma and urine proteomes, which closely represent the protein compositions of the input and output of the kidney, respectively, have been profiled in much greater detail by different research teams. Many datasets have been accumulated to form "reference profiles" of the plasma and urine proteomes. Comparing these two proteomes may help us understand the protein handling aspect of kidney function in a way, however, which has been unavailable until the recent advances in proteomics technology. METHODOLOGY/PRINCIPAL FINDINGS: After removing secreted proteins downstream of the kidney, 2611 proteins in plasma and 1522 in urine were identified with high confidence and compared based on available proteomic data to generate three subproteomes, the plasma-only subproteome, the plasma-and-urine subproteome, and the urine-only subproteome, and they correspond to three groups of proteins that are handled in three different ways by the kidney. The available experimental molecular weights of the proteins in the three subproteomes were collected and analyzed. Since the functions of the overrepresented proteins in the plasma-and-urine subproteome are probably the major functions that can be routinely regulated by excretion from the kidney in physiological conditions, Gene Ontology term enrichment in the plasma-and-urine subproteome versus the whole plasma proteome was analyzed. Protease activity, calcium and growth factor binding proteins, and coagulation and immune response-related proteins were found to be enriched. CONCLUSION/SIGNIFICANCE: The comparison method described in this paper provides an illustration of a new approach for studying organ functions with a proteomics methodology. Because of its distinctive input (plasma and output (urine, it is reasonable to predict that the kidney will be the first organ whose functions are further elucidated by proteomic methods in the near future. It

  17. In-depth analysis of low abundant proteins in bovine colostrum using different fractionation techniques

    DEFF Research Database (Denmark)

    Nissen, Asger; Bendixen, Emøke; Ingvartsen, Klaus Lønne

    2012-01-01

    Bovine colostrum is well known for its large content of bioactive components and its importance for neonatal survival. Unfortunately, the colostrum proteome is complicated by a wide dynamic range, because of a few dominating proteins that hamper sensitivity and proteome coverage achieved on low...... abundant proteins. Moreover, the composition of colostrum is complex and the proteins are located within different physical fractions that make up the colostrum. To gain a more exhaustive picture of the bovine colostrum proteome and gather information on protein location, we performed an extensive pre......-analysis fractionation of colostrum prior to 2D-LC-MS/MS analysis. Physical and chemical properties of the proteins and colostrum were used alone or in combination for the separation of proteins. ELISA was used to quantify and verify the presence of proteins in colostrum. In total, 403 proteins were identified...

  18. 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.

  19. Physico-Pathologic Mechanisms Involved in Neurodegeneration: Misfolded Protein-Plasma Membrane Interactions.

    Science.gov (United States)

    Shrivastava, Amulya Nidhi; Aperia, Anita; Melki, Ronald; Triller, Antoine

    2017-07-05

    Several neurodegenerative disorders, such as Alzheimer's and Parkinson's disease, are characterized by prominent loss of synapses and neurons associated with the presence of abnormally structured or misfolded protein assemblies. Cell-to-cell transfer of misfolded proteins has been proposed for the intra-cerebral propagation of these diseases. When released, misfolded proteins diffuse in the 3D extracellular space before binding to the plasma membrane of neighboring cells, where they diffuse on a 2D plane. This reduction in diffusion dimension and the cell surface molecular crowding promote deleterious interactions with native membrane proteins, favoring clustering and further aggregation of misfolded protein assemblies. These processes open up new avenues for therapeutics development targeting the initial interactions of deleterious proteins with the plasma membrane or the subsequent pathological signaling. Copyright © 2017 Elsevier Inc. All rights reserved.

  20. Multi-protein assemblies underlie the mesoscale organization of the plasma membrane

    Science.gov (United States)

    Saka, Sinem K.; Honigmann, Alf; Eggeling, Christian; Hell, Stefan W.; Lang, Thorsten; Rizzoli, Silvio O.

    2014-01-01

    Most proteins have uneven distributions in the plasma membrane. Broadly speaking, this may be caused by mechanisms specific to each protein, or may be a consequence of a general pattern that affects the distribution of all membrane proteins. The latter hypothesis has been difficult to test in the past. Here, we introduce several approaches based on click chemistry, through which we study the distribution of membrane proteins in living cells, as well as in membrane sheets. We found that the plasma membrane proteins form multi-protein assemblies that are long lived (minutes), and in which protein diffusion is restricted. The formation of the assemblies is dependent on cholesterol. They are separated and anchored by the actin cytoskeleton. Specific proteins are preferentially located in different regions of the assemblies, from their cores to their edges. We conclude that the assemblies constitute a basic mesoscale feature of the membrane, which affects the patterning of most membrane proteins, and possibly also their activity. PMID:25060237

  1. 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. Copyright © 2014 Elsevier Ltd. All rights reserved.

  2. 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

    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

  3. Enrichment and Identification of the Most Abundant Zinc Binding Proteins in Developing Barley Grains by Zinc-IMAC Capture and Nano LC-MS/MS

    Directory of Open Access Journals (Sweden)

    Giuseppe Dionisio

    2018-01-01

    Full Text Available Background: Zinc accumulates in the embryo, aleurone, and subaleurone layers at different amounts in cereal grains. Our hypothesis is that zinc could be stored bound, not only to low MW metabolites/proteins, but also to high MW proteins as well. Methods: In order to identify the most abundant zinc binding proteins in different grain tissues, we microdissected barley grains into (1 seed coats; (2 aleurone/subaleurone; (3 embryo; and (4 endosperm. Initial screening for putative zinc binding proteins from the different tissue types was performed by fractionating proteins according to solubility (Osborne fractionation, and resolving those via Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE followed by polyvinylidene fluoride (PVDF membrane blotting and dithizone staining. Selected protein fractions were subjected to Zn2+-immobilized metal ion affinity chromatography, and the captured proteins were identified using nanoscale liquid chromatography coupled to tandem mass spectrometry (nanoLC-MS/MS. Results: In the endosperm, the most abundant zinc binding proteins were the storage protein B-hordeins, gamma-, and D-hordeins, while in the embryo, 7S globulins storage proteins exhibited zinc binding. In the aleurone/subaleurone, zinc affinity captured proteins were late abundant embryogenesis proteins, dehydrins, many isoforms of non-specific lipid transfer proteins, and alpha amylase trypsin inhibitor. Conclusions: We have shown evidence that abundant barley grain proteins have been captured by Zn-IMAC, and their zinc binding properties in relationship to the possibility of zinc storage is discussed.

  4. The cell-based L-glutathione protection assays to study endocytosis and recycling of plasma membrane proteins.

    Science.gov (United States)

    Cihil, Kristine M; Swiatecka-Urban, Agnieszka

    2013-12-13

    Membrane trafficking involves transport of proteins from the plasma membrane to the cell interior (i.e. endocytosis) followed by trafficking to lysosomes for degradation or to the plasma membrane for recycling. The cell based L-glutathione protection assays can be used to study endocytosis and recycling of protein receptors, channels, transporters, and adhesion molecules localized at the cell surface. The endocytic assay requires labeling of cell surface proteins with a cell membrane impermeable biotin containing a disulfide bond and the N-hydroxysuccinimide (NHS) ester at 4 ºC - a temperature at which membrane trafficking does not occur. Endocytosis of biotinylated plasma membrane proteins is induced by incubation at 37 ºC. Next, the temperature is decreased again to 4 ºC to stop endocytic trafficking and the disulfide bond in biotin covalently attached to proteins that have remained at the plasma membrane is reduced with L-glutathione. At this point, only proteins that were endocytosed remain protected from L-glutathione and thus remain biotinylated. After cell lysis, biotinylated proteins are isolated with streptavidin agarose, eluted from agarose, and the biotinylated protein of interest is detected by western blotting. During the recycling assay, after biotinylation cells are incubated at 37 °C to load endocytic vesicles with biotinylated proteins and the disulfide bond in biotin covalently attached to proteins remaining at the plasma membrane is reduced with L-glutathione at 4 ºC as in the endocytic assay. Next, cells are incubated again at 37 °C to allow biotinylated proteins from endocytic vesicles to recycle to the plasma membrane. Cells are then incubated at 4 ºC, and the disulfide bond in biotin attached to proteins that recycled to the plasma membranes is reduced with L-glutathione. The biotinylated proteins protected from L-glutathione are those that did not recycle to the plasma membrane.

  5. Proteomic profiling of human plasma exosomes identifies PPARγ as an exosome-associated protein

    International Nuclear Information System (INIS)

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

    2009-01-01

    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-γ (PPARγ), 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 PPARγ as a component of human plasma exosomes identifies a potential new pathway for the paracrine transfer of nuclear receptors.

  6. 2 D gel based analysis of biological variability of the human plasma proteome

    DEFF Research Database (Denmark)

    Rentsch, Maria Louise; Jessen, Flemming

    individuals and within an individual changes will also happen over time (e.g. after meal intake). Thus, the aim of the present study was to examine the inter-individual variability of plasma protein levels in humans after meal intake. Five subjects consumed three single meals in a randomised order separated...... by one-week interval. Blood samples were drawn before the meal intake and five times during 24 hours for proteome analysis. Plasma was fractionated by use of IgY-12 spin column depleting the 12 highly abundant proteins and further processed for two-dimensional gel electrophoresis. The plasma proteome...

  7. Looking Deep Inside: Detection of Low-Abundance Proteins in Leaf Extracts of Arabidopsis and Phloem Exudates of Pumpkin1[W

    Science.gov (United States)

    Fröhlich, Andreas; Gaupels, Frank; Sarioglu, Hakan; Holzmeister, Christian; Spannagl, Manuel; Durner, Jörg; Lindermayr, Christian

    2012-01-01

    The field of proteomics suffers from the immense complexity of even small proteomes and the enormous dynamic range of protein concentrations within a given sample. Most protein samples contain a few major proteins, which hamper in-depth proteomic analysis. In the human field, combinatorial hexapeptide ligand libraries (CPLL; such as ProteoMiner) have been used for reduction of the dynamic range of protein concentrations; however, this technique is not established in plant research. In this work, we present the application of CPLL to Arabidopsis (Arabidopsis thaliana) leaf proteins. One- and two-dimensional gel electrophoresis showed a decrease in high-abundance proteins and an enrichment of less abundant proteins in CPLL-treated samples. After optimization of the CPLL protocol, mass spectrometric analyses of leaf extracts led to the identification of 1,192 proteins in control samples and an additional 512 proteins after the application of CPLL. Upon leaf infection with virulent Pseudomonas syringae DC3000, CPLL beads were also used for investigating the bacterial infectome. In total, 312 bacterial proteins could be identified in infected Arabidopsis leaves. Furthermore, phloem exudates of pumpkin (Cucurbita maxima) were analyzed. CPLL prefractionation caused depletion of the major phloem proteins 1 and 2 and improved phloem proteomics, because 67 of 320 identified proteins were detectable only after CPLL treatment. In sum, our results demonstrate that CPLL beads are a time- and cost-effective tool for reducing major proteins, which often interfere with downstream analyses. The concomitant enrichment of less abundant proteins may facilitate a deeper insight into the plant proteome. PMID:22555880

  8. Thioredoxin h regulates calcium dependent protein kinases in plasma membranes.

    Science.gov (United States)

    Ueoka-Nakanishi, Hanayo; Sazuka, Takashi; Nakanishi, Yoichi; Maeshima, Masayoshi; Mori, Hitoshi; Hisabori, Toru

    2013-07-01

    Thioredoxin (Trx) is a key player in redox homeostasis in various cells, modulating the functions of target proteins by catalyzing a thiol-disulfide exchange reaction. Target proteins of cytosolic Trx-h of higher plants were studied, particularly in the plasma membrane, because plant plasma membranes include various functionally important protein molecules such as transporters and signal receptors. Plasma membrane proteins from Arabidopsis thaliana cell cultures were screened using a resin Trx-h1 mutant-immobilized, and a total of 48 candidate proteins obtained. These included two calcium-sensing proteins: a phosphoinositide-specific phospholipase 2 (AtPLC2) and a calcium-dependent protein kinase 21 (AtCPK21). A redox-dependent change in AtCPK21 kinase activity was demonstrated in vitro. Oxidation of AtCPK21 resulted in a decrease in kinase activity to 19% of that of untreated AtCPK21, but Trx-h1 effectively restored the activity to 90%. An intramolecular disulfide bond (Cys97-Cys108) that is responsible for this redox modulation was then identified. In addition, endogenous AtCPK21 was shown to be oxidized in vivo when the culture cells were treated with H2 O2 . These results suggest that redox regulation of AtCPK21 by Trx-h in response to external stimuli is important for appropriate cellular responses. The relationship between the redox regulation system and Ca(2+) signaling pathways is discussed. © 2013 The Authors. FEBS Journal published by John Wiley & Sons Ltd on behalf of FEBS.

  9. On the variability of the salting-out curves of proteins of normal human plasma and serum

    NARCIS (Netherlands)

    Steyn-Parvé, Elizabeth P.; Hout, A.J. van den

    1953-01-01

    Salting-out curves of proteins of normal human plasma reflect the influence of a number of other factors besides the protein composition: the manner of obtaining the blood, the nature of the anti-coagulant used, the non-protein components of the plasma. Diagrams of serum and plasma obtained from

  10. Trace Elements (Zn & Cu) And Plasma Proteins Status In Mauritian ...

    African Journals Online (AJOL)

    A commercially available kit was used for the direct determination of zinc while copper assay was performed by atomic absorption spectrophotometry. Total plasma proteins, albumin and globulin levels were also measured by colorimetric method using commercially available kits. Plasma zinc and copper levels in pregnant ...

  11. Role of plasma adiponectin /C-reactive protein ratio in obesity and ...

    African Journals Online (AJOL)

    African Health Sciences ... Objective(s): We examined relations between fasting plasma adiponectin (ADIP), C-reactive protein (CRP) ... Methods: Fasting plasma ADIP, CRP, Insulin (IN), HOMA-IR, lipid profiles, body fat percent (%BF), waist ...

  12. Mannan-binding proteins from boar seminal plasma

    Czech Academy of Sciences Publication Activity Database

    Jelínková-Slavíčková, Petra; Liberda, J.; Maňásková, Pavla; Ryšlavá, H.; Jonáková, Věra; Tichá, M.

    2004-01-01

    Roč. 62, 1-2 (2004), s. 167-182 ISSN 0165-0378. [Congress of the European Society for Reproductive & Developmental Immunology /4./. Rhodes, 04.06.2003-06.06.2003] R&D Projects: GA ČR GA303/02/0433; GA ČR GP303/02/P069; GA MŠk VS96141; GA MZd NJ7463 Institutional research plan: CEZ:AV0Z5052915 Keywords : boar seminal plasma proteins * mannan-binding proteins * oviductal epithelium Subject RIV: CE - Biochemistry Impact factor: 2.726, year: 2004

  13. The role of plasma proteins in formation of obstructive protamine complexes

    International Nuclear Information System (INIS)

    De Paulis, R.; Mohammad, S.F.; Chiariello, L.; Morea, M.; Olsen, D.B.

    1991-01-01

    Formation of complexes between heparin and protamine (in saline), or heparin, plasma proteins, and protamine (in plasma) was assessed by measurements of light transmission through different test solutions. To examine the formation of these complexes, 125I-labeled protamine was used. Addition of 125I-protamine to plasma or blood resulted in the sedimentation of 125I-protamine in the form of insoluble complexes. This complex formation was not affected by the presence of heparin, suggesting that protamine-plasma protein interaction may be primarily responsible for precipitation of 125I-protamine. To assess the capability of these complexes to obstruct the pulmonary circulation, an in vitro experimental model was developed. Citrated serum, plasma, blood, or saline were allowed to flow through a glass bead column with the help of a peristaltic pump. A pressure transducer positioned before the column allowed pressure measurements at a constant flow rate during the experiment. Mixing of protamine with plasma or blood prior to their passage through the glass bead column resulted in a significant increase in pressure suggesting that the column was being clogged with insoluble complexes. The increase in pressure occurred both in the presence and absence of heparin in plasma or blood. Under identical experimental conditions, the increase in pressure was insignificant when protamine was added to saline or serum regardless of whether heparin was present or absent. This was further confirmed by the use of 125I-protamine. These observations suggest that protamine forms insoluble complexes with certain plasma proteins. Based on these observations, it is hypothesized that following intravenous administration, protamine immediately forms complexes in circulating blood

  14. Deep Coverage Proteomics Identifies More Low-Abundance Missing Proteins in Human Testis Tissue with Q-Exactive HF Mass Spectrometer.

    Science.gov (United States)

    Wei, Wei; Luo, Weijia; Wu, Feilin; Peng, Xuehui; Zhang, Yao; Zhang, Manli; Zhao, Yan; Su, Na; Qi, YingZi; Chen, Lingsheng; Zhang, Yangjun; Wen, Bo; He, Fuchu; Xu, Ping

    2016-11-04

    Since 2012, missing proteins (MPs) investigation has been one of the critical missions of Chromosome-Centric Human Proteome Project (C-HPP) through various biochemical strategies. On the basis of our previous testis MPs study, faster scanning and higher resolution mass-spectrometry-based proteomics might be conducive to MPs exploration, especially for low-abundance proteins. In this study, Q-Exactive HF (HF) was used to survey proteins from the same testis tissues separated by two separating methods (tricine- and glycine-SDS-PAGE), as previously described. A total of 8526 proteins were identified, of which more low-abundance proteins were uniquely detected in HF data but not in our previous LTQ Orbitrap Velos (Velos) reanalysis data. Further transcriptomics analysis showed that these uniquely identified proteins by HF also had lower expression at the mRNA level. Of the 81 total identified MPs, 74 and 39 proteins were listed as MPs in HF and Velos data sets, respectively. Among the above MPs, 47 proteins (43 neXtProt PE2 and 4 PE3) were ranked as confirmed MPs after verifying with the stringent spectra match and isobaric and single amino acid variants filtering. Functional investigation of these 47 MPs revealed that 11 MPs were testis-specific proteins and 7 MPs were involved in spermatogenesis process. Therefore, we concluded that higher scanning speed and resolution of HF might be factors for improving the low-abundance MP identification in future C-HPP studies. All mass-spectrometry data from this study have been deposited in the ProteomeXchange with identifier PXD004092.

  15. Glucose rapidly decreases plasma membrane GLUT4 content in rat skeletal muscle.

    Science.gov (United States)

    Marette, A; Dimitrakoudis, D; Shi, Q; Rodgers, C D; Klip, A; Vranic, M

    1999-02-01

    We have previously demonstrated that chronic hyperglycemia per se decreases GLUT4 glucose transporter expression and plasma membrane content in mildly streptozotocin- (STZ) diabetic rats (Biochem. J. 284, 341-348, 1992). In the present study, we investigated the effect of an acute rise in glycemia on muscle GLUT4 and GLUT1 protein contents in the plasma membrane, in the absence of insulin elevation. Four experimental groups of rats were analyzed in the postabsorptive state: 1. Control rats. 2. Hyperglycemic STZ-diabetic rats with moderately reduced fasting insulin levels. 3. STZ-diabetic rats made normoglycemic with phlorizin treatment. 4. Phlorizin-treated (normoglycemic) STZ-diabetic rats infused with glucose for 40 min. The uniqueness of the latter model is that glycemia can be rapidly raised without any concomitant increase in plasma insulin levels. Plasma membranes were isolated from hindlimb muscle and GLUT1 and GLUT4 proteins amounts determined by Western blot analysis. As predicted, STZ-diabetes caused a significant decrease in the abundance of GLUT4 in the isolated plasma membranes. Normalization of glycemia for 3 d with phlorizin treatment restored plasma membrane GLUT4 content in muscle of STZ-diabetic rats. A sudden rise in glycemia over a period of 40 min caused the GLUT4 levels in the plasma membrane fraction to decrease to those of nontreated STZ-diabetic rats. In contrast to the GLUT4 transporter, plasma membrane GLUT1 abundance was not changed by the acute glucose challenge. It is concluded that glucose can have regulatory effect by acutely reducing plasma membrane GLUT4 protein contents in rat skeletal muscle. We hypothesize that this glucose-induced downregulation of plasma membrane GLUT4 could represent a protective mechanism against excessive glucose uptake under hyperglycemic conditions accompanied by insulin resistance.

  16. Relationships between protein-encoding gene abundance and corresponding process are commonly assumed yet rarely observed

    Science.gov (United States)

    Rocca, Jennifer D.; Hall, Edward K.; Lennon, Jay T.; Evans, Sarah E.; Waldrop, Mark P.; Cotner, James B.; Nemergut, Diana R.; Graham, Emily B.; Wallenstein, Matthew D.

    2015-01-01

    For any enzyme-catalyzed reaction to occur, the corresponding protein-encoding genes and transcripts are necessary prerequisites. Thus, a positive relationship between the abundance of gene or transcripts and corresponding process rates is often assumed. To test this assumption, we conducted a meta-analysis of the relationships between gene and/or transcript abundances and corresponding process rates. We identified 415 studies that quantified the abundance of genes or transcripts for enzymes involved in carbon or nitrogen cycling. However, in only 59 of these manuscripts did the authors report both gene or transcript abundance and rates of the appropriate process. We found that within studies there was a significant but weak positive relationship between gene abundance and the corresponding process. Correlations were not strengthened by accounting for habitat type, differences among genes or reaction products versus reactants, suggesting that other ecological and methodological factors may affect the strength of this relationship. Our findings highlight the need for fundamental research on the factors that control transcription, translation and enzyme function in natural systems to better link genomic and transcriptomic data to ecosystem processes.

  17. Radiation induced changes in plasma total protein nitrogen and urinary total nitrogen in desert rodent and albino rats subjected to dietary protein deficiency

    International Nuclear Information System (INIS)

    Roushdy, H.; El-Husseini, M.; Saleh, F.

    1986-01-01

    The effect of gamma-irradiation on plasma total protein nitrogen and urinary total nitrogen was studied in the desert rodent, psammomy obesus obesus and albino rats subjected to dietary protein deficiency. In albino rats kept on high protein diet, the radiation syndrome resulted in urine retention, while in those kept on non-protein diet, such phenomenon was recorded only with the high radiation level of 1170r. Radiation exposure to 780 and 1170r caused remarkable diuresis in psammomys obesus obesus whereas they induced significant urine retention in albino rats. The levels of plasma total protein nitrogen and urinary total nitrogen were higher in albino rats maintained on high protein diet than in those kept on non-protein diet. Radiation exposure caused an initial drop in plasma total protein nitrogen concentration, concomitant with an initial rise in total urinary nitrogen, radiation exposure of psammomys obesus obesus caused significant increase in the levels of plasma protein nitrogen and urinary total nitrogen. Psammomys obesus obesus seemed to be more affected by radiation exposure than did the albino rats

  18. Gonadal cell surface receptor for plasma retinol-binding protein

    International Nuclear Information System (INIS)

    Krishna Bhat, M.; Cama, H.R.

    1979-01-01

    A specific membrane receptor for plasma retinol-binding protein has been demonstrated in testicular cells. Prealbumin-2 did not show any specific binding to the membrane. The affinity of retinol-binding protein for receptor drastically decreases upon delivery of retinol and the retinol-binding protein does not enter the cell. The mechanism of delivery of retinol to the target cell by plasma retinol-binding protein has been investigated. The process involves two steps; direct binding of retinol-binding protein to the receptor and uptake of retinol by the target cell with a concomitant drastic reduction in the affinity of the retinol-binding protein to the receptor. Probably the second step of the process needs a cytosolic factor, possibly the cellular retinol-binding protein or an enzyme. The binding of retinol-binding protein to the receptor is saturable and reversible. The interaction shows a Ksub(d) value of 2.1x10 -10 . The specific binding of a retinol-binding protein with great affinity has been employed in the development of a method for radioassay of the receptor. The receptor level of the gonadal cell has been found to vary with the stage of differentiation. The receptor concentrations in 11-week-old birds and adult birds are comparable. Testosterone treatment of 11-week-old birds produced a substantial increase in the receptor concentration over control, while the protein content increased marginally, indicating that, probably, synthesis of the receptor is specifcally induced by testosterone during spermatogenesis, and the concentration of receptor is relatively higher before the formation of the acrosome. (Auth.)

  19. 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

    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......-trap-Fourier transform (LTQ-FT) and a linear ion trap-Orbitrap (LTQ-Orbitrap) for mass spectrometry (MS) analysis. Both instruments allow the measurement of peptide masses in the low ppm range. Furthermore, we employed a statistical score that allows database peptide identification searching using the products of two...... consecutive stages of tandem mass spectrometry (MS3). The combination of MS3 with very high mass accuracy in the parent peptide allows peptide identification with orders of magnitude more confidence than that typically achieved. RESULTS: Herein we established a high confidence set of 697 blood plasma proteins...

  20. Plasma Amino Acid Responses After Consumption of Beverages with Varying Protein Type

    Science.gov (United States)

    2009-07-01

    saturated fat Protein Total earbohydT;ltes dietary fiber soluble fiber total sugar lactose sucrose other carbohydrates (maltodextrin. cocoa powder...provided by the manufacturer, 6 Plasma Amino Acids 7 coagulate for 30 min before being centrifuged for 10 min at 3,600 rpm. Resulting plasma and serum...Therefore, consumers should be aware that these products might not contain the specific protein sources being advertised. Acknowledgment The authors

  1. Protein-centric N-glycoproteomics analysis of membrane and plasma membrane proteins.

    Science.gov (United States)

    Sun, Bingyun; Hood, Leroy

    2014-06-06

    The advent of proteomics technology has transformed our understanding of biological membranes. The challenges for studying membrane proteins have inspired the development of many analytical and bioanalytical tools, and the techniques of glycoproteomics have emerged as an effective means to enrich and characterize membrane and plasma-membrane proteomes. This Review summarizes the development of various glycoproteomics techniques to overcome the hurdles formed by the unique structures and behaviors of membrane proteins with a focus on N-glycoproteomics. Example contributions of N-glycoproteomics to the understanding of membrane biology are provided, and the areas that require future technical breakthroughs are discussed.

  2. Morphological changes of plasma membrane and protein assembly during clathrin-mediated endocytosis

    Science.gov (United States)

    Yoshida, Aiko; Sakai, Nobuaki; Uekusa, Yoshitsugu; Imaoka, Yuka; Itagaki, Yoshitsuna; Suzuki, Yuki

    2018-01-01

    Clathrin-mediated endocytosis (CME) proceeds through a series of morphological changes of the plasma membrane induced by a number of protein components. Although the spatiotemporal assembly of these proteins has been elucidated by fluorescence-based techniques, the protein-induced morphological changes of the plasma membrane have not been fully clarified in living cells. Here, we visualize membrane morphology together with protein localizations during CME by utilizing high-speed atomic force microscopy (HS-AFM) combined with a confocal laser scanning unit. The plasma membrane starts to invaginate approximately 30 s after clathrin starts to assemble, and the aperture diameter increases as clathrin accumulates. Actin rapidly accumulates around the pit and induces a small membrane swelling, which, within 30 s, rapidly covers the pit irreversibly. Inhibition of actin turnover abolishes the swelling and induces a reversible open–close motion of the pit, indicating that actin dynamics are necessary for efficient and irreversible pit closure at the end of CME. PMID:29723197

  3. Lateral Organization of Influenza Virus Proteins in the Budozone Region of the Plasma Membrane.

    Science.gov (United States)

    Leser, George P; Lamb, Robert A

    2017-05-01

    Influenza virus assembles and buds at the plasma membrane of virus-infected cells. The viral proteins assemble at the same site on the plasma membrane for budding to occur. This involves a complex web of interactions among viral proteins. Some proteins, like hemagglutinin (HA), NA, and M2, are integral membrane proteins. M1 is peripherally membrane associated, whereas NP associates with viral RNA to form an RNP complex that associates with the cytoplasmic face of the plasma membrane. Furthermore, HA and NP have been shown to be concentrated in cholesterol-rich membrane raft domains, whereas M2, although containing a cholesterol binding motif, is not raft associated. Here we identify viral proteins in planar sheets of plasma membrane using immunogold staining. The distribution of these proteins was examined individually and pairwise by using the Ripley K function, a type of nearest-neighbor analysis. Individually, HA, NA, M1, M2, and NP were shown to self-associate in or on the plasma membrane. HA and M2 are strongly coclustered in the plasma membrane; however, in the case of NA and M2, clustering depends upon the expression system used. Despite both proteins being raft resident, HA and NA occupy distinct but adjacent membrane domains. M2 and M1 strongly cocluster, but the association of M1 with HA or NA is dependent upon the means of expression. The presence of HA and NP at the site of budding depends upon the coexpression of other viral proteins. Similarly, M2 and NP occupy separate compartments, but an association can be bridged by the coexpression of M1. IMPORTANCE The complement of influenza virus proteins necessary for the budding of progeny virions needs to accumulate at budozones. This is complicated by HA and NA residing in lipid raft-like domains, whereas M2, although an integral membrane protein, is not raft associated. Other necessary protein components such as M1 and NP are peripherally associated with the membrane. Our data define spatial relationships

  4. Detection of cervical cancer biomarker patterns in blood plasma and urine by differential scanning calorimetry and mass spectrometry.

    Directory of Open Access Journals (Sweden)

    Nichola C Garbett

    Full Text Available Improved methods for the accurate identification of both the presence and severity of cervical intraepithelial neoplasia (CIN and extent of spread of invasive carcinomas of the cervix (IC are needed. Differential scanning calorimetry (DSC has recently been shown to detect specific changes in the thermal behavior of blood plasma proteins in several diseases. This methodology is being explored to provide a complementary approach for screening of cervical disease. The present study evaluated the utility of DSC in differentiating between healthy controls, increasing severity of CIN and early and advanced IC. Significant discrimination was apparent relative to the extent of disease with no clear effect of demographic factors such as age, ethnicity, smoking status and parity. Of most clinical relevance, there was strong differentiation of CIN from healthy controls and IC, and amongst patients with IC between FIGO Stage I and advanced cancer. The observed disease-specific changes in DSC profiles (thermograms were hypothesized to reflect differential expression of disease biomarkers that subsequently bound to and affected the thermal behavior of the most abundant plasma proteins. The effect of interacting biomarkers can be inferred from the modulation of thermograms but cannot be directly identified by DSC. To investigate the nature of the proposed interactions, mass spectrometry (MS analyses were employed. Quantitative assessment of the low molecular weight protein fragments of plasma and urine samples revealed a small list of peptides whose abundance was correlated with the extent of cervical disease, with the most striking plasma peptidome data supporting the interactome theory of peptide portioning to abundant plasma proteins. The combined DSC and MS approach in this study was successful in identifying unique biomarker signatures for cervical cancer and demonstrated the utility of DSC plasma profiles as a complementary diagnostic tool to evaluate

  5. Detection of cervical cancer biomarker patterns in blood plasma and urine by differential scanning calorimetry and mass spectrometry.

    Science.gov (United States)

    Garbett, Nichola C; Merchant, Michael L; Helm, C William; Jenson, Alfred B; Klein, Jon B; Chaires, Jonathan B

    2014-01-01

    Improved methods for the accurate identification of both the presence and severity of cervical intraepithelial neoplasia (CIN) and extent of spread of invasive carcinomas of the cervix (IC) are needed. Differential scanning calorimetry (DSC) has recently been shown to detect specific changes in the thermal behavior of blood plasma proteins in several diseases. This methodology is being explored to provide a complementary approach for screening of cervical disease. The present study evaluated the utility of DSC in differentiating between healthy controls, increasing severity of CIN and early and advanced IC. Significant discrimination was apparent relative to the extent of disease with no clear effect of demographic factors such as age, ethnicity, smoking status and parity. Of most clinical relevance, there was strong differentiation of CIN from healthy controls and IC, and amongst patients with IC between FIGO Stage I and advanced cancer. The observed disease-specific changes in DSC profiles (thermograms) were hypothesized to reflect differential expression of disease biomarkers that subsequently bound to and affected the thermal behavior of the most abundant plasma proteins. The effect of interacting biomarkers can be inferred from the modulation of thermograms but cannot be directly identified by DSC. To investigate the nature of the proposed interactions, mass spectrometry (MS) analyses were employed. Quantitative assessment of the low molecular weight protein fragments of plasma and urine samples revealed a small list of peptides whose abundance was correlated with the extent of cervical disease, with the most striking plasma peptidome data supporting the interactome theory of peptide portioning to abundant plasma proteins. The combined DSC and MS approach in this study was successful in identifying unique biomarker signatures for cervical cancer and demonstrated the utility of DSC plasma profiles as a complementary diagnostic tool to evaluate cervical cancer

  6. 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 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...

  7. Transfer of plasma lipoprotein components and of plasma proteins into aortas of cholesterol-fed rabbits. Molecular size as a determinant of plasma lipoprotein influx

    International Nuclear Information System (INIS)

    Stender, S.; Zilversmit, D.B.

    1981-01-01

    The arterial influx of esterified and free cholesterol from low density lipoproteins and very low density lipoproteins in 20 hypercholesterolemic rabbits was measured simultaneously by the use of lipoproteins labeled in vivo with [ 3 H]- and [ 14 C]-cholesterol. The simultaneous arterial influx of either [ 3 H]-leucine-labeled very low density lipoproteins, low density lipoproteins, high density lipoproteins, or plasma proteins was also measured in each rabbit. The arterial influx was calculated as intimal clearance, i.e., the influx of a given fraction divided by its plasma concentration. The intimal clearance of low density lipoprotein esterified cholesterol was equal to that for the apolipoproteins of that fraction, which is compatible with an arterial influx of intact low density lipoprotein molecules. The intimal clearance of very low density apolipoprotein or cholesteryl ester was less than that for low density lipoprotein, whereas high density lipoprotein and albumin clearances exceeded low density lipoprotein clearance by 1.5- to 3-fold. The intimal clearances of plasma proteins, high density, low density, and very low density lipoproteins decreased linearly with the logarithm of the macromolecular diameter. This indicates that the arterial influx of three plasma lipoprotein fractions and of plasma proteins proceeds by similar mechanisms. Apparently the relative intimal clearances of lipoproteins are more dependent on their size relative to pores or vesicular diameters at the plasma-artery interface than on specific interactions between lipoproteins and the arterial intimal surface

  8. Preparation of synaptic plasma membrane and postsynaptic density proteins using a discontinuous sucrose gradient.

    Science.gov (United States)

    Bermejo, Marie Kristel; Milenkovic, Marija; Salahpour, Ali; Ramsey, Amy J

    2014-09-03

    Neuronal subcellular fractionation techniques allow the quantification of proteins that are trafficked to and from the synapse. As originally described in the late 1960's, proteins associated with the synaptic plasma membrane can be isolated by ultracentrifugation on a sucrose density gradient. Once synaptic membranes are isolated, the macromolecular complex known as the post-synaptic density can be subsequently isolated due to its detergent insolubility. The techniques used to isolate synaptic plasma membranes and post-synaptic density proteins remain essentially the same after 40 years, and are widely used in current neuroscience research. This article details the fractionation of proteins associated with the synaptic plasma membrane and post-synaptic density using a discontinuous sucrose gradient. Resulting protein preparations are suitable for western blotting or 2D DIGE analysis.

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

    Science.gov (United States)

    Larsen, M; Røntved, C M; Theil, P K; Khatun, M; Lauridsen, C; Kristensen, N B

    2017-05-01

    The effect of experimentally increasing the postpartum protein supply on plasma protein synthesis, rumen tissue proliferation, and immune homeostasis was studied using 8 periparturient Holstein cows in a complete randomized design. At calving, cows were assigned to abomasal infusion of water (CTRL) or casein (CAS) in addition to a lactation diet. Casein infusion was gradually decreased from 696 ± 1 g/d at +2 d relative to calving (DRTC) to 212 ± 10 g/d at +29 DRTC to avoid excessive supply. Synthesis rate of plasma proteins was measured at -14, +4, +15, and +29 DRTC by measuring [C]Phe isotopic enrichment in arterial plasma free Phe, total plasma proteins, and albumin after 3, 5, and 7 h of jugular ring[C]Phe infusion. Plasma volume was determined at +4 and +29 DRTC by dilution of a [I]BSA dose. Synthesis rate of tissue protein in biopsied rumen papillae was determined by measuring [C]Phe isotopic enrichment, and mRNA expression of selected genes was measured by real-time qPCR. Total and differential leukocyte counts were performed and immune responsiveness of monocytes was evaluated by tumor necrosis factor ɑ (TNFɑ) concentration on ex vivo whole blood stimulation with Escherichia coli lipopolysaccharide (LPS) and responsiveness of T-lymphocytes by interferon γ (IFNγ) concentration on stimulation with Staphylococcus aureus enterotoxin β (SEB). Further, ELISA plasma concentrations of IgM, IgA, and IgG were determined. The DRTC affected the majority of investigated parameters as expected. The CAS treatment increased milk protein yield (P = 0.04), and tended to lower TNFɑ (P = 0.06), and lowered IFNγ (P = 0.03) responsiveness per monocyte and lymphocyte, respectively, compared with CTRL. Further, fractional synthesis rate of albumin was greater at +4 DRTC for CAS compared with CTRL but did not differ by +29 DRTC (interaction: P = 0.01). In rumen papillae, synthesis rate of tissue protein was greater for CAS compared with CTRL (P protein supply seem to

  10. The relative importance of kinetic mechanisms and variable enzyme abundances for the regulation of hepatic glucose metabolism--insights from mathematical modeling.

    Science.gov (United States)

    Bulik, Sascha; Holzhütter, Hermann-Georg; Berndt, Nikolaus

    2016-03-02

    Adaptation of the cellular metabolism to varying external conditions is brought about by regulated changes in the activity of enzymes and transporters. Hormone-dependent reversible enzyme phosphorylation and concentration changes of reactants and allosteric effectors are the major types of rapid kinetic enzyme regulation, whereas on longer time scales changes in protein abundance may also become operative. Here, we used a comprehensive mathematical model of the hepatic glucose metabolism of rat hepatocytes to decipher the relative importance of different regulatory modes and their mutual interdependencies in the hepatic control of plasma glucose homeostasis. Model simulations reveal significant differences in the capability of liver metabolism to counteract variations of plasma glucose in different physiological settings (starvation, ad libitum nutrient supply, diabetes). Changes in enzyme abundances adjust the metabolic output to the anticipated physiological demand but may turn into a regulatory disadvantage if sudden unexpected changes of the external conditions occur. Allosteric and hormonal control of enzyme activities allow the liver to assume a broad range of metabolic states and may even fully reverse flux changes resulting from changes of enzyme abundances alone. Metabolic control analysis reveals that control of the hepatic glucose metabolism is mainly exerted by enzymes alone, which are differently controlled by alterations in enzyme abundance, reversible phosphorylation, and allosteric effects. In hepatic glucose metabolism, regulation of enzyme activities by changes of reactants, allosteric effects, and reversible phosphorylation is equally important as changes in protein abundance of key regulatory enzymes.

  11. 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.

  12. Variations of plasma protein electrophoresis in healthy captive Green Iguanas (Iguana iguana).

    Science.gov (United States)

    Musilová, Anna; Knotková, Zora; Pinterová, Kateřina; Knotek, Zdeněk

    2015-06-01

    Serum or plasma protein electrophoresis is used as a routine test for health assessment in veterinary medicine, but there are only a limited number of studies regarding clinical use of electrophoresis in reptile species. The goals of this study were to establish reference intervals for plasma protein electrophoresis in the Green Iguana (Iguana iguana), compare values between males and females, and to identify season-related changes. Plasma samples were obtained from 21 healthy captive male and female Green Iguanas. Agarose gel electrophoresis was performed using an automated Hydrasys system. Four main protein fractions were observed: albumin, α globulins, β globulins, and γ globulins. Bisalbuminemia was observed in 4 of 21 healthy iguanas. Minimum and maximum values were reported for healthy Green Iguanas in March, June, September, and December. Seasonal changes in albumin were determined between March and December, and in γ globulins between June and September. Differences between males and females were seen in albumin concentration in September. Reference intervals of the plasma protein fractions according to electrophoresis in the Green Iguana can be affected by seasonal changes and sex of animals. It should be taken into account when clinical evaluation is performed. © 2015 American Society for Veterinary Clinical Pathology.

  13. Triton X-114 cloud point extraction to subfractionate blood plasma proteins for two-dimensional gel electrophoresis

    DEFF Research Database (Denmark)

    Jessen, Flemming; Wulff, Tune

    2015-01-01

    -sterol acyltransferase, serum amyloid A, and serum paraoxonase/arylesterase 1, which are proteins of a hydrophobic nature, as in plasma they relate to lipoprotein particles. Thus, Triton X-114-based CPE is a simple plasma prefractionation tool, attractive for detailed 2DE studies of hydrophobic plasma proteins...

  14. Effect of whole body gamma-irradiation and/or dietary protein deficiency on the levels of plasma non-protein nitrogen and amino acids; plasma and urinary ammonia and urea in desert rodent and albino rats

    International Nuclear Information System (INIS)

    Roushdy, H.M.; El-Husseini, M.; Saleh, F.

    1984-01-01

    The effect of gamma-irradiation exposure on the levels of non-protein nitrogen (N.P.N.) and amino acids in plasma; ammonia and urea in plasma and urine was studied in the desert rodent, Psammomys obesus obesus and albino rats subjected to dietary protein deficiency, N.P.N. and amino acids in plasma were shown to increase by irradiation exposure. The effect of radiation on blood ammonia was less marked, but it caused a significant increase in ammonia excretion in urine. Radiation exposure in albino rats caused a marked increase in urea concentration in plasma of animals fed the high protein diet and irradiated at 780 r. In urine, the tested radiation levels caused an initial increase in urea concentration followed by a subsequent decrease. In psammomys, radiation exposure exerted a little effect on the plasma urea level, whereas significant increase in the daily urea excretion was recorded. It seems that urea level in plasma is more stabilized in psammomys than in albino rats

  15. Novel effects of hormonal contraceptive use on the plasma proteome.

    Directory of Open Access Journals (Sweden)

    Andrea R Josse

    Full Text Available Hormonal contraceptive (HC use may increase cardiometabolic risk; however, the effect of HC on emerging cardiometabolic and other disease risk factors is not clear.To determine the association between HC use and plasma proteins involved in established and emerging disease risk pathways.Concentrations of 54 high-abundance plasma proteins were measured simultaneously by LC-MRM/MS in 783 women from the Toronto Nutrigenomics and Health Study. C-reactive protein (CRP was measured separately. ANCOVA was used to test differences in protein concentrations between users and non-users, and among HC users depending on total hormone dose. Linear regression was used to test the association between duration (years of HC use and plasma protein concentrations. Principal components analysis (PCA was used to identify plasma proteomic profiles in users and non-users.After Bonferroni correction, 19 proteins involved in inflammation, innate immunity, coagulation and blood pressure regulation were significantly different between users and non-users (P<0.0009. These differences were replicated across three distinct ethnocultural groups. Traditional markers of glucose and lipid metabolism were also significantly higher among HC users. Neither hormone dose nor duration of use affected protein concentrations. PCA identified 4 distinct proteomic profiles in users and 3 in non-users.HC use was associated with different concentrations of plasma proteins along various disease-related pathways, and these differences were present across different ethnicities. Aside from the known effect of HC on traditional biomarkers of cardiometabolic risk, HC use also affects numerous proteins that may be biomarkers of dysregulation in inflammation, coagulation and blood pressure.

  16. 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...

  17. A Physiologically Based Pharmacokinetic Model to Predict the Pharmacokinetics of Highly Protein-Bound Drugs and Impact of Errors in Plasma Protein Binding

    Science.gov (United States)

    Ye, Min; Nagar, Swati; Korzekwa, Ken

    2015-01-01

    Predicting the pharmacokinetics of highly protein-bound drugs is difficult. Also, since historical plasma protein binding data was often collected using unbuffered plasma, the resulting inaccurate binding data could contribute to incorrect predictions. This study uses a generic physiologically based pharmacokinetic (PBPK) model to predict human plasma concentration-time profiles for 22 highly protein-bound drugs. Tissue distribution was estimated from in vitro drug lipophilicity data, plasma protein binding, and blood: plasma ratio. Clearance was predicted with a well-stirred liver model. Underestimated hepatic clearance for acidic and neutral compounds was corrected by an empirical scaling factor. Predicted values (pharmacokinetic parameters, plasma concentration-time profile) were compared with observed data to evaluate model accuracy. Of the 22 drugs, less than a 2-fold error was obtained for terminal elimination half-life (t1/2, 100% of drugs), peak plasma concentration (Cmax, 100%), area under the plasma concentration-time curve (AUC0–t, 95.4%), clearance (CLh, 95.4%), mean retention time (MRT, 95.4%), and steady state volume (Vss, 90.9%). The impact of fup errors on CLh and Vss prediction was evaluated. Errors in fup resulted in proportional errors in clearance prediction for low-clearance compounds, and in Vss prediction for high-volume neutral drugs. For high-volume basic drugs, errors in fup did not propagate to errors in Vss prediction. This is due to the cancellation of errors in the calculations for tissue partitioning of basic drugs. Overall, plasma profiles were well simulated with the present PBPK model. PMID:26531057

  18. Palmitoylation of POTE family proteins for plasma membrane targeting

    International Nuclear Information System (INIS)

    Das, Sudipto; Ise, Tomoko; Nagata, Satoshi; Maeda, Hiroshi; Bera, Tapan K.; Pastan, Ira

    2007-01-01

    The POTE gene family is composed of 13 paralogs and likely evolved by duplications and remodeling of the human genome. One common property of POTE proteins is their localization on the inner aspect of the plasma membrane. To determine the structural elements required for membrane localization, we expressed mutants of different POTEs in 293T cells as EGFP fusion proteins. We also tested their palmitoylation by a biotin-switch assay. Our data indicate that the membrane localizations of different POTEs are mediated by similar 3-4 short cysteine rich repeats (CRRs) near the amino-terminuses and that palmitoylation on paired cysteine residues in each CRR motif is responsible for the localization. Multiple palmitoylation in the small CRRs can result in the strong association of whole POTEs with plasma membrane

  19. Viral protein Nef is detected in plasma of half of HIV-infected adults with undetectable plasma HIV RNA.

    Directory of Open Access Journals (Sweden)

    Jana Ferdin

    Full Text Available To address the role of translationally active HIV reservoir in chronic inflammation and non-AIDS related disorders, we first need a simple and accurate assay to evaluate viral protein expression in virally suppressed subjects.We optimized an HIV Nef enzyme-linked immunosorbent assay (ELISA and used it to quantify plasma Nef levels as an indicator of the leaky HIV reservoir in an HIV-infected cohort.This study accessed 134 plasma samples from a well-characterized cohort study of HIV-infected and uninfected adults in San Francisco (the SCOPE cohort. We optimized an ELISA for detection of plasma Nef in HIV-negative subjects and HIV-infected non-controllers, and evaluated its utility to quantify plasma Nef levels in a cross-sectional study of ART-suppressed and elite controller HIV-infected subjects.Here, we describe the performance of an optimized HIV Nef ELISA. When we applied this assay to the study cohort we found that plasma Nef levels were correlated with plasma HIV RNA levels in untreated disease. However, we were able to detect Nef in plasma of approximately half of subjects on ART or with elite control, despite the lack of detectable plasma HIV RNA levels using standard assays. Plasma Nef levels were not consistently associated with CD4+ T-cell count, CD8+ T-cell count, self-reported nadir CD4+ T-cell count or the CD4+/CD8+ T-cell ratio in HIV-infected subjects.Since plasma HIV RNA levels are undetectable in virally suppressed subjects, it is reasonable to assume that viral protein expression in leaky reservoir, and not plasma virions, is the source of Nef accumulating in plasma. To examine this further, improvements of the assay sensitivity, by lowering the background through improvements in the quality of Nef antibodies, and detailed characterization of the HIV reservoirs are needed.

  20. Inhibition of the plasma SCUBE1, a novel platelet adhesive protein, protects mice against thrombosis.

    Science.gov (United States)

    Wu, Meng-Ying; Lin, Yuh-Charn; Liao, Wei-Ju; Tu, Cheng-Fen; Chen, Ming-Huei; Roffler, Steve R; Yang, Ruey-Bing

    2014-07-01

    Signal peptide-CUB-EGF domain-containing protein 1 (SCUBE1), a secreted and surface-exposed glycoprotein on activated platelets, promotes platelet-platelet interaction and supports platelet-matrix adhesion. Its plasma level is a biomarker of platelet activation in acute thrombotic diseases. However, the exact roles of plasma SCUBE1 in vivo remain undefined. We generated new mutant (Δ) mice lacking the soluble but retaining the membrane-bound form of SCUBE1. Plasma SCUBE1-depleted Δ/Δ mice showed normal hematologic and coagulant features and expression of major platelet receptors, but Δ/Δ platelet-rich plasma showed impaired platelet aggregation in response to ADP and collagen treatment. The addition of purified recombinant SCUBE1 protein restored the aggregation of platelets in Δ/Δ platelet-rich plasma and further enhanced platelet aggregation in +/+ platelet-rich plasma. Plasma deficiency of SCUBE1 diminished arterial thrombosis in mice and protected against lethal thromboembolism induced by collagen-epinephrine treatment. Last, antibodies directed against the epidermal growth factor-like repeats of SCUBE1, which are involved in trans-homophilic protein-protein interactions, protected mice against fatal thromboembolism without causing bleeding in vivo. We conclude that plasma SCUBE1 participates in platelet aggregation by bridging adjacent activated platelets in thrombosis. Blockade of soluble SCUBE1 might represent a novel antithrombotic strategy. © 2014 American Heart Association, Inc.

  1. Experimental determination of net protein charge, [A]tot, and Ka of nonvolatile buffers in bird plasma.

    Science.gov (United States)

    Stämpfli, Henry; Taylor, Michael; McNicoll, Carl; Gancz, Ady Y; Constable, Peter D

    2006-06-01

    The quantitative mechanistic acid-base approach to clinical assessment of acid-base status requires species-specific values for [A]tot (the total concentration of nonvolatile buffers in plasma) and Ka (the effective dissociation constant for weak acids in plasma). The aim of this study was to determine [A]tot and Ka values for plasma in domestic pigeons. Plasma from 12 healthy commercial domestic pigeons was tonometered with 20% CO2 at 37 degrees C. Plasma pH, Pco2, and plasma concentrations of strong cations (Na, K, Ca), strong anions (Cl, L-lactate), and nonvolatile buffer ions (total protein, albumin, phosphate) were measured over a pH range of 6.8-7.7. Strong ion difference (SID) (SID5=Na+K+Ca-Cl-lactate) was used to calculate [A]tot and Ka from the measured pH and Pco2 and SID5. Mean (+/-SD) values for bird plasma were as follows: [A]tot=7.76+/-2.15 mmol/l (equivalent to 0.32 mmol/g of total protein, 0.51 mmol/g of albumin, 0.23 mmol/g of total solids); Ka=2.15+/-1.15x10(-7); and pKa=6.67. The net protein charge at normal pH (7.43) was estimated to be 6 meq/l; this value indicates that pigeon plasma has a much lower anion gap value than mammals after adjusting for high mean L-lactate concentrations induced by restraint during blood sampling. This finding indicates that plasma proteins in pigeons have a much lower net anion charge than mammalian plasma protein. An incidental finding was that total protein concentration measured by a multianalyzer system was consistently lower than the value for total solids measured by refractometer.

  2. Plasma membrane lipid–protein interactions affect signaling processes in sterol-biosynthesis mutants in Arabidopsis thaliana

    Science.gov (United States)

    Zauber, Henrik; Burgos, Asdrubal; Garapati, Prashanth; Schulze, Waltraud X.

    2014-01-01

    The plasma membrane is an important organelle providing structure, signaling and transport as major biological functions. Being composed of lipids and proteins with different physicochemical properties, the biological functions of membranes depend on specific protein–protein and protein–lipid interactions. Interactions of proteins with their specific sterol and lipid environment were shown to be important factors for protein recruitment into sub-compartmental structures of the plasma membrane. System-wide implications of altered endogenous sterol levels for membrane functions in living cells were not studied in higher plant cells. In particular, little is known how alterations in membrane sterol composition affect protein and lipid organization and interaction within membranes. Here, we conducted a comparative analysis of the plasma membrane protein and lipid composition in Arabidopsis sterol-biosynthesis mutants smt1 and ugt80A2;B1. smt1 shows general alterations in sterol composition while ugt80A2;B1 is significantly impaired in sterol glycosylation. By systematically analyzing different cellular fractions and combining proteomic with lipidomic data we were able to reveal contrasting alterations in lipid–protein interactions in both mutants, with resulting differential changes in plasma membrane signaling status. PMID:24672530

  3. Plasma-treated polystyrene film that enhances binding efficiency for sensitive and label-free protein biosensing

    Energy Technology Data Exchange (ETDEWEB)

    Guo, Bihong [National Center for NanoScience and Technology, No. 11 Beiyitiao, Zhongguancun, Beijing 100190 (China); Li, Shaopeng [National Center for NanoScience and Technology, No. 11 Beiyitiao, Zhongguancun, Beijing 100190 (China); Department of Chemistry, Tsinghua University, Beijing 100084 (China); Song, Lusheng [National Center for NanoScience and Technology, No. 11 Beiyitiao, Zhongguancun, Beijing 100190 (China); Yang, Mo; Zhou, Wenfei; Tyagi, Deependra [National Center for NanoScience and Technology, No. 11 Beiyitiao, Zhongguancun, Beijing 100190 (China); University of Chinese Academy of Sciences, Yuquan Rd., 19(A), Beijing 100049 (China); Zhu, Jinsong, E-mail: jizhu88@gmail.com [National Center for NanoScience and Technology, No. 11 Beiyitiao, Zhongguancun, Beijing 100190 (China)

    2015-08-01

    Highlights: • A simple and robust plasma-treated ultrathin polystyrene film surface was developed for protein biosensing. • The surface was optimized by evaluating up to 120 types of fabrication parameters with high-throughput analytical methods. • The optimized surface showed a 620% improvement of the protein detection signal and 210% protein binding per immobilized protein ligand compared with a self-assembled monolayer surface. - Abstract: A plasma-treated ultrathin polystyrene (PS) film surface was explored as a simple, robust, and low-cost surface chemistry solution for protein biosensing applications. This surface could dramatically improve the binding efficiency of the protein–protein interactions, which is defined as the binding signal per immobilized ligand. The PS-modified protein biosensor was readily fabricated by spin coating and plasma treatment. Various parameters for fabrication, including the concentration of the PS solution, rate of spin coating, and duration of plasma treatment, were systematically optimized based on the improvement of fluorescence signal yielded by the microfluidic network-aided fluorescence immunoassay. The performance of the label-free protein detection on the optimized surfaces was further evaluated by surface plasmon resonance imaging (SPRi). PS surfaces with optimal fabrication parameters exhibited up to an 620% enhancement of the protein binding response and approximately 210% of the protein binding per immobilized protein ligand compared with a self-assembled monolayer (SAM) surface of 11-mercapto undecanoic acid (MUA). The relationship between the fabrication parameters used and changes to the surface chemistry and the morphological properties were characterized with atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS), and Fourier transform infrared spectroscopy (FTIR). It was revealed that the morphological changes observed in the plasma-treated PS film were the dominant factor for the

  4. The dynamic changes of the plasma membrane proteins and the protective roles of nitric oxide in rice subjected to heavy metal cadmium stress

    Directory of Open Access Journals (Sweden)

    Liming eYang

    2016-02-01

    Full Text Available The heavy metal cadmium is a common environmental contaminant in soils and has adverse effects on crop growth and development. The signaling processes in plants that initiate cellular responses to environmental stress have been shown to be located in the plasma membrane (PM. A better understanding of the PM proteome in response to environmental stress might provide new insights for improving stress-tolerant crops. Nitric oxide (NO is reported to be involved in the plant response to cadmium (Cd stress. To further investigate how NO modulates protein changes in the plasma membrane during Cd stress, a quantitative proteomics approach based on isobaric tags for relative and absolute quantification (iTRAQ was used to identify differentially regulated proteins from the rice plasma membrane after Cd or Cd and NO treatment. Sixty-six differentially expressed proteins were identified, of which, many function as transporters, ATPases, kinases, metabolic enzymes, phosphatases and phospholipases. Among these, the abundance of phospholipase D (PLD was altered substantially after the treatment of both Cd and Cd and NO. Transient expression of the PLD fused with green fluorescent peptide (GFP in rice protoplasts showed that the Cd and NO treatment promoted the accumulation of PLD in the plasma membrane. Addition of NO also enhanced Cd-induced PLD activity and the accumulation of phosphatidic acid (PA produced through PLD activity. Meanwhile, NO elevated the activities of antioxidant enzymes and caused the accumulation of glutathione both which function to reduce Cd-induced H2O2 accumulation. Taken together, we suggest that NO signaling is associated with the accumulation of antioxidant enzymes, glutathione and PA which increases cadmium tolerance in rice via the antioxidant defense system.

  5. Longitudinal changes in C-reactive protein, proform of eosinophil major basic protein, and pregnancy-associated plasma protein-A during weight changes in obese children

    DEFF Research Database (Denmark)

    Lausten-Thomsen, Ulrik; Gamborg, Michael; Bøjsøe, Christine

    2015-01-01

    BACKGROUND: Childhood obesity is associated with several complications, including cardiovascular comorbidity. Several biomarkers, such as high-sensitive C-reactive protein (hs-CRP), proform of eosinophil major basic protein (Pro-MBP) and pregnancy associated plasma protein-A (PAPP-A), have equally...

  6. 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.

  7. Concentration of Proteins and Protein Fractions in Blood Plasma of Chickens Hatched from Eggs Irradiated with Low Level Gamma Rays

    International Nuclear Information System (INIS)

    Kraljevic, P.; Vilic, M.; Simpraga, M.; Matisic, D.; Miljanic, S.

    2011-01-01

    In literature there are many results which have shown that low dose radiation can stimulate many physiological processes of living organism. In our earlier paper it was shown that low dose of gamma radiation has a stimulative effect upon metabolic process in chickens hatched from eggs irradiated before incubation. This was proved by increase of body weight gain and body weight, as well as by increase of two enzymes activities in blood plasma (aspartate aminotransferase and alanine aminotransferase) which play an important role in protein metabolism. Therefore, an attempt was made to determine the effect of eggs irradiation by low dose gamma rays upon concentration of total proteins and protein fractions in the blood plasma of chickens hatched from irradiated eggs. The eggs of heavy breed chickens were irradiated with a dose of 0.15 Gy gamma radiation (60Co) before incubation. Along with the chickens which were hatched from irradiated eggs, there was a control group of chickens hatched from nonirradiated eggs. All other conditions were the same for both groups of chickens. Blood samples were taken from the right jugular vein on the 1 s t and 3 r d day, or from the wing vein on days 5 and 7 after hatching. The total proteins concentration in the blood plasma was determined by the biuret method using Boehringer Mannheim GmbH optimized kits. The protein fractions (albumin, α 1 -globulin, α 2 -globulin, β- and γ-globulins) were estimated electrophoretically on Cellogel strips. The total proteins concentration was significantly decreased in blood plasma of chickens hatched from irradiated eggs on days 3 (P t h day (P 2 -globulin was decreased on days 1 (P t h day of life. Obtained results indicate that low dose of gamma radiation has mostly inhibitory effect upon concentration of total proteins and protein fractions in the blood plasma of chickens hatched from irradiated eggs before incubation. (author)

  8. A fluorogenic probe for SNAP-tagged plasma membrane proteins based on the solvatochromic molecule Nile Red.

    Science.gov (United States)

    Prifti, Efthymia; Reymond, Luc; Umebayashi, Miwa; Hovius, Ruud; Riezman, Howard; Johnsson, Kai

    2014-03-21

    A fluorogenic probe for plasma membrane proteins based on the dye Nile Red and SNAP-tag is introduced. It takes advantage of Nile Red, a solvatochromic molecule highly fluorescent in an apolar environment, such as cellular membranes, but almost dark in a polar aqueous environment. The probe possesses a tuned affinity for membranes allowing its Nile Red moiety to insert into the lipid bilayer of the plasma membrane, becoming fluorescent, only after its conjugation to a SNAP-tagged plasma membrane protein. The fluorogenic character of the probe was demonstrated for different SNAP-tag fusion proteins, including the human insulin receptor. This work introduces a new approach for generating a powerful turn-on probe for "no-wash" labeling of plasma membrane proteins with numerous applications in bioimaging.

  9. Pregnancy Associated Plasma Protein-A in Type 2 Diabetic Patient with Peripheral Neuropathy

    International Nuclear Information System (INIS)

    Nosseir, N.M.

    2011-01-01

    Metabolic changes induced by hyperglycemia lead to dysregulation of cytokines control, subclinical inflammation together with oxidative stress associated with diabetes. The aim of this study is to correlate the role of type 2 diabetic neuropathy on serum pregnancy associated plasma protein-A,interleukin-6 and c-reactive protein .The results denoted that both pregnancy associated plasma protein-A and interleukin-6 were significantly increased in those patients with diabetic neuropathy compared with those without neuropathy but while c-reactive proteins showed significant differences between the three groups, the results lead to the conclusion that PAPP-A,IL-6 are useful tests in monitoring the neuropathic complications associated with type 2 diabetes

  10. A cell-free assay to determine the stoichiometry of plasma membrane proteins.

    Science.gov (United States)

    Trigo, Cesar; Vivar, Juan P; Gonzalez, Carlos B; Brauchi, Sebastian

    2013-04-01

    Plasma membrane receptors, transporters, and ion channel molecules are often found as oligomeric structures that participate in signaling cascades essential for cell survival. Different states of protein oligomerization may play a role in functional control and allosteric regulation. Stochastic GFP-photobleaching (SGP) has emerged as an affordable and simple method to determine the stoichiometry of proteins at the plasma membrane. This non-invasive optical approach can be useful for total internal reflection of fluorescence microscopy (TIRFM), where signal-to-noise ratio is very high at the plasma membrane. Here, we report an alternative methodology implemented on a standard laser scanning confocal microscope (LSCM). The simplicity of our method will allow for its implementation in any epifluorescence microscope of choice.

  11. GLUT-4 content in plasma membrane of muscle from patients with non-insulin-dependent diabetes mellitus

    DEFF Research Database (Denmark)

    Lund, S; Vestergaard, H; Andersen, P H

    1993-01-01

    The abundance of GLUT-4 protein in both total crude membrane and plasma membrane fractions of vastus lateralis muscle from 13 obese non-insulin-dependent diabetes mellitus (NIDDM) patients and 14 healthy subjects were examined in the fasting state and after supraphysiological hyperinsulinemia....... In the basal state the immunoreactive mass of GLUT-4 protein both in the crude membrane preparation and in the plasma membrane fraction was similar in NIDDM patients and control subjects. Moreover, in vivo insulin exposure neither for 30 min nor for 4 h had any impact on the content of GLUT-4 protein in plasma...... membranes. With the use of the same methodology, antibody, and achieving the same degree of plasma membrane purification and recovery, we found, however, that intraperitoneal administration of insulin to 7-wk-old rats within 30 min increased the content of GLUT-4 protein more than twofold (P

  12. Influence of Acute High Glucose on Protein Abundance Changes in Murine Glomerular Mesangial Cells

    Directory of Open Access Journals (Sweden)

    Michelle T. Barati

    2016-01-01

    Full Text Available The effects of acute exposure to high glucose levels as experienced by glomerular mesangial cells in postprandial conditions and states such as in prediabetes were investigated using proteomic methods. Two-dimensional gel electrophoresis and matrix assisted laser desorption ionization time of flight mass spectrometry methods were used to identify protein expression patterns in immortalized rat mesangial cells altered by 2 h high glucose (HG growth conditions as compared to isoosmotic/normal glucose control (NG⁎ conditions. Unique protein expression changes at 2 h HG treatment were measured for 51 protein spots. These proteins could be broadly grouped into two categories: (1 proteins involved in cell survival/cell signaling and (2 proteins involved in stress response. Immunoblot experiments for a protein belonging to both categories, prohibitin (PHB, supported a trend for increased total expression as well as significant increases in an acidic PHB isoform. Additional studies confirmed the regulation of proteasomal subunit alpha-type 2 and the endoplasmic reticulum chaperone and oxidoreductase PDI (protein disulfide isomerase, suggesting altered ER protein folding capacity and proteasomal function in response to acute HG. We conclude that short term high glucose induces subtle changes in protein abundances suggesting posttranslational modifications and regulation of pathways involved in proteostasis.

  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. Plasma levels of leptin, omentin, collagenous repeat-containing sequence of 26-kDa protein (CORS-26 and adiponectin before and after oral glucose uptake in slim adults

    Directory of Open Access Journals (Sweden)

    Schäffler Andreas

    2007-02-01

    Full Text Available Abstract Background Adipose tissue secreted proteins are collectively named adipocytokines and include leptin, adiponectin, resistin, collagenous repeat-containing sequence of 26-kDa protein (CORS-26 and omentin. Several of these adipocytokines influence insulin sensitivity and glucose metabolism and therefore systemic levels may be affected by oral glucose uptake. Whereas contradictory results have been published for leptin and adiponectin, resistin has not been extensively investigated and no reports on omentin and CORS-26 do exist. Methods Therefore the plasma levels of these proteins before and 120 min after an oral glucose load were analyzed in 20 highly-insulin sensitive, young adults by ELISA or immunoblot. Results Circulating leptin was reduced 2 h after glucose uptake whereas adiponectin and resistin levels are not changed. Distribution of adiponectin and CORS-26 isoforms were similar before and after glucose ingestion. Omentin is highly abundant in plasma and immunoblot analysis revealed no alterations when plasma levels before and 2 h after glucose intake were compared. Conclusion Taken together our data indicate that only leptin is reduced by glucose uptake in insulin-sensitive probands whereas adiponectin and resistin are not altered. CORS-26 was demonstrated for the first time to circulate as high molecular weight form in plasma and like omentin was not influenced by oral glucose load. Omentin was shown to enhance insulin-stimulated glucose uptake but systemic levels are not correlated to postprandial blood glucose.

  15. ER-to-plasma membrane tethering proteins regulate cell signaling and ER morphology.

    Science.gov (United States)

    Manford, Andrew G; Stefan, Christopher J; Yuan, Helen L; Macgurn, Jason A; Emr, Scott D

    2012-12-11

    Endoplasmic reticulum-plasma membrane (ER-PM) junctions are conserved structures defined as regions of the ER that tightly associate with the plasma membrane. However, little is known about the mechanisms that tether these organelles together and why such connections are maintained. Using a quantitative proteomic approach, we identified three families of ER-PM tethering proteins in yeast: Ist2 (related to mammalian TMEM16 ion channels), the tricalbins (Tcb1/2/3, orthologs of the extended synaptotagmins), and Scs2 and Scs22 (vesicle-associated membrane protein-associated proteins). Loss of all six tethering proteins results in the separation of the ER from the PM and the accumulation of cytoplasmic ER. Importantly, we find that phosphoinositide signaling is misregulated at the PM, and the unfolded protein response is constitutively activated in the ER in cells lacking ER-PM tether proteins. These results reveal critical roles for ER-PM contacts in cell signaling, organelle morphology, and ER function. Copyright © 2012 Elsevier Inc. All rights reserved.

  16. 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.

  17. Unfoldomics of prostate cancer: on the abundance and roles of intrinsically disordered proteins in prostate cancer

    Science.gov (United States)

    Landau, Kevin S; Na, Insung; Schenck, Ryan O; Uversky, Vladimir N

    2016-01-01

    Prostatic diseases such as prostate cancer and benign prostatic hyperplasia are highly prevalent among men. The number of studies focused on the abundance and roles of intrinsically disordered proteins in prostate cancer is rather limited. The goal of this study is to analyze the prevalence and degree of disorder in proteins that were previously associated with the prostate cancer pathogenesis and to compare these proteins to the entire human proteome. The analysis of these datasets provides means for drawing conclusions on the roles of disordered proteins in this common male disease. We also hope that the results of our analysis can potentially lead to future experimental studies of these proteins to find novel pathways associated with this disease. PMID:27453073

  18. Radioimmunoassay of human plasma protein C

    International Nuclear Information System (INIS)

    Wang Bocheng; Li Jinquan; Jing Jian; Zhang Manda

    1995-01-01

    A radioimmunoassay method for the measurement of human plasma protein C (PC) is established. PC was isolated and purified from human plasma. The antisera against PC was obtained by immunizing rabbits. Iodination of PC was carried out with chroramine-T. The sensitivity was 3.94% μg/L, and the assay covered 6.25∼1024 μg/L for PC. The intra-assay and inter-assay CV were 4.4% and 9.68% respectively, with a recovery rate of 104.28%. There was no cross reaction with factor II. The normal value was 3.84 +- 0.34 mg/L in 36 normal persons. Value of 1.03 +-0.41 mg/L was found in 16 patients with fulminating hepatitis complicated with coagulation disturbance. It is an effective approach for the diagnosis of hereditary or acquired PC deficiency and also for the study of thrombotic diseases

  19. Triple SILAC quantitative proteomic analysis reveals differential abundance of cell signaling proteins between normal and lung cancer-derived exosomes.

    Science.gov (United States)

    Clark, David J; Fondrie, William E; Yang, Austin; Mao, Li

    2016-02-05

    Exosomes are 30-100 nm sized membrane vesicles released by cells into the extracellular space that mediate intercellular communication via transfer of proteins and other biological molecules. To better understand the role of these microvesicles in lung carcinogenesis, we employed a Triple SILAC quantitative proteomic strategy to examine the differential protein abundance between exosomes derived from an immortalized normal bronchial epithelial cell line and two non-small cell lung cancer (NSCLC) cell lines harboring distinct activating mutations in the cell signaling molecules: Kirsten rat sarcoma viral oncogene homolog (KRAS) or epidermal growth factor receptor (EGFR). In total, we were able to quantify 721 exosomal proteins derived from the three cell lines. Proteins associated with signal transduction, including EGFR, GRB2 and SRC, were enriched in NSCLC exosomes, and could actively regulate cell proliferation in recipient cells. This study's investigation of the NSCLC exosomal proteome has identified enriched protein cargo that can contribute to lung cancer progression, which may have potential clinical implications in biomarker development for patients with NSCLC. The high mortality associated with lung cancer is a result of late-stage diagnosis of the disease. Current screening techniques used for early detection of lung cancer lack the specificity for accurate diagnosis. Exosomes are nano-sized extracellular vesicles, and the increased abundance of select protein cargo in exosomes derived from cancer cells may be used for diagnostic purposes. In this paper, we applied quantitative proteomic analysis to elucidate abundance differences in exosomal protein cargo between two NSCLC cell lines with distinctive oncogene mutations and an immortalized normal bronchial epithelial cell line. This study revealed proteins associated with cell adhesion, the extracellular matrix, and a variety of signaling molecules were enriched in NSCLC exosomes. The present data reveals

  20. Characterization of plasma protein binding dissociation with online SPE-HPLC

    OpenAIRE

    Li, Ping; Fan, Yiran; Wang, Yunlong; Lu, Yaxin; Yin, Zheng

    2015-01-01

    A novel parameter of relative recovery (Rre) was defined and determined by online SPE-HPLC to characterize plasma protein binding (PPB) kinetics of highly plasma binding drugs. The proportional relationship of Rre with koff of PPB has been established with a new SPE model. A rapid, easy to use method could potentially be used to categorize PK properties of the drug candidates in the decision process of drug discovery and development.

  1. HK2 Proximal Tubule Epithelial Cells Synthesize and Secrete Plasma Proteins Predominantly Through the Apical Surface.

    Science.gov (United States)

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

    2017-04-01

    Renal proximal tubule epithelial cells (PTECs) are known to reabsorb salts and small plasma proteins filtered through Bowman's capsule. Following acute kidney injury, PTECs assume some characteristics of hepatocytes in producing various plasma proteins. We now demonstrate that even at a resting state, a PTEC cell line, HK2 expresses mRNAs for and synthesizes and secretes plasma proteins in a complex with complement C3, an α 2 -macroglobulin family chaperone, including albumin, transferrin, α 1 -antitrypsin, α 1 -antichymotrypsin, α 2 -HS-glycoprotein, ceruloplasmin, haptoglobin, C1-inhibitor, secreted phosphoprotein-24, and insulin-like growth factor-1. When grown on transwell inserts, HK2 cells predominantly secrete (∼90%) plasma proteins into the apical side and a smaller fraction into the basolateral side as determined by ELISA assays. When cultured in the presence of exogenous cytokines such as IL1β, IL6, TNFα, BMP2, or TGFβ1, HK2 cell mRNA expressions for plasma proteins were variably affected whereas basolateral secretions were elevated to or in excess of those of the apical level. In addition, HK2 cells produce proTGFβ1 with its intact N-terminal latency associated peptide and latent-TGF-β-binding proteins. The complex cannot be dissociated under conditions of SDS, heating, and electrophoresis. Moreover, HK2 cells maintain their ability to quickly uptake exogenously added serum proteins from the culture medium, as if they are recognized differently by the endocytic receptors. These results provide new insight into the hepatization of PTECs. In addition to their unique uptake of plasma proteins and salts from the filtrate, they are a source of urinary proteins under normal conditions as wells as in chronic and acute kidney diseases. J. Cell. Biochem. 118: 924-933, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  2. 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.

  3. Plasma proteins of Barbus holubi and Clarias gariepinus

    African Journals Online (AJOL)

    The plasma proteins of the teleosts, Barbus holubl and ClariDs gariepllUl3 were investigated by means of cel1u1ose-acetate and polyacrylamide gel electrophoresis. Characteristic patterns were obtained with both methods for each species. It was found that the application of human nomenclature to the patterns obtained in ...

  4. Plasma Signaling Proteins in Persons at Genetic Risk for Alzheimer Disease

    Science.gov (United States)

    Ringman, John M.; Elashoff, David; Geschwind, Daniel H.; Welsh, Brian T.; Gylys, Karen H.; Lee, Cathy; Cummings, Jeffrey L.; Cole, Greg M.

    2013-01-01

    Objective To study the effect of familial Alzheimer disease (FAD) mutations and APOE genotype on plasma signaling protein levels. Design Cross-sectional comparison of plasma levels of 77 proteins measured using multiplex immune assays. Setting A tertiary referral dementia research center. Participants Thirty-three persons from families harboring PSEN1 or APP mutations, aged 19 to 59 years. Main Outcome Measures Protein levels were compared between FAD mutation carriers (MCs) and non-carriers (NCs) and among APOE genotype groups, using multiple linear regression models. Results Twenty-one participants were FAD MCs and 12 were NCs. Six had the APOE ε2/3, 6 had the ε3/4, and 21 had the ε3/3 genotype. Levels of 17 proteins differed among APOE genotype groups, and there were significant interactions between age and APOE genotype for 12 proteins. Plasma levels of apolipoprotein E and superoxide dismutase 1 were highest in the ε2 carriers, lowest in ε4 carriers, and intermediate in the ε3 carriers. Levels of multiple interleukins showed the opposite pattern and, among the ε4 carriers, demonstrated significant negative correlations with age. Although there were no significant differences between FAD MCs and NCs, there were interactions between mutation status and APOE genotype for 13 proteins. Conclusions We found different patterns of inflammatory markers in young and middle-aged persons among APOE genotype groups. The APOE ε4 carriers had the lowest levels of apolipoprotein E. Young ε4 carriers have increased inflammatory markers that diminish with age. We demonstrated altered inflammatory responses in young and middle adulthood in ε4 carriers that may relate to AD risk later in life. PMID:22689192

  5. Stimulated synthesis of plasma protein species in Q fever and endotoxemia

    Energy Technology Data Exchange (ETDEWEB)

    Picking, W.D.; Ershadi, M.; Hackstadt, T.; Paretsky, D.

    1987-05-01

    Q fever stimulates hepatic transcription and translation. Products of stimulated transcription have been identified, but not of translation. Protein (Pr) synthesis and rPr S6 phosphorylation correlated. The authors now report stimulated synthesis of plasma Pr species in early febrile responses to Q fever and Coxiella burnetii lipopolysaccharide (LPS). Guinea pigs received 400 g LPS intraperitoneally and 7 hr later 250 Ci L-(TVS)met, then sacrificed 3 hr later. Plasma Pr sp act (cpm/mg Pr) increased 2.3X over controls (N). Exptl plasma Pr PAGE autorads showed intensified Pr bands at M/sub r/ 55K. Guinea pigs infected with C. burnetii (Inf) received 400 Ci (TVS)met 84 hr p.i. and were sacrificed 3 hr later. Inf plasma Pr 1D-PAGE showed bands at 55K similar to that found with LPS, with lower albumin concn. Coomassie stain and autorads of 2-D PAGEs showed intensified or new acidic peptide species in Inf plasma. PAGE autorads in vitro translations using liver mRNA and ribosomes showed major species in Inf systems at 49K (4+) and 62K (2+) compared to N. The data indicate acute phase protein induction by LPS or rickettsial infection.

  6. Stimulated synthesis of plasma protein species in Q fever and endotoxemia

    International Nuclear Information System (INIS)

    Picking, W.D.; Ershadi, M.; Hackstadt, T.; Paretsky, D.

    1987-01-01

    Q fever stimulates hepatic transcription and translation. Products of stimulated transcription have been identified, but not of translation. Protein (Pr) synthesis and rPr S6 phosphorylation correlated. The authors now report stimulated synthesis of plasma Pr species in early febrile responses to Q fever and Coxiella burnetii lipopolysaccharide (LPS). Guinea pigs received 400 μg LPS intraperitoneally and 7 hr later 250 μCi L-[ 35 S]met, then sacrificed 3 hr later. Plasma Pr sp act (cpm/mg Pr) increased 2.3X over controls (N). Exptl plasma Pr PAGE autorads showed intensified Pr bands at M/sub r/ 55K. Guinea pigs infected with C. burnetii (Inf) received 400 μCi [ 35 S]met 84 hr p.i. and were sacrificed 3 hr later. Inf plasma Pr 1D-PAGE showed bands at 55K similar to that found with LPS, with lower albumin concn. Coomassie stain and autorads of 2-D PAGEs showed intensified or new acidic peptide species in Inf plasma. PAGE autorads in vitro translations using liver mRNA and ribosomes showed major species in Inf systems at 49K (4+) and 62K (2+) compared to N. The data indicate acute phase protein induction by LPS or rickettsial infection

  7. Variations in riboflavin binding by human plasma: identification of immunoglobulins as the major proteins responsible

    International Nuclear Information System (INIS)

    Innis, W.S.; McCormick, D.B.; Merrill, A.H. Jr.

    1985-01-01

    Riboflavin binding by plasma proteins from healthy human subjects was examined by equilibrium dialysis using a physiological concentration of [2-14C]riboflavin (0.04 microM). Binding ranged from 0.080 to 0.917 pmole of riboflavin/mg of protein (with a mean +/- SD of 0.274 +/- 0.206), which corresponded to 4.14 to 49.4 pmole/ml of plasma (15.5 +/- 11.0) (N = 34). Males and females yielded similar results. Upon fractionation of plasma by gel filtration, the major riboflavin-binding components eluted with albumin and gamma-globulins. Albumin was purified and found to bind riboflavin only very weakly (Kd = 3.8 to 10.4 mM), although FMN and photochemical degradation products (e.g., lumiflavine and lumichrome) were more tightly bound. Binding in the gamma-globulin fraction was attributed to IgG and IGA because the binding protein(s) and immunoglobulins copurified using various methods were removed by treatment of plasma with protein A-agarose, and were coincident upon immunoelectrophoresis followed by autoradiography to detect [2-14C]riboflavin. Differences among the plasma samples correlated with the binding recovered with the immunoglobulins. Binding was not directly related to the total IgG or IgA levels of subjects. Hence, it appears that the binding is due to a subfraction of these proteins. These findings suggest that riboflavin-binding immunoglobulins are a major cause of variations in riboflavin binding in human circulation, and may therefore affect the utilization of this micronutrient

  8. TEMPERATURE DEPENDENT PHASE BEHAVIOR AND PROTEIN PARTITIONING IN GIANT PLASMA MEMBRANE VESICLES

    OpenAIRE

    Johnson, SA; Stinson, BM; Go, M; Carmona, LM; Reminick, JI; Fang, X; Baumgart, T

    2010-01-01

    Liquid-ordered (Lo) and liquid-disordered (Ld) phase coexistence has been suggested to partition the plasma membrane of biological cells into lateral compartments, allowing for enrichment or depletion of functionally relevant molecules. This dynamic partitioning might be involved in fine-tuning cellular signaling fidelity through coupling to the plasma membrane protein and lipid composition. In earlier work, giant plasma membrane vesicles, obtained by chemically induced blebbing from cultured...

  9. Flow induced dispersion analysis rapidly quantifies proteins in human plasma samples

    DEFF Research Database (Denmark)

    Poulsen, Nicklas N; Andersen, Nina Z; Østergaard, Jesper

    2015-01-01

    Rapid and sensitive quantification of protein based biomarkers and drugs is a substantial challenge in diagnostics and biopharmaceutical drug development. Current technologies, such as ELISA, are characterized by being slow (hours), requiring relatively large amounts of sample and being subject...... to cumbersome and expensive assay development. In this work a new approach for quantification based on changes in diffusivity is presented. The apparent diffusivity of an indicator molecule interacting with the protein of interest is determined by Taylor Dispersion Analysis (TDA) in a hydrodynamic flow system...... in a blood plasma matrix), fully automated, and being subject to a simple assay development. FIDA is demonstrated for quantification of the protein Human Serum Albumin (HSA) in human plasma as well as for quantification of an antibody against HSA. The sensitivity of the FIDA assay depends on the indicator...

  10. A physiologically based pharmacokinetic model to predict the pharmacokinetics of highly protein-bound drugs and the impact of errors in plasma protein binding.

    Science.gov (United States)

    Ye, Min; Nagar, Swati; Korzekwa, Ken

    2016-04-01

    Predicting the pharmacokinetics of highly protein-bound drugs is difficult. Also, since historical plasma protein binding data were often collected using unbuffered plasma, the resulting inaccurate binding data could contribute to incorrect predictions. This study uses a generic physiologically based pharmacokinetic (PBPK) model to predict human plasma concentration-time profiles for 22 highly protein-bound drugs. Tissue distribution was estimated from in vitro drug lipophilicity data, plasma protein binding and the blood: plasma ratio. Clearance was predicted with a well-stirred liver model. Underestimated hepatic clearance for acidic and neutral compounds was corrected by an empirical scaling factor. Predicted values (pharmacokinetic parameters, plasma concentration-time profile) were compared with observed data to evaluate the model accuracy. Of the 22 drugs, less than a 2-fold error was obtained for the terminal elimination half-life (t1/2 , 100% of drugs), peak plasma concentration (Cmax , 100%), area under the plasma concentration-time curve (AUC0-t , 95.4%), clearance (CLh , 95.4%), mean residence time (MRT, 95.4%) and steady state volume (Vss , 90.9%). The impact of fup errors on CLh and Vss prediction was evaluated. Errors in fup resulted in proportional errors in clearance prediction for low-clearance compounds, and in Vss prediction for high-volume neutral drugs. For high-volume basic drugs, errors in fup did not propagate to errors in Vss prediction. This is due to the cancellation of errors in the calculations for tissue partitioning of basic drugs. Overall, plasma profiles were well simulated with the present PBPK model. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

  11. Interactions of Ras proteins with the plasma membrane and their roles in signaling.

    Science.gov (United States)

    Eisenberg, Sharon; Henis, Yoav I

    2008-01-01

    The complex dynamic structure of the plasma membrane plays critical roles in cellular signaling; interactions with the membrane lipid milieu, spatial segregation within and between cellular membranes and/or targeting to specific membrane-associated scaffolds are intimately involved in many signal transduction pathways. In this review, we focus on the membrane interactions of Ras proteins. These small GTPases play central roles in the regulation of cell growth and proliferation, and their excessive activation is commonly encountered in human tumors. Ras proteins associate with the membrane continuously via C-terminal lipidation and additional interactions in both their inactive and active forms; this association, as well as the targeting of specific Ras isoforms to plasma membrane microdomains and to intracellular organelles, have recently been implicated in Ras signaling and oncogenic potential. We discuss biochemical and biophysical evidence for the roles of specific domains of Ras proteins in mediating their association with the plasma membrane, and consider the potential effects of lateral segregation and interactions with membrane-associated protein assemblies on the signaling outcomes.

  12. Steric exclusion and protein conformation determine the localization of plasma membrane transporters.

    Science.gov (United States)

    Bianchi, Frans; Syga, Łukasz; Moiset, Gemma; Spakman, Dian; Schavemaker, Paul E; Punter, Christiaan M; Seinen, Anne-Bart; van Oijen, Antoine M; Robinson, Andrew; Poolman, Bert

    2018-02-05

    The plasma membrane (PM) of Saccharomyces cerevisiae contains membrane compartments, MCC/eisosomes and MCPs, named after the protein residents Can1 and Pma1, respectively. Using high-resolution fluorescence microscopy techniques we show that Can1 and the homologous transporter Lyp1 are able to diffuse into the MCC/eisosomes, where a limited number of proteins are conditionally trapped at the (outer) edge of the compartment. Upon addition of substrate, the immobilized proteins diffuse away from the MCC/eisosomes, presumably after taking a different conformation in the substrate-bound state. Our data indicate that the mobile fraction of all integral plasma membrane proteins tested shows extremely slow Brownian diffusion through most of the PM. We also show that proteins with large cytoplasmic domains, such as Pma1 and synthetic chimera of Can1 and Lyp1, are excluded from the MCC/eisosomes. We hypothesize that the distinct localization patterns found for these integral membrane proteins in S. cerevisiae arises from a combination of slow lateral diffusion, steric exclusion, and conditional trapping in membrane compartments.

  13. Plasma and liver acetaminophen-protein adduct levels in mice after acetaminophen treatment: Dose–response, mechanisms, and clinical implications

    International Nuclear Information System (INIS)

    McGill, Mitchell R.; Lebofsky, Margitta; Norris, Hye-Ryun K.; Slawson, Matthew H.; Bajt, Mary Lynn; Xie, Yuchao; Williams, C. David; Wilkins, Diana G.; Rollins, Douglas E.; Jaeschke, Hartmut

    2013-01-01

    At therapeutic doses, acetaminophen (APAP) is a safe and effective analgesic. However, overdose of APAP is the principal cause of acute liver failure in the West. Binding of the reactive metabolite of APAP (NAPQI) to proteins is thought to be the initiating event in the mechanism of hepatotoxicity. Early work suggested that APAP-protein binding could not occur without glutathione (GSH) depletion, and likely only at toxic doses. Moreover, it was found that protein-derived APAP-cysteine could only be detected in serum after the onset of liver injury. On this basis, it was recently proposed that serum APAP-cysteine could be used as diagnostic marker of APAP overdose. However, comprehensive dose–response and time course studies have not yet been done. Furthermore, the effects of co-morbidities on this parameter have not been investigated. We treated groups of mice with APAP at multiple doses and measured liver GSH and both liver and plasma APAP-protein adducts at various timepoints. Our results show that protein binding can occur without much loss of GSH. Importantly, the data confirm earlier work that showed that protein-derived APAP-cysteine can appear in plasma without liver injury. Experiments performed in vitro suggest that this may involve multiple mechanisms, including secretion of adducted proteins and diffusion of NAPQI directly into plasma. Induction of liver necrosis through ischemia–reperfusion significantly increased the plasma concentration of protein-derived APAP-cysteine after a subtoxic dose of APAP. While our data generally support the measurement of serum APAP-protein adducts in the clinic, caution is suggested in the interpretation of this parameter. - Highlights: • Extensive GSH depletion is not required for APAP-protein binding in the liver. • APAP-protein adducts appear in plasma at subtoxic doses. • Proteins are adducted in the cell and secreted out. • Coincidental liver injury increases plasma APAP-protein adducts at subtoxic doses

  14. PLASMA PROTEIN AND HEMOGLOBIN PRODUCTION : DELETION OF INDIVIDUAL AMINO ACIDS FROM GROWTH MIXTURE OF TEN ESSENTIAL AMINO ACIDS. SIGNIFICANT CHANGES IN URINARY NITROGEN.

    Science.gov (United States)

    Robscheit-Robbins, F S; Miller, L L; Whipple, G H

    1947-02-28

    Given healthy dogs fed abundant iron and protein-free or low protein diets with sustained anemia and hypoproteinemia, we can study the capacity of these animals to produce simultaneously new hemoglobin and plasma protein. Reserve stores of blood protein-building materials are measurably depleted and levels of 6 to 8 gm. per cent for hemoglobin and 4 to 5 gm. per cent for plasma protein can be maintained for weeks or months depending upon the intake of food proteins or amino acid mixtures. These dogs are very susceptible to infection and various poisons. Dogs tire of these diets and loss of appetite terminates many experiments. Under these conditions (double depletion) standard growth mixtures of essential amino acids are tested to show the response in blood protein output and urinary nitrogen balance. As a part of each tabulated experiment one of the essential amino acids is deleted from the complete growth mixture to compare such response with that of the whole mixture. Methionine, threonine, phenylalanine, and tryptophane when singly eliminated from the complete amino acid mixture do effect a sharp rise in urinary nitrogen. This loss of urinary nitrogen is corrected when the individual amino acid is replaced in the mixture. Histidine, lysine, and valine have a moderate influence upon urinary nitrogen balance toward nitrogen conservation. Leucine, isoleucine, and arginine have minimal or no effect upon urinary nitrogen balance when these individual amino acids are deleted from the complete growth mixture of amino acids during 3 to 4 week periods. Tryptophane and to a less extent phenylalanine and threonine when returned to the amino acid mixture are associated with a conspicuous preponderance of plasma protein output over the hemoglobin output (Table 4). Arginine, lysine, and histidine when returned to the amino acid mixture are associated with a large preponderance of hemoglobin output. Various amino acid mixtures under these conditions may give a positive

  15. Deficiency in plasma protein synthesis caused by x-ray-induced lethal albino alleles in mouse

    International Nuclear Information System (INIS)

    Garland, R.C.; Satrustegui, J.; Gluecksohn-Waelsch, S.; Cori, C.F.

    1976-01-01

    Plasma protein synthesis was studied in mice bearing x-ray induced lethal mutations at the albino locus. Newborn albino mutants showed a decrease in each of the three principal plasma proteins, albumin, α-fetoprotein, and transferrin, when compared with colored littermate controls. Incorporation of [ 14 C] leucine into plasma proteins of the newborn albinos 30 min after injection was only 1 / 5 that of the controls, but incorporation into total liver protein was only slightly diminished. Incorporation of [ 14 C] leucine into an albumin fraction obtained by immunoprecipitation from livers incubated in vitro in an amino acid mixture was also strongly diminished. Thus, the liver of 18-day-old albino fetuses incorporated into this fraction 1 / 3 and that of newborn albinos 1 / 8 as much as the controls, but in both cases the incorporation into total liver protein was only 25 percent less than in the respective controls. These results indicate that the rather severe structural abnormalities observed in the mutants in the endoplasmic reticulum and the Golgi apparatus are not associated with a general deficiency of hepatic protein synthesis. Instead the data from this and previous work show that the progressive deficiency from fetal life to birth involves certain specific proteins represented by several perinatally developing enzymes and by plasma proteins. It is suggested that the mutational effects observed in these mice are due to deletions involving regulatory rather than structural genes at or near the albino locus

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

    International Nuclear Information System (INIS)

    Zhang, Zhiyu; Ma, Fang; Cai, Zhengdong; Zhang, Lijun; Hua, Yingqi; Jia, Xiaofang; Li, Jian; Hu, Shuo; Peng, Xia; Yang, Pengyuan; Sun, Mengxiong

    2010-01-01

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

  17. Study on the interaction between active components from traditional Chinese medicine and plasma proteins.

    Science.gov (United States)

    Jiao, Qishu; Wang, Rufeng; Jiang, Yanyan; Liu, Bin

    2018-05-04

    Traditional Chinese medicine (TCM), as a unique form of natural medicine, has been used in Chinese traditional therapeutic systems over two thousand years. Active components in Chinese herbal medicine are the material basis for the prevention and treatment of diseases. Research on drug-protein binding is one of the important contents in the study of early stage clinical pharmacokinetics of drugs. Plasma protein binding study has far-reaching influence on the pharmacokinetics and pharmacodynamics of drugs and helps to understand the basic rule of drug effects. It is important to study the binding characteristics of the active components in Chinese herbal medicine with plasma proteins for the medical science and modernization of TCM. This review summarizes the common analytical methods which are used to study the active herbal components-protein binding and gives the examples to illustrate their application. Rules and influence factors of the binding between different types of active herbal components and plasma proteins are summarized in the end. Finally, a suggestion on choosing the suitable technique for different types of active herbal components is provided, and the prospect of the drug-protein binding used in the area of TCM research is also discussed.

  18. Endogenous Plasma Peptide Detection and Identification in the Rat by a Combination of Fractionation Methods and Mass Spectrometry

    Directory of Open Access Journals (Sweden)

    Fabrice Bertile

    2007-01-01

    Full Text Available Mass spectrometry-based analyses are essential tools in the field of biomarker research. However, detection and characterization of plasma low abundance and/or low molecular weight peptides is challenged by the presence of highly abundant proteins, salts and lipids. Numerous strategies have already been tested to reduce the complexity of plasma samples. The aim of this study was to enrich the low molecular weight fraction of rat plasma. To this end, we developed and compared simple protocols based on membrane filtration, solid phase extraction, and a combination of both. As assessed by UV absorbance, an albumin depletion 99% was obtained. The multistep fractionation strategy (including reverse phase HPLC allowed detection, in a reproducible manner (CV [1] 30%–35%, of more than 450 peaks below 3000 Da by MALDI-TOF/MS. A MALDI-TOF/MS-determined LOD as low as 1 fmol/μL was obtained, thus allowing nanoLC-Chip/ MS/MS identification of spiked peptides representing ∼10–6% of total proteins, by weight. Signal peptide recovery ranged between 5%–100% according to the spiked peptide considered. Tens of peptide sequence tags from endogenous plasma peptides were also obtained and high confidence identifications of low abundance fibrinopeptide A and B are reported here to show the efficiency of the protocol. It is concluded that the fractionation protocol presented would be of particular interest for future differential (high throughput analyses of the plasma low molecular weight fraction.

  19. Systematic study of plasma and serum proteins in the pig

    International Nuclear Information System (INIS)

    Daburon, F.; Nizza, P.; Hatchikian, C.; Schmidt, J.-P.

    1966-01-01

    This work has been carried out in the framework of the determination of the physiological constants of a normal pig. The aim was to study the serum and plasma proteins of this animal species, the ultimate object being to discover whether the qualitative and quantitative changes in these proteins can make a significant contribution to the establishment of a biological dosimetry for irradiated pigs. The serum and plasma from a normal pig were analyzed first by various simple electrophoretic methods and then by immuno-electrophoresis. As a result of the particular characteristics of pig serum we have gradually been led to make numerous modifications to the techniques used for human serums or for those of small laboratory animals. Much careful work and patience were required in order to obtain reproducible results. (authors) [fr

  20. Plasma Proteome Biomarkers of Inflammation in School Aged Children in Nepal.

    Directory of Open Access Journals (Sweden)

    Sun Eun Lee

    Full Text Available Inflammation is a condition stemming from complex host defense and tissue repair mechanisms, often simply characterized by plasma levels of a single acute reactant. We attempted to identify candidate biomarkers of systemic inflammation within the plasma proteome. We applied quantitative proteomics using isobaric mass tags (iTRAQ tandem mass spectrometry to quantify proteins in plasma of 500 Nepalese children 6-8 years of age. We evaluated those that co-vary with inflammation, indexed by α-1-acid glycoprotein (AGP, a conventional biomarker of inflammation in population studies. Among 982 proteins quantified in >10% of samples, 99 were strongly associated with AGP at a family-wise error rate of 0.1%. Magnitude and significance of association varied more among proteins positively (n = 41 than negatively associated (n = 58 with AGP. The former included known positive acute phase proteins including C-reactive protein, serum amyloid A, complement components, protease inhibitors, transport proteins with anti-oxidative activity, and numerous unexpected intracellular signaling molecules. Negatively associated proteins exhibited distinct differences in abundance between secretory hepatic proteins involved in transporting or binding lipids, micronutrients (vitamin A and calcium, growth factors and sex hormones, and proteins of largely extra-hepatic origin involved in the formation and metabolic regulation of extracellular matrix. With the same analytical approach and the significance threshold, seventy-two out of the 99 proteins were commonly associated with CRP, an established biomarker of inflammation, suggesting the validity of the identified proteins. Our findings have revealed a vast plasma proteome within a free-living population of children that comprise functional biomarkers of homeostatic and induced host defense, nutrient metabolism and tissue repair, representing a set of plasma proteins that may be used to assess dynamics and extent of

  1. The molecular mechanisms of plant plasma membrane intrinsic proteins trafficking and stress response.

    Science.gov (United States)

    Wang, Xing; Zhang, Ji-long; Feng, Xiu-xiu; Li, Hong-jie; Zhang, Gen-fa

    2017-04-20

    Plasma membrane intrinsic proteins (PIPs) are plant channel proteins located on the plasma membrane. PIPs transfer water, CO 2 and small uncharged solutes through the plasma membrane. PIPs have high selectivity to substrates, suggestive of a central role in maintaining cellular water balance. The expression, activity and localization of PIPs are regulated at the transcriptional and post-translational levels, and also affected by environmental factors. Numerous studies indicate that the expression patterns and localizations of PIPs can change in response to abiotic stresses. In this review, we summarize the mechanisms of PIP trafficking, transcriptional and post-translational regulations, and abiotic stress responses. Moreover, we also discuss the current research trends and future directions on PIPs.

  2. Free-Flow Electrophoresis of Plasma Membrane Vesicles Enriched by Two-Phase Partitioning Enhances the Quality of the Proteome from Arabidopsis Seedlings.

    Science.gov (United States)

    de Michele, Roberto; McFarlane, Heather E; Parsons, Harriet T; Meents, Miranda J; Lao, Jeemeng; González Fernández-Niño, Susana M; Petzold, Christopher J; Frommer, Wolf B; Samuels, A Lacey; Heazlewood, Joshua L

    2016-03-04

    The plant plasma membrane is the interface between the cell and its environment undertaking a range of important functions related to transport, signaling, cell wall biosynthesis, and secretion. Multiple proteomic studies have attempted to capture the diversity of proteins in the plasma membrane using biochemical fractionation techniques. In this study, two-phase partitioning was combined with free-flow electrophoresis to produce a population of highly purified plasma membrane vesicles that were subsequently characterized by tandem mass spectroscopy. This combined high-quality plasma membrane isolation technique produced a reproducible proteomic library of over 1000 proteins with an extended dynamic range including plasma membrane-associated proteins. The approach enabled the detection of a number of putative plasma membrane proteins not previously identified by other studies, including peripheral membrane proteins. Utilizing multiple data sources, we developed a PM-confidence score to provide a value indicating association to the plasma membrane. This study highlights over 700 proteins that, while seemingly abundant at the plasma membrane, are mostly unstudied. To validate this data set, we selected 14 candidates and transiently localized 13 to the plasma membrane using a fluorescent tag. Given the importance of the plasma membrane, this data set provides a valuable tool to further investigate important proteins. The mass spectrometry data are available via ProteomeXchange, identifier PXD001795.

  3. Easy measurement of diffusion coefficients of EGFP-tagged plasma membrane proteins using k-space Image Correlation Spectroscopy

    DEFF Research Database (Denmark)

    Christensen, Eva Arnspang; Koffman, Jennifer Skaarup; Marlar, Saw

    2014-01-01

    Lateral diffusion and compartmentalization of plasma membrane proteins are tightly regulated in cells and thus, studying these processes will reveal new insights to plasma membrane protein function and regulation. Recently, k-Space Image Correlation Spectroscopy (kICS)1 was developed to enable...... routine measurements of diffusion coefficients directly from images of fluorescently tagged plasma membrane proteins, that avoided systematic biases introduced by probe photophysics. Although the theoretical basis for the analysis is complex, the method can be implemented by nonexperts using a freely...... to the correlation function yields the diffusion coefficient. This paper provides a step-by-step guide to the image analysis and measurement of diffusion coefficients via kICS. First, a high frame rate image sequence of a fluorescently labeled plasma membrane protein is acquired using a fluorescence microscope Then...

  4. Proteomic analysis of a pleistocene mammoth femur reveals more than one hundred ancient bone proteins

    DEFF Research Database (Denmark)

    Cappellini, Enrico; Jensen, Lars Juhl; Szklarczyk, Damian Milosz

    2012-01-01

    We used high-sensitivity, high-resolution tandem mass spectrometry to shotgun sequence ancient protein remains extracted from a 43 000 year old woolly mammoth (Mammuthus primigenius) bone preserved in the Siberian permafrost. For the first time, 126 unique protein accessions, mostly low-abundance......We used high-sensitivity, high-resolution tandem mass spectrometry to shotgun sequence ancient protein remains extracted from a 43 000 year old woolly mammoth (Mammuthus primigenius) bone preserved in the Siberian permafrost. For the first time, 126 unique protein accessions, mostly low......-abundance extracellular matrix and plasma proteins, were confidently identified by solid molecular evidence. Among the best characterized was the carrier protein serum albumin, presenting two single amino acid substitutions compared to extant African (Loxodonta africana) and Indian (Elephas maximus) elephants. Strong...

  5. Affinity proteomics reveals elevated muscle proteins in plasma of children with cerebral malaria.

    Directory of Open Access Journals (Sweden)

    Julie Bachmann

    2014-04-01

    Full Text Available Systemic inflammation and sequestration of parasitized erythrocytes are central processes in the pathophysiology of severe Plasmodium falciparum childhood malaria. However, it is still not understood why some children are more at risks to develop malaria complications than others. To identify human proteins in plasma related to childhood malaria syndromes, multiplex antibody suspension bead arrays were employed. Out of the 1,015 proteins analyzed in plasma from more than 700 children, 41 differed between malaria infected children and community controls, whereas 13 discriminated uncomplicated malaria from severe malaria syndromes. Markers of oxidative stress were found related to severe malaria anemia while markers of endothelial activation, platelet adhesion and muscular damage were identified in relation to children with cerebral malaria. These findings suggest the presence of generalized vascular inflammation, vascular wall modulations, activation of endothelium and unbalanced glucose metabolism in severe malaria. The increased levels of specific muscle proteins in plasma implicate potential muscle damage and microvasculature lesions during the course of cerebral malaria.

  6. 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

    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

  7. 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. Copyright © 2015. Published by Elsevier B.V.

  8. PARTIAL PURIFICATION AND IMMUNO-BIOCHEMICAL CHARACTERISATION OF FERTILITY ASSOCIATED PROTEIN OF KARAN FRIES BULL SEMINAL PLASMA

    Directory of Open Access Journals (Sweden)

    Brajesh Raman

    2014-06-01

    Full Text Available The objective of the present study was detection, isolation, partial purification and immunobiochemical characterization of fertility associated protein in the seminal plasma of high prolific Karan fries bull. Seminal plasma of Karan Fries bull was partially purified by gel filtration chromatography and analyzed by 10% SDS-PAGE for their polypeptide profile. PAGE analysis revealed major band of 55 kDa, and 26 kDa. Hyperimmune serum was raised in rabbit against crude seminal plasma protein. Single precipitin line was observed in DID test when each of the partially purified 26 kDa and 55 kDa proteins were reacted with hyperimmune serum. These proteins were also found to be immunoreactive against hyperimmune serum in Western blot technique.

  9. Caveolae as plasma membrane sensors, protectors and organizers.

    Science.gov (United States)

    Parton, Robert G; del Pozo, Miguel A

    2013-02-01

    Caveolae are submicroscopic, plasma membrane pits that are abundant in many mammalian cell types. The past few years have seen a quantum leap in our understanding of the formation, dynamics and functions of these enigmatic structures. Caveolae have now emerged as vital plasma membrane sensors that can respond to plasma membrane stresses and remodel the extracellular environment. Caveolae at the plasma membrane can be removed by endocytosis to regulate their surface density or can be disassembled and their structural components degraded. Coat proteins, called cavins, work together with caveolins to regulate the formation of caveolae but also have the potential to dynamically transmit signals that originate in caveolae to various cellular destinations. The importance of caveolae as protective elements in the plasma membrane, and as membrane organizers and sensors, is highlighted by links between caveolae dysfunction and human diseases, including muscular dystrophies and cancer.

  10. Integration of multi-omics data of a genome-reduced bacterium: Prevalence of post-transcriptional regulation and its correlation with protein abundances

    Science.gov (United States)

    Chen, Wei-Hua; van Noort, Vera; Lluch-Senar, Maria; Hennrich, Marco L.; H. Wodke, Judith A.; Yus, Eva; Alibés, Andreu; Roma, Guglielmo; Mende, Daniel R.; Pesavento, Christina; Typas, Athanasios; Gavin, Anne-Claude; Serrano, Luis; Bork, Peer

    2016-01-01

    We developed a comprehensive resource for the genome-reduced bacterium Mycoplasma pneumoniae comprising 1748 consistently generated ‘-omics’ data sets, and used it to quantify the power of antisense non-coding RNAs (ncRNAs), lysine acetylation, and protein phosphorylation in predicting protein abundance (11%, 24% and 8%, respectively). These factors taken together are four times more predictive of the proteome abundance than of mRNA abundance. In bacteria, post-translational modifications (PTMs) and ncRNA transcription were both found to increase with decreasing genomic GC-content and genome size. Thus, the evolutionary forces constraining genome size and GC-content modify the relative contributions of the different regulatory layers to proteome homeostasis, and impact more genomic and genetic features than previously appreciated. Indeed, these scaling principles will enable us to develop more informed approaches when engineering minimal synthetic genomes. PMID:26773059

  11. The 14-3-3 protein interacts directly with the C-terminal region of the plant plasma membrane H(+)-ATPase

    DEFF Research Database (Denmark)

    Jahn, T.; Fuglsang, A.T.; Olsson, A.

    1997-01-01

    Accumulating evidence suggests that 14-3-3 proteins are involved in the regulation of plant plasma membrane H(+)-ATPase activity. However, it is not known whether the 14-3-3 protein interacts directly or indirectly with the H(+)-ATPase. In this study, detergent-solubilized plasma membrane H...... plasma membrane H(+)-ATPase. We propose that the 14-3-3 protein is a natural ligand of the plasma membrane H(+)-ATPase, regulating proton pumping by displacing the C-terminal autoinhibitory domain of the H(+)-ATPase....

  12. Resolving mixed mechanisms of protein subdiffusion at the T cell plasma membrane

    Science.gov (United States)

    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.

  13. Proteomic analysis reveals differential accumulation of small heat shock proteins and late embryogenesis abundant proteins between ABA-deficient mutant vp5 seeds and wild-type Vp5 seeds in maize

    Directory of Open Access Journals (Sweden)

    Xiaolin eWu

    2015-01-01

    Full Text Available ABA is a major plant hormone that plays important roles during many phases of plant life cycle, including seed development, maturity and dormancy, and especially the acquisition of desiccation tolerance. Understanding of the molecular basis of ABA-mediated plant response to stress is of interest not only in basic research on plant adaptation but also in applied research on plant productivity. Maize mutant viviparous-5 (vp5, deficient in ABA biosynthesis in seeds, is a useful material for studying ABA-mediated response in maize. Due to carotenoid deficiency, vp5 endosperm is white, compared to yellow Vp5 endosperm. However, the background difference at proteome level between vp5 and Vp5 seeds is unclear. This study aimed to characterize proteome alterations of maize vp5 seeds and to identify ABA-dependent proteins during seed maturation. We compared the embryo and endosperm proteomes of vp5 and Vp5 seeds by gel-based proteomics. Up to 46 protein spots, most in embryos, were found to be differentially accumulated between vp5 and Vp5. The identified proteins included small heat shock proteins (sHSPs, late embryogenesis abundant (LEA proteins, stress proteins, storage proteins and enzymes among others. However, EMB564, the most abundant LEA protein in maize embryo, accumulated in comparable levels between vp5 and Vp5 embryos, which contrasted to previously characterized, greatly lowered expression of emb564 mRNA in vp5 embryos. Moreover, LEA proteins and sHSPs displayed differential accumulations in vp5 embryos: six out of eight identified LEA proteins decreased while nine sHSPs increased in abundance. Finally, we discussed the possible causes of global proteome alterations, especially the observed differential accumulation of identified LEA proteins and sHSPs in vp5 embryos. The data derived from this study provides new insight into ABA-dependent proteins and ABA-mediated response during maize seed maturation.

  14. Novel platelet-agglutinating protein from a thrombotic thrombocytopenic purpura plasma.

    OpenAIRE

    Siddiqui, F A; Lian, E C

    1985-01-01

    A novel platelet-agglutinating protein (PAP) was purified approximately 2,000-fold from the plasma of a patient with thrombotic thrombocytopenic purpura (TTP) by ammonium sulfate fractionation, DEAE-Sephacel and concanavalin A-Sepharose chromatographies. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with and without reduction, this preparation revealed a major protein band with a molecular weight of 37,000, and a minor band with a molecular weight of 32,000-34,000. After eluti...

  15. Reticulomics: Protein-Protein Interaction Studies with Two Plasmodesmata-Localized Reticulon Family Proteins Identify Binding Partners Enriched at Plasmodesmata, Endoplasmic Reticulum, and the Plasma Membrane.

    Science.gov (United States)

    Kriechbaumer, Verena; Botchway, Stanley W; Slade, Susan E; Knox, Kirsten; Frigerio, Lorenzo; Oparka, Karl; Hawes, Chris

    2015-11-01

    The endoplasmic reticulum (ER) is a ubiquitous organelle that plays roles in secretory protein production, folding, quality control, and lipid biosynthesis. The cortical ER in plants is pleomorphic and structured as a tubular network capable of morphing into flat cisternae, mainly at three-way junctions, and back to tubules. Plant reticulon family proteins (RTNLB) tubulate the ER by dimerization and oligomerization, creating localized ER membrane tensions that result in membrane curvature. Some RTNLB ER-shaping proteins are present in the plasmodesmata (PD) proteome and may contribute to the formation of the desmotubule, the axial ER-derived structure that traverses primary PD. Here, we investigate the binding partners of two PD-resident reticulon proteins, RTNLB3 and RTNLB6, that are located in primary PD at cytokinesis in tobacco (Nicotiana tabacum). Coimmunoprecipitation of green fluorescent protein-tagged RTNLB3 and RTNLB6 followed by mass spectrometry detected a high percentage of known PD-localized proteins as well as plasma membrane proteins with putative membrane-anchoring roles. Förster resonance energy transfer by fluorescence lifetime imaging microscopy assays revealed a highly significant interaction of the detected PD proteins with the bait RTNLB proteins. Our data suggest that RTNLB proteins, in addition to a role in ER modeling, may play important roles in linking the cortical ER to the plasma membrane. © 2015 American Society of Plant Biologists. All Rights Reserved.

  16. 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

  17. Association between pregnancy-associated plasma protein-A levels in the first trimester and gestational diabetes mellitus in Chinese women.

    Science.gov (United States)

    Cheuk, Q Ky; Lo, T K; Wong, S F; Lee, C P

    2016-02-01

    Several studies have shown that women with pre-existing diabetes mellitus have significantly lower pregnancy-associated plasma protein-A levels than those without. This study aimed to evaluate whether first-trimester pregnancy-associated plasma protein-A multiple of median is associated with gestational diabetes mellitus in Chinese pregnant women. This prospectively collected case series was conducted in a regional hospital in Hong Kong. All consecutive Chinese women with a singleton pregnancy who attended the hospital for their first antenatal visit (before 14 weeks' gestation) from April to July 2014 were included. Pregnancy-associated plasma protein-A multiple of median was compared between the gestational diabetic (especially for early-onset gestational diabetes) and non-diabetic groups. The correlation between pregnancy-associated plasma protein-A level and glycosylated haemoglobin level in women with gestational diabetes was also examined. Of the 520 women recruited, gestational diabetes was diagnosed in 169 (32.5%). Among them, 43 (25.4%) had an early diagnosis, and 167 (98.8%) with the disease were managed by diet alone. The gestational diabetic group did not differ significantly to the non-diabetic group in pregnancy-associated plasma protein-A (0.97 vs 0.99, P=0.40) or free β-human chorionic gonadotrophin multiple of median (1.05 vs 1.02, P=0.29). Compared with the non-gestational diabetic group, women with early diagnosis of gestational diabetes had a non-significant reduction in pregnancy-associated plasma protein-A multiple of median (median, interquartile range: 0.86, 0.57-1.23 vs 0.99, 0.67-1.44; P=0.11). Pregnancy-associated plasma protein-A and glycosylated haemoglobin levels were not correlated in women with gestational diabetes (r=0.027; P=0.74). Chinese women with non-insulin-dependent gestational diabetes did not exhibit significant changes to pregnancy-associated plasma protein-A multiple of median nor a correlation between pregnancy

  18. Impact of an ionic liquid on protein thermodynamics in the presence of cold atmospheric plasma and gamma rays.

    Science.gov (United States)

    Attri, Pankaj; Kim, Minsup; Choi, Eun Ha; Cho, Art E; Koga, Kazunori; Shiratani, Masaharu

    2017-09-27

    Cold atmospheric plasma and gamma rays are known to have anticancer properties, even though their specific mechanisms and roles as co-solvents during their action are still not clearly understood. Despite the use of gamma rays in cancer therapy, they have oncogenic potential, whereas this has not been observed for plasma treatment (to date). To gain a better understanding, we studied the action of dielectric barrier discharge (DBD) plasma and gamma rays on the myoglobin protein. We analyzed the secondary structure and thermodynamic properties of myoglobin after both treatments. In addition, in the last few years, ammonium ionic liquids (ILs) have revealed their important role in protein folding as co-solvents. In this work, we treated the protein with ammonium ILs such as triethylammonium methanesulfonate (TEMS) and tetrabutylammonium methanesulfonate (TBMS) and later treated this IL-protein solution with DBD plasma and gamma rays. In this study, we show the chemical and thermal denaturation of the protein after plasma and gamma treatments in the presence and absence of ILs using circular dichroism (CD) and UV-vis spectroscopy. Furthermore, we also show the influence of plasma and gamma rays on the secondary structure of myoglobin in the absence and presence of ILs or ILs + urea using CD. Finally, molecular dynamic simulations were conducted to gain deeper insight into how the ILs behave to protect the protein against the hydrogen peroxide generated by the DBD plasma and gamma rays.

  19. Application of epifluorescence scanning for monitoring the efficacy of protein removal by RF gas-plasma decontamination

    Energy Technology Data Exchange (ETDEWEB)

    Baxter, Helen C; Richardson, Patricia R; Campbell, Gaynor A; Jones, Anita C; Baxter, Robert L [School of Chemistry, Joseph Black Chemistry Building, University of Edinburgh, West Mains Road, Edinburgh EH9 3JJ (United Kingdom); Kovalev, Valeri I; Maier, Robert; Barton, James S [School of Engineering and Physical Science, Heriot-Watt University, Edinburgh EH14 4AS (United Kingdom); DeLarge, Greg [Plasma Etch Inc, 3522 Arrowhead Drive, Carson City, NV 89706 (United States); Casey, Mark [Sterile Services Department, Royal Infirmary of Edinburgh, Edinburgh EH16 4AS (United Kingdom)], E-mail: r.baxter@ed.ac.uk

    2009-11-15

    The development of methods for measuring the efficiency of gas-plasma decontamination has lagged far behind application. An approach to measuring the efficiency of protein removal from solid surfaces using fluorescein-labelled bovine serum albumin and epifluorescence scanning (EFSCAN) is described. A method for fluorescently labelling proteins, which are adsorbed and denatured on metal surfaces, has been developed. Both approaches have been used to evaluate the efficiency of radio frequency (RF) gas-plasma decontamination protocols. Examples with 'real' surgical instruments demonstrate that an argon-oxygen RF gas-plasma treatment can routinely reduce the protein load by about three orders of magnitude beyond that achieved by current decontamination methods.

  20. Application of epifluorescence scanning for monitoring the efficacy of protein removal by RF gas-plasma decontamination

    International Nuclear Information System (INIS)

    Baxter, Helen C; Richardson, Patricia R; Campbell, Gaynor A; Jones, Anita C; Baxter, Robert L; Kovalev, Valeri I; Maier, Robert; Barton, James S; DeLarge, Greg; Casey, Mark

    2009-01-01

    The development of methods for measuring the efficiency of gas-plasma decontamination has lagged far behind application. An approach to measuring the efficiency of protein removal from solid surfaces using fluorescein-labelled bovine serum albumin and epifluorescence scanning (EFSCAN) is described. A method for fluorescently labelling proteins, which are adsorbed and denatured on metal surfaces, has been developed. Both approaches have been used to evaluate the efficiency of radio frequency (RF) gas-plasma decontamination protocols. Examples with 'real' surgical instruments demonstrate that an argon-oxygen RF gas-plasma treatment can routinely reduce the protein load by about three orders of magnitude beyond that achieved by current decontamination methods.

  1. Aggregated forms of bull seminal plasma proteins and their heparin-binding activity

    Czech Academy of Sciences Publication Activity Database

    Jelínková, Petra; Ryšlavá, H.; Liberda, J.; Jonáková, Věra; Tichá, M.

    2004-01-01

    Roč. 69, - (2004), s. 616-630 ISSN 0010-0765 R&D Projects: GA ČR GA303/02/0433; GA ČR GP303/02/P069; GA MZd NJ7463 Institutional research plan: CEZ:AV0Z5052915; CEZ:MSM 113100001 Keywords : bull seminal plasma proteins * heparin-binding proteins * aggregated forms of proteins Subject RIV: CE - Biochemistry Impact factor: 1.062, year: 2004

  2. Pregnancy-associated plasma protein-A, a marker for outcome in patients suspected for acute coronary syndrome

    DEFF Research Database (Denmark)

    Iversen, Kasper K; Dalsgaard, Morten; Teisner, Ane S

    2010-01-01

    To examine if pregnancy-associated plasma protein-A (PAPP-A) in patients with chest pain, could identify patients at risk for death or myocardial infarction.......To examine if pregnancy-associated plasma protein-A (PAPP-A) in patients with chest pain, could identify patients at risk for death or myocardial infarction....

  3. Plant glycosylphosphatidylinositol (GPI) anchored proteins at the plasma membrane-cell wall nexus.

    Science.gov (United States)

    Yeats, Trevor H; Bacic, Antony; Johnson, Kim L

    2018-04-18

    Approximately 1% of plant proteins are predicted to be post-translationally modified with a glycosylphosphatidylinositol (GPI) anchor that tethers the polypeptide to the outer leaflet of the plasma membrane. While the synthesis and structure of GPI anchors is largely conserved across eukaryotes, the repertoire of functional domains present in the GPI-anchored proteome has diverged substantially. In plants, this includes a large fraction of the GPI-anchored proteome being further modified with plant-specific arabinogalactan (AG) O-glycans. The importance of the GPI-anchored proteome to plant development is underscored by the fact that GPI biosynthetic null mutants exhibit embryo lethality. Mutations in genes encoding specific GPI-anchored proteins (GAPs) further supports their contribution to diverse biological processes occurring at the interface of the plasma membrane and cell wall, including signaling, cell wall metabolism, cell wall polymer cross-linking, and plasmodesmatal transport. Here, we review the literature concerning plant GPI-anchored proteins in the context of their potential to act as molecular hubs that mediate interactions between the plasma membrane and the cell wall and their potential to transduce the signal into the protoplast and thereby activate signal transduction pathways. This article is protected by copyright. All rights reserved.

  4. Development and validation of a high-performance liquid chromatography method for the quantification of talazoparib in rat plasma: Application to plasma protein binding studies.

    Science.gov (United States)

    Hidau, Mahendra Kumar; Kolluru, Srikanth; Palakurthi, Srinath

    2018-02-01

    A sensitive and selective RP-HPLC method has been developed and validated for the quantification of a highly potent poly ADP ribose polymerase inhibitor talazoparib (TZP) in rat plasma. Chromatographic separation was performed with isocratic elution method. Absorbance for TZP was measured with a UV detector (SPD-20A UV-vis) at a λ max of 227 nm. Protein precipitation was used to extract the drug from plasma samples using methanol-acetonitrile (65:35) as the precipitating solvent. The method proved to be sensitive and reproducible over a 100-2000 ng/mL linearity range with a lower limit of quantification (LLQC) of 100 ng/mL. TZP recovery was found to be >85%. Following analytical method development and validation, it was successfully employed to determine the plasma protein binding of TZP. TZP has a high level of protein binding in rat plasma (95.76 ± 0.38%) as determined by dialysis method. Copyright © 2017 John Wiley & Sons, Ltd.

  5. Intake of Meat Proteins Substantially Increased the Relative Abundance of Genus Lactobacillus in Rat Feces

    Science.gov (United States)

    Zhu, Yingying; Lin, Xisha; Li, He; Li, Yingqiu; Shi, Xuebin; Zhao, Fan; Xu, Xinglian; Li, Chunbao; Zhou, Guanghong

    2016-01-01

    Diet has been shown to have a critical influence on gut bacteria and host health, and high levels of red meat in diet have been shown to increase colonic DNA damage and thus be harmful to gut health. However, previous studies focused more on the effects of meat than of meat proteins. In order to investigate whether intake of meat proteins affects the composition and metabolic activities of gut microbiota, feces were collected from growing rats that were fed with either meat proteins (from beef, pork or fish) or non-meat proteins (casein or soy) for 14 days. The resulting composition of gut microbiota was profiled by sequencing the V4-V5 region of the 16S ribosomal RNA genes and the short chain fatty acids (SCFAs) were analyzed using gas chromatography. The composition of gut microbiota and SCFA levels were significantly different between the five diet groups. At a recommended dose of 20% protein in the diet, meat protein-fed rats had a higher relative abundance of the beneficial genus Lactobacillus, but lower levels of SCFAs and SCFA-producing bacteria including Fusobacterium, Bacteroides and Prevotella, compared with the soy protein-fed group. Further work is needed on the regulatory pathways linking dietary protein intake to gut microbiota. PMID:27042829

  6. Intake of Meat Proteins Substantially Increased the Relative Abundance of Genus Lactobacillus in Rat Feces.

    Directory of Open Access Journals (Sweden)

    Yingying Zhu

    Full Text Available Diet has been shown to have a critical influence on gut bacteria and host health, and high levels of red meat in diet have been shown to increase colonic DNA damage and thus be harmful to gut health. However, previous studies focused more on the effects of meat than of meat proteins. In order to investigate whether intake of meat proteins affects the composition and metabolic activities of gut microbiota, feces were collected from growing rats that were fed with either meat proteins (from beef, pork or fish or non-meat proteins (casein or soy for 14 days. The resulting composition of gut microbiota was profiled by sequencing the V4-V5 region of the 16S ribosomal RNA genes and the short chain fatty acids (SCFAs were analyzed using gas chromatography. The composition of gut microbiota and SCFA levels were significantly different between the five diet groups. At a recommended dose of 20% protein in the diet, meat protein-fed rats had a higher relative abundance of the beneficial genus Lactobacillus, but lower levels of SCFAs and SCFA-producing bacteria including Fusobacterium, Bacteroides and Prevotella, compared with the soy protein-fed group. Further work is needed on the regulatory pathways linking dietary protein intake to gut microbiota.

  7. Intake of Meat Proteins Substantially Increased the Relative Abundance of Genus Lactobacillus in Rat Feces.

    Science.gov (United States)

    Zhu, Yingying; Lin, Xisha; Li, He; Li, Yingqiu; Shi, Xuebin; Zhao, Fan; Xu, Xinglian; Li, Chunbao; Zhou, Guanghong

    2016-01-01

    Diet has been shown to have a critical influence on gut bacteria and host health, and high levels of red meat in diet have been shown to increase colonic DNA damage and thus be harmful to gut health. However, previous studies focused more on the effects of meat than of meat proteins. In order to investigate whether intake of meat proteins affects the composition and metabolic activities of gut microbiota, feces were collected from growing rats that were fed with either meat proteins (from beef, pork or fish) or non-meat proteins (casein or soy) for 14 days. The resulting composition of gut microbiota was profiled by sequencing the V4-V5 region of the 16S ribosomal RNA genes and the short chain fatty acids (SCFAs) were analyzed using gas chromatography. The composition of gut microbiota and SCFA levels were significantly different between the five diet groups. At a recommended dose of 20% protein in the diet, meat protein-fed rats had a higher relative abundance of the beneficial genus Lactobacillus, but lower levels of SCFAs and SCFA-producing bacteria including Fusobacterium, Bacteroides and Prevotella, compared with the soy protein-fed group. Further work is needed on the regulatory pathways linking dietary protein intake to gut microbiota.

  8. 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.

  9. Comparative analysis of Brassica napus plasma membrane proteins under phosphorus deficiency using label-free and MaxQuant-based proteomics approaches.

    Science.gov (United States)

    Chen, Shuisen; Luo, Ying; Ding, Guangda; Xu, Fangsen

    2016-02-05

    Phosphorus (P) deficiency is a primary constraint for plant growth in terrestrial ecosystems. To better understand the genotypic differences in the adaptation mechanism of Brassica napus to P deficiency, we purified the plasma membrane (PM) from the roots of two genotypes: P-efficient "Eyou Changjia" and P-inefficient "B104-2". Combining label-free quantitative proteomics with the MaxQuant approach, a total of 71 proteins that significantly changed in abundances were identified in the two genotypes in response to P-free starvation, including 31 in "Eyou Changjia" and 40 in "B104-2". Based on comparative genomics study, 28 proteins were mapped to the confidence intervals of quantitative trait loci (QTLs) for P efficiency related traits. Seven decreased proteins with transporter activity were found to be located in the PM by subcellular localization analyses. These proteins involved in intracellular protein transport and ATP hydrolysis coupled proton transport were mapped to the QTL for P content and dry weight. Compared with "B104-2", more decreased proteins referring to transporter activity were found in "Eyou Changjia", showing that substance exchange was decreased in response to short-term P-free starvation. Together with the finding, more decreased proteins functioning in signal transduction and protein synthesis/degradation suggested that "Eyou Changjia" could slow the progression of growth and save more P in response to short-term P-free starvation. P deficiency seriously limits the production and quality of B. napus. Roots absorb water and nutrients and anchor the plant in the soil. Therefore, to study root PM proteome under P stress would be helpful to understand the adaptation mechanism for P deficiency. However, PM proteome analysis in B. napus has been seldom reported due to the high hydrophobicity and low abundance of PM. Thus, we herein investigated the PM proteome alteration of roots in two B. napus genotypes, with different P deficient tolerances, in

  10. Experimental determination of net protein charge and A(tot) and K(a) of nonvolatile buffers in canine plasma.

    Science.gov (United States)

    Constable, Peter D; Stämpfli, Henry R

    2005-01-01

    Acid-base abnormalities frequently are present in sick dogs. The mechanism for an acid-base disturbance can be determined with the simplified strong ion approach, which requires accurate values for the total concentration of plasma nonvolatile buffers (A(tot)) and the effective dissociation constant for plasma weak acids (K(a)). The aims of this study were to experimentally determine A(tot) and K(a) values for canine plasma. Plasma was harvested from 10 healthy dogs; the concentrations of quantitatively important strong ions (Na+, K+, Ca2+, Mg2+, Cl-, L-lactate) and nonvolatile buffer ions (total protein, albumin, phosphate) were determined; and the plasma was tonometered with CO2 at 37 degrees C. Strong ion difference (SID) was calculated from the measured strong ion concentrations, and nonlinear regression was used to estimate values for A(tot) and K(a), which were validated with data from an in vitro and in vivo study. Mean (+/- SD) values for canine plasma were A(tot) = (17.4 +/- 8.6) mM (equivalent to 0.273 mmol/g of total protein or 0.469 mmol/g of albumin); K(a) = (0.17 +/- 0.11) x 10(-7); pK(a) = 7.77. The calculated SID for normal canine plasma (pH = 7.40; P(CO2) = 37 mm Hg; [total protein] = 64 g/L) was 27 mEq/L. The net protein charge for normal canine plasma was 0.25 mEq/g of total protein or 0.42 mEq/g of albumin. Application of the experimentally determined values for A(tot), K(a), and net protein charge should improve understanding of the mechanism for complex acid-base disturbances in dogs.

  11. Effect of Maillard browning reaction on protein utilization and plasma amino acid response by rainbow trout (Salmo gairdneri).

    Science.gov (United States)

    Plakas, S M; Lee, T C; Wolke, R E; Meade, T L

    1985-12-01

    The effect of the Maillard browning reaction in the diet of rainbow trout (Salmo gairdneri) on growth and amino acid availability was investigated. Chemical and enzymatic hydrolysis methods were applied for the detection of the losses of amino acids in a model protein browning system. Arginine and lysine exhibited the greatest losses in the mixture of fish protein isolate and glucose stored for 40 d at 37 degrees C. The apparent digestibility and absorption of individual amino acids, particularly lysine, was lower in trout fed browned protein than in those fed the control protein. Plasma lysine levels were significantly depressed, while the plasma levels of glucose and most other amino acids were elevated in relation to the loss in nutritive value of dietary protein after browning. The early Maillard reaction derivative of lysine, epsilon-deoxy-fructosyl-lysine, was recovered from browned protein (by using the in vitro enzymatic hydrolysis procedure) and from the plasma of trout fed browned protein. Analysis of plasma free amino acids provided an indication of lysine bioavailability and identified lysine as the first-limiting amino acid in the diets containing browned protein.

  12. Whey protein supplementation does not alter plasma branched-chained amino acid profiles but results in unique metabolomics patterns in obese women enrolled in an 8-week weight loss trial.

    Science.gov (United States)

    Piccolo, Brian D; Comerford, Kevin B; Karakas, Sidika E; Knotts, Trina A; Fiehn, Oliver; Adams, Sean H

    2015-04-01

    It has been suggested that perturbations in branched-chain amino acid (BCAA) catabolism are associated with insulin resistance and contribute to elevated systemic BCAAs. Evidence in rodents suggests dietary protein rich in BCAAs can increase BCAA catabolism, but there is limited evidence in humans. We hypothesize that a diet rich in BCAAs will increase BCAA catabolism, which will manifest in a reduction of fasting plasma BCAA concentrations. The metabolome of 27 obese women with metabolic syndrome before and after weight loss was investigated to identify changes in BCAA metabolism using GC-time-of-flight mass spectrometry. Subjects were enrolled in an 8-wk weight-loss study including either a 20-g/d whey (whey group, n = 16) or gelatin (gelatin group, n = 11) protein supplement. When matched for total protein by weight, whey protein has 3 times the amount of BCAAs compared with gelatin protein. Postintervention plasma abundances of Ile (gelatin group: 637 ± 18, quantifier ion peak height ÷ 100; whey group: 744 ± 65), Leu (gelatin group: 1210 ± 33; whey group: 1380 ± 79), and Val (gelatin group: 2080 ± 59; whey group: 2510 ± 230) did not differ between treatment groups. BCAAs were significantly correlated with homeostasis model assessment of insulin resistance at baseline (r = 0.52, 0.43, and 0.49 for Leu, Ile, and Val, respectively; all, P BCAA metabolism is, at best, only modestly affected at a whey protein supplementation dose of 20 g/d. Furthermore, the loss of an association between postintervention BCAA and homeostasis model assessment suggests that factors associated with calorie restriction or protein intake affect how plasma BCAAs relate to insulin sensitivity. This trial was registered at clinicaltrials.gov as NCT00739479. © 2015 American Society for Nutrition.

  13. Effect of electric charge on the transperitoneal transport of plasma proteins during CAPD

    NARCIS (Netherlands)

    Buis, B.; Koomen, G. C.; Imholz, A. L.; Struijk, D. G.; Reddingius, R. E.; Arisz, L.; Krediet, R. T.

    1996-01-01

    BACKGROUND: Controversy exists as to whether electric charges of plasma proteins influence their transport across the peritoneal membrane during CAPD. Fixed negative charges in the peritoneal membrane are diminished during peritonitis in rats. METHODS: Peritoneal clearances of 10 proteins and their

  14. Ovulation-inducing factor: a protein component of llama seminal plasma

    Directory of Open Access Journals (Sweden)

    Huanca Wilfredo

    2010-05-01

    Full Text Available Abstract Background Previously, we documented the presence of ovulation-inducing factor (OIF in the seminal plasma of llamas and alpacas. The purpose of the study was to define the biochemical characteristics of the molecule(s in seminal plasma responsible for inducing ovulation. Methods In Experiment 1, llama seminal plasma was centrifuged using filtration devices with nominal molecular mass cut-offs of 30, 10 and 5 kDa. Female llamas (n = 9 per group were treated i.m. with whole seminal plasma (positive control, phosphate-buffered saline (negative control, or the fraction of seminal plasma equal or higher than 30 kDa, 10 to 30 kDa, 5 to 10 kDa, or Results In Experiment 1, all llamas in the equal or higher than 30 kDa and positive control groups ovulated (9/9 in each, but none ovulated in the other groups (P Conclusions We conclude that ovulation-inducing factor (OIF in llama seminal plasma is a protein molecule that is resistant to heat and enzymatic digestion with proteinase K, and has a molecular mass of approximately equal or higher than 30 kDa.

  15. Fish protein hydrolysate elevates plasma bile acids and reduces visceral adipose tissue mass in rats

    DEFF Research Database (Denmark)

    Liaset, Bjørn; Madsen, Lise; Hao, Qin

    2009-01-01

    levels relative to rats fed soy protein or casein. Concomitantly, the saithe FPH fed rats had reduced liver lipids and fasting plasma TAG levels. Furthermore, visceral adipose tissue mass was reduced and expression of genes involved in fatty acid oxidation and energy expenditure was induced in perirenal....../retroperitoneal adipose tissues of rats fed saithe FPH. Our results provide the first evidence that dietary protein sources with different amino acid compositions can modulate the level of plasma bile acids and our data suggest potential novel mechanisms by which dietary protein sources can affect energy metabolism....

  16. Plasma Constraints on the Cosmological Abundance of Magnetic Monopoles and the Origin of Cosmic Magnetic Fields

    Science.gov (United States)

    Medvedev, Mikhail; Loeb, Abraham

    2017-10-01

    Existing theoretical and observational constraints on the abundance of magnetic monopoles are limited. Here we demonstrate that an ensemble of monopoles forms a plasma whose properties are well determined and whose collective effects place new tight constraints on the cosmological abundance of monopoles. In particular, the existence of micro-Gauss magnetic fields in galaxy clusters and radio relics implies that the scales of these structures are below the Debye screening length, thus setting an upper limit on the cosmological density parameter of monopoles, ΩM <= 3 ×10-4 , which precludes them from being the dark matter. Future detection of Gpc-scale coherent magnetic fields could improve this limit by a few orders of magnitude. In addition, we predict the existence of magnetic Langmuir waves and turbulence which may appear on the sky as ``zebra patterns'' of an alternating magnetic field with k . B ≠ 0 . We also show that magnetic monopole Langmuir turbulence excited near the accretion shock of galaxy clusters may be an efficient mechanism for generating the observed intracluster magnetic fields. The authors acknowledge DOE partial support via Grant DE-SC0016368.

  17. Plasma constraints on the cosmological abundance of magnetic monopoles and the origin of cosmic magnetic fields

    Energy Technology Data Exchange (ETDEWEB)

    Medvedev, Mikhail V.; Loeb, Abraham, E-mail: mmedvedev@cfa.harvard.edu, E-mail: aloeb@cfa.harvard.edu [Department of Astronomy, Harvard University, Cambridge, MA 02138 (United States)

    2017-06-01

    Existing theoretical and observational constraints on the abundance of magnetic monopoles are limited. Here we demonstrate that an ensemble of monopoles forms a plasma whose properties are well determined and whose collective effects place new tight constraints on the cosmological abundance of monopoles. In particular, the existence of micro-Gauss magnetic fields in galaxy clusters and radio relics implies that the scales of these structures are below the Debye screening length, thus setting an upper limit on the cosmological density parameter of monopoles, Ω {sub M} {sub ∼<} {sub 3} {sub ×} {sub 10}{sup −4}, which precludes them from being the dark matter. Future detection of Gpc-scale coherent magnetic fields could improve this limit by a few orders of magnitude. In addition, we predict the existence of magnetic Langmuir waves and turbulence which may appear on the sky as ''zebra patterns'' of an alternating magnetic field with k·B ≠ 0. We also show that magnetic monopole Langmuir turbulence excited near the accretion shock of galaxy clusters may be an efficient mechanism for generating the observed intracluster magnetic fields.

  18. Establishment of reference intervals for plasma protein electrophoresis in Indo-Pacific green sea turtles, Chelonia mydas.

    Science.gov (United States)

    Flint, Mark; Matthews, Beren J; Limpus, Colin J; Mills, Paul C

    2015-01-01

    Biochemical and haematological parameters are increasingly used to diagnose disease in green sea turtles. Specific clinical pathology tools, such as plasma protein electrophoresis analysis, are now being used more frequently to improve our ability to diagnose disease in the live animal. Plasma protein reference intervals were calculated from 55 clinically healthy green sea turtles using pulsed field electrophoresis to determine pre-albumin, albumin, α-, β- and γ-globulin concentrations. The estimated reference intervals were then compared with data profiles from clinically unhealthy turtles admitted to a local wildlife hospital to assess the validity of the derived intervals and identify the clinically useful plasma protein fractions. Eighty-six per cent {19 of 22 [95% confidence interval (CI) 65-97]} of clinically unhealthy turtles had values outside the derived reference intervals, including the following: total protein [six of 22 turtles or 27% (95% CI 11-50%)], pre-albumin [two of five, 40% (95% CI 5-85%)], albumin [13 of 22, 59% (95% CI 36-79%)], total albumin [13 of 22, 59% (95% CI 36-79%)], α- [10 of 22, 45% (95% CI 24-68%)], β- [two of 10, 20% (95% CI 3-56%)], γ- [one of 10, 10% (95% CI 0.3-45%)] and β-γ-globulin [one of 12, 8% (95% CI 0.2-38%)] and total globulin [five of 22, 23% (8-45%)]. Plasma protein electrophoresis shows promise as an accurate adjunct tool to identify a disease state in marine turtles. This study presents the first reference interval for plasma protein electrophoresis in the Indo-Pacific green sea turtle.

  19. D-fructose-binding proteins in bull seminal plasma: Isolation and characterization

    Czech Academy of Sciences Publication Activity Database

    Liberda, J.; Kraus, Marek; Ryšlavá, H.; Vlasáková, M.; Jonáková, Věra; Tichá, M.

    2001-01-01

    Roč. 47, č. 4 (2001), s. 113-119 ISSN 0015-5500 R&D Projects: GA ČR GA303/99/0357; GA ČR GV524/96/K162 Institutional research plan: CEZ:AV0Z5052915 Keywords : bull seminal plasma * non-heparin-binding and heparin-binding proteins * D-fructose-binding proteins Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 0.519, year: 2001

  20. Modulation of the Plasma Kallikrein-Kinin System Proteins Performed by Heparan Sulfate Proteoglycans

    OpenAIRE

    Motta, Guacyara; Tersariol, Ivarne L. S.

    2017-01-01

    Human plasma kallikrein-kinin system proteins are related to inflammation through bradykinin. In the proximity of its target cells, high molecular weight kininogen (H-kininogen) is the substrate of plasma kallikrein, which releases bradykinin from H-kininogen. Heparan sulfate proteoglycans (HSPGs) play a critical role in either recruiting kinin precursors from the plasma, or in the assembly of kallikrein-kinin system components on the cell surface. Furthermore, HSPGs mediate the endocytosis a...

  1. A Phospholipidomic Analysis of All Defined Human Plasma Lipoproteins

    Science.gov (United States)

    Dashti, Monireh; Kulik, Willem; Hoek, Frans; Veerman, Enno C.; Peppelenbosch, Maikel P.; Rezaee, Farhad

    2011-01-01

    Since plasma lipoproteins contain both protein and phospholipid components, either may be involved in processes such as atherosclerosis. In this study the identification of plasma lipoprotein-associated phospholipids, which is essential for understanding these processes at the molecular level, are performed. LC-ESI/MS, LC-ESI-MS/MS and High Performance Thin Layer Chromatography (HPTLC) analysis of different lipoprotein fractions collected from pooled plasma revealed the presence of phosphatidylethanolamine (PE), phosphatidylinositol (PI), and sphingomyeline (SM) only on lipoproteins and phosphatidylcholine (PC), Lyso-PC on both lipoproteins and plasma lipoprotein free fraction (PLFF). Cardiolipin, phosphatidylglycerol (PG) and Phosphatidylserine (PS) were observed neither in the lipoprotein fractions nor in PLFF. All three approaches led to the same results regarding phospholipids occurrence in plasma lipoproteins and PLFF. A high abundancy of PE and SM was observed in VLDL and LDL fractions respectively. This study provides for the first time the knowledge about the phospholipid composition of all defined plasma lipoproteins. PMID:22355656

  2. 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...

  3. Affinity proteomic profiling of plasma for proteins associated to area-based mammographic breast density.

    Science.gov (United States)

    Byström, Sanna; Eklund, Martin; Hong, Mun-Gwan; Fredolini, Claudia; Eriksson, Mikael; Czene, Kamila; Hall, Per; Schwenk, Jochen M; Gabrielson, Marike

    2018-02-14

    Mammographic breast density is one of the strongest risk factors for breast cancer, but molecular understanding of how breast density relates to cancer risk is less complete. Studies of proteins in blood plasma, possibly associated with mammographic density, are well-suited as these allow large-scale analyses and might shed light on the association between breast cancer and breast density. Plasma samples from 1329 women in the Swedish KARMA project, without prior history of breast cancer, were profiled with antibody suspension bead array (SBA) assays. Two sample sets comprising 729 and 600 women were screened by two different SBAs targeting a total number of 357 proteins. Protein targets were selected through searching the literature, for either being related to breast cancer or for being linked to the extracellular matrix. Association between proteins and absolute area-based breast density (AD) was assessed by quantile regression, adjusting for age and body mass index (BMI). Plasma profiling revealed linear association between 20 proteins and AD, concordant in the two sets of samples (p density and processes of tissue homeostasis, DNA repair, cancer development and/or progression in breast cancer. Further validation and follow-up studies of the shortlisted protein candidates in independent cohorts will be needed to infer their role in breast density and its progression in premenopausal and postmenopausal women.

  4. Pregnancy associated plasma protein-A (PAPP-A) is not a marker of the vulnerable atherosclerotic plaque

    DEFF Research Database (Denmark)

    Iversen, Kasper; Teisner, Ane; Dalager, Soren

    2011-01-01

    To investigate if pregnancy associated plasma protein-A (PAPP-A) was present in the vulnerable plaque, and if not, to find alternative hypothesis for the release of PAPP-A.......To investigate if pregnancy associated plasma protein-A (PAPP-A) was present in the vulnerable plaque, and if not, to find alternative hypothesis for the release of PAPP-A....

  5. Validation of cold plasma treatment for protein inactivation: a surface plasmon resonance-based biosensor study

    International Nuclear Information System (INIS)

    Bernard, C; Leduc, A; Barbeau, J; Saoudi, B; Yahia, L'H; Crescenzo, G De

    2006-01-01

    Gas plasma is being proposed as an interesting and promising tool to achieve sterilization. The efficacy of gas plasma to destroy bacterial spores (the most resistant living microorganisms) has been demonstrated and documented over the last ten years. In addition to causing damage to deoxyribonucleic acid by UV radiation emitted by excited species originating from the plasma, gas plasma has been shown to promote erosion of the microorganism in addition to possible oxidation reactions within the microorganism. In this work, we used lysozyme as a protein model to assess the effect of gas plasma on protein inactivation. Lysozyme samples have been subjected to the flowing afterglow of a gas discharge achieved in a nitrogen-oxygen mixture. The efficiency of this plasma treatment on lysozyme has been tested by two different assays. These are an enzyme-linked immunosorbent assay (ELISA) and a surface plasmon resonance (SPR)-based biosensor assay. The two methods showed that exposure to gas plasma can abrogate lysozyme interactions with lysozyme-specific antibodies, more likely by destroying the epitopes responsible for the interaction. More specifically, two SPR-based assays were developed since our ELISA approach did not allow us to discriminate between background and low, but still intact, quantities of lysozyme epitope after plasma treatment. Our SPR results clearly demonstrated that significant protein destruction or desorption was achieved when amounts of lysozyme less than 12.5 ng had been deposited in polystyrene 96-well ELISA plates. At higher lysozyme amounts, traces of available lysozyme epitopes were detected by SPR through indirect measurements. Finally, we demonstrated that a direct SPR approach in which biosensor-immobilized lysozyme activity is directly measured prior and after plasma treatment is more sensitive, and thus, more appropriate to define plasma treatment efficacy with more certainty

  6. Validation of cold plasma treatment for protein inactivation: a surface plasmon resonance-based biosensor study

    Science.gov (United States)

    Bernard, C.; Leduc, A.; Barbeau, J.; Saoudi, B.; Yahia, L'H.; DeCrescenzo, G.

    2006-08-01

    Gas plasma is being proposed as an interesting and promising tool to achieve sterilization. The efficacy of gas plasma to destroy bacterial spores (the most resistant living microorganisms) has been demonstrated and documented over the last ten years. In addition to causing damage to deoxyribonucleic acid by UV radiation emitted by excited species originating from the plasma, gas plasma has been shown to promote erosion of the microorganism in addition to possible oxidation reactions within the microorganism. In this work, we used lysozyme as a protein model to assess the effect of gas plasma on protein inactivation. Lysozyme samples have been subjected to the flowing afterglow of a gas discharge achieved in a nitrogen-oxygen mixture. The efficiency of this plasma treatment on lysozyme has been tested by two different assays. These are an enzyme-linked immunosorbent assay (ELISA) and a surface plasmon resonance (SPR)-based biosensor assay. The two methods showed that exposure to gas plasma can abrogate lysozyme interactions with lysozyme-specific antibodies, more likely by destroying the epitopes responsible for the interaction. More specifically, two SPR-based assays were developed since our ELISA approach did not allow us to discriminate between background and low, but still intact, quantities of lysozyme epitope after plasma treatment. Our SPR results clearly demonstrated that significant protein destruction or desorption was achieved when amounts of lysozyme less than 12.5 ng had been deposited in polystyrene 96-well ELISA plates. At higher lysozyme amounts, traces of available lysozyme epitopes were detected by SPR through indirect measurements. Finally, we demonstrated that a direct SPR approach in which biosensor-immobilized lysozyme activity is directly measured prior and after plasma treatment is more sensitive, and thus, more appropriate to define plasma treatment efficacy with more certainty.

  7. Nifedipine effect on the labelling of blood cells and plasma proteins with Tc-99m

    International Nuclear Information System (INIS)

    Gutfilen, B.; Boasquevisque, E.M.; Bernardo Filho, M.

    1988-01-01

    The labeling of red blood cells (RBC) with Tc-99m depends on the presence of stannous ion (Sn) that helps this radionuclide's fixation on the hemoglobin molecule. Nifedipine is an agent capable to block a specific way where calcius (Ca) ion acrosses the cellular membrane and to bind itself on plasma proteins. The effect of nifedipine in the labeling of RBC and plasma proteins with Tc-99m was studied because of similarities between Ca and Sn ions. Blood with anticoagulant was treated with nifedipine concentration of 10 -6 M for 15 min at 37 0 C. The labeling of RBC with Tc-99m was done incubating with Sn ion solution (3 uM) for different times. The % of radioactivity in RBC was determined. Samples of plasma were precipited with trichloroacetic acid and the % of radiocctivity in insoluble fraction was calculated. The same procedure was done using different nifedipine concentrations and the blood was incubated for 60 min with Sn ion. The determination of the % of Tc-99m labeled in RBC and plasma proteins showed that this drug does not have the capability to alter this incorporation because the results are similar to control. It is suggested that the Sn ions passage across RBC is not altered by nifedipine although this drug could bind to plasma protein, it does not modify the Tc-99m fixation on it. (author) [pt

  8. Plasma levels of C-Reactive Protein and Fibrinogen in Pulmonary ...

    African Journals Online (AJOL)

    In this study, we determined the changes in plasma C- reactive protein (C-RP) and Fibrinogen levels in Drug sensitive Tuberculosis (DSTB) patients at diagnosis, Multi drug resistant tuberculosis (MDRTB) patients at diagnosis and during chemotherapy. Twenty-four (24) patients MDRTB patients and 24 newly diagnosed ...

  9. Exercise-intensity dependent alterations in plasma redox status do not reflect skeletal muscle redox-sensitive protein signaling.

    Science.gov (United States)

    Parker, Lewan; Trewin, Adam; Levinger, Itamar; Shaw, Christopher S; Stepto, Nigel K

    2018-04-01

    Redox homeostasis and redox-sensitive protein signaling play a role in exercise-induced adaptation. The effects of sprint-interval exercise (SIE), high-intensity interval exercise (HIIE) and continuous moderate-intensity exercise (CMIE), on post-exercise plasma redox status are unclear. Furthermore, whether post-exercise plasma redox status reflects skeletal muscle redox-sensitive protein signaling is unknown. In a randomized crossover design, eight healthy adults performed a cycling session of HIIE (5×4min at 75% W max ), SIE (4×30s Wingate's), and CMIE work-matched to HIIE (30min at 50% of W max ). Plasma hydrogen peroxide (H 2 O 2 ), thiobarbituric acid reactive substances (TBARS), superoxide dismutase (SOD) activity, and catalase activity were measured immediately post, 1h, 2h and 3h post-exercise. Plasma redox status biomarkers were correlated with phosphorylation of skeletal muscle p38-MAPK, JNK, NF-κB, and IκBα protein content immediately and 3h post-exercise. Plasma catalase activity was greater with SIE (56.6±3.8Uml -1 ) compared to CMIE (42.7±3.2, pexercise plasma TBARS and SOD activity significantly (pexercise protocol. A significant positive correlation was detected between plasma catalase activity and skeletal muscle p38-MAPK phosphorylation 3h post-exercise (r=0.40, p=0.04). No other correlations were detected (all p>0.05). Low-volume SIE elicited greater post-exercise plasma catalase activity compared to HIIE and CMIE, and greater H 2 O 2 compared to CMIE. Plasma redox status did not, however, adequately reflect skeletal muscle redox-sensitive protein signaling. Copyright © 2017 Sports Medicine Australia. Published by Elsevier Ltd. All rights reserved.

  10. A simple microfluidic platform to study age-dependent protein abundance and localization changes in Saccharomyces cerevisiae

    Directory of Open Access Journals (Sweden)

    Margarita Cabrera

    2017-04-01

    Full Text Available The budding yeast Saccharomyces cerevisiae divides asymmetrically, with a smaller daughter cell emerging from its larger mother cell. While the daughter lineage is immortal, mother cells age with each cell division and have a finite lifespan. The replicative ageing of the yeast mother cell has been used as a model to study the ageing of mitotically active human cells. Several microfluidic platforms, which use fluid flow to selectively remove daughter cells, have recently been developed that can monitor cell physiology as mother cells age. However, these platforms are not trivial to set up and users often require many hours of training. In this study, we have developed a simple system, which combines a commercially available microfluidic platform (the CellASIC ONIX Microfluidic Platform and a genetic tool to prevent the proliferation of daughter cells (the Mother Enrichment Program, to monitor protein abundance and localization changes during approximately the first half of the yeast replicative lifespan. We validated our system by observing known age-dependent changes, such as decreased Sir2 abundance, and have identified a protein with a previously unknown age-dependent change in localization.

  11. Plasma membrane of a marine T cell lymphoma: surface labelling, membrane isolation, separation of membrane proteins and distribution of surface label amongst these proteins

    International Nuclear Information System (INIS)

    Crumpton, M.J.; Marchalonis, J.J.; Haustein, D.; Atwell, J.L.; Harris, A.W.

    1976-01-01

    Two established techniques for analysis of plasma membranes, namely, lactoperoxidase catalyzed surface radioiodination of intact cells and bulk membrane isolation following disruption of cells by shear forces, were applied in studies of membrane proteins of continuously cultured cells of the monoclonal T lymphoma line WEHI-22. It was found that macromolecular 125 I-iodide incorporated into plasma membrane proteins of intact cells was at least as good a marker for the plasma as was the commonly used enzyme 5'-nucleotidase, T lymphoma plasma membrane proteins were complex when analysed by polyacrylamide gel electrophoresis in sodium dodecylsulphate-containing buffers and more than thirty distinct components were resolved. More than fifteen of the components observed on a mass basis were also labelled with 125 I-iodide. Certain bands, however, exhibited a degree of label disproportionate to their staining properties with Coomassie Blue. This was interpreted in terms of their accessibility to the solvent in the intact cells. (author)

  12. Development stage-specific proteomic profiling uncovers small, lineage specific proteins most abundant in the Aspergillus Fumigatus conidial proteome

    Directory of Open Access Journals (Sweden)

    Suh Moo-Jin

    2012-04-01

    Full Text Available Abstract Background The pathogenic mold Aspergillus fumigatus is the most frequent infectious cause of death in severely immunocompromised individuals such as leukemia and bone marrow transplant patients. Germination of inhaled conidia (asexual spores in the host is critical for the initiation of infection, but little is known about the underlying mechanisms of this process. Results To gain insights into early germination events and facilitate the identification of potential stage-specific biomarkers and vaccine candidates, we have used quantitative shotgun proteomics to elucidate patterns of protein abundance changes during early fungal development. Four different stages were examined: dormant conidia, isotropically expanding conidia, hyphae in which germ tube emergence has just begun, and pre-septation hyphae. To enrich for glycan-linked cell wall proteins we used an alkaline cell extraction method. Shotgun proteomic resulted in the identification of 375 unique gene products with high confidence, with no evidence for enrichment of cell wall-immobilized and secreted proteins. The most interesting discovery was the identification of 52 proteins enriched in dormant conidia including 28 proteins that have never been detected in the A. fumigatus conidial proteome such as signaling protein Pil1, chaperones BipA and calnexin, and transcription factor HapB. Additionally we found many small, Aspergillus specific proteins of unknown function including 17 hypothetical proteins. Thus, the most abundant protein, Grg1 (AFUA_5G14210, was also one of the smallest proteins detected in this study (M.W. 7,367. Among previously characterized proteins were melanin pigment and pseurotin A biosynthesis enzymes, histones H3 and H4.1, and other proteins involved in conidiation and response to oxidative or hypoxic stress. In contrast, expanding conidia, hyphae with early germ tubes, and pre-septation hyphae samples were enriched for proteins responsible for

  13. Transgenic overexpression of pregnancy-associated plasma protein-A in murine arterial smooth muscle accelerates atherosclerotic lesion development

    DEFF Research Database (Denmark)

    Conover, Cheryl A.; Mason, Megan A.; Bale, Laurie K.

    2010-01-01

    Pregnancy-associated plasma protein-A (PAPP-A) increases local IGF-I bioavailability through cleavage of inhibitory IGF binding protein (IGFBP)-4 in a variety of systems, including the cardiovascular system. To test the hypothesis that expression of PAPP-A promotes the development of atherosclero......Pregnancy-associated plasma protein-A (PAPP-A) increases local IGF-I bioavailability through cleavage of inhibitory IGF binding protein (IGFBP)-4 in a variety of systems, including the cardiovascular system. To test the hypothesis that expression of PAPP-A promotes the development...

  14. Exoproteome analysis reveals higher abundance of proteins linked to alkaline stress in persistent Listeria monocytogenes strains.

    Science.gov (United States)

    Rychli, Kathrin; Grunert, Tom; Ciolacu, Luminita; Zaiser, Andreas; Razzazi-Fazeli, Ebrahim; Schmitz-Esser, Stephan; Ehling-Schulz, Monika; Wagner, Martin

    2016-02-02

    The foodborne pathogen Listeria monocytogenes, responsible for listeriosis a rare but severe infection disease, can survive in the food processing environment for month or even years. So-called persistent L. monocytogenes strains greatly increase the risk of (re)contamination of food products, and are therefore a great challenge for food safety. However, our understanding of the mechanism underlying persistence is still fragmented. In this study we compared the exoproteome of three persistent strains with the reference strain EGDe under mild stress conditions using 2D differential gel electrophoresis. Principal component analysis including all differentially abundant protein spots showed that the exoproteome of strain EGDe (sequence type (ST) 35) is distinct from that of the persistent strain R479a (ST8) and the two closely related ST121 strains 4423 and 6179. Phylogenetic analyses based on multilocus ST genes showed similar grouping of the strains. Comparing the exoproteome of strain EGDe and the three persistent strains resulted in identification of 22 differentially expressed protein spots corresponding to 16 proteins. Six proteins were significantly increased in the persistent L. monocytogenes exoproteomes, among them proteins involved in alkaline stress response (e.g. the membrane anchored lipoprotein Lmo2637 and the NADPH dehydrogenase NamA). In parallel the persistent strains showed increased survival under alkaline stress, which is often provided during cleaning and disinfection in the food processing environments. In addition, gene expression of the proteins linked to stress response (Lmo2637, NamA, Fhs and QoxA) was higher in the persistent strain not only at 37 °C but also at 10 °C. Invasion efficiency of EGDe was higher in intestinal epithelial Caco2 and macrophage-like THP1 cells compared to the persistent strains. Concurrently we found higher expression of proteins involved in virulence in EGDe e.g. the actin-assembly-inducing protein ActA and the

  15. The effect of polycarboxylate shell of magnetite nanoparticles on protein corona formation in blood plasma

    Energy Technology Data Exchange (ETDEWEB)

    Szekeres, Márta, E-mail: szekeres@chem.u-szeged.hu [Department of Physical Chemistry and Materials Sciences, University of Szeged, Hungary, 1 Aradi vt, 6720 Szeged (Hungary); Tóth, Ildikó Y. [Department of Physical Chemistry and Materials Sciences, University of Szeged, Hungary, 1 Aradi vt, 6720 Szeged (Hungary); Turcu, R. [National Institute R& D for Isotopic and Molecular Technology, Cluj-Napoca 400293 (Romania); Tombácz, Etelka [Department of Physical Chemistry and Materials Sciences, University of Szeged, Hungary, 1 Aradi vt, 6720 Szeged (Hungary)

    2017-04-01

    The development of protein corona around nanoparticles upon administration to the human body is responsible in a large part for their biodistribution, cell-internalization and toxicity or biocompatibility. We studied the influence of the chemical composition of polyelectrolyte shells (citric acid (CA) and poly(acrylic-co-maleic acid) (PAM)) of core-shell magnetite nanoparticles (MNPs) on the evolution of protein corona in human plasma (HP). The aggregation state and zeta potential of the particles were measured in the range of HP concentration between 1 and 80 (v/v)% 3 min and 20 h after dispersing the particles in HP diluted with Tris buffered saline. Naked MNPs aggregated in HP solution, but the carboxylated MNPs became stabilized colloidally at higher plasma concentrations. Significant differences were observed at low plasma concentration. CA@MNPs aggregated instantly while the hydrodynamic diameter of PAM@MNP increased only slightly at 1–3 v/v % HP concentrations. The observed differences in protein corona formation can be explained by the differences in the steric effects of the polycarboxylate shells. It is interesting that relatively small but systematic changes in zeta potential alter the aggregation state significantly. - Highlights: • Human plasma protein corona cannot stabilize naked and citrate-coated magnetite nanoparticles. • Polycarboxylic acid (PAM) coated MNPs are well stabilized with HP protein corona. • Stability pattern of naked, CA and PAM-coated MNPs is not predicted by zeta potential.

  16. Skeletal muscle protein synthesis and the abundance of the mRNA translation initiation repressor PDCD4 are inversely regulated by fasting and refeeding in rats.

    Science.gov (United States)

    Zargar, Sana; Moreira, Tracy S; Samimi-Seisan, Helena; Jeganathan, Senthure; Kakade, Dhanshri; Islam, Nushaba; Campbell, Jonathan; Adegoke, Olasunkanmi A J

    2011-06-01

    Optimal skeletal muscle mass is vital to human health, because defects in muscle protein metabolism underlie or exacerbate human diseases. The mammalian target of rapamycin complex 1 is critical in the regulation of mRNA translation and protein synthesis. These functions are mediated in part by the ribosomal protein S6 kinase 1 (S6K1) through mechanisms that are poorly understood. The tumor suppressor programmed cell death 4 (PDCD4) has been identified as a novel substrate of S6K1. Here, we examined 1) the expression of PDCD4 in skeletal muscle and 2) its regulation by feed deprivation (FD) and refeeding. Male rats (~100 g; n = 6) were subjected to FD for 48 h; some rats were refed for 2 h. FD suppressed muscle fractional rates of protein synthesis and Ser(67) phosphorylation of PDCD4 (-50%) but increased PDCD4 abundance (P muscle fractional rates of protein synthesis and reduced PDCD4 abundance relative to FD. Finally, when myoblasts were grown in amino acid- and serum-free medium, phenylalanine incorporation into proteins in cells depleted of PDCD4 more than doubled the values in cells with a normal level of PDCD4 (P skeletal muscle in parallel with the reduction of the abundance of this mRNA translation inhibitor.

  17. Influence of hemodilution of plasma proteins on erythrocyte aggregability : An in vivo study in patients undergoing cardiopulmonary bypass

    NARCIS (Netherlands)

    Gu, YJ; Graaff, R; de Hoog, E; Veeger, NJGM; Panday, G; Boonstra, PW; van Oeveren, W

    2005-01-01

    Erythrocyte aggregation is known to be affected by a number of factors including the concentration of various plasma proteins. This study was performed to examine the in vivo effect of hemodilution of plasma proteins on erythrocyte aggregation in patients undergoing cardiopulmonary bypass (CPB)

  18. Sequential extraction results in improved proteome profiling of medicinal plant Pinellia ternata tubers, which contain large amounts of high-abundance proteins.

    Directory of Open Access Journals (Sweden)

    Xiaolin Wu

    Full Text Available Pinellia ternata tuber is one of the well-known Chinese traditional medicines. In order to understand the pharmacological properties of tuber proteins, it is necessary to perform proteome analysis of P. ternata tubers. However, a few high-abundance proteins (HAPs, mainly mannose-binding lectin (agglutinin, exist in aggregates of various sizes in the tubers and seriously interfere with proteome profiling by two-dimensional electrophoresis (2-DE. Therefore, selective depletion of these HAPs is a prerequisite for enhanced proteome analysis of P. ternata tubers. Based on differential protein solubility, we developed a novel protocol involving two sequential extractions for depletion of some HAPs and prefractionation of tuber proteins prior to 2-DE. The first extraction using 10% acetic acid selectively extracted acid-soluble HAPs and the second extraction using the SDS-containing buffer extracted remaining acid-insoluble proteins. After application of the protocol, 2-DE profiles of P. ternata tuber proteins were greatly improved and more protein spots were detected, especially low-abundance proteins. Moreover, the subunit composition of P. ternata lectin was analyzed by electrophoresis. Native lectin consists of two hydrogen-bonded subunits (11 kDa and 25 kDa and the 11 kDa subunit was a glycoprotein. Subsequently, major HAPs in the tubers were analyzed by mass spectrometry, with nine protein spots being identified as lectin isoforms. The methodology was easy to perform and required no specialized apparatus. It would be useful for proteome analysis of other tuber plants of Araceae.

  19. Free radical-mediated stimulation of tyrosine-specific protein kinase in rat liver plasma membrane

    International Nuclear Information System (INIS)

    Chan, T.M.; Tatoyan, A.; Cheng, E.; Shargill, N.S.; Pleta, M.

    1986-01-01

    Incorporation of 32 P from (γ- 32 P)-ATP into endogenous proteins of plasma membranes isolated from rat liver was significantly increased by several naphthoquinones including menadione. This apparent stimulation of membrane-associated protein kinase activity by these compounds was most striking (up to 6-7 fold) when the synthetic copolymers containing glutamate and tyrosine residues (4:1) was used as substrate. Since tyrosine residues are the only possible phosphate acceptor in the copolymers, the quinone-stimulated liver membrane protein kinase is most likely tyrosine specific. Although not required for protein kinase activity, dithiothreitol (DTT) was necessary for its stimulation by these quinonoid compounds. Hydrolysis of ATP was not significantly affected by quinones under the experimental conditions. Both menadione and vitamin k 5 increased phosphorylation of plasma membrane proteins of molecular weight 45 and 60 kd. The stimulatory effect of menadione on protein phosphorylation was prevented by the addition of superoxide dismutase. Dihydroxyfumerate, which spontaneously produces various radical species, and H 2 O 2 , also stimulated tyrosine-specific protein phosphorylation. DTT was also required for their full effect. It, therefore, appears that quinonone stimulation of tyrosine-specific protein phosphorylation is mediated by oxygen radicals

  20. Development of a lectin binding assay to differentiate between recombinant and endogenous proteins in pharmacokinetic studies of protein-biopharmaceuticals.

    Science.gov (United States)

    Weber, Alfred; Minibeck, Eva; Scheiflinger, Friedrich; Turecek, Peter L

    2015-04-10

    Human glycoproteins, expressed in hamster cell lines, show similar glycosylation patterns to naturally occurring human molecules except for a minute difference in the linkage of terminal sialic acid: both cell types lack α2,6-galactosyl-sialyltransferase, abundantly expressed in human hepatocytes and responsible for the α2,6-sialylation of circulating glycoproteins. This minute difference, which is currently not known to have any physiological relevance, was the basis for the selective measurement of recombinant glycoproteins in the presence of their endogenous counterparts. The assay is based on using the lectin Sambucus nigra agglutinin (SNA), selectively binding to α2,6-sialylated N-glycans. Using von Willebrand factor (VWF), factor IX (FIX), and factor VIIa (FVIIa), it was demonstrated that (i) the plasma-derived proteins, but not the corresponding recombinant proteins, specifically bind to SNA and (ii) this binding can be used to deplete the plasma-derived proteins. The feasibility of this approach was confirmed in spike-recovery studies for all three recombinant coagulation proteins in human plasma and for recombinant VWF (rVWF) in macaque plasma. Analysis of plasma samples from macaques after administration of recombinant and a plasma-derived VWF demonstrated the suitability and robustness of this approach. Data showed that rVWF could be selectively measured without changing the ELISAs and furthermore revealed the limitations of baseline adjustment using a single measurement of the predose concentration only. The SNA gel-based depletion procedure can easily be integrated in existing procedures as a specific sample pre-treatment step. While ELISA-based methods were used to measure the recombinant coagulation proteins in the supernatants obtained by depletion, this procedure is applicable for all biochemical analyses. Copyright © 2015 Elsevier B.V. All rights reserved.

  1. Identification of low abundance cyclophilins in human plasma.

    Science.gov (United States)

    Schumann, Michael; Ihling, Christian H; Prell, Erik; Schierhorn, Angelika; Sinz, Andrea; Fischer, Gunter; Schiene-Fischer, Cordelia; Malešević, Miroslav

    2016-11-01

    Cylophilins (Cyps) belong to the ubiquitously distributed enzyme class of peptidyl prolyl cis/trans isomerases (EC5.2.1.8), which are foldases capable of accelerating slow steps in the refolding of denatured proteins. At least 20 different Cyp isoenzymes are broadly distributed among all organs and cellular compartments in humans. Extracellularly localized Cyps came into the scientific focus recently because of their involvement in the control of inflammatory diseases, as well as viral and bacterial infections. However, detailed insights into Cyp functions are often hampered by the lack of sensitive detection methods. We present an improved method for affinity purification and detection of Cyp in biotic samples in this manuscript. The procedure takes advantage of two novel cyclosporine A derivatives. Derivative 1 was used to capture Cyps from the sample while derivative 2 was applied for selective release from the affinity matrix. Using this approach, eight different Cyp (CypA, CypB, CypC, Cyp40 (PPID), CypE, CypD (PPIF), CypH, and CypL1) were unambiguously detected in healthy human blood plasma. Moreover, extracellular CypA was found to be partially modified by N ε acetylation on residues Lys44, Lys133, Lys155, as well as N α  acetylation at the N-terminal Val residue. N α  acetylation of Ser2 residue was also found for Cyp40. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. The Enzymology of Protein Translocation across the Escherichia coli Plasma Membrane

    NARCIS (Netherlands)

    Wickner, William; Driessen, Arnold J.M.; Hartl, Franz-Ulrich

    1991-01-01

    Converging physiological, genetic, and biochemical studies have established the salient features of preprotein translocation across the plasma membrane of Escherichia coli. Translocation is catalyzed by two proteins, a soluble chaperone and a membrane-bound translocase. SecB, the major chaperone for

  3. Plasma treatment of paper for protein immobilization on paper-based chemiluminescence immunodevice.

    Science.gov (United States)

    Zhao, Mei; Li, Huifang; Liu, Wei; Guo, Yumei; Chu, Weiru

    2016-05-15

    A novel protein immobilization method based on plasma treatment of paper on the low-cost paper-based immunodevice was established in this work. By using a benchtop plasma cleaner, the paper microzone was treated by oxygen plasma treatment for 4 min and then the antibody can be directly immobilized on the paper surface. Aldehyde group was produced after the plasma treatment, which can be verified from the fourier transform infrared spectroscopy (FT-IR) spectra and x-ray photoelectron spectroscopy (XPS) spectra. By linked to aldehyde group, the antibody can be immobilized on the paper surface without any other pretreatment. A paper-based immunodevice was introduced here through this antibody immobilization method. With sandwich chemiluminescence (CL) immunoassay method, the paper-based immunodevice was successfully performed for carcinoembryonic antigen (CEA) detection in human serum with a linear range of 0.1-80.0 ng/mL. The detection limit was 0.03 ng/mL, which was 30 times lower than the clinical CEA level. Comparing to the other protein immobilization methods on paper-based device, this strategy was faster and simpler and had potential applications in point-of-care testing, public health and environmental monitoring. Copyright © 2015 Elsevier B.V. All rights reserved.

  4. Effects of Dietary Crude Protein Levels and Cysteamine Supplementation on Protein Synthetic and Degradative Signaling in Skeletal Muscle of Finishing Pigs.

    Directory of Open Access Journals (Sweden)

    Ping Zhou

    Full Text Available Dietary protein levels and cysteamine (CS supplementation can affect growth performance and protein metabolism of pigs. However, the influence of dietary protein intake on the growth response of CS-treated pigs is unclear, and the mechanisms involved in protein metabolism remain unknown. Hence, we investigated the interactions between dietary protein levels and CS supplementation and the effects of dietary crude protein levels and CS supplementation on protein synthetic and degradative signaling in skeletal muscle of finishing pigs. One hundred twenty barrows (65.84 ± 0.61 kg were allocated to a 2 × 2 factorial arrangement with five replicates of six pigs each. The primary variations were dietary crude protein (CP levels (14% or 10% and CS supplemental levels (0 or 700 mg/kg. The low-protein (LP diets (10% CP were supplemented with enough essential amino acids (EAA to meet the NRC AA requirements of pigs and maintain the balanced supply of eight EAA including lysine, methionine, threonine, tryptophan, valine, phenylalanine, isoleucine, and leucine. After 41 days, 10 pigs per treatment were slaughtered. We found that LP diets supplemented with EAA resulted in decreased concentrations of plasma somatostatin (SS (P<0.01 and plasma urea nitrogen (PUN (P<0.001, while dietary protein levels did not affect other traits. However, CS supplementation increased the average daily gain (P<0.001 and lean percentage (P<0.05, and decreased the feed conversion ratio (P<0.05 and back fat (P<0.05. CS supplementation also increased the concentrations of plasma insulin-like growth factor 1 (IGF-1 (P<0.001, and reduced the concentrations of leptin, SS, and PUN (P<0.001. Increased mRNA abundance of Akt1 and IGF-1 signaling (P<0.001 and decreased mRNA abundance of Forkhead Box O (FOXO 4 (P<0.01 and muscle atrophy F-box (P<0.001 were observed in pigs receiving CS. Additionally, CS supplementation increased the protein levels for the phosphorylated mammalian target of

  5. Abundance patterns in the interstellar medium of early-type galaxies observed with Suzaku

    International Nuclear Information System (INIS)

    Konami, Saori; Matsushita, Kyoko; Tamagawa, Toru; Nagino, Ryo

    2014-01-01

    We have analyzed 17 early-type galaxies, 13 ellipticals and 4 S0 galaxies, observed with Suzaku, and investigated metal abundances (O, Mg, Si, and Fe) and abundance ratios (O/Fe, Mg/Fe, and Si/Fe) in the interstellar medium (ISM). The emission from each on-source region, which is four times the effective radius, r e , is reproduced with one-temperature (1T) or two-temperature (2T) thermal plasma models as well as a multi-temperature model, using APEC plasma code version 2.0.1. The multi-temperature model gave almost the same abundances and abundance ratios with the 1T or 2T models. The weighted averages of the O, Mg, Si, and Fe abundances of all the sample galaxies derived from the multi-temperature model fits are 0.83 ± 0.04, 0.93 ± 0.03, 0.80 ± 0.02, and 0.80 ± 0.02 solar, respectively, in solar units according to the solar abundance table by Lodders in 2003. These abundances show no significant dependence on the morphology and environment. The systematic differences in the derived metal abundances between versions 2.0.1 and 1.3.1 of the APEC plasma codes were investigated. The derived O and Mg abundances in the ISM agree with the stellar metallicity within an aperture with a radius of one r e derived from optical spectroscopy. From these results, we discuss the past and present Type Ia supernova rates and star formation histories in early-type galaxies.

  6. Protein profiling reveals inter-individual protein homogeneity of arachnoid cyst fluid and high qualitative similarity to cerebrospinal fluid

    Directory of Open Access Journals (Sweden)

    Berle Magnus

    2011-05-01

    the majority of abundant proteins in AC fluid also can be found in CSF. Compared to plasma, as many as 104 proteins in AC were not found in the list of 3017 plasma proteins. Conclusions Based on the protein content of AC fluid, our data indicate that temporal AC is a homogenous condition, pointing towards a similar AC filling mechanism for the 14 patients examined. Most of the proteins identified in AC fluid have been identified in CSF, indicating high similarity in the qualitative protein content of AC to CSF, whereas this was not the case between AC and plasma. This indicates that AC is filled with a liquid similar to CSF. As far as we know, this is the first proteomics study that explores the AC fluid proteome.

  7. Modulation of the Plasma Kallikrein-Kinin System Proteins Performed by Heparan Sulfate Proteoglycans

    Directory of Open Access Journals (Sweden)

    Guacyara Motta

    2017-07-01

    Full Text Available Human plasma kallikrein-kinin system proteins are related to inflammation through bradykinin. In the proximity of its target cells, high molecular weight kininogen (H-kininogen is the substrate of plasma kallikrein, which releases bradykinin from H-kininogen. Heparan sulfate proteoglycans (HSPGs play a critical role in either recruiting kinin precursors from the plasma, or in the assembly of kallikrein-kinin system components on the cell surface. Furthermore, HSPGs mediate the endocytosis and activation of H-kininogen and plasma prekallikrein. In the presence of HSPGs (Chinese hamster ovary cell, CHO-K1, wild type cells both heparin and heparan sulfate strongly inhibit the H-kininogen interaction with the cell membrane. H-kininogen is internalized in endosomal acidic vesicles in CHO-K1 but not in CHO-745 cells (mutant cells deficient in glycosaminoglycan biosynthesis. The endocytosis process is lipid raft-mediated and is dependent on caveolae. Both types of CHO cells do not internalize bradykinin-free H-kininogen. At pH 7.35, bradykinin is released from H-kininogen on the surface of CHO-745 cells only by serine proteases; however, in CHO-K1 cells either serine or cysteine proteases are found to be involved. The CHO-K1 cell lysate contains different kininogenases. Plasma prekallikrein endocytosis in CHO-K1 cells is independent of H-kininogen, and also prekallikrein is not internalized by CHO-745 cells. Plasma prekallikrein cleavage/activation is independent of glycosaminoglycans but plasma kallikrein formation is more specific on H-kininogen assembled on the cell surface through glycosaminoglycans. In this mini-review, the importance of HSPGs in the regulation of plasma kallikrein-kinin system proteins is shown.

  8. 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.

  9. Association of plasma protein C levels and coronary artery disease ...

    African Journals Online (AJOL)

    Several studies have shown the risk factor causes of coronary heart disease. In this study we tested the hypothesis that plasma protein C level might be used as a biomarker for coronary heart disease and myocardial infarction. The study included 60 men that were classified into 3 groups according to clinical examination; ...

  10. Effects of ranavirus infection of red-eared sliders (Trachemys scripta elegans) on plasma proteins.

    Science.gov (United States)

    Moore, A Russell; Allender, Matthew C; MacNeill, Amy L

    2014-06-01

    Ranavirus is an emerging disease that infects fish, amphibians, and reptiles. Ranavirus induces an inflammatory response leading to death in many susceptible species. Red-eared sliders (RES; Trachemys scripta elegans) are vulnerable to ranavirus infection and are economically significant chelonians kept in the pet trade and utilized in research. Early identification of RES with inflammatory diseases would allow for isolation of affected individuals and subsequent disease investigation, including molecular testing for ranavirus. Validation of an inexpensive, clinically relevant, and reproducible diagnostic test that detects inflammation in turtles is needed. Although commonly used, plasma protein electrophoresis to detect an inflammatory acute-phase protein response has not been evaluated in a controlled environment in turtles with experimentally induced inflammatory disease. The objective of this study was to measure plasma protein fractions by electrophoresis to determine if an acute-phase protein response occurs in RES during infection with a frog virus 3-like ranavirus (FV3-like virus) isolated from a chelonian. A Bradford assay and agarose gel electrophoresis (AGE) were performed using plasma collected during a study of the effect of temperature on the pathogenesis of ranavirus in RES. In RES at the time of viremia, total albumin (ALB(mg/ml)) and albumin to globulin ratio were significantly lower and beta-globulin percentage was significantly higher in RES exposed to ranavirus (n = 4) as compared to matched, uninfected RES (n = 8). In the last sample collected prior to death, total protein (TP(mg/ml)), ALB(mg/ml), alpha-globulin percentage, and total alpha-globulin (alpha(mg/ml)) were significantly lower in RES exposed to ranavirus (n = 4) than control individuals (n = 8). In summary, FV3-like virus induces a decrease in plasma albumin concentration at the onset ofviremia and decreases in TP(mg/ml, ALB(mg/ml), and alpha(mg/ml) concentrations prior to death in

  11. Genome analysis of Excretory/Secretory proteins in Taenia solium reveals their Abundance of Antigenic Regions (AAR).

    Science.gov (United States)

    Gomez, Sandra; Adalid-Peralta, Laura; Palafox-Fonseca, Hector; Cantu-Robles, Vito Adrian; Soberón, Xavier; Sciutto, Edda; Fragoso, Gladis; Bobes, Raúl J; Laclette, Juan P; Yauner, Luis del Pozo; Ochoa-Leyva, Adrián

    2015-05-19

    Excretory/Secretory (ES) proteins play an important role in the host-parasite interactions. Experimental identification of ES proteins is time-consuming and expensive. Alternative bioinformatics approaches are cost-effective and can be used to prioritize the experimental analysis of therapeutic targets for parasitic diseases. Here we predicted and functionally annotated the ES proteins in T. solium genome using an integration of bioinformatics tools. Additionally, we developed a novel measurement to evaluate the potential antigenicity of T. solium secretome using sequence length and number of antigenic regions of ES proteins. This measurement was formalized as the Abundance of Antigenic Regions (AAR) value. AAR value for secretome showed a similar value to that obtained for a set of experimentally determined antigenic proteins and was different to the calculated value for the non-ES proteins of T. solium genome. Furthermore, we calculated the AAR values for known helminth secretomes and they were similar to that obtained for T. solium. The results reveal the utility of AAR value as a novel genomic measurement to evaluate the potential antigenicity of secretomes. This comprehensive analysis of T. solium secretome provides functional information for future experimental studies, including the identification of novel ES proteins of therapeutic, diagnosis and immunological interest.

  12. An investigation into the population abundance distribution of mRNAs, proteins, and metabolites in biological systems.

    Science.gov (United States)

    Lu, Chuan; King, Ross D

    2009-08-15

    Distribution analysis is one of the most basic forms of statistical analysis. Thanks to improved analytical methods, accurate and extensive quantitative measurements can now be made of the mRNA, protein and metabolite from biological systems. Here, we report a large-scale analysis of the population abundance distributions of the transcriptomes, proteomes and metabolomes from varied biological systems. We compared the observed empirical distributions with a number of distributions: power law, lognormal, loglogistic, loggamma, right Pareto-lognormal (PLN) and double PLN (dPLN). The best-fit for mRNA, protein and metabolite population abundance distributions was found to be the dPLN. This distribution behaves like a lognormal distribution around the centre, and like a power law distribution in the tails. To better understand the cause of this observed distribution, we explored a simple stochastic model based on geometric Brownian motion. The distribution indicates that multiplicative effects are causally dominant in biological systems. We speculate that these effects arise from chemical reactions: the central-limit theorem then explains the central lognormal, and a number of possible mechanisms could explain the long tails: positive feedback, network topology, etc. Many of the components in the central lognormal parts of the empirical distributions are unidentified and/or have unknown function. This indicates that much more biology awaits discovery.

  13. High-fat diet-induced plasma protein and liver changes in obese rats can be attenuated by melatonin supplementation.

    Science.gov (United States)

    Wongchitrat, Prapimpun; Klosen, Paul; Pannengpetch, Supitcha; Kitidee, Kuntida; Govitrapong, Piyarat; Isarankura-Na-Ayudhya, Chartchalerm

    2017-06-01

    Obesity triggers changes in protein expression in various organs that might participate in the pathogenesis of obesity. Melatonin has been reported to prevent or attenuate such pathological protein changes in several chronic diseases. However, such melatonin effects on plasma proteins have not yet been studied in an obesity model. Using a proteomic approach, we investigated the effect of melatonin on plasma protein profiles after rats were fed a high-fat diet (HFD) to induce obesity. We hypothesized that melatonin would attenuate abnormal protein expression in obese rats. After 10weeks of the HFD, animals displayed increased body weight and fat accumulation as well as increased glucose levels, indicating an obesity-induced prediabetes mellitus-like state. Two-dimensional gel electrophoresis and liquid chromatography-mass spectrometry/mass spectrometry revealed 12 proteins whose expression was altered in response to the HFD and the melatonin treatment. The altered proteins are related to the development of liver pathology, such as cirrhosis (α1-antiproteinase), thrombosis (fibrinogen, plasminogen), and inflammation (mannose-binding protein A, complement C4, complement factor B), contributing to liver steatosis or hepatic cell death. Melatonin treatment most probably reduced the severity of the HFD-induced obesity by reducing the amplitude of HFD-induced plasma protein changes. In conclusion, we identified several potential biomarkers associated with the progression of obesity and its complications, such as liver damage. Furthermore, our findings reveal melatonin's beneficial effect of attenuating plasma protein changes and liver pathogenesis in obese rats. Copyright © 2017 Elsevier Inc. All rights reserved.

  14. 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...

  15. Protein profiles of CCL5, HPGDS, and NPSR1 in plasma reveal association with childhood asthma.

    Science.gov (United States)

    Hamsten, C; Häggmark, A; Grundström, J; Mikus, M; Lindskog, C; Konradsen, J R; Eklund, A; Pershagen, G; Wickman, M; Grunewald, J; Melén, E; Hedlin, G; Nilsson, P; van Hage, M

    2016-09-01

    Asthma is a common chronic childhood disease with many different phenotypes that need to be identified. We analyzed a broad range of plasma proteins in children with well-characterized asthma phenotypes to identify potential markers of childhood asthma. Using an affinity proteomics approach, plasma levels of 362 proteins covered by antibodies from the Human Protein Atlas were investigated in a total of 154 children with persistent or intermittent asthma and controls. After screening, chemokine ligand 5 (CCL5) hematopoietic prostaglandin D synthase (HPGDS) and neuropeptide S receptor 1 (NPSR1) were selected for further investigation. Significantly lower levels of both CCL5 and HPGDS were found in children with persistent asthma, while NPSR1 was found at higher levels in children with mild intermittent asthma compared to healthy controls. In addition, the protein levels were investigated in another respiratory disease, sarcoidosis, showing significantly higher NPSR1 levels in sera from sarcoidosis patients compared to healthy controls. Immunohistochemical staining of healthy tissues revealed high cytoplasmic expression of HPGDS in mast cells, present in stroma of both airway epithelia, lung as well as in other organs. High expression of NPSR1 was observed in neuroendocrine tissues, while no expression was observed in airway epithelia or lung. In conclusion, we have utilized a broad-scaled affinity proteomics approach to identify three proteins with altered plasma levels in asthmatic children, representing one of the first evaluations of HPGDS and NPSR1 protein levels in plasma. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  16. Systematic study of plasma and serum proteins in the pig; Etude systematique des proteines plasmatiques et seriques du porc

    Energy Technology Data Exchange (ETDEWEB)

    Daburon, F; Nizza, P; Hatchikian, C; Schmidt, J -P [Commissariat a l' Energie Atomique (France)

    1966-07-01

    This work has been carried out in the framework of the determination of the physiological constants of a normal pig. The aim was to study the serum and plasma proteins of this animal species, the ultimate object being to discover whether the qualitative and quantitative changes in these proteins can make a significant contribution to the establishment of a biological dosimetry for irradiated pigs. The serum and plasma from a normal pig were analyzed first by various simple electrophoretic methods and then by immuno-electrophoresis. As a result of the particular characteristics of pig serum we have gradually been led to make numerous modifications to the techniques used for human serums or for those of small laboratory animals. Much careful work and patience were required in order to obtain reproducible results. (authors) [French] Ce travail se situe dans le cadre de la determination des constantes physiologiques du porc normal. il s'agissait de proceder a l'etude des proteines seriques et plasmatiques de cette espece animale, le but ulterieur etant de savoir si les modifications qualitatives et quantitatives de ces proteines pourront representer une contribution valable a l'etablissement d'une dosimetrie biologique chez le porc irradie. Le serum et le plasma du porc normal ont ete analyses d'abord par diverses methodes electrophoretiques simples puis par immunoelectrophorese. Les caracteristiques particulieres du serum de porc nous ont conduits a apporter progressivement de nombreuses modifications aux techniques utilisees pour des serums humains ou de petits animaux de laboratoire. L'obtention de resultats reproductible a exige beaucoup de patience et de minutie. (auteurs)

  17. 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.

  18. Proteomic analysis of plasma membrane proteins in wheat roots exposed to phenanthrene.

    Science.gov (United States)

    Shen, Yu; Du, Jiangxue; Yue, Le; Zhan, Xinhua

    2016-06-01

    Polycyclic aromatic hydrocarbons (PAHs) are potentially carcinogenic and toxic to humans through ingestion of contaminated food crops. PAHs can enter crop roots through proton/PAH symporters; however, to date, the symporter remains unclear. Here we reveal, for the first time, the plasma membrane proteome of Triticum aestivum seedling roots in response to phenanthrene (a model PAH) exposure. Two-dimensional gel electrophoresis (2-DE) coupled with MALDI-TOF/TOF-MS and protein database search engines were employed to analyze and identify phenanthrene-responsive proteins. Over 192 protein spots are reproducibly detected in each gel, while 8 spots are differentially expressed under phenanthrene treatment. Phenanthrene induces five up-regulated proteins distinguished as 5-methyltetrahydropteroyltriglutamate-homocysteine methyltransferase 2, enolase, heat shock protein 80-2, probable mediator of RNA polymerase II transcription subunit 37e (heat shock 70-kDa protein 1), and lactoylglutathione lyase. Three proteins identified as adenosine kinase 2, 4-hydroxy-7-methoxy-3-oxo-3,4-dihydro-2H-1,4-benzoxazin-2-yl glucoside beta-D-glucosidase 1c, and glyceraldehyde-3-phosphate dehydrogenase 3 are down-regulated under exposure to phenanthrene. The up-regulated proteins are related to plant defense response, antioxidant system, and glycolysis. The down-regulated proteins involve the metabolism of high-energy compounds and plant growth. Magnesium, which is able to bind to enolase, can enhance the transport of phenanthrene into wheat roots. Therefore, it is concluded that phenanthrene can induce differential expression of proteins in relation to carbohydrate metabolism, self-defense, and plant growth on wheat root plasma membrane. This study not only provides novel insights into PAH uptake by plant roots and PAH stress responses, but is also a good starting point for further determination and analyses of their functions using genetic and other approaches.

  19. Lowering Low-Density Lipoprotein Particles in Plasma Using Dextran Sulphate Co-Precipitates Procoagulant Extracellular Vesicles

    Directory of Open Access Journals (Sweden)

    Jiong-Wei Wang

    2017-12-01

    Full Text Available Plasma extracellular vesicles (EVs are lipid membrane vesicles involved in several biological processes including coagulation. Both coagulation and lipid metabolism are strongly associated with cardiovascular events. Lowering very-low- and low-density lipoprotein ((VLDL particles via dextran sulphate LDL apheresis also removes coagulation proteins. It remains unknown, however, how coagulation proteins are removed in apheresis. We hypothesize that plasma EVs that contain high levels of coagulation proteins are concomitantly removed with (VLDL particles by dextran sulphate apheresis. For this, we precipitated (VLDL particles from human plasma with dextran sulphate and analyzed the abundance of coagulation proteins and EVs in the precipitate. Coagulation pathway proteins, as demonstrated by proteomics and a bead-based immunoassay, were over-represented in the (VLDL precipitate. In this precipitate, both bilayer EVs and monolayer (VLDL particles were observed by electron microscopy. Separation of EVs from (VLDL particles using density gradient centrifugation revealed that almost all coagulation proteins were present in the EVs and not in the (VLDL particles. These EVs also showed a strong procoagulant activity. Our study suggests that dextran sulphate used in LDL apheresis may remove procoagulant EVs concomitantly with (VLDL particles, leading to a loss of coagulation proteins from the blood.

  20. Plasma membrane associated membranes (PAM) from Jurkat cells contain STIM1 protein is PAM involved in the capacitative calcium entry?

    Science.gov (United States)

    Kozieł, Katarzyna; Lebiedzinska, Magdalena; Szabadkai, Gyorgy; Onopiuk, Marta; Brutkowski, Wojciech; Wierzbicka, Katarzyna; Wilczyński, Grzegorz; Pinton, Paolo; Duszyński, Jerzy; Zabłocki, Krzysztof; Wieckowski, Mariusz R

    2009-12-01

    A proper cooperation between the plasma membrane, the endoplasmic reticulum and the mitochondria seems to be essential for numerous cellular processes involved in Ca(2+) signalling and maintenance of Ca(2+) homeostasis. A presence of microsomal and mitochondrial proteins together with those characteristic for the plasma membrane in the fraction of the plasma membrane associated membranes (PAM) indicates a formation of stabile interactions between these three structures. We isolated the plasma membrane associated membranes from Jurkat cells and found its significant enrichment in the plasma membrane markers including plasma membrane Ca(2+)-ATPase, Na(+), K(+)-ATPase and CD3 as well as sarco/endoplasmic reticulum Ca(2+) ATPase as a marker of the endoplasmic reticulum membranes. In addition, two proteins involved in the store-operated Ca(2+) entry, Orai1 located in the plasma membrane and an endoplasmic reticulum protein STIM1 were found in this fraction. Furthermore, we observed a rearrangement of STIM1-containing protein complexes isolated from Jurkat cells undergoing stimulation by thapsigargin. We suggest that the inter-membrane compartment composed of the plasma membrane and the endoplasmic reticulum, and isolated as a stabile plasma membrane associated membranes fraction, might be involved in the store-operated Ca(2+) entry, and their formation and rebuilding have an important regulatory role in cellular Ca(2+) homeostasis.

  1. Deglycosylation of serum vitamin D3-binding protein by alpha-N-acetylgalactosaminidase detected in the plasma of patients with systemic lupus erythematosus.

    Science.gov (United States)

    Yamamoto, N; Naraparaju, V R; Moore, M; Brent, L H

    1997-03-01

    A serum glycoprotein, Gc protein (vitamin D3-binding protein), can be converted by beta-galactosidase of B cells and sialidase of T cells to a potent macrophage-activating factor (MAF), a protein with N-acetylgalactosamine as the remaining sugar moiety. Thus, Gc protein is the precursor for MAF. Treatment of Gc protein with immobilized beta-galactosidase and sialidase generates a remarkably high titered macrophage-activating factor (GcMAF). When peripheral blood monocytes/ macrophages (designated macrophages) of 33 systemic lupus erythematosus patients were incubated with GcMAF (100 pg/ml), the macrophages of all patients were activated as determined by superoxide generation. However, the precursor activity of patient plasma Gc protein was lost or reduced in these patients. Loss of the precursor activity was the result of deglycosylation of plasma Gc protein by alpha-N-acetylgalactosaminidase activity found in the patient plasma. Levels of plasma alpha-N-acetylgalactosaminidase activity in individual patients had an inverse correlation with the MAF precursor activity of their plasma Gc protein. Deglycosylated Gc protein cannot be converted to macro-phage-activating factor. The resulting defect in macro-phage activation may lead to an inability to clear pathogenic immune complexes. Thus, elevated plasma alpha-N-acetylgalactosaminidase activity resulting in the loss of MAF precursor activity and reduced macro-phage activity may play a role in the pathogenesis of systemic lupus erythematosus.

  2. Neutrophil glycoprotein Mo1 is an integral membrane protein of plasma membranes and specific granules

    International Nuclear Information System (INIS)

    Stevenson, K.B.; Nauseef, W.M.; Clark, R.A.

    1987-01-01

    The glucoprotein Mo1 has previously been demonstrated to be on the cell surface and in the specific granule fraction of neutrophils and to be translocated to the cell surface during degranulation. It is not known, however, whether Mo1 is an integral membrane protein or a soluble, intragranular constituent loosely associated with the specific granule membrane. Purified neutrophils were disrupted by nitrogen cavitation and separated on Percoll density gradients into four fractions enriched for azurophilic granules, specific granules, plasma membrane, and cytosol, respectively. The glycoproteins in these fractions were labeled with 3 H-borohydride reduction, extracted with Triton X-114, and immunoprecipitated with 60.3, an anti-Mo1 monoclonal antibody. Mo1 was detected only in the specific granule and plasma membrane fractions and partitioned exclusively into the detergent-rich fraction consistent with Mo1 being an integral membrane protein. In addition, treatment of specific granule membranes with a high salt, high urea buffer to remove adsorbed or peripheral proteins failed to dissociate Mo1. These data support the hypothesis that Mo1 is an integral membrane protein of plasma and specific granule membranes in human neutrophils

  3. Adsorption of proteins from plasma to a series of hydrophilic-hydrophobic copolymers. I. Analysis with the in situ radioiodination technique

    International Nuclear Information System (INIS)

    Horbett, T.A.; Weathersby, P.K.

    1981-01-01

    The adsorption of proteins affects cellular interactions with foreign surfaces and thus plays an important role in determining the biocompatibility of implants. Previous studies have indicated differences in the affinity of various proteins for a given polymer, and differences in the affinity of fibrinogen for a series of polymers varying in hydrophilicity. These studies suggest that differences in the composition of the protein layer adsorbed to polymers from plasma might exist. To examine this hypothesis, the proteins adsorbed from plasma to a series of polymers varying in hydrophilicity were analyzed with the iodogram technique. Copolymers of hydroxyethyl methacrylate and ethyl methacrylate made by the radiation grafting technique were exposed to plasma for 0.5 or 150 min. The adsorbed proteins were iodinated, eluted with SDS, and separated with polyacrylamide gel electrophoresis. Fibrinogen, immunoglobulin G, hemoglobin, and a peak tentatively ascribed to prothrombin were the major proteins detected. Very little iodine was incorporated into adsorbed albumin, even though it was shown to be present by a separate experiment using dye binding. The fraction of total radioactivity associated with each of nine proteins was found to vary markedly and systematically among the surfaces. The distribution of radioactivity into the proteins was very different on 0.5 and 150-min plasma exposed polymers. The results reflect both compositional differences in the adsorbed protein layer on the polymers and differences in the accessibility of proteins to the labeling reagent in the adsorbed state. Differences in the organization of the adsorbed protein layer may play a key role in determining whether cell surface receptors can come in contact with the specific plasma protein able to further stimulate the cell

  4. Probing plasma membrane microdomains in cowpea protoplasts using lipidated GFP-fusion proteins and multimode FRET microscopy

    NARCIS (Netherlands)

    Vermeer, J.E.M.; van Munster, E.B.; Vischer, N.O.; Gadella, T.

    2004-01-01

    Multimode fluorescence resonance energy transfer (FRET) microscopy was applied to study the plasma membrane organization using different lipidated green fluorescent protein (GFP)-fusion proteins co-expressed in cowpea protoplasts. Cyan fluorescent protein (CFP) was fused to the hyper variable region

  5. Comprehensive Analysis of Low-Molecular-Weight Human Plasma Proteome Using Top-Down Mass Spectrometry.

    Science.gov (United States)

    Cheon, Dong Huey; Nam, Eun Ji; Park, Kyu Hyung; Woo, Se Joon; Lee, Hye Jin; Kim, Hee Cheol; Yang, Eun Gyeong; Lee, Cheolju; Lee, Ji Eun

    2016-01-04

    While human plasma serves as a great source for disease diagnosis, low-molecular-weight (LMW) proteome (mass spectrometry to analyze the LMW proteoforms present in four types of human plasma samples pooled from three healthy controls (HCs) without immunoaffinity depletion and with depletion of the top two, six, and seven high-abundance proteins. The LMW proteoforms were first fractionated based on molecular weight using gel-eluted liquid fraction entrapment electrophoresis (GELFrEE). Then, the GELFrEE fractions containing up to 30 kDa were subjected to nanocapillary-LC-MS/MS, and the high-resolution MS and MS/MS data were processed using ProSightPC 3.0. As a result, a total of 442 LMW proteins and cleaved products, including those with post-translational modifications and single amino acid variations, were identified. From additional comparative analysis of plasma samples without immunoaffinity depletion between HCs and colorectal cancer (CRC) patients via top-down approach, tens of LMW proteoforms, including platelet factor 4, were found to show >1.5-fold changes between the plasma samples of HCs and CRC patients, and six of the LMW proteins were verified by Western blot analysis.

  6. Comparative proteome analysis of cryopreserved flagella and head plasma membrane proteins from sea bream spermatozoa: effect of antifreeze proteins.

    Science.gov (United States)

    Zilli, Loredana; Beirão, José; Schiavone, Roberta; Herraez, Maria Paz; Gnoni, Antonio; Vilella, Sebastiano

    2014-01-01

    Cryopreservation induces injuries to fish spermatozoa that in turn affect sperm quality in terms of fertilization ability, motility, DNA and protein integrity and larval survival. To reduce the loss of sperm quality due to freezing-thawing, it is necessary to improve these procedures. In the present study we investigated the ability of two antifreeze proteins (AFPI and AFPIII) to reduce the loss of quality of sea bream spermatozoa due to cryopreservation. To do so, we compared viability, motility, straight-line velocity and curvilinear velocity of fresh and (AFPs)-cryopreserved spermatozoa. AFPIII addition to cryopreservation medium improved viability, motility and straight-line velocity with respect to DMSO or DMSO plus AFPI. To clarify the molecular mechanism(s) underlying these findings, the protein profile of two different cryopreserved sperm domains, flagella and head plasma membranes, was analysed. The protein profiles differed between fresh and frozen-thawed semen and results of the image analysis demonstrated that, after cryopreservation, out of 270 proteins 12 were decreased and 7 were increased in isolated flagella, and out of 150 proteins 6 showed a significant decrease and 4 showed a significant increase in head membranes. Mass spectrometry analysis identified 6 proteins (4 from isolated flagella and 2 present both in flagella and head plasma membranes) within the protein spots affected by the freezing-thawing procedure. 3 out of 4 proteins from isolated flagella were involved in the sperm bioenergetic system. Our results indicate that the ability of AFPIII to protect sea bream sperm quality can be, at least in part, ascribed to reducing changes in the sperm protein profile occurring during the freezing-thawing procedure. Our results clearly demonstrated that AFPIII addition to cryopreservation medium improved the protection against freezing respect to DMSO or DMSO plus AFPI. In addition we propose specific proteins of spermatozoa as markers related to

  7. Isoelectrofocusing analysis of plasma proteins in dogs irradiated with γ-rays in different doses

    International Nuclear Information System (INIS)

    Li Jinping; Ma Liren

    1986-01-01

    The plasma proteins in dogs irradiated with 2.65, 3.75, 5 and 10 Gy of γ-rays were analysed by isoelectrofocusing. Two groups of proteins, which the author named acute phase reactive proteins (A 1 pI = 4.3, A 2 pI = 4.8), were increased during the acute phase of disease. The levels of these proteins were found to be relative to the ultimate fate of the dogs. The causes of the changes of these proteins are discussed

  8. Standardized 15N tracer methods for the evaluation of the plasma protein turnover in clinical practice. 1

    International Nuclear Information System (INIS)

    Bornhak, H.

    1984-01-01

    Methods for quantitative isolation of plasma proteins or groups of proteins (total plasma or serum proteins, fibrin, total globulines, α, β, γ-globolines, albumin) are described based on combination of chromatography with precipitation and extraction techniques. These methods are adapted to the special requirements of 15 N analysis. They can be performed in clinic-chemical standard laboratories without special apparatuses or devices. The described procedures are the biochemico-analytical basis for the quantitative evaluation of tracer kinetics data by means of mathematic modelling. (author)

  9. Detection of lung cancer using plasma protein profiling by matrix-assisted laser desorption/ionization mass spectrometry.

    Science.gov (United States)

    Shevchenko, Valeriy E; Arnotskaya, Natalia E; Zaridze, David G

    2010-01-01

    There are no satisfactory plasma biomarkers which are available for the early detection and monitoring of lung cancer, one of the most frequent cancers worldwide. The aim of this study is to explore the application of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF MS) to plasma proteomic patterns to distinguish lung cancer patients from healthy individuals. The EDTA plasma samples have been pre-fractionated using magnetic bead kits functionalized with weak cation exchange coatings. We compiled MS protein profiles for 90 patients with squamous cell carcinomas (SCC) and compared them with profiles from 187 healthy controls. The MALDI-ToF spectra were analyzed statistically using ClinProTools bioinformatics software. Depending on the sample used, up to 441 peaks/spectrum could be detected in a mass range of 1000-20,000 Da; 33 of these proteins had statistically differential expression levels between SCC and control plasma (P 90%) in external validation test. These results suggest that plasma MALDI-ToF MS protein profiling can distinguish patients with SCC and also from healthy individuals with relatively high sensitivity and specificity and that MALDI- ToF MS is a potential tool for the screening of lung cancer.

  10. The effects of eating marine- or vegetable-fed farmed trout on the human plasma proteome profiles of healthy men

    DEFF Research Database (Denmark)

    Rentsch, Maria Louise; Lametsch, René; Bügel, Susanne

    2015-01-01

    Most human intervention studies have examined the effects on a subset of risk factors, some of which may require long-term exposure. The plasma proteome may reflect the underlying changes in protein expression and activation, and this could be used to identify early risk markers. The aim of the p......Most human intervention studies have examined the effects on a subset of risk factors, some of which may require long-term exposure. The plasma proteome may reflect the underlying changes in protein expression and activation, and this could be used to identify early risk markers. The aim...... of the present study was to evaluate the impact of regular fish intake on the plasma proteome. We recruited thirty healthy men aged 40 to 70 years, who were randomly allocated to a daily meal of chicken or trout raised on vegetable or marine feeds. Blood samples were collected before and after 8 weeks...... of intervention, and after the removal of the twelve most abundant proteins, plasma proteins were separated by two-dimensional gel electrophoresis. Protein spots 4·3 visualised by silver staining were matched by two-dimensional imaging software. Within-subject changes in spots were compared...

  11. Plasma protein corona modulates the vascular wall interaction of drug carriers in a material and donor specific manner.

    Directory of Open Access Journals (Sweden)

    Daniel J Sobczynski

    Full Text Available The nanoscale plasma protein interaction with intravenously injected particulate carrier systems is known to modulate their organ distribution and clearance from the bloodstream. However, the role of this plasma protein interaction in prescribing the adhesion of carriers to the vascular wall remains relatively unknown. Here, we show that the adhesion of vascular-targeted poly(lactide-co-glycolic-acid (PLGA spheres to endothelial cells is significantly inhibited in human blood flow, with up to 90% reduction in adhesion observed relative to adhesion in simple buffer flow, depending on the particle size and the magnitude and pattern of blood flow. This reduced PLGA adhesion in blood flow is linked to the adsorption of certain high molecular weight plasma proteins on PLGA and is donor specific, where large reductions in particle adhesion in blood flow (>80% relative to buffer is seen with ∼60% of unique donor bloods while others exhibit moderate to no reductions. The depletion of high molecular weight immunoglobulins from plasma is shown to successfully restore PLGA vascular wall adhesion. The observed plasma protein effect on PLGA is likely due to material characteristics since the effect is not replicated with polystyrene or silica spheres. These particles effectively adhere to the endothelium at a higher level in blood over buffer flow. Overall, understanding how distinct plasma proteins modulate the vascular wall interaction of vascular-targeted carriers of different material characteristics would allow for the design of highly functional delivery vehicles for the treatment of many serious human diseases.

  12. Regulatory crosstalk by protein kinases on CFTR trafficking and activity

    Science.gov (United States)

    Farinha, Carlos Miguel; Swiatecka-Urban, Agnieszka; Brautigan, David; Jordan, Peter

    2016-01-01

    Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a member of the ATP binding cassette (ABC) transporter superfamily that functions as a cAMP-activated chloride ion channel in fluid-transporting epithelia. There is abundant evidence that CFTR activity (i.e. channel opening and closing) is regulated by protein kinases and phosphatases via phosphorylation and dephosphorylation. Here, we review recent evidence for the role of protein kinases in regulation of CFTR delivery to and retention in the plasma membrane. We review this information in a broader context of regulation of other transporters by protein kinases because the overall functional output of transporters involves the integrated control of both their number at the plasma membrane and their specific activity. While many details of the regulation of intracellular distribution of CFTR and other transporters remain to be elucidated, we hope that this review will motivate research providing new insights into how protein kinases control membrane transport to impact health and disease.

  13. Adsorption of plasma proteins : adsorption behaviour on apolar surfaces and effect on colloid stability

    NARCIS (Netherlands)

    van der Scheer, Albert

    1978-01-01

    In this thesis the adsorption of some plasma proteins (human albumin (HSA) and fibrinogen (HFb)) on non polar surfaces is studied, together with the influence of these proteins on the stability of polystyrene latices. The aim of these investigations is a better understanding of the processes

  14. A pilot study to evaluate the application of a generic protein standard panel for quality control of biomarker detection technologies

    Directory of Open Access Journals (Sweden)

    Valdivia Hernan J

    2011-08-01

    Full Text Available Abstract Background Protein biomarker studies are currently hampered by a lack of measurement standards to demonstrate quality, reliability and comparability across multiple assay platforms. This is especially pertinent for immunoassays where multiple formats for detecting target analytes are commonly used. Findings In this pilot study a generic panel of six non-human protein standards (50 - 10^7 pg/mL of varying abundance was prepared as a quality control (QC material. Simulated "normal" and "diseased" panels of proteins were prepared in pooled human plasma and incorporated into immunoassays using the Meso Scale Discovery® (MSD® platform to illustrate reliable detection of the component proteins. The protein panel was also evaluated as a spike-in material for a model immunoassay involving detection of ovarian cancer biomarkers within individual human plasma samples. Our selected platform could discriminate between two panels of the proteins exhibiting small differences in abundance. Across distinct experiments, all component proteins exhibited reproducible signal outputs in pooled human plasma. When individual donor samples were used, half the proteins produced signals independent of matrix effects. These proteins may serve as a generic indicator of platform reliability. Each of the remaining proteins exhibit differential signals across the distinct samples, indicative of sample matrix effects, with the three proteins following the same trend. This subset of proteins may be useful for characterising the degree of matrix effects associated with the sample which may impact on the reliability of quantifying target diagnostic biomarkers. Conclusions We have demonstrated the potential utility of this panel of standards to act as a generic QC tool for evaluating the reproducibility of the platform for protein biomarker detection independent of serum matrix effects.

  15. Interactions between lipids and proteins are critical for organization of plasma membrane-ordered domains in tobacco BY-2 cells.

    Science.gov (United States)

    Grosjean, Kevin; Der, Christophe; Robert, Franck; Thomas, Dominique; Mongrand, Sébastien; Simon-Plas, Françoise; Gerbeau-Pissot, Patricia

    2018-06-27

    The laterally heterogeneous plant plasma membrane (PM) is organized into finely controlled specialized areas that include membrane-ordered domains. Recently, the spatial distribution of such domains within the PM has been identified as playing a key role in cell responses to environmental challenges. To examine membrane order at a local level, BY-2 tobacco suspension cell PMs were labelled with an environment-sensitive probe (di-4-ANEPPDHQ). Four experimental models were compared to identify mechanisms and cell components involved in short-term (1 h) maintenance of the ordered domain organization in steady-state cell PMs: modulation of the cytoskeleton or the cell wall integrity of tobacco BY-2 cells; and formation of giant vesicles using either a lipid mixture of tobacco BY-2 cell PMs or the original lipid and protein combinations of the tobacco BY-2 cell PM. Whilst inhibiting phosphorylation or disrupting either the cytoskeleton or the cell wall had no observable effects, we found that lipids and proteins significantly modified both the abundance and spatial distribution of ordered domains. This indicates the involvement of intrinsic membrane components in the local physical state of the plant PM. Our findings support a major role for the 'lipid raft' model, defined as the sterol-dependent ordered assemblies of specific lipids and proteins in plant PM organization.

  16. Age- and gender-related alteration in plasma advanced oxidation protein products (AOPP) and glycosaminoglycan (GAG) concentrations in physiological ageing.

    Science.gov (United States)

    Komosinska-Vassev, Katarzyna; Olczyk, Pawel; Winsz-Szczotka, Katarzyna; Kuznik-Trocha, Kornelia; Klimek, Katarzyna; Olczyk, Krystyna

    2012-02-13

    The authors studied the role of increased oxidative stress in the development of oxidative protein damage and extracellular matrix (ECM) components in ageing. The age- and gender-associated disturbances in connective tissue metabolism were evaluated by the plasma chondroitin sulphated glycosaminoglycans (CS-GAG) and non-sulphated GAG-hyaluronan (HA) measurements. Plasma concentration of advanced oxidation protein products (AOPP) was analysed in order to assess oxidative protein damage and evaluate the possible deleterious role of oxidative phenomenon on tissue proteoglycans' metabolism during the physiological ageing process. Sulphated and non-sulphated GAGs as well as AOPP were quantified in plasma samples from 177 healthy volunteers. A linear age-related decline of plasma CS-GAG level was found in this study (r=-0.46; page (r=0.44; page-dependent relationship has been shown in regard to AOPP. AOPP levels significantly increased with age (r=0.63; pphysiological ageing. A significant correlation was found between the concentrations of AOPP and both CS-GAG (r=-0.31; page changes in the ECM are reflected by CS-GAG and HA plasma levels. Strong correlations between AOPP and ECM components indicate that oxidative stress targets protein and non-protein components of the connective tissue matrix during human ageing.

  17. Annexin A2 Mediates the Localization of Measles Virus Matrix Protein at the Plasma Membrane.

    Science.gov (United States)

    Koga, Ritsuko; Kubota, Marie; Hashiguchi, Takao; Yanagi, Yusuke; Ohno, Shinji

    2018-02-28

    Annexins are a family of structurally related proteins that bind negatively charged membrane phospholipids in a Ca 2+ -dependent manner. Annexin A2 (AnxA2), a member of the family, has been implicated in a variety of cellular functions including the organization of membrane domains, vesicular trafficking and cell-cell adhesion. AnxA2 generally forms the heterotetrameric complex with a small Ca 2+ -binding protein S100A10. Measles virus (MV), a member of the family Paramyxoviridae , is an enveloped virus with a nonsegmented negative strand RNA genome. Knockdown of AnxA2 greatly reduced MV growth in cells, without affecting its entry and viral RNA production. In MV-infected, AnxA2-knockdown cells, the expression level of the matrix (M) protein, but not other viral proteins, was reduced compared with that in control cells, and the distribution of the M protein at the plasma membrane was decreased. The M protein lines the inner surface of the envelope and plays an important role in virus assembly by connecting the nucleocapsid to the envelope proteins. The M protein bound to AnxA2 independently of AnxA2's phosphorylation or its association with S100A10, and was co-localized with AnxA2 within cells. Truncation of the N-terminal 10 amino acid residues, but not the N-terminal 5 residues, compromised the ability of the M protein to interact with AnxA2 and localize at the plasma membrane. These results indicate that AnxA2 mediates the localization of the MV M protein at the plasma membrane by interacting with its N-terminal region (especially residues at positions 6-10), thereby aiding in MV assembly. IMPORTANCE Measles virus (MV) is an important human pathogen, still claiming ∼ 100,000 lives per year despite the presence of effective vaccines, and causes occasional outbreaks even in developed countries. Replication of viruses largely relies on the functions of host cells. Our study revealed that the reduction of the host protein annexin A2 compromises the replication of

  18. Increased reactive oxygen species production and lower abundance of complex I subunits and carnitine palmitoyltransferase 1B protein despite normal mitochondrial respiration in insulin-resistant human skeletal muscle.

    Science.gov (United States)

    Lefort, Natalie; Glancy, Brian; Bowen, Benjamin; Willis, Wayne T; Bailowitz, Zachary; De Filippis, Elena A; Brophy, Colleen; Meyer, Christian; Højlund, Kurt; Yi, Zhengping; Mandarino, Lawrence J

    2010-10-01

    The contribution of mitochondrial dysfunction to skeletal muscle insulin resistance remains elusive. Comparative proteomics are being applied to generate new hypotheses in human biology and were applied here to isolated mitochondria to identify novel changes in mitochondrial protein abundance present in insulin-resistant muscle. Mitochondria were isolated from vastus lateralis muscle from lean and insulin-sensitive individuals and from obese and insulin-resistant individuals who were otherwise healthy. Respiration and reactive oxygen species (ROS) production rates were measured in vitro. Relative abundances of proteins detected by mass spectrometry were determined using a normalized spectral abundance factor method. NADH- and FADH(2)-linked maximal respiration rates were similar between lean and obese individuals. Rates of pyruvate and palmitoyl-DL-carnitine (both including malate) ROS production were significantly higher in obesity. Mitochondria from obese individuals maintained higher (more negative) extramitochondrial ATP free energy at low metabolic flux, suggesting that stronger mitochondrial thermodynamic driving forces may underlie the higher ROS production. Tandem mass spectrometry identified protein abundance differences per mitochondrial mass in insulin resistance, including lower abundance of complex I subunits and enzymes involved in the oxidation of branched-chain amino acids (BCAA) and fatty acids (e.g., carnitine palmitoyltransferase 1B). We provide data suggesting normal oxidative capacity of mitochondria in insulin-resistant skeletal muscle in parallel with high rates of ROS production. Furthermore, we show specific abundance differences in proteins involved in fat and BCAA oxidation that might contribute to the accumulation of lipid and BCAA frequently associated with the pathogenesis of insulin resistance.

  19. Numerical calculation on a two-step subdiffusion behavior of lateral protein movement in plasma membranes

    Science.gov (United States)

    Sumi, Tomonari; Okumoto, Atsushi; Goto, Hitoshi; Sekino, Hideo

    2017-10-01

    A two-step subdiffusion behavior of lateral movement of transmembrane proteins in plasma membranes has been observed by using single-molecule experiments. A nested double-compartment model where large compartments are divided into several smaller ones has been proposed in order to explain this observation. These compartments are considered to be delimited by membrane-skeleton "fences" and membrane-protein "pickets" bound to the fences. We perform numerical simulations of a master equation using a simple two-dimensional lattice model to investigate the heterogeneous diffusion dynamics behavior of transmembrane proteins within plasma membranes. We show that the experimentally observed two-step subdiffusion process can be described using fence and picket models combined with decreased local diffusivity of transmembrane proteins in the vicinity of the pickets. This allows us to explain the two-step subdiffusion behavior without explicitly introducing nested double compartments.

  20. Majority of cellular fatty acid acylated proteins are localized to the cytoplasmic surface of the plasma membrane

    International Nuclear Information System (INIS)

    Wilcox, C.A.; Olson, E.N.

    1987-01-01

    The BC 2 Hl muscle cell line was previously reported to contain a broad array of fatty acid acylated proteins. Palmitate was shown to be attached to membrane proteins posttranslationally through thiol ester linkages, whereas myristate was attached cotranslationally, or within seconds thereafter, to soluble and membrane-bound proteins through amide linkages. The temporal and subcellular differences between palmitate and myristate acylation suggested that these two classes of acyl proteins might follow different intracellular pathways to distinct subcellular membrane systems or organelles. In this study, the authors examined the subcellular localization of the major fatty acylated proteins in BC 4 Hl cells. Palmitate-containing proteins were localized to the plasma membrane, but only a subset of myristate-containing proteins was localized to this membrane fraction. The majority of acyl proteins were nonglycosylated and resistant to digestion with extracellular proteases, suggesting that they were not exposed to the external surface of the plasma membrane. Many proteins were, however, digested during incubation of isolated membranes with proteases, which indicates that these proteins were, however, digested during incubation of isolated membranes with proteases, which indicates that these proteins face the cytoplasm. Two-dimensional gel electrophoresis of proteins labeled with [ 3 H]palmitate and [ 3 H]myristate revealed that individual proteins were modified by only one of the two fatty acids and did not undergo both N-linked myristylation and ester-linked palmitylation. Together, these results suggest that the majority of cellular acyl proteins are routed to the cytoplasmic surface of the plasma membrane, and they raise the possibility that fatty acid acylation may play a role in intracellular sorting of nontransmembranous, nonglycosylated membrane proteins

  1. A 39-kD plasma membrane protein (IP39) is an anchor for the unusual membrane skeleton of Euglena gracilis

    International Nuclear Information System (INIS)

    Rosiere, T.K.; Marrs, J.A.; Bouck, G.B.

    1990-01-01

    The major integral plasma membrane protein (IP39) of Euglena gracilis was radiolabeled, peptide mapped, and dissected with proteases to identify cytoplasmic domains that bind and anchor proteins of the cell surface. When plasma membranes were radioiodinated and extracted with octyl glucoside, 98% of the extracted label was found in IP39 or the 68- and 110-kD oligomers of IP39. The octyl glucoside extracts were incubated with unlabeled cell surface proteins immobilized on nitrocellulose (overlays). Radiolabel from the membrane extract bound one (80 kD) of the two (80 and 86 kD) major membrane skeletal protein bands. Resolubilization of the bound label yielded a radiolabeled polypeptide identical in Mr to IP39. Intact plasma membranes were also digested with papain before or after radioiodination, thereby producing a cytoplasmically truncated IP39. The octyl glucoside extract of truncated IP39 no longer bound to the 80-kD membrane skeletal protein in the nitrocellulose overlays. EM of intact or trypsin digested plasma membranes incubated with membrane skeletal proteins under stringent conditions similar to those used in the nitrocellulose overlays revealed a partially reformed membrane skeletal layer. Little evidence of a membrane skeletal layer was found, however, when plasma membranes were predigested with papain before reassociation. A candidate 80-kD binding domain of IP39 has been tentatively identified as a peptide fragment that was present after trypsin digestion of plasma membranes, but was absent after papain digestion in two-dimensional peptide maps of IP39. Together, these data suggest that the unique peripheral membrane skeleton of Euglena binds to the plasma membrane through noncovalent interactions between the major 80-kD membrane skeletal protein and a small, papain sensitive cytoplasmic domain of IP39

  2. The association of 83 plasma proteins with CHD mortality, BMI, HDL-, and total-cholesterol in men: applying multivariate statistics to identify proteins with prognostic value and biological relevance.

    Science.gov (United States)

    Heidema, A Geert; Thissen, Uwe; Boer, Jolanda M A; Bouwman, Freek G; Feskens, Edith J M; Mariman, Edwin C M

    2009-06-01

    In this study, we applied the multivariate statistical tool Partial Least Squares (PLS) to analyze the relative importance of 83 plasma proteins in relation to coronary heart disease (CHD) mortality and the intermediate end points body mass index, HDL-cholesterol and total cholesterol. From a Dutch monitoring project for cardiovascular disease risk factors, men who died of CHD between initial participation (1987-1991) and end of follow-up (January 1, 2000) (N = 44) and matched controls (N = 44) were selected. Baseline plasma concentrations of proteins were measured by a multiplex immunoassay. With the use of PLS, we identified 15 proteins with prognostic value for CHD mortality and sets of proteins associated with the intermediate end points. Subsequently, sets of proteins and intermediate end points were analyzed together by Principal Components Analysis, indicating that proteins involved in inflammation explained most of the variance, followed by proteins involved in metabolism and proteins associated with total-C. This study is one of the first in which the association of a large number of plasma proteins with CHD mortality and intermediate end points is investigated by applying multivariate statistics, providing insight in the relationships among proteins, intermediate end points and CHD mortality, and a set of proteins with prognostic value.

  3. Interleukin (IL)-1 in rat parturition: IL-1 receptors 1 and 2 and accessory proteins abundance in pregnant rat uterus at term - regulation by progesterone.

    Science.gov (United States)

    Ishiguro, Tomohito; Takeda, Jun; Fang, Xin; Bronson, Heather; Olson, David M

    2016-07-01

    The role of interleukin-1 (IL-1), a pro-inflammatory cytokine, in parturition is typically noted by changes in its concentrations. Studying the expression of its receptor family, IL-1 receptor (IL-1R) 1, IL-1R2, IL-1R accessory protein (IL-1RAcP), and its predominantly brain isoform, IL-1RAcPb, during late gestation in the uterus in the Long-Evans rat is another. We assessed changes in their mRNA and protein relative abundance in the uterus and compared IL-1RAcP and IL-1RAcPb mRNA abundance in uterus, cervix, ovaries, placenta, and whole blood of Long-Evans rats during late gestation or in RU486 and progesterone-treated dams using quantitative real-time PCR and western immunoblotting. IL-1R1, IL-1RAcP, and IL-1RAcPb mRNA abundance significantly increased in the uterus at delivery whereas IL-1R2 mRNA abundance significantly decreased. IL-1R1 protein increased at term and IL-1R2 protein decreased at term compared to nonpregnant uteri. IL1-RAcPb mRNA abundance was less than IL-1RAcP, but in the lower uterine segment it was the highest of all tissues examined. RU486 stimulated preterm delivery and an increase in IL-1R1 mRNA abundance whereas progesterone administration extended pregnancy and suppressed the increase in IL-1R1. These data suggest that changes in uterine sensitivity to IL-1 occur during late gestation and suggest another level of regulation for the control of delivery. The roles for IL-1RAcP and IL-1RAcPb need to be determined, but may relate to different intracellular signaling pathways. © 2016 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society.

  4. Removal of model proteins by means of low-pressure inductively coupled plasma discharge

    International Nuclear Information System (INIS)

    Kylian, O; Rauscher, H; Gilliland, D; Bretagnol, F; Rossi, F

    2008-01-01

    Surgical instruments are intended to come into direct contact with the patients' tissues and thus interact with their first immune defence system. Therefore they have to be cleaned, sterilized and decontaminated, in order to prevent any kind of infections and inflammations or to exclude the possibility of transmission of diseases. From this perspective, the removal of protein residues from their surfaces constitutes new challenges, since certain proteins exhibit high resistance to commonly used sterilization and decontamination techniques and hence are difficult to remove without inducing major damages to the object treated. Therefore new approaches must be developed for that purpose and the application of non-equilibrium plasma discharges represents an interesting option. The possibility to effectively remove model proteins (bovine serum albumin, lysozyme and ubiquitin) from surfaces of different materials (Si wafer, glass, polystyrene and gold) by means of inductively coupled plasma discharges sustained in different argon containing mixtures is demonstrated and discussed in this paper

  5. Removal of model proteins by means of low-pressure inductively coupled plasma discharge

    Energy Technology Data Exchange (ETDEWEB)

    Kylian, O; Rauscher, H; Gilliland, D; Bretagnol, F; Rossi, F [European Commission, Joint Research Centre, Institute for Health and Consumer Protection, Via E Fermi 2749, 21027 Ispra (Italy)], E-mail: francois.rossi@jrc.it

    2008-05-07

    Surgical instruments are intended to come into direct contact with the patients' tissues and thus interact with their first immune defence system. Therefore they have to be cleaned, sterilized and decontaminated, in order to prevent any kind of infections and inflammations or to exclude the possibility of transmission of diseases. From this perspective, the removal of protein residues from their surfaces constitutes new challenges, since certain proteins exhibit high resistance to commonly used sterilization and decontamination techniques and hence are difficult to remove without inducing major damages to the object treated. Therefore new approaches must be developed for that purpose and the application of non-equilibrium plasma discharges represents an interesting option. The possibility to effectively remove model proteins (bovine serum albumin, lysozyme and ubiquitin) from surfaces of different materials (Si wafer, glass, polystyrene and gold) by means of inductively coupled plasma discharges sustained in different argon containing mixtures is demonstrated and discussed in this paper.

  6. Effect of whey protein on plasma amino acids in diabetic mice

    OpenAIRE

    HAN, TING; CAI, DONGLIAN; GENG, SHANSHAN; WANG, YING; ZHEN, HUI; WU, PEIYING

    2013-01-01

    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 ...

  7. Induction of hepatic protein synthesis by a peptide in blood plasma of patients with sepsis and trauma.

    Science.gov (United States)

    Loda, M; Clowes, G H; Dinarello, C A; George, B C; Lane, B; Richardson, W

    1984-08-01

    Accelerated release of amino acids from muscle and their uptake for protein synthesis by liver and other visceral tissues are characteristic of trauma or sepsis. Experimentally, this response is induced by interleukin-1 (IL-1) generated by activated macrophages in vitro. However, IL-1 has not been demonstrated in human blood. A small 4000-dalton peptide recently isolated from plasma of patients with sepsis and trauma induces muscle proteolysis and is called "proteolysis-inducing factor" (PIF). To test whether this agent has the ability also to induce hepatic protein synthesis, a series of animal experiments and clinical observations were undertaken. The structural and secretory (acute-phase reactants) in vitro protein synthesis in livers of normal rats injected intraperitoneally with IL-1 or PIF was significantly greater than that of normal rats or those injected with Ringer's lactate (p less than 0.01). In patients with sepsis and trauma the central plasma clearance rate of amino acids, a measure of visceral (principally hepatic) amino acid uptake, was elevated and correlated with the rates of protein synthesis in incubated liver slices obtained by biopsy at operation from the same patients (p less than 0.05). Both in vivo measured central plasma clearance rate of amino acids and in vitro measured hepatic protein synthesis correlated with plasma levels of PIF in the same patients (p less than 0.01 and p less than 0.05, respectively). We conclude that since PIF, and not IL-1, is present in human plasma and both are produced by activated macrophages, PIF seems to be the stable circulating cleavage product of IL-1, which induces not only muscle proteolysis but also hepatic protein synthesis, principally in the form of acute-phase reactants during infection and other states in which inflammation is present.

  8. [Screening differentially expressed plasma proteins in cold stress rats based on iTRAQ combined with mass spectrometry technology].

    Science.gov (United States)

    Liu, Yan-zhi; Guo, Jing-ru; Peng, Meng-ling; Ma, Li; Zhen, Li; Ji, Hong; Yang, Huan-min

    2015-09-01

    Isobaric tags for relative and absolute quantitation (iTRAQ) combined with mass spectrometry were used to screen differentially expressed plasma proteins in cold stress rats. Thirty health SPF Wistar rats were randomly divided into cold stress group A and control group B, then A and B were randomly divided into 3 groups (n = 5): A1, A2, A3 and B1, B2, B3. The temperature of room raising was (24.0 +/- 0.1) degrees C, and the cold stress temperature was (4.0 +/- 0.1) degrees C. The rats were treated with different temperatures until 12 h. The abdominal aortic blood was collected with heparin anticoagulation suction tube. Then, the plasma was separated for protein extraction, quantitative, enzymolysis, iTHAQ labeling, scx fractionation and mass spectrometry analysis. Totally, 1085 proteins were identified in the test, 39 differentially expressed proteins were screened, including 29 up-regulated proteins and 10 down-regulated proteins. Three important differentially expressed proteins related to cold stress were screened by bioinfonnatics analysis (Minor histocompatihility protein HA-1, Has-related protein Rap-1b, Integrin beta-1). In the experiment, the differentially expressed plasma proteins were successfully screened in cold stress rats. iTRAQ technology provided a good platform to screen protein diaguostic markers on cold stress rats, and laid a good foundation for further. study on animal cold stress mechanism.

  9. Systematic study of plasma and serum proteins in the pig; Etude systematique des proteines plasmatiques et seriques du porc

    Energy Technology Data Exchange (ETDEWEB)

    Daburon, F.; Nizza, P.; Hatchikian, C.; Schmidt, J.-P. [Commissariat a l' Energie Atomique (France)

    1966-07-01

    This work has been carried out in the framework of the determination of the physiological constants of a normal pig. The aim was to study the serum and plasma proteins of this animal species, the ultimate object being to discover whether the qualitative and quantitative changes in these proteins can make a significant contribution to the establishment of a biological dosimetry for irradiated pigs. The serum and plasma from a normal pig were analyzed first by various simple electrophoretic methods and then by immuno-electrophoresis. As a result of the particular characteristics of pig serum we have gradually been led to make numerous modifications to the techniques used for human serums or for those of small laboratory animals. Much careful work and patience were required in order to obtain reproducible results. (authors) [French] Ce travail se situe dans le cadre de la determination des constantes physiologiques du porc normal. il s'agissait de proceder a l'etude des proteines seriques et plasmatiques de cette espece animale, le but ulterieur etant de savoir si les modifications qualitatives et quantitatives de ces proteines pourront representer une contribution valable a l'etablissement d'une dosimetrie biologique chez le porc irradie. Le serum et le plasma du porc normal ont ete analyses d'abord par diverses methodes electrophoretiques simples puis par immunoelectrophorese. Les caracteristiques particulieres du serum de porc nous ont conduits a apporter progressivement de nombreuses modifications aux techniques utilisees pour des serums humains ou de petits animaux de laboratoire. L'obtention de resultats reproductible a exige beaucoup de patience et de minutie. (auteurs)

  10. Elevated pre-treatment levels of plasma C-reactive protein are associated with poor prognosis after breast cancer

    DEFF Research Database (Denmark)

    Allin, Kristine H; Nordestgaard, Børge G; Flyger, Henrik

    2011-01-01

    We examined whether plasma C-reactive protein (CRP) levels at the time of diagnosis of breast cancer are associated with overall survival, disease-free survival, death from breast cancer, and recurrence of breast cancer.......We examined whether plasma C-reactive protein (CRP) levels at the time of diagnosis of breast cancer are associated with overall survival, disease-free survival, death from breast cancer, and recurrence of breast cancer....

  11. Knowns and unknowns of plasma membrane protein degradation in plants.

    Science.gov (United States)

    Liu, Chuanliang; Shen, Wenjin; Yang, Chao; Zeng, Lizhang; Gao, Caiji

    2018-07-01

    Plasma membrane (PM) not only creates a physical barrier to enclose the intracellular compartments but also mediates the direct communication between plants and the ever-changing environment. A tight control of PM protein homeostasis by selective degradation is thus crucial for proper plant development and plant-environment interactions. Accumulated evidences have shown that a number of plant PM proteins undergo clathrin-dependent or membrane microdomain-associated endocytic routes to vacuole for degradation in a cargo-ubiquitination dependent or independent manner. Besides, several trans-acting determinants involved in the regulation of endocytosis, recycling and multivesicular body-mediated vacuolar sorting have been identified in plants. More interestingly, recent findings have uncovered the participation of selective autophagy in PM protein turnover in plants. Although great progresses have been made to identify the PM proteins that undergo dynamic changes in subcellular localizations and to explore the factors that control the membrane protein trafficking, several questions remain to be answered regarding the molecular mechanisms of PM protein degradation in plants. In this short review article, we briefly summarize recent progress in our understanding of the internalization, sorting and degradation of plant PM proteins. More specifically, we focus on discussing the elusive aspects underlying the pathways of PM protein degradation in plants. Copyright © 2018 Elsevier B.V. All rights reserved.

  12. [Blood plasma protein adsorption capacity of perfluorocarbon emulsion stabilized by proxanol 268 (in vitro and in vivo studies)].

    Science.gov (United States)

    Sklifas, A N; Zhalimov, V K; Temnov, A A; Kukushkin, N I

    2012-01-01

    The adsorption abilities of the perfluorocarbon emulsion stabilized by Proxanol 268 were investigated in vitro and in vivo. In vitro, the saturation point for the blood plasma proteins was nearly reached after five minutes of incubation of the emulsion with human/rabbit blood plasma and was stable for all incubation periods studied. The decrease in volume ratio (emulsion/plasma) was accompanied by the increase in the adsorptive capacity of the emulsion with maximal values at 1/10 (3.2 and 1.5 mg of proteins per 1 ml of the emulsion, for human and rabbit blood plasma, respectively) that was unchanged at lower ratios. In vivo, in rabbits, intravenously injected with the emulsion, the proteins with molecular masses of 12, 25, 32, 44, 55, 70, and 200 kDa were adsorbed by the emulsion (as in vitro) if it was used 6 hours or less before testing. More delayed testing (6 h) revealed elimination of proteins with molecular masses of 25 and 44 kDa and an additional pool of adsorpted new ones of 27, 50, and 150 kDa. Specific adsorptive capacity of the emulsion enhanced gradually after emulsion injection and reached its maximum (3.5-5 mg of proteins per 1 ml of the emulsion) after 24 hours.

  13. Lactotransferrin in Asian elephant (Elephas maximus seminal plasma correlates with semen quality.

    Directory of Open Access Journals (Sweden)

    Wendy K Kiso

    Full Text Available Asian elephants (Elephas maximus have highly variable ejaculate quality within individuals, greatly reducing the efficacy of artificial insemination and making it difficult to devise a sperm cryopreservation protocol for this endangered species. Because seminal plasma influences sperm function and physiology, including sperm motility, the objectives of this study were to characterize the chemistry and protein profiles of Asian elephant seminal plasma and to determine the relationships between seminal plasma components and semen quality. Ejaculates exhibiting good sperm motility (≥65% expressed higher percentages of spermatozoa with normal morphology (80.3±13.0 vs. 44.9±30.8% and positive Spermac staining (51.9±14.5 vs. 7.5±14.4%, in addition to higher total volume (135.1±89.6 vs. 88.8±73.1 ml and lower sperm concentration (473.0±511.2 vs. 1313.8±764.7×10⁶ cells ml⁻¹ compared to ejaculates exhibiting poor sperm motility (≤10%; P<0.05. Comparison of seminal plasma from ejaculates with good versus poor sperm motility revealed significant differences in concentrations of creatine phosphokinase, alanine aminotransferase, phosphorus, sodium, chloride, magnesium, and glucose. These observations suggest seminal plasma influences semen quality in elephants. One- and two-dimensional (2D gel electrophoresis revealed largely similar compositional profiles of seminal plasma proteins between good and poor motility ejaculates. However, a protein of ∼80 kDa was abundant in 85% of ejaculates with good motility, and was absent in 90% of poor motility ejaculates (P<0.05. We used mass spectrometry to identify this protein as lactotransferrin, and immunoblot analysis to confirm this identification. Together, these findings lay a functional foundation for understanding the contributions of seminal plasma in the regulation of Asian elephant sperm motility, and for improving semen collection and storage in this endangered species.

  14. Analysis of plasma protein adsorption onto DC-Chol-DOPE cationic liposomes by HPLC-CHIP coupled to a Q-TOF mass spectrometer

    KAUST Repository

    Capriotti, Anna Laura; Caracciolo, Giulio; Caruso, Giuseppe; Cavaliere, Chiara; Pozzi, Daniela; Samperi, Roberto; Laganà , Aldo

    2010-01-01

    Plasma protein adsorption is regarded as a key factor in the in vivo organ distribution of intravenously administered drug carriers, and strongly depends on vector surface characteristics. The present study aimed to characterize the "protein corona" absorbed onto DC-Chol-DOPE cationic liposomes. This system was chosen because it is one of the most efficient and widely used non-viral formulations in vitro and a potential candidate for in vivo transfection of genetic material. After incubation of human plasma with cationic liposomes, nanoparticle-protein complex was separated from plasma by centrifugation. An integrated approach based on protein separation by one-dimensional 12% polyacrylamide gel electrophoresis followed by the automated HPLC-Chip technology coupled to a high-resolution mass spectrometer was employed for protein corona characterization. Thirty gel lanes, approximately 2 mm, were cut, digested and analyzed by HPLC-MS/MS. Fifty-eight human plasma proteins adsorbed onto DC-Chol-DOPE cationic liposomes were identified. The knowledge of the interactions of proteins with liposomes can be exploited for future controlled design of colloidal drug carriers and possibly in the controlled creation of biocompatible surfaces of other devices that come into contact with proteins in body fluids. © 2010 Springer-Verlag.

  15. Analysis of plasma protein adsorption onto DC-Chol-DOPE cationic liposomes by HPLC-CHIP coupled to a Q-TOF mass spectrometer

    KAUST Repository

    Capriotti, Anna Laura

    2010-09-22

    Plasma protein adsorption is regarded as a key factor in the in vivo organ distribution of intravenously administered drug carriers, and strongly depends on vector surface characteristics. The present study aimed to characterize the "protein corona" absorbed onto DC-Chol-DOPE cationic liposomes. This system was chosen because it is one of the most efficient and widely used non-viral formulations in vitro and a potential candidate for in vivo transfection of genetic material. After incubation of human plasma with cationic liposomes, nanoparticle-protein complex was separated from plasma by centrifugation. An integrated approach based on protein separation by one-dimensional 12% polyacrylamide gel electrophoresis followed by the automated HPLC-Chip technology coupled to a high-resolution mass spectrometer was employed for protein corona characterization. Thirty gel lanes, approximately 2 mm, were cut, digested and analyzed by HPLC-MS/MS. Fifty-eight human plasma proteins adsorbed onto DC-Chol-DOPE cationic liposomes were identified. The knowledge of the interactions of proteins with liposomes can be exploited for future controlled design of colloidal drug carriers and possibly in the controlled creation of biocompatible surfaces of other devices that come into contact with proteins in body fluids. © 2010 Springer-Verlag.

  16. NEU3 Sialidase Protein Interactors in the Plasma Membrane and in the Endosomes*

    Science.gov (United States)

    Cirillo, Federica; Ghiroldi, Andrea; Fania, Chiara; Piccoli, Marco; Torretta, Enrica; Tettamanti, Guido; Gelfi, Cecilia; Anastasia, Luigi

    2016-01-01

    NEU3 sialidase has been shown to be a key player in many physio- and pathological processes, including cell differentiation, cellular response to hypoxic stress, and carcinogenesis. The enzyme, peculiarly localized on the outer leaflet of the plasma membrane, has been shown to be able to remove sialic acid residues from the gangliosides present on adjacent cells, thus creating cell to cell interactions. Nonetheless, herein we report that the enzyme localization is dynamically regulated between the plasma membrane and the endosomes, where a substantial amount of NEU3 is stored with low enzymatic activity. However, under opportune stimuli, NEU3 is shifted from the endosomes to the plasma membrane, where it greatly increases the sialidase activity. Finally, we found that NEU3 possesses also the ability to interact with specific proteins, many of which are different in each cell compartment. They were identified by mass spectrometry, and some selected ones were also confirmed by cross-immunoprecipitation with the enzyme, supporting NEU3 involvement in the cell stress response, protein folding, and intracellular trafficking. PMID:26987901

  17. Quantitative, multiplexed workflow for deep analysis of human blood plasma and biomarker discovery by mass spectrometry.

    Science.gov (United States)

    Keshishian, Hasmik; Burgess, Michael W; Specht, Harrison; Wallace, Luke; Clauser, Karl R; Gillette, Michael A; Carr, Steven A

    2017-08-01

    Proteomic characterization of blood plasma is of central importance to clinical proteomics and particularly to biomarker discovery studies. The vast dynamic range and high complexity of the plasma proteome have, however, proven to be serious challenges and have often led to unacceptable tradeoffs between depth of coverage and sample throughput. We present an optimized sample-processing pipeline for analysis of the human plasma proteome that provides greatly increased depth of detection, improved quantitative precision and much higher sample analysis throughput as compared with prior methods. The process includes abundant protein depletion, isobaric labeling at the peptide level for multiplexed relative quantification and ultra-high-performance liquid chromatography coupled to accurate-mass, high-resolution tandem mass spectrometry analysis of peptides fractionated off-line by basic pH reversed-phase (bRP) chromatography. The overall reproducibility of the process, including immunoaffinity depletion, is high, with a process replicate coefficient of variation (CV) of 4,500 proteins are detected and quantified per patient sample on average, with two or more peptides per protein and starting from as little as 200 μl of plasma. The approach can be multiplexed up to 10-plex using tandem mass tags (TMT) reagents, further increasing throughput, albeit with some decrease in the number of proteins quantified. In addition, we provide a rapid protocol for analysis of nonfractionated depleted plasma samples analyzed in 10-plex. This provides ∼600 quantified proteins for each of the ten samples in ∼5 h of instrument time.

  18. The Sur7 protein regulates plasma membrane organization and prevents intracellular cell wall growth in Candida albicans.

    Science.gov (United States)

    Alvarez, Francisco J; Douglas, Lois M; Rosebrock, Adam; Konopka, James B

    2008-12-01

    The Candida albicans plasma membrane plays important roles in cell growth and as a target for antifungal drugs. Analysis of Ca-Sur7 showed that this four transmembrane domain protein localized to stable punctate patches, similar to the plasma membrane subdomains known as eisosomes or MCC that were discovered in S. cerevisiae. The localization of Ca-Sur7 depended on sphingolipid synthesis. In contrast to S. cerevisiae, a C. albicans sur7Delta mutant displayed defects in endocytosis and morphogenesis. Septins and actin were mislocalized, and cell wall synthesis was very abnormal, including long projections of cell wall into the cytoplasm. Several phenotypes of the sur7Delta mutant are similar to the effects of inhibiting beta-glucan synthase, suggesting that the abnormal cell wall synthesis is related to activation of chitin synthase activity seen under stress conditions. These results expand the roles of eisosomes by demonstrating that Sur7 is needed for proper plasma membrane organization and cell wall synthesis. A conserved Cys motif in the first extracellular loop of fungal Sur7 proteins is similar to a characteristic motif of the claudin proteins that form tight junctions in animal cells, suggesting a common role for these tetraspanning membrane proteins in forming specialized plasma membrane domains.

  19. LeCPK1, a Calcium-Dependent Protein Kinase from Tomato. Plasma Membrane Targeting and Biochemical Characterization1

    Science.gov (United States)

    Rutschmann, Frank; Stalder, Urs; Piotrowski, Markus; Oecking, Claudia; Schaller, Andreas

    2002-01-01

    The cDNA of LeCPK1, a calcium-dependent protein kinase, was cloned from tomato (Lycopersicon esculentum Mill.). LeCPK1 was expressed in Escherichia coli and purified from bacterial extracts. The recombinant protein was shown to be a functional protein kinase using a synthetic peptide as the substrate (syntide-2, Km = 85 μm). Autophosphorylation of LeCPK1 was observed on threonine and serine residues, one of which was identified as serine-439. Kinase activity was shown to be Ca2+ dependent and required the C-terminal, calmodulin-like domain of LeCPK1. Two classes of high- and low-affinity Ca2+-binding sites were observed, exhibiting dissociation constants of 0.6 and 55 μm, respectively. LeCPK1 was found to phosphorylate the regulatory C-terminal domain of the plasma membrane H+-ATPase in vitro. A potential role in the regulation of proton pump activity is corroborated by the apparent colocalization of the plasma membrane H+-ATPase and LeCPK1 in vivo. Upon transient expression in suspension-cultured cells, a C-terminal fusion of LeCPK1 with the green fluorescent protein was targeted to the plasma membrane. Myristoylation of the LeCPK1 N terminus was found to be required for plasma membrane targeting. PMID:12011347

  20. The Sur7 Protein Regulates Plasma Membrane Organization and Prevents Intracellular Cell Wall Growth in Candida albicans

    Science.gov (United States)

    Alvarez, Francisco J.; Douglas, Lois M.; Rosebrock, Adam

    2008-01-01

    The Candida albicans plasma membrane plays important roles in cell growth and as a target for antifungal drugs. Analysis of Ca-Sur7 showed that this four transmembrane domain protein localized to stable punctate patches, similar to the plasma membrane subdomains known as eisosomes or MCC that were discovered in S. cerevisiae. The localization of Ca-Sur7 depended on sphingolipid synthesis. In contrast to S. cerevisiae, a C. albicans sur7Δ mutant displayed defects in endocytosis and morphogenesis. Septins and actin were mislocalized, and cell wall synthesis was very abnormal, including long projections of cell wall into the cytoplasm. Several phenotypes of the sur7Δ mutant are similar to the effects of inhibiting β-glucan synthase, suggesting that the abnormal cell wall synthesis is related to activation of chitin synthase activity seen under stress conditions. These results expand the roles of eisosomes by demonstrating that Sur7 is needed for proper plasma membrane organization and cell wall synthesis. A conserved Cys motif in the first extracellular loop of fungal Sur7 proteins is similar to a characteristic motif of the claudin proteins that form tight junctions in animal cells, suggesting a common role for these tetraspanning membrane proteins in forming specialized plasma membrane domains. PMID:18799621

  1. Convective transport of highly plasma protein bound drugs facilitates direct penetration into deep tissues after topical application

    Science.gov (United States)

    Dancik, Yuri; Anissimov, Yuri G; Jepps, Owen G; Roberts, Michael S

    2012-01-01

    AIMS To relate the varying dermal, subcutaneous and muscle microdialysate concentrations found in man after topical application to the nature of the drug applied and to the underlying physiology. METHODS We developed a physiologically based pharmacokinetic model in which transport to deeper tissues was determined by tissue diffusion, blood, lymphatic and intersitial flow transport and drug properties. The model was applied to interpret published human microdialysis data, estimated in vitro dermal diffusion and protein binding affinity of drugs that have been previously applied topically in vivo and measured in deep cutaneous tissues over time. RESULTS Deeper tissue microdialysis concentrations for various drugs in vivo vary widely. Here, we show that carriage by the blood to the deeper tissues below topical application sites facilitates the transport of highly plasma protein bound drugs that penetrate the skin, leading to rapid and significant concentrations in those tissues. Hence, the fractional concentration for the highly plasma protein bound diclofenac in deeper tissues is 0.79 times that in a probe 4.5 mm below a superficial probe whereas the corresponding fractional concentration for the poorly protein bound nicotine is 0.02. Their corresponding estimated in vivo lag times for appearance of the drugs in the deeper probes were 1.1 min for diclofenac and 30 min for nicotine. CONCLUSIONS Poorly plasma protein bound drugs are mainly transported to deeper tissues after topical application by tissue diffusion whereas the transport of highly plasma protein bound drugs is additionally facilitated by convective blood, lymphatic and interstitial transport to deep tissues. PMID:21999217

  2. Robustness of solvent/detergent treatment of plasma derivatives: a data collection from Plasma Protein Therapeutics Association member companies.

    Science.gov (United States)

    Dichtelmüller, Herbert O; Biesert, Lothar; Fabbrizzi, Fabrizio; Gajardo, Rodrigo; Gröner, Albrecht; von Hoegen, Ilka; Jorquera, Juan I; Kempf, Christoph; Kreil, Thomas R; Pifat, Dominique; Osheroff, Wendy; Poelsler, Gerhard

    2009-09-01

    Solvent/detergent (S/D) treatment is an established virus inactivation technology that has been applied in the manufacture of medicinal products derived from human plasma for more than 20 years. Data on the inactivation of enveloped viruses by S/D treatment collected from seven Plasma Protein Therapeutics Association member companies demonstrate the robustness, reliability, and efficacy of this virus inactivation method. The results from 308 studies reflecting production conditions as well as technical variables significantly beyond the product release specification were evaluated for virus inactivation, comprising different combinations of solvent and detergent (tri(n-butyl) phosphate [TNBP]/Tween 80, TNBP/Triton X-100, TNBP/Na-cholate) and different products (Factor [F]VIII, F IX, and intravenous and intramuscular immunoglobulins). Neither product class, process temperature, protein concentration, nor pH value has a significant impact on virus inactivation. A variable that did appear to be critical was the concentration of solvent and detergent. The data presented here demonstrate the robustness of virus inactivation by S/D treatment for a broad spectrum of enveloped test viruses and process variables. Our data substantiate the fact that no transmission of viruses such as human immunodeficiency virus, hepatitis B virus, hepatitis C virus, or of other enveloped viruses was reported for licensed plasma derivatives since the introduction of S/D treatment.

  3. Clinical value of indicators of cationic proteins, leukocytes myeloperoxidase and fibronectin blood plasma in viral meningitis in children

    Directory of Open Access Journals (Sweden)

    O. G. Kimirilova

    2017-01-01

    Full Text Available Objective: was to establish clinical and diagnostic value of cytochemical indices of peripheral blood leukocytes (cationic protein and myeloperoxidase, fibronectin blood plasma to assess the severity, predict the course and outcome of viral meningitis in children.Subjects and methods. In 450 patients with viral meningitis (enterovirus, arbovirus, parotitic, herpesviral, adenovirus etiology at the age of 14 years, the parameters of the microbicidal system of leukocytes (cation proteins, myeloperoxidase and fibronectin blood plasma were determined. Etiological diagnosis of meningitis was confirmed by release of viral RNA from blood and cerebrospinal fluid by the polymerase chain reaction, enzyme-linked immunosorbent assay (ELISA.The results and conclusion. Found that severe, prolonged duration, lethal outcome of viral meningitis in children are accompanied by sugnificant suppression of cationic proteins, myeloperoxidase, fibronectin blood plasma, maximally expressed in lethal outcomes, compared with the severe form, but with a favorable outcome and control. Settings imbalance cationic proteins, myeloperoxidase, fibronectin blood plasma are objective criteria of the adaptation syndrome that reflects the state of the phagocytosis system in viral meningitis in children and can be considered as additional criteria for predicting the course and outcome of disease.

  4. 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 corresp...

  5. G-protein activity in Percoll-purified plasma membranes, bulk plasma membranes, and low-density plasma membranes isolated from rat cerebral cortex

    Czech Academy of Sciences Publication Activity Database

    Bouřová, Lenka; Stöhr, Jiří; Lisý, Václav; Rudajev, Vladimír; Novotný, Jiří; Svoboda, Petr

    2009-01-01

    Roč. 15, č. 4 (2009), BR111-BR122 ISSN 1234-1010 R&D Projects: GA MŠk(CZ) LC554; GA MŠk(CZ) LC06063; GA ČR(CZ) GA309/06/0121; GA AV ČR(CZ) IAA500110606 Institutional research plan: CEZ:AV0Z50110509 Keywords : rat cerebral cortex * plasma membrane * G-protein activity Subject RIV: CE - Biochemistry Impact factor: 1.543, year: 2009

  6. 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

  7. iTRAQ based investigation of plasma proteins in HIV infected and HIV/HBV coinfected patients - C9 and KLK are related to HIV/HBV coinfection.

    Science.gov (United States)

    Sun, Tao; Liu, Li; Wu, Ao; Zhang, Yujiao; Jia, Xiaofang; Yin, Lin; Lu, Hongzhou; Zhang, Lijun

    2017-10-01

    Human immunodeficiency virus (HIV) and hepatitis B virus (HBV) share similar routes of transmission, and rapid progression of hepatic and immunodeficiency diseases has been observed in coinfected individuals. Our main objective was to investigate the molecular mechanism of HIV/HBV coinfections. We selected HIV infected and HIV/HBV coinfected patients with and without Highly Active Antiretroviral Therapy (HAART). Low abundance proteins enriched using a multiple affinity removal system (MARS) were labeled with isobaric tags for relative and absolute quantitation (iTRAQ) kits and analyzed using liquid chromatography-mass spectrometry (LC-MS). The differential proteins were analyzed by Gene Ontology (GO) database. A total of 41 differential proteins were found in HIV/HBV coinfected patients as compared to HIV mono-infected patients with or without HAART treatment, including 7 common HBV-regulated proteins. The proteins involved in complement and coagulation pathways were significantly enriched, including plasma kallikrein (KLK) and complement component C9 (C9). C9 and KLK were verified to be down-regulated in HIV/HBV coinfected patients through ELISA analysis. The present iTRAQ based proteomic analyses identified 7 proteins that are related to HIV/HBV coinfection. HBV might influence hepatic and immune functions by deregulating complement and coagulation pathways. C9 and KLK could potentially be used as targets for the treatment of HIV/HBV coinfections. Copyright © 2017. Published by Elsevier Ltd.

  8. Steric exclusion and protein conformation determine the localization of plasma membrane transporters

    NARCIS (Netherlands)

    Bianchi, Frans; Syga, Łukasz; Moiset, Gemma; Spakman, Dian; Schavemaker, Paul E; Punter, Christiaan M; Seinen, Anne-Bart; van Oijen, Antoine M; Robinson, Andrew; Poolman, Bert

    2018-01-01

    The plasma membrane (PM) of Saccharomyces cerevisiae contains membrane compartments, MCC/eisosomes and MCPs, named after the protein residents Can1 and Pma1, respectively. Using high-resolution fluorescence microscopy techniques we show that Can1 and the homologous transporter Lyp1 are able to

  9. Plasma phospholipid transfer protein activity is related to insulin resistance : impaired acute lowering by insulin in obese Type II diabetic patients

    NARCIS (Netherlands)

    Riemens, SC; van Tol, A; Sluiter, WJ; Dullaart, RPF

    Cholesteryl ester transfer protein (CETP) and phospholipid transfer protein (PLTP) have important functions in high density lipoprotein (HDL) metabolism. We determined the association of plasma CETP and PLTP activities (measured with exogenous' substrate assays) with insulin resistance, plasma

  10. Plasma membrane fatty acid-binding protein and mitochondrial glutamic-oxaloacetic transaminase of rat liver are related

    International Nuclear Information System (INIS)

    Berk, P.D.; Potter, B.J.; Sorrentino, D.; Zhou, S.L.; Isola, L.M.; Stump, D.; Kiang, C.L.; Thung, S.; Wada, H.; Horio, Y.

    1990-01-01

    The hepatic plasma membrane fatty acid-binding protein (h-FABP PM ) and the mitochondrial isoenzyme of glutamic-oxaloacetic transaminase (mGOT) of rat liver have similar amino acid compositions and identical amino acid sequences for residues 3-24. Both proteins migrate with an apparent molecular mass of 43 kDa on SDS/polyacrylamide gel electrophoresis, have a similar pattern of basic charge isomers on isoelectric focusing, are eluted similarly from four different high-performance liquid chromatographic columns, have absorption maxima at 435 nm under acid conditions and 354 nm at pH 8.3, and bind oleate. Sinusoidally enriched liver plasma membranes and purified h-FABP PM have GOT enzymatic activity. Monospecific rabbit antiserum against h-FABP PM reacts on Western blotting with mGOT, and vice versa. Antisera against both proteins produce plasma membrane immunofluorescence in rat hepatocytes and selectively inhibit the hepatocellular uptake of [ 3 H]oleate but not that of [ 35 S]sulfobromophthalein or [ 14 C]taurocholate. The inhibition of oleate uptake produced by anti-h-FABP PM can be eliminated by preincubation of the antiserum with mGOT; similarly, the plasma membrane immunofluorescence produced by either antiserum can be eliminated by preincubation with the other antigen. These data suggest that h-FABP PM and mGOT are closely related

  11. Body Position Modulates Gastric Emptying and Affects the Post-Prandial Rise in Plasma Amino Acid Concentrations Following Protein Ingestion in Humans

    Directory of Open Access Journals (Sweden)

    Andrew M. Holwerda

    2016-04-01

    Full Text Available Dietary protein digestion and amino acid absorption kinetics determine the post-prandial muscle protein synthetic response. Body position may affect gastrointestinal function and modulate the post-prandial rise in plasma amino acid availability. We aimed to assess the impact of body position on gastric emptying rate and the post-prandial rise in plasma amino acid concentrations following ingestion of a single, meal-like amount of protein. In a randomized, cross-over design, eight healthy males (25 ± 2 years, 23.9 ± 0.8 kg·m−2 ingested 22 g protein and 1.5 g paracetamol (acetaminophen in an upright seated position (control and in a −20° head-down tilted position (inversion. Blood samples were collected during a 240-min post-prandial period and analyzed for paracetamol and plasma amino acid concentrations to assess gastric emptying rate and post-prandial amino acid availability, respectively. Peak plasma leucine concentrations were lower in the inversion compared with the control treatment (177 ± 15 vs. 236 ± 15 mmol·L−1, p < 0.05, which was accompanied by a lower plasma essential amino acid (EAA response over 240 min (31,956 ± 6441 vs. 50,351 ± 4015 AU; p < 0.05. Peak plasma paracetamol concentrations were lower in the inversion vs. control treatment (5.8 ± 1.1 vs. 10.0 ± 0.6 mg·L−1, p < 0.05. Gastric emptying rate and post-prandial plasma amino acid availability are significantly decreased after protein ingestion in a head-down tilted position. Therefore, upright body positioning should be considered when aiming to augment post-prandial muscle protein accretion in both health and disease.

  12. Effects of Acute Endurance Exercise on Plasma Protein Profiles of Endurance-Trained and Untrained Individuals over Time

    Directory of Open Access Journals (Sweden)

    Marius Schild

    2016-01-01

    Full Text Available Acute physical exercise and repeated exercise stimuli affect whole-body metabolic and immunologic homeostasis. The aim of this study was to determine plasma protein profiles of trained (EET, n=19 and untrained (SED, n=17 individuals at rest and in response to an acute bout of endurance exercise. Participants completed a bicycle exercise test at an intensity corresponding to 80% of their VO2max. Plasma samples were taken before, directly after, and three hours after exercise and analyzed using multiplex immunoassays. Seventy-eight plasma variables were included in the final analysis. Twenty-nine variables displayed significant acute exercise effects in both groups. Seven proteins differed between groups, without being affected by acute exercise. Among these A2Macro and IL-5 were higher in EET individuals while leptin showed elevated levels in SED individuals. Fifteen variables revealed group and time differences with elevated levels for IL-3, IL-7, IL-10, and TNFR2 in EET individuals. An interaction effect could be observed for nine variables including IL-6, MMP-2, MMP-3, and muscle damage markers. The proteins that differ between groups indicate a long-term exercise effect on plasma protein concentrations. These findings might be of importance in the development of exercise-based strategies in the prevention and therapy of chronic metabolic and inflammatory diseases and for training monitoring.

  13. Plasma signaling proteins in persons at genetic risk for Alzheimer disease: influence of APOE genotype.

    Science.gov (United States)

    Ringman, John M; Elashoff, David; Geschwind, Daniel H; Welsh, Brian T; Gylys, Karen H; Lee, Cathy; Cummings, Jeffrey L; Cole, Greg M

    2012-06-01

    To study the effect of familial Alzheimer disease (FAD) mutations and APOE genotype on plasma signaling protein levels. Cross-sectional comparison of plasma levels of 77 proteins measured using multiplex immune assays. A tertiary referral dementia research center. Thirty-three persons from families harboring PSEN1 or APP mutations, aged 19 to 59 years. Protein levels were compared between FAD mutation carriers (MCs) and noncarriers (NCs) and among APOE genotype groups, using multiple linear regression models. Twenty-one participants were FAD MCs and 12 were NCs. Six had the APOE ε2/3, 6 had the ε3/4, and 21 had the ε3/3 genotype. Levels of 17 proteins differed among APOE genotype groups, and there were significant interactions between age and APOE genotype for 12 proteins. Plasma levels of apolipoprotein E and superoxide dismutase 1 were highest in the ε2 carriers, lowest in ε4 carriers, and intermediate in the ε3 carriers. Levels of multiple interleukins showed the opposite pattern and, among the ε4 carriers, demonstrated significant negative correlations with age. Although there were no significant differences between FAD MCs and NCs, there were interactions between mutation status and APOE genotype for 13 proteins. We found different patterns of inflammatory markers in young and middle-aged persons among APOE genotype groups. The APOE ε4 carriers had the lowest levels of apolipoprotein E. Young ε4 carriers have increased inflammatory markers that diminish with age. We demonstrated altered inflammatory responses in young and middle adulthood in ε4 carriers that may relate to AD risk later in life.

  14. Plasma myelin basic protein assay using Gilford enzyme immunoassay cuvettes.

    Science.gov (United States)

    Groome, N P

    1981-10-01

    The assay of myelin basic protein in body fluids has potential clinical importance as a routine indicator of demyelination. Preliminary details of a competitive enzyme immunoassay for this protein have previously been published by the author (Groome, N. P. (1980) J. Neurochem. 35, 1409-1417). The present paper now describes the adaptation of this assay for use on human plasma and various aspects of routine data processing. A commercially available cuvette system was found to have advantages over microtitre plates but required a permuted arrangement of sample replicates for consistent results. For dose interpolation, the standard curve could be fitted to a three parameter non-linear equation by regression analysis or linearised by the logit/log transformation.

  15. Differential proteomics of human seminal plasma: A potential target for searching male infertility marker proteins.

    Science.gov (United States)

    Tomar, Anil Kumar; Sooch, Balwinder Singh; Singh, Sarman; Yadav, Savita

    2012-04-01

    The clinical fertility tests, available in the market, fail to define the exact cause of male infertility in almost half of the cases and point toward a crucial need of developing better ways of infertility investigations. The protein biomarkers may help us toward better understanding of unknown cases of male infertility that, in turn, can guide us to find better therapeutic solutions. Many clinical attempts have been made to identify biomarkers of male infertility in sperm proteome but only few studies have targeted seminal plasma. Human seminal plasma is a rich source of proteins that are essentially required for development of sperm and successful fertilization. This viewpoint article highlights the importance of human seminal plasma proteome in reproductive physiology and suggests that differential proteomics integrated with functional analysis may help us in searching potential biomarkers of male infertility. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. Membrane proteins involved in transport, vesicle traffic and Ca(2+) signaling increase in beetroots grown in saline soils.

    Science.gov (United States)

    Lino, Bárbara; Chagolla, Alicia; E González de la Vara, Luis

    2016-07-01

    By separating plasma membrane proteins according to their hydropathy from beetroots grown in saline soils, several proteins probably involved in salt tolerance were identified by mass spectrometry. Beetroots, as a salt-tolerant crop, have developed mechanisms to cope with stresses associated with saline soils. To observe which plasma membrane (PM) proteins were more abundant in beet roots grown in saline soils, beet root plants were irrigated with water or 0.2 M NaCl. PM-enriched membrane preparations were obtained from these plants, and their proteins were separated according to their hydropathy by serial phase partitioning with Triton X-114. Some proteins whose abundance increased visibly in membranes from salt-grown beetroots were identified by mass spectrometry. Among them, there was a V-type H(+)-ATPase (probably from contaminating vacuolar membranes), which increased with salt at all stages of beetroots' development. Proteins involved in solute transport (an H(+)-transporting PPase and annexins), vesicle traffic (clathrin and synaptotagmins), signal perception and transduction (protein kinases and phospholipases, mostly involved in calcium signaling) and metabolism, appeared to increase in salt-grown beetroot PM-enriched membranes. These results suggest that PM and vacuolar proteins involved in transport, metabolism and signal transduction increase in beet roots adapted to saline soils. In addition, these results show that serial phase partitioning with Triton X-114 is a useful method to separate membrane proteins for their identification by mass spectrometry.

  17. Plasma phospholipid transfer protein activity is independently determined by obesity and insulin resistance in non-diabetic subjects

    NARCIS (Netherlands)

    de Vries, Rindert; Kappelle, Paul J. W. H.; Dallinga-Thie, Geesje M.; Dullaart, Robin P. F.

    2011-01-01

    Phospholipid transfer protein (PLTP) is an emerging cardio-metabolic risk factor which is intricately involved in lipoprotein metabolism. Elevated plasma PLTP activity levels are reported in obesity and diabetes mellitus, but the relative contributions of obesity and insulin resistance to plasma

  18. Effects of dietary nitrogen concentration on messenger RNA expression and protein abundance of urea transporter-B and aquaporins in ruminal papillae from lactating Holstein cows

    DEFF Research Database (Denmark)

    Røjen, Betina Amdisen; Poulsen, Søren Brandt; Theil, Peter Kappel

    2011-01-01

    To test the hypothesis that dietary N concentrations affect gut epithelial urea transport by modifying the expression of urea transporter B (UT-B) and aquaporins (AQP), the mRNA expression and protein abundance of UT-B and AQP3, AQP7, AQP8, and AQP10 were investigated in ruminal papillae from 9...... lactating dairy cows. Ruminal papillae were harvested from cows fed low N (12.9% crude protein) and high N (17.1% crude protein) diets in a crossover design with 21-d periods. The mRNA expression was determined by real-time reverse transcription-PCR and protein abundance by immunoblotting. The m......RNA expression of UT-B was not affected by dietary treatment, whereas mRNA expression of AQP3, 7, and 10 were greater in the high N compared with the low N fed cows. Using peptide-derived rabbit antibodies to cow AQP3, 7, and 8, immunoblotting revealed bands of approximately 27, 27, and 24 kDa in ruminal...

  19. High-dose thiamine therapy counters dyslipidemia and advanced glycation of plasma protein in streptozotocin-induced diabetic rats.

    Science.gov (United States)

    Karachalias, Nikolaos; Babaei-Jadidi, Roya; Kupich, Christian; Ahmed, Naila; Thornalley, Paul J

    2005-06-01

    The streptozotocin-induced (STZ) diabetic rat experimental model of diabetes on insulin maintenance therapy exhibits dyslipidemia, mild thiamine deficiency, and increased plasma protein advanced glycation end products (AGEs). The reversal of thiamine deficiency by high-dose thiamine and S-benzoylthiamine monophosphate (benfotiamine) prevented the development of incipient nephropathy. Recently, we reported that high-dose thiamine (but not benfotiamine) countered diabetic dyslipidemia. To understand further the differences between the effects of thiamine and benfotiamine therapy, we quantified the levels of the AGEs in plasma protein. We found hydroimidazolone AGE residues derived from glyoxal and methylglyoxal, G-H1 and MG-H1, were increased 115% and 68% in STZ diabetic rats, with respect to normal controls, and were normalized by both thiamine and benfotiamine; whereas N-carboxymethyl-lysine (CML) and N-carboxyethyl-lysine (CEL) residues were increased 74% and 118% in STZ diabetic rats and were normalized by thiamine only. The lack of effect of benfotiamine on plasma CML and CEL residue concentrations suggests there may be important precursors of plasma protein CML and CEL residues other than glyoxal and methylglyoxal. These are probably lipid-derived aldehydes.

  20. LEAping to conclusions: a computational reanalysis of late embryogenesis abundant proteins and their possible roles.

    Science.gov (United States)

    Wise, Michael J

    2003-10-29

    The late embryogenesis abundant (LEA) proteins cover a number of loosely related groups of proteins, originally found in plants but now being found in non-plant species. Their precise function is unknown, though considerable evidence suggests that LEA proteins are involved in desiccation resistance. Using a number of statistically-based bioinformatics tools the classification of a large set of LEA proteins, covering all Groups, is reexamined together with some previous findings. Searches based on peptide composition return proteins with similar composition to different LEA Groups; keyword clustering is then applied to reveal keywords and phrases suggestive of the Groups' properties. Previous research has suggested that glycine is characteristic of LEA proteins, but it is only highly over-represented in Groups 1 and 2, while alanine, thought characteristic of Group 2, is over-represented in Group 3, 4 and 6 but under-represented in Groups 1 and 2. However, for LEA Groups 1 2 and 3 it is shown that glutamine is very significantly over-represented, while cysteine, phenylalanine, isoleucine, leucine and tryptophan are significantly under-represented. There is also evidence that the Group 4 LEA proteins are more appropriately redistributed to Group 2 and Group 3. Similarly, Group 5 is better found among the Group 3 LEA proteins. There is evidence that Group 2 and Group 3 LEA proteins, though distinct, might be related. This relationship is also evident in the overlapping sets of keywords for the two Groups, emphasising alpha-helical structure and, at a larger scale, filaments, all of which fits well with experimental evidence that proteins from both Groups are natively unstructured, but become structured under stress conditions. The keywords support localisation of LEA proteins both in the nucleus and associated with the cytoskeleton, and a mode of action similar to chaperones, perhaps the cold shock chaperones, via a role in DNA-binding. In general, non-globular and

  1. LEAping to conclusions: A computational reanalysis of late embryogenesis abundant proteins and their possible roles

    Directory of Open Access Journals (Sweden)

    Wise Michael J

    2003-10-01

    Full Text Available Abstract Background The late embryogenesis abundant (LEA proteins cover a number of loosely related groups of proteins, originally found in plants but now being found in non-plant species. Their precise function is unknown, though considerable evidence suggests that LEA proteins are involved in desiccation resistance. Using a number of statistically-based bioinformatics tools the classification of a large set of LEA proteins, covering all Groups, is reexamined together with some previous findings. Searches based on peptide composition return proteins with similar composition to different LEA Groups; keyword clustering is then applied to reveal keywords and phrases suggestive of the Groups' properties. Results Previous research has suggested that glycine is characteristic of LEA proteins, but it is only highly over-represented in Groups 1 and 2, while alanine, thought characteristic of Group 2, is over-represented in Group 3, 4 and 6 but under-represented in Groups 1 and 2. However, for LEA Groups 1 2 and 3 it is shown that glutamine is very significantly over-represented, while cysteine, phenylalanine, isoleucine, leucine and tryptophan are significantly under-represented. There is also evidence that the Group 4 LEA proteins are more appropriately redistributed to Group 2 and Group 3. Similarly, Group 5 is better found among the Group 3 LEA proteins. Conclusions There is evidence that Group 2 and Group 3 LEA proteins, though distinct, might be related. This relationship is also evident in the overlapping sets of keywords for the two Groups, emphasising alpha-helical structure and, at a larger scale, filaments, all of which fits well with experimental evidence that proteins from both Groups are natively unstructured, but become structured under stress conditions. The keywords support localisation of LEA proteins both in the nucleus and associated with the cytoskeleton, and a mode of action similar to chaperones, perhaps the cold shock chaperones

  2. Dietary modulation of plasma angiopoietin-like protein 4 concentrations in healthy volunteers and in patients with type 2 diabetes

    NARCIS (Netherlands)

    Jonker, Jacqueline T.; Smit, Johannes W. A.; Hammer, Sebastiaan; Snel, Marieke; van der Meer, Rutger W.; Lamb, Hildo J.; Mattijssen, Frits; Mudde, Karin; Jazet, Ingrid M.; Dekkers, Olaf M.; de Roos, Albert; Romijn, Johannes A.; Kersten, Sander; Rensen, Patrick C. N.

    2013-01-01

    Angiopoietin-like protein 4 (ANGPTL4) has been identified as an inhibitor of lipoprotein lipase. Preliminary data suggest that plasma nonesterified fatty acids (NEFAs) raise plasma ANGPTL4 concentrations in humans. The objective was to assess plasma ANGPTL4 concentrations after various nutritional

  3. Dietary modulation of plasma angiopoietin-like protein 4 concentrations in healthy volunteers and in patients with type 2 diabetes

    NARCIS (Netherlands)

    Jonker, J.T.; Smit, J.W.A.; Hammer, S.; Snel, M.; Meer, R.W. van der; Lamb, H.J.; Mattijssen, F.; Mudde, K.; Jazet, I.M.; Dekkers, O.M.; Roos, A. de; Romijn, J.A.; Kersten, S.; Rensen, P.C.

    2013-01-01

    BACKGROUND: Angiopoietin-like protein 4 (ANGPTL4) has been identified as an inhibitor of lipoprotein lipase. Preliminary data suggest that plasma nonesterified fatty acids (NEFAs) raise plasma ANGPTL4 concentrations in humans. OBJECTIVE: The objective was to assess plasma ANGPTL4 concentrations

  4. Dietary modulation of plasma angiopoietin-like protein 4 concentrations in healthy volunteers and in patients with type 2 diabetes

    NARCIS (Netherlands)

    Jonker, J.T.; Smit, J.W.A.; Hammer, S.; Snel, M.; Meer, van der R.; Lamb, H.J.; Mattijssen, F.B.J.; Mudde, C.M.; Jazet, I.M.; Dekkers, O.M.; Roos, de A.; Romijn, J.A.; Kersten, A.H.; Rensen, P.C.N.

    2013-01-01

    Background: Angiopoietin-like protein 4 (ANGPTL4) has been identified as an inhibitor of lipoprotein lipase. Preliminary data suggest that plasma nonesterified fatty acids (NEFAs) raise plasma ANGPTL4 concentrations in humans. Objective: The objective was to assess plasma ANGPTL4 concentrations

  5. Protein precipitation: an expedient procedure for the routine analysis of the plasma metabolites of [123I]IBZM

    International Nuclear Information System (INIS)

    Zea-Ponce, Yolanda; Laruelle, Marc

    1999-01-01

    Plasma metabolite analysis of the single photon emission computed tomography (SPECT) D 2 /D 3 receptor radiotracer (S)(-)-N-[(1-ethyl-2-pyrrolidinyl)methyl]-2-hydroxy-3-[ 123 I] iodo-6-methoxyb enzamide ([ 123 I]IBZM) is needed for the equilibrium analysis of the SPECT data, in brain imaging studies involving bolus plus constant infusion paradigm. The purpose of these experiments was to find an appropriate procedure to expedite this analysis during routine determinations. The procedure was applied to the plasma analysis of 22 human subjects. Each plasma sample was subjected to acetonitrile protein precipitation. After separation of the pellet, the acetonitrile fraction contained 91%±2% (n=88) of the mixture of labeled metabolites and parent compound. The recovery coefficient of unmetabolized [ 123 I]IBZM determined with an standard plasma sample was 95%±2% (n=22). The percent parent compound present in the extracted fraction, measured by high performance liquid chromatography, was 16%±9% (n=85) and the percent metabolites was 84%±9% (n=85). Free fraction determination (f 1 , fraction of radiotracer unbound to protein), was 4%±0.8% (n=22). Free fraction of parent was 15%±8% (n=85). The results indicate that acetonitrile protein precipitation is an adequate method for the analysis of the [ 123 I]IBZM plasma metabolites

  6. Concentration of total proteins in blood plasma of chickens hatched from irradiated eggs with low dose gamma radiation

    International Nuclear Information System (INIS)

    Vilic, M.; Kraljevic, P.; Miljanic, S.; Simpraga, M.

    2005-01-01

    It is known that low-dose ionising radiation may have stimulating effects on chickens. Low doses may also cause changes in the concentration of blood plasma total proteins, glucose and cholesterol in chickens. This study investigates the effects of low dose gamma-radiation on the concentration of total proteins in the blood plasma of chickens hatched from eggs irradiated with a dose of 0.15 Gy on incubation days 7 and 19. Results were compared with the control group (chickens hatched from non-irradiated eggs). After hatching, all other conditions were the same for both groups. Blood samples were drawn from the heart, and later from the wing vein on days 1, 3, 5, 7,10, 20, 30 and 42. The concentration of total proteins was determined spectrophotometrically using Boehringer Mannheim GmbH optimised kits. The concentration of total proteins in blood plasma in chickens hatched from eggs irradiated with 0.15 Gy on incubation day 7 showed a statistically significant decrease on the sampling day 3 (P less than 0.05) and 7 (P less than 0.01). The concentration of total proteins in blood plasma in chickens hatched from eggs irradiated with 0.15 Gy on incubation day 19 showed a statistically significant increase only on sampling day 1 (P less than 0.05). These results suggest that exposure of eggs to 0.15 Gy of gamma-radiation on the 7th and 19th day of incubation could produce different effects on the protein metabolism in chickens.(author)

  7. Reporter-Based Synthetic Genetic Array Analysis: A Functional Genomics Approach for Investigating Transcript or Protein Abundance Using Fluorescent Proteins in Saccharomyces cerevisiae.

    Science.gov (United States)

    Göttert, Hendrikje; Mattiazzi Usaj, Mojca; Rosebrock, Adam P; Andrews, Brenda J

    2018-01-01

    Fluorescent reporter genes have long been used to quantify various cell features such as transcript and protein abundance. Here, we describe a method, reporter synthetic genetic array (R-SGA) analysis, which allows for the simultaneous quantification of any fluorescent protein readout in thousands of yeast strains using an automated pipeline. R-SGA combines a fluorescent reporter system with standard SGA analysis and can be used to examine any array-based strain collection available to the yeast community. This protocol describes the R-SGA methodology for screening different arrays of yeast mutants including the deletion collection, a collection of temperature-sensitive strains for the assessment of essential yeast genes and a collection of inducible overexpression strains. We also present an alternative pipeline for the analysis of R-SGA output strains using flow cytometry of cells in liquid culture. Data normalization for both pipelines is discussed.

  8. Screening of plasma biomarkers in patients with unstable angina pectoris with proteomics analysis

    Directory of Open Access Journals (Sweden)

    Shui-wang HU

    2017-08-01

    Full Text Available Objective To analyze and compare the differentially expressed plasma proteins between patients with stable angina pectoris (SAP and unstable angina pectoris (UAP, and search for the biomarkers that maybe used for early diagnosis of UAP. Methods Sixty plasma samples were collected respectively from normal controls group (N group, SAP group and UAP group during Jun. 2014 to Apr. 2015 from the Third Affiliated Hospital of Southern Medical University. Ten samples (100μl of each group were selected randomly to pool into 3 groups severally. After removing high-abundance proteins from plasma, two- dimensional difference gel electrophoresis (DIGE was used to isolate the total proteins, and then the protein spots with more than 2-fold changes between UAP and SAP were picked up after the differential software analysis. Afterward, the varied proteins were identified by matrix assisted laser desorption ionization-time of flight/time of flight (MALDI-TOF/TOF mass spectrometry (MS. Finally, 40 plasma samples were collected respectively from N, SAP and UAP group, and the UAP specific differential proteins were selected to be verified by ELISA. Results A total of 10 varied protein spots with more than 2-fold changes in UAP and SAP were found including 9 up-regulated proteins and 1 down-regulated one. MS identification indicated that the up-regulated proteins included fibrinogen gamma chain (FGG, complement C4-B (C4B, immunoglobulin (Ig kappa chain C region (IGKC and hemoglobin subunit alpha (HBA1, whereas the down-regulated one was haptoglobin (HP. After comparing the varied proteins with that in N group, 2 specifically UAP-related proteins, IGKC and HP, were detected totally. IGKC was selected to validate by ELISA, and the corresponding results showed that IGKC was increased specifically in UAP plasma (P<0.05 when compared with N and SAP group, which was consistent with DIGE. Conclusion IGKC and HP have been detected as specifically related proteins to UAP

  9. Plasma Protein Profiles Differ Between Women Diagnosed with Cervical Intraepithelial Neoplasia (CIN 1 and 3

    Directory of Open Access Journals (Sweden)

    Edward E. Partridge

    2006-01-01

    Full Text Available Early detection of precancerous cells in the cervix and their clinical management is the main purpose of cervical cancer prevention and treatment programs. Cytological findings or testing for high risk (HR-human papillomavirus (HPV are inadequately sensitive for use in triage of women at high risk for cervical cancer. The current study is an exploratory study to identify candidate surface-enhanced laser desorption/ionization (SELDI time of flight (TOF mass spectrometry (MS protein profiles in plasma that may distinguish cervical intraepithelial neoplasia (CIN 3 from CIN 1 among women infected with HR-HPV. We evaluated the SELDI-TOF-MS plasma protein profiles of HR-HPV positive 32 women with CIN 3 (cases and 28 women with CIN1 (controls. Case-control status was kept blinded and triplicates of each sample and quality control plasma samples were randomized and after robotic sample preparations were run on WCX2 chips. After alignment of mass/charge (m-z values, an iterative method was used to develop a classifier on a training data set that had 28 cases and 22 controls. The classifier developed was used to classify the subjects in a test data set that has six cases and six controls. The classifier separated the cases from controls in the test set with 100% sensitivity and 100% specificity suggesting the possibility of using plasma SELDI protein profiles to identify women who are likely to have CIN 3 lesions.

  10. Experimental determination of net protein charge and A(tot) and K(a) of nonvolatile buffers in human plasma.

    Science.gov (United States)

    Staempfli, Henry R; Constable, Peter D

    2003-08-01

    The mechanism for an acid-base disturbance can be determined by using the strong ion approach, which requires species-specific values for the total concentration of plasma nonvolatile buffers (Atot) and the effective dissociation constant for plasma weak acids (Ka). The aim of this study was to experimentally determine Atot and Ka values for human plasma by using in vitro CO2 tonometry. Plasma Pco2 was systematically varied from 25 to 145 Torr at 37 degrees C, thereby altering plasma pH over the physiological range of 6.90-7.55, and plasma pH, Pco2, and concentrations of quantitatively important strong ions (Na+, K+, Ca2+, Mg2+, Cl-, lactate) and buffer ions (total protein, albumin, phosphate) were measured. Strong ion difference was estimated, and nonlinear regression was used to calculate Atot and Ka from the measured pH and Pco2 and estimated strong ion difference; the Atot and Ka values were then validated by using a published data set (Figge J, Rossing TH, and Fencl V, J Lab Clin Med 117: 453-467, 1991). The values (mean +/- SD) were as follows: Atot = 17.2 +/- 3.5 mmol/l (equivalent to 0.224 mmol/g of protein or 0.378 mmol/g of albumin); Ka = 0.80 +/- 0.60 x 10-7; negative log of Ka = 7.10. Mean estimates were obtained for strong ion difference (37 meq/l) and net protein charge (13+.0 meq/l). The experimentally determined values for Atot, Ka, and net protein charge should facilitate the diagnosis and treatment of acid-base disturbances in critically ill humans.

  11. Increased reactive oxygen species production and lower abundance of complex I subunits and carnitine palmitoyltransferase 1B protein despite normal mitochondrial respiration in insulin-resistant human skeletal muscle

    DEFF Research Database (Denmark)

    Lefort, Natalie; Glancy, Brian; Bowen, Benjamin

    2010-01-01

    the higher ROS production. Tandem mass spectrometry identified protein abundance differences per mitochondrial mass in insulin resistance, including lower abundance of complex I subunits and enzymes involved in the oxidation of branched-chain amino acids (BCAA) and fatty acids (e.g., carnitine...

  12. Molecular characterization of three Rhesus glycoproteins from the gills of the African lungfish, Protopterus annectens, and effects of aestivation on their mRNA expression levels and protein abundance.

    Directory of Open Access Journals (Sweden)

    You R Chng

    Full Text Available African lungfishes are ammonotelic in water. They can aestivate for long periods on land during drought. During aestivation, the gills are covered with dried mucus and ammonia excretion ceases. In fishes, ammonia excretion through the gills involves Rhesus glycoproteins (RhGP/Rhgp. This study aimed to obtain the complete cDNA coding sequences of rhgp from the gills of Protopterus annectens, and to determine their branchial mRNA and protein expression levels during the induction, maintenance and arousal phases of aestivation. Three isoforms of rhgp (rhag, rhbg and rhcg were obtained in the gills of P. annectens. Their complete cDNA coding sequences ranged between 1311 and 1398 bp, coding for 436 to 465 amino acids with estimated molecular masses between 46.8 and 50.9 kDa. Dendrogramic analyses indicated that Rhag was grouped closer to fishes, while Rhbg and Rhcg were grouped closer to tetrapods. During the induction phase, the protein abundance of Rhag, but not its transcript level, was down-regulated in the gills, suggesting that there could be a decrease in the release of ammonia from the erythrocytes to the plasma. Furthermore, the branchial transcript levels of rhbg and rhcg decreased significantly, in preparation for the subsequent shutdown of gill functions. During the maintenance phase, the branchial expression levels of rhag/Rhag, rhbg/Rhbg and rhcg/Rhcg decreased significantly, indicating that their transcription and translation were down-regulated. This could be part of an overall mechanism to shut down branchial functions and save metabolic energy used for transcription and translation. It could also be regarded as an adaptive response to stop ammonia excretion. During the arousal phase, it is essential for the lungfish to regain the ability to excrete ammonia. Indeed, the protein abundance of Rhag, Rhbg and Rhcg recovered to the corresponding control levels after 1 day or 3 days of recovery from 6 months of aestivation.

  13. 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...... the two dietary treatment groups correlated largely with the amino acid content of the two diets except for methionine, lysine and arginine, where the differences were more extreme than what would be expected from differences in dietary concentrations. The apparent protein digestibility coefficient...

  14. Characterization of the late embryogenesis abundant (LEA) proteins family and their role in drought stress tolerance in upland cotton.

    Science.gov (United States)

    Magwanga, Richard Odongo; Lu, Pu; Kirungu, Joy Nyangasi; Lu, Hejun; Wang, Xingxing; Cai, Xiaoyan; Zhou, Zhongli; Zhang, Zhenmei; Salih, Haron; Wang, Kunbo; Liu, Fang

    2018-01-15

    Late embryogenesis abundant (LEA) proteins are large groups of hydrophilic proteins with major role in drought and other abiotic stresses tolerance in plants. In-depth study and characterization of LEA protein families have been carried out in other plants, but not in upland cotton. The main aim of this research work was to characterize the late embryogenesis abundant (LEA) protein families and to carry out gene expression analysis to determine their potential role in drought stress tolerance in upland cotton. Increased cotton production in the face of declining precipitation and availability of fresh water for agriculture use is the focus for breeders, cotton being the backbone of textile industries and a cash crop for many countries globally. In this work, a total of 242, 136 and 142 LEA genes were identified in G. hirsutum, G. arboreum and G. raimondii respectively. The identified genes were classified into eight groups based on their conserved domain and phylogenetic tree analysis. LEA 2 were the most abundant, this could be attributed to their hydrophobic character. Upland cotton LEA genes have fewer introns and are distributed in all chromosomes. Majority of the duplicated LEA genes were segmental. Syntenic analysis showed that greater percentages of LEA genes are conserved. Segmental gene duplication played a key role in the expansion of LEA genes. Sixty three miRNAs were found to target 89 genes, such as miR164, ghr-miR394 among others. Gene ontology analysis revealed that LEA genes are involved in desiccation and defense responses. Almost all the LEA genes in their promoters contained ABRE, MBS, W-Box and TAC-elements, functionally known to be involved in drought stress and other stress responses. Majority of the LEA genes were involved in secretory pathways. Expression profile analysis indicated that most of the LEA genes were highly expressed in drought tolerant cultivars Gossypium tomentosum as opposed to drought susceptible, G. hirsutum. The tolerant

  15. Collisionless plasmas in astrophysics

    CERN Document Server

    Belmont, Gerard; Mottez, Fabrice; Pantellini, Filippo; Pelletier, Guy

    2013-01-01

    Collisionless Plasmas in Astrophysics examines the unique properties of media without collisions in plasma physics. Experts in this field, the authors present the first book to concentrate on collisionless conditions in plasmas, whether close or not to thermal equilibrium. Filling a void in scientific literature, Collisionless Plasmas in Astrophysics explains the possibilities of modeling such plasmas, using a fluid or a kinetic framework. It also addresses common misconceptions that even professionals may possess, on phenomena such as "collisionless (Landau) damping". Abundant illustrations

  16. Standard test method for isotopic abundance analysis of uranium hexafluoride and uranyl nitrate solutions by multi-collector, inductively coupled plasma-mass spectrometry

    CERN Document Server

    American Society for Testing and Materials. Philadelphia

    2014-01-01

    1.1 This test method covers the isotopic abundance analysis of 234U, 235U, 236U and 238U in samples of hydrolysed uranium hexafluoride (UF6) by inductively coupled plasma source, multicollector, mass spectrometry (ICP-MC-MS). The method applies to material with 235U abundance in the range of 0.2 to 6 % mass. This test method is also described in ASTM STP 1344. 1.2 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard. 1.3 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use.

  17. Determination of plasma albumin concentration in healthy and diseased turtles: a comparison of protein electrophoresis and the bromcresol green dye-binding method.

    Science.gov (United States)

    Müller, Kerstin; Brunnberg, Leo

    2010-03-01

    In reptile medicine, plasma chemistry analysis is widely used for the evaluation of an individual's health status. The standard method for the determination of plasma albumin concentration is protein electrophoresis combined with the determination of total protein concentration, but the bromcresol green (BCG) dye-binding method is also used. The reliability of the BCG method for the measurement of albumin concentration in reptiles is unknown. The aim of this study was to compare the plasma albumin values of turtles obtained by protein electrophoresis and the BCG method. Between March 2008 and September 2008, heparinized plasma samples from 16 clinically healthy and 10 diseased turtles of different species were collected. Plasma albumin concentrations were measured by protein electrophoresis and by the BCG method. The results of the 2 methods were compared using Passing-Bablok regression and Bland-Altman plots. Albumin concentration measured by BCG was weakly correlated with the corresponding protein electrophoretic values in all turtles (r(s)=.610, Palbumin concentration measured with the 2 different methods differed significantly in all turtles (P=.009; Wilcoxon's test) and in healthy turtles (P=.005) but not in diseased animals (P=.241). In the Bland-Altman plot a systematic error was found between the 2 methods in diseased turtles. Measurement of albumin by the BCG dye-binding method may lead to inaccurate results for plasma albumin concentration, especially in ill turtles. Therefore, for health assessment in turtles, albumin should be measured by protein electrophoresis.

  18. Circulating growth hormone (GH)-binding protein complex: a major constituent of plasma GH in man

    International Nuclear Information System (INIS)

    Baumann, G.; Amburn, K.; Shaw, M.A.

    1988-01-01

    The recent discovery of a specific binding protein for human GH (hGH) in human plasma suggests that hGH circulates in part as a complex in association with the binding protein(s). However, the magnitude of the complexed fraction prevailing under physiological conditions is unknown because of 1) dissociation of the complex during analysis and 2) potential differences in the binding characteristics of radiolabeled and native hGH. We conducted experiments designed to minimize dissociation during analysis (gel filtration in prelabeled columns, frontal analysis, and batch molecular sieving) with both native and radioiodinated hGH. All three methods yielded similar estimates for the complexed fraction. In normal plasma the bound fraction for 22 K hGH averaged 50.1% (range, 39-59%), that for 20 K hGH averaged 28.5% (range, 26-31%). Above a hGH level of about 20 ng/ml the bound fraction declines in concentration-dependent manner due to saturation of the binding protein. We conclude that a substantial part of circulating hGH is complexed with carrier proteins. This concept has important implications for the metabolism, distribution, and biological activity of hGH

  19. Investigation of SnSPR1, a novel and abundant surface protein of Sarcocystis neurona merozoites.

    Science.gov (United States)

    Zhang, Deqing; Howe, Daniel K

    2008-04-15

    An expressed sequence tag (EST) sequencing project has produced over 15,000 partial cDNA sequences from the equine pathogen Sarcocystis neurona. While many of the sequences are clear homologues of previously characterized genes, a significant number of the S. neurona ESTs do not exhibit similarity to anything in the extensive sequence databases that have been generated. In an effort to characterize parasite proteins that are novel to S. neurona, a seemingly unique gene was selected for further investigation based on its abundant representation in the collection of ESTs and the predicted presence of a signal peptide and glycolipid anchor addition on the encoded protein. The gene was expressed in E. coli, and monospecific polyclonal antiserum against the recombinant protein was produced by immunization of a rabbit. Characterization of the native protein in S. neurona merozoites and schizonts revealed that it is a low molecular weight surface protein that is expressed throughout intracellular development of the parasite. The protein was designated Surface Protein 1 (SPR1) to reflect its display on the outer surface of merozoites and to distinguish it from the ubiquitous SAG/SRS surface antigens of the heteroxenous Coccidia. Interestingly, infection assays in the presence of the polyclonal antiserum suggested that SnSPR1 plays some role in attachment and/or invasion of host cells by S. neurona merozoites. The work described herein represents a general template for selecting and characterizing the various unidentified gene sequences that are plentiful in the EST databases for S. neurona and other apicomplexans. Furthermore, this study illustrates the value of investigating these novel sequences since it can offer new candidates for diagnostic or vaccine development while also providing greater insight into the biology of these parasites.

  20. Effects of previous protein intake on rectal temperature, blood glucose, plasma thyroid hormone and minerals by laying hens during a forced molt

    International Nuclear Information System (INIS)

    Rodrigues, G.A.; Moraes, V.M.B.; Cherici, I; Furlan, R.L.; Macari, M.

    1991-01-01

    The effects of forced molting on blood glucose, rectal temperature, plasma T4, T3 and minerals were studied in hens previously fed rations with different protein contents (14, 17 and 20% crude protein). Blood samples were obtained from brachial veins for blood glucose, T4 and T3 were measured by radioimmunoassay, and plasma minerals were determined by atomic absorption spectroscopy. Blood glucose and rectal temperature were reduced during fasting regardless of previous protein intake. Pre molting T4 plasma level was higher in laying hens fed higher protein ration, but feed deprivation reduced T 4 and T 3 concentrations irrespective of protein intake, except T 4 level for 14% crude protein fed birds that increased during fasting. The data obtained in this experiment suggest that previous protein intake does not interfere with the metabolic changes during forced molt. (author). 19 refs, 1 fig, 4 tabs

  1. Effects of previous protein intake on rectal temperature, blood glucose, plasma thyroid hormone and minerals by laying hens during a forced molt

    Energy Technology Data Exchange (ETDEWEB)

    Rodrigues, G A; Moraes, V M.B.; Cherici, I; Furlan, R L; Macari, M [UNESP, Jaboticabal, SP (Brazil). Faculdade de Ciencias Agrarias e Veterinarias

    1991-12-01

    The effects of forced molting on blood glucose, rectal temperature, plasma T4, T3 and minerals were studied in hens previously fed rations with different protein contents (14, 17 and 20% crude protein). Blood samples were obtained from brachial veins for blood glucose, T4 and T3 were measured by radioimmunoassay, and plasma minerals were determined by atomic absorption spectroscopy. Blood glucose and rectal temperature were reduced during fasting regardless of previous protein intake. Pre molting T4 plasma level was higher in laying hens fed higher protein ration, but feed deprivation reduced T{sub 4} and T{sub 3} concentrations irrespective of protein intake, except T{sub 4} level for 14% crude protein fed birds that increased during fasting. The data obtained in this experiment suggest that previous protein intake does not interfere with the metabolic changes during forced molt. (author). 19 refs, 1 fig, 4 tabs.

  2. Cucumber metal tolerance protein CsMTP9 is a plasma membrane H⁺-coupled antiporter involved in the Mn²⁺ and Cd²⁺ efflux from root cells.

    Science.gov (United States)

    Migocka, Magdalena; Papierniak, Anna; Kosieradzka, Anna; Posyniak, Ewelina; Maciaszczyk-Dziubinska, Ewa; Biskup, Robert; Garbiec, Arnold; Marchewka, Tadeusz

    2015-12-01

    Members of the plant metal tolerance protein (MTP) family have been classified into three major groups - Zn-CDF, Mn-CDF and Zn/Fe-CDF - however, the selectivity of most of the MTPs has not been confirmed yet. Cucumber gene CsMTP9 encoding a putative CDF transporter homologous to members of the Mn-CDF cluster is expressed exclusively in roots. The relative abundance of CsMTP9 transcript and protein in roots is significantly increased under Mn excess and Cd. Immunolocalization with specific antibodies revealed that CsMTP9 is a plasma membrane transporter that localizes to the inner PM domain of root endodermal cells. The plasma membrane localization of CsMTP9 was confirmed by the expression of the fusion proteins of GFP (green fluorescent protein) and CsMTP9 in yeast and protoplasts prepared from Arabidopsis cells. In yeast, CsMTP9 transports Mn(2+) and Cd(2+) via a proton-antiport mechanism with an apparent Km values of approximately 10 μm and 2.5 μm for Mn(2+) and Cd(2+) , respectively. In addition, CsMTP9 expression in yeast rescues the Mn- and Cd-hypersensitive phenotypes through the enhanced efflux of Mn(2+) and Cd(2+) from yeast cells. Similarly, the overexpression of CsMTP9 in A. thaliana confers increased resistance of plants to Mn excess and Cd but not to other heavy metals and leads to the enhanced translocation of manganese and cadmium from roots to shoots. These findings indicate that CsMTP9 is a plasma membrane H(+) -coupled Mn(2+) and Cd(2+) antiporter involved in the efflux of manganese and cadmium from cucumber root cells by the transport of both metals from endodermis into vascular cylinder. © 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd.

  3. Plasma membrane fatty acid-binding protein and mitochondrial glutamic-oxaloacetic transaminase of rat liver are related

    Energy Technology Data Exchange (ETDEWEB)

    Berk, P.D.; Potter, B.J.; Sorrentino, D.; Zhou, S.L.; Isola, L.M.; Stump, D.; Kiang, C.L.; Thung, S. (Mount Sinai School of Medicine, New York, NY (USA)); Wada, H.; Horio, Y. (Univ. of Osaka (Japan))

    1990-05-01

    The hepatic plasma membrane fatty acid-binding protein (h-FABP{sub PM}) and the mitochondrial isoenzyme of glutamic-oxaloacetic transaminase (mGOT) of rat liver have similar amino acid compositions and identical amino acid sequences for residues 3-24. Both proteins migrate with an apparent molecular mass of 43 kDa on SDS/polyacrylamide gel electrophoresis, have a similar pattern of basic charge isomers on isoelectric focusing, are eluted similarly from four different high-performance liquid chromatographic columns, have absorption maxima at 435 nm under acid conditions and 354 nm at pH 8.3, and bind oleate. Sinusoidally enriched liver plasma membranes and purified h-FABP{sub PM} have GOT enzymatic activity. Monospecific rabbit antiserum against h-FABP{sub PM} reacts on Western blotting with mGOT, and vice versa. Antisera against both proteins produce plasma membrane immunofluorescence in rat hepatocytes and selectively inhibit the hepatocellular uptake of ({sup 3}H)oleate but not that of ({sup 35}S)sulfobromophthalein or ({sup 14}C)taurocholate. The inhibition of oleate uptake produced by anti-h-FABP{sub PM} can be eliminated by preincubation of the antiserum with mGOT; similarly, the plasma membrane immunofluorescence produced by either antiserum can be eliminated by preincubation with the other antigen. These data suggest that h-FABP{sub PM} and mGOT are closely related.

  4. Estradiol-mediated internalisation of the non-activated estrogen receptor from the goat uterine plasma membrane: identification of the proteins involved.

    Science.gov (United States)

    Sreeja, S; Thampan, Raghava Varman

    2004-04-01

    An indirect approach has been made to study the molecular details associated with the estradiol-induced internalisation of the non-activated estrogen receptor (naER) from the goat uterine plasma membrane. The internalisation of naER appears to be an energy dependent process. Exposure of the plasma membrane to estradiol results in the activation of a Mg2+ dependent ATPase associated with the membrane fraction. Presence of quercetin in the medium prevented the activation of the Mg2+ ATPase as well as the dissociation of naER from the plasma membrane. Using isolated plasma membrane preparations it has been possible to identify the proteins which interact with naER during various stages of its internalisation. The main proteins identified are: (1) a 58 kDa protein, p58, which apparently recognizes the nuclear localization signals on the naER and transports it to the nucleus: (2) hsp70: (3) hsp90, the functional roles of which remain unknown at this stage; (4) a 50 kDa protein associated with the clathrin coated vesicles, presumed to be involved in recognizing the tyrosine based internalisation signals on the naER; (5) actin which mediates the plasma membrane-to-nucleus movement of the naER-p58 complex.

  5. 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

    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...... 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...

  6. Vesicle-associated membrane protein 2 mediates trafficking of α5β1 integrin to the plasma membrane

    International Nuclear Information System (INIS)

    Hasan, Nazarul; Hu, Chuan

    2010-01-01

    Integrins are major receptors for cell adhesion to the extracellular matrix (ECM). As transmembrane proteins, the levels of integrins at the plasma membrane or the cell surface are ultimately determined by the balance between two vesicle trafficking events: endocytosis of integrins at the plasma membrane and exocytosis of the vesicles that transport integrins. Here, we report that vesicle-associated membrane protein 2 (VAMP2), a SNARE protein that mediates vesicle fusion with the plasma membrane, is involved in the trafficking of α5β1 integrin. VAMP2 was present on vesicles containing endocytosed β1 integrin. Small interfering RNA (siRNA) silencing of VAMP2 markedly reduced cell surface α5β1 and inhibited cell adhesion and chemotactic migration to fibronectin, the ECM ligand of α5β1, without altering cell surface expression of α2β1 integrin or α3β1 integrin. By contrast, silencing of VAMP8, another SNARE protein, had no effect on cell surface expression of the integrins or cell adhesion to fibronectin. In addition, VAMP2-mediated trafficking is involved in cell adhesion to collagen but not to laminin. Consistent with disruption of integrin functions in cell proliferation and survival, VAMP2 silencing diminished proliferation and triggered apoptosis. Collectively, these data indicate that VAMP2 mediates the trafficking of α5β1 integrin to the plasma membrane and VAMP2-dependent integrin trafficking is critical in cell adhesion, migration and survival.

  7. Plasma urea nitrogen and progesterone concentrations and follicular dynamics in ewes fed proteins of different degradability

    Directory of Open Access Journals (Sweden)

    Gustavo Bianchi Lazarin

    2012-07-01

    Full Text Available The effects of overfeeding with protein of different degradability on body condition, plasma urea nitrogen and progesterone concentrations, ovulation number and follicular dynamics were assessed in Santa Ines ewes. Twelve ewes were assigned to a randomized block design according to body weight and received overfeeding with soybean meal or with corn gluten meal or maintenance diet for 28 days before ovulation and during the next estrous cycle. Blood samples were taken on days 7, 14, 21, and 28 after the beginning of treatments for analysis of plasma urea nitrogen and on days 3, 6, 9, 12, and 15 into the estrous cycle for analysis of plasma urea nitrogen and progesterone. Follicular dynamics was monitored daily by ultrasound during one estrous cycle. Dry matter and crude protein intake, weight gain, plasma urea nitrogen concentration before ovulation, number of ovulations, diameter of the largest follicle of the 1st and of the 2nd waves and the growth rate of the largest follicle of the 1st wave were higher in the ewes that received overfeeding. The growth rate of the largest follicle of the 3rd wave was higher in the ewes fed maintenance diet. The back fat thickness, plasma urea nitrogen before ovulation and progesterone concentrations, diameter of the largest follicle of the 2nd wave and growth rate of the largest follicle of the 3rd wave were higher in ewes that received overfeeding with soybean meal. The growth rate of the largest follicle of the 1st wave was higher in ewes that received overfeeding with corn gluten meal. Overfeeding with protein-rich feeds may increase the ovulation number and with soybean meal, it may be effective in increasing plasma progesterone concentration in ewes.

  8. An evaluation of multiplex bead-based analysis of cytokines and soluble proteins in archived lithium heparin plasma, EDTA plasma and serum samples

    DEFF Research Database (Denmark)

    Brøndum, Line; Sørensen, Brita Singers; Eriksen, Jesper Grau

    2016-01-01

    -13, and VEGF, LH plasma levels vs. EDTA plasma: IL-2 and IL-4. CONCLUSION: Stored serum, LH plasma, and EDTA plasma from clinical trials can be used for analysis of circulating cytokines and proteins. Variations in measurements occur, but are within reasonable ranges. The optimal type of media...... in plasma and serum from 86 head and neck cancer patients and 33 controls were evaluated: EGFR, leptin, OPN, VEGFR-1, VEGFR-2, IL-2, IL-13, PDGF-bb, TNF, PAI-1, SDF-1a, IL-4, IL-6, IL-8, eotaxin, G-CSF, VEGF, GRO-a, and HGF. RESULTS: The correlation between measurements of the same samples analyzed...... on different dates was reasonable. However, samples run on different dates could exhibit different absolute values. The 75th percentile of the fold differences for samples run on different dates was 2.2. No significant difference was found between one and four freeze-thaw cycles (except for HGF...

  9. High Protein Intake Does Not Prevent Low Plasma Levels of Conditionally Essential Amino Acids in Very Preterm Infants Receiving Parenteral Nutrition.

    Science.gov (United States)

    Morgan, Colin; Burgess, Laura

    2017-03-01

    We have shown that increasing protein intake using a standardized, concentrated, added macronutrients parenteral (SCAMP) nutrition regimen improves head growth in very preterm infants (VPIs) compared with a control parenteral nutrition (PN) regimen. VPIs are at risk of conditionally essential amino acid (CEAA) deficiencies because of current neonatal PN amino acid (AA) formulations. We hypothesized that the SCAMP regimen would prevent low plasma levels of CEAAs. To compare the plasma AA profiles at approximately day 9 of life in VPIs receiving SCAMP vs a control PN regimen. VPIs (parenteral and enteral protein, energy, and individual AA intake and the first plasma AA profile. Plasma profiles of the 20 individual protogenic AA levels were measured using ion exchange chromatography. Plasma AA profiles were obtained at median (interquartile range [IQR]) age of 9 (8-10) days in both SCAMP (n = 59) and control (n = 67) groups after randomizing 150 VPIs. Median (IQR) plasma levels of individual essential AAs were higher than the reference population mean (RPM) in both groups, especially for threonine. SCAMP infants had higher plasma levels of essential AAs than did the controls. Median (IQR) plasma levels of glutamine, arginine, and cysteine (CEAAs) were lower than the RPM in both groups. Plasma AA levels in PN-dependent VPIs indicate there is an imbalance in essential and CEAA provision in neonatal PN AA formulations that is not improved by increasing protein intake.

  10. Comparative study of the Ar and He atmospheric pressure plasmas on E-cadherin protein regulation for plasma-mediated transdermal drug delivery

    Science.gov (United States)

    Lee, Hyun Young; Hae Choi, Jeong; Hong, Jin Woo; Kim, Gyoo Cheon; Lee, Hae June

    2018-05-01

    The effects of argon plasma (ArP) and helium plasma (HeP) jets on E-cadherin protein function have been tested in order to choose the working gas for a better plasma-mediated transdermal drug delivery. The plasma-mediated changes of the E-cadherin function and the skin penetration efficacies of epidermal growth factor (EGF) were monitored in vitro using HaCaT human keratinocytes and in vivo using hairless mice. The ArP showed higher efficacy for E-cadherin regulation and EGF absorption than HeP under the same applied voltage and the same gas flow rate. The ArP generates higher volume power density, higher discharge current peak, and more reactive species than HeP, especially for OH with the same operating parameters. Moreover, the effect of ArP on E-cadherin function was blocked by the use of a grounded metal mesh. Taken together, this study presents the possibility that the synergetic effect of negative charges with radicals plays an important role in plasma-mediated E-cadherin regulation, which leads to enhanced transdermal drug delivery.

  11. Measurements of protein turnover in man with nitrogen-15

    International Nuclear Information System (INIS)

    Garlick, P.J.; Waterlow, J.C.

    1977-01-01

    Past and present methods of measuring total body protein turnover with 15 N-labelled compounds are reviewed, and a short assessment made of results achieved. Protein turnover in man was first measured by Sprinson and Rittenberg (1949) using 15 N glycine as tracer. The rate of turnover was calculated from the fraction of the dose excreted in urine in the days following a single oral dose of the isotope. In the following years a number of similar methods were devised but interest waned, perhaps because consistent changes in turnover rate under different conditions were not detected. A revival of interest came with the measurement of changes in protein turnover in malnourished and recovering children by Picou and Taylor-Roberts (1969). Nitrogen-15-glycine was infused and measurements were made of the abundance of 15 N in urinary urea, which reached a plateau in about 24 hours. Turnover rates were calculated from the proportion of the infused dose excreted in urea after the plateau had been reached. This method has also been used successfully on adults when the isotope has been given as three-hourly oral doses. In adults, however, plateau labelling in urea is not usually reached for two days. In our laboratory the use of urinary ammonia, which reaches a plateau more quickly than urea, has been examined. In addition, comparisons have been made between constant infusion and single dose of isotope. Halliday and McKeran (1975) infused 15 N-lysine and measured the 15 N abundance in the free lysine of plasma, which reached a plateau in about 12 hours. Turnover in the whole body and turnover of muscle proteins and of plasma albumin were estimated separately. These methods of measuring protein turnover with 15 N are described and results reviewed of the effects of food intake, nutritional status and age. (author)

  12. Blimp-1 controls plasma cell function through regulation of immunoglobulin secretion and the unfolded protein response

    Science.gov (United States)

    Tellier, Julie; Shi, Wei; Minnich, Martina; Liao, Yang; Crawford, Simon; Smyth, Gordon K; Kallies, Axel; Busslinger, Meinrad; Nutt, Stephen L

    2015-01-01

    Plasma cell differentiation requires silencing of B cell transcription, while establishing antibody-secretory function and long-term survival. The transcription factors Blimp-1 and IRF4 are essential for plasma cell generation, however their function in mature plasma cells has remained elusive. We have found that while IRF4 was essential for plasma cell survival, Blimp-1 was dispensable. Blimp-1-deficient plasma cells retained their transcriptional identity, but lost the ability to secrete antibody. Blimp-1 regulated many components of the unfolded protein response (UPR), including XBP-1 and ATF6. The overlap of Blimp-1 and XBP-1 function was restricted to the UPR, with Blimp-1 uniquely regulating mTOR activity and plasma cell size. Thus, Blimp-1 is required for the unique physiological capacity of plasma cells that enables the secretion of protective antibody. PMID:26779600

  13. Study of factors that interfere in the labelling process of erythrocytes and plasma proteins with Technetium-99m

    International Nuclear Information System (INIS)

    Gutfilen, Bianca

    1989-01-01

    The labelling of red blood cells (RBC) with technetium-99m (Tc-99m) depends on several factors, as the stannous ion (Sn++) concentration, time, temperature, the presence of plasma proteins (PP) and others. However the Sn++ concentration seems to be the most important factor; probably because the uptake of this reducing agent by RBC is limited. The excess of Sn++ in extracellular medium can determine the labelling of PP. the modifications of RBC at 50 deg C described in the literature, the possibility of labelling RBC with Tc-99m at this temperature and experimental results obtained made it possible to perform spleen selective scintigraphy through a simple technique with few manipulations. The effect of gentamicin, nifedipine and verapamil in the labelling of RBC and plasma proteins with Tc-99m was studied because of similarities between Ca++ and Sn++. The results show that, under some conditions, these drugs are capable to alter this Tc-99m incorporation. The modification of the ionic distribution determined by these drugs or the blockage of Sn++ and/or Tc-99m or the fact that they bind theirselves to plasma proteins, or the possibility of the labelling of these drugs, are factors that can interfere in the labelling process of red blood cells and plasma proteins with Tc-99m. (author)

  14. Chernobyl seed project. Advances in the identification of differentially abundant proteins in a radio-contaminated environment.

    Science.gov (United States)

    Rashydov, Namik M; Hajduch, Martin

    2015-01-01

    Plants have the ability to grow and successfully reproduce in radio-contaminated environments, which has been highlighted by nuclear accidents at Chernobyl (1986) and Fukushima (2011). The main aim of this article is to summarize the advances of the Chernobyl seed project which has the purpose to provide proteomic characterization of plants grown in the Chernobyl area. We present a summary of comparative proteomic studies on soybean and flax seeds harvested from radio-contaminated Chernobyl areas during two successive generations. Using experimental design developed for radio-contaminated areas, altered abundances of glycine betaine, seed storage proteins, and proteins associated with carbon assimilation into fatty acids were detected. Similar studies in Fukushima radio-contaminated areas might complement these data. The results from these Chernobyl experiments can be viewed in a user-friendly format at a dedicated web-based database freely available at http://www.chernobylproteomics.sav.sk.

  15. Nitric oxide circulates in mammalian plasma primarily as an S-nitroso adduct of serum albumin.

    Science.gov (United States)

    Stamler, J S; Jaraki, O; Osborne, J; Simon, D I; Keaney, J; Vita, J; Singel, D; Valeri, C R; Loscalzo, J

    1992-01-01

    We have recently shown that nitric oxide or authentic endothelium-derived relaxing factor generated in a biologic system reacts in the presence of specific protein thiols to form S-nitrosoprotein derivatives that have endothelium-derived relaxing factor-like properties. The single free cysteine of serum albumin, Cys-34, is particularly reactive toward nitrogen oxides (most likely nitrosonium ion) under physiologic conditions, primarily because of its anomalously low pK; given its abundance in plasma, where it accounts for approximately 0.5 mM thiol, we hypothesized that this plasma protein serves as a reservoir for nitric oxide produced by the endothelial cell. To test this hypothesis, we developed a methodology, which involves UV photolytic cleavage of the S--NO bond before reaction with ozone for chemiluminescence detection, with which to measure free nitric oxide, S-nitrosothiols, and S-nitrosoproteins in biologic systems. We found that human plasma contains approximately 7 microM S-nitrosothiols, of which 96% are S-nitrosoproteins, 82% of which is accounted for by S-nitroso-serum albumin. By contrast, plasma levels of free nitric oxide are only in the 3-nM range. In rabbits, plasma S-nitrosothiols are present at approximately 1 microM; 60 min after administration of NG-monomethyl-L-arginine at 50 mg/ml, a selective and potent inhibitor of nitric oxide synthetases, S-nitrosothiols decreased by approximately 40% (greater than 95% of which were accounted for by S-nitrosoproteins, and approximately 80% of which was S-nitroso-serum albumin); this decrease was accompanied by a concomitant increase in mean arterial blood pressure of 22%. These data suggest that naturally produced nitric oxide circulates in plasma primarily complexed in S-nitrosothiol species, principal among which is S-nitroso-serum albumin. This abundant, relatively long-lived adduct likely serves as a reservoir with which plasma levels of highly reactive, short-lived free nitric oxide can be

  16. Informing the Human Plasma Protein Binding of ...

    Science.gov (United States)

    The free fraction of a xenobiotic in plasma (Fub) is an important determinant of chemical adsorption, distribution, metabolism, elimination, and toxicity, yet experimental plasma protein binding data is scarce for environmentally relevant chemicals. The presented work explores the merit of utilizing available pharmaceutical data to predict Fub for environmentally relevant chemicals via machine learning techniques. Quantitative structure-activity relationship (QSAR) models were constructed with k nearest neighbors (kNN), support vector machines (SVM), and random forest (RF) machine learning algorithms from a training set of 1045 pharmaceuticals. The models were then evaluated with independent test sets of pharmaceuticals (200 compounds) and environmentally relevant ToxCast chemicals (406 total, in two groups of 238 and 168 compounds). The selection of a minimal feature set of 10-15 2D molecular descriptors allowed for both informative feature interpretation and practical applicability domain assessment via a bounded box of descriptor ranges and principal component analysis. The diverse pharmaceutical and environmental chemical sets exhibit similarities in terms of chemical space (99-82% overlap), as well as comparable bias and variance in constructed learning curves. All the models exhibit significant predictability with mean absolute errors (MAE) in the range of 0.10-0.18 Fub. The models performed best for highly bound chemicals (MAE 0.07-0.12), neutrals (MAE 0

  17. Foetal life protein restriction in male mink (Neovison vison) kits lowers post-weaning protein oxidation and the relative abundance of hepatic fructose-1,6-bisphosphatase mRNA

    DEFF Research Database (Denmark)

    Matthiesen, Connie Marianne Frank; Blache, D.; Thomsen, Preben Dybdahl

    2012-01-01

    Foetal life malnutrition has been studied intensively in a number of animal models. Results show that especially foetal life protein malnutrition can lead to metabolic changes later in life. This might be of particular importance for strict carnivores, for example, cat and mink (Neovison vison...... born to mothers fed either a low-protein diet (LP), that is, 14% of metabolizable energy (ME) from protein (foetal low – FL), n = 16, or an adequate-protein (AP) diet, that is, 29% of ME from protein (foetal adequate – FA), n = 16) in the last 16.3 ± 1.8 days of pregnancy were used. The FL offspring...... had lower birth weight and lower relative abundance of fructose-1,6-bisphosphatase (Fru-1,6-P2ase) and pyruvate kinase mRNA in foetal hepatic tissue than FA kits. The mothers were fed a diet containing adequate protein until weaning. At weaning (7 weeks of age), half of the kits from each foetal...

  18. Effects of Soy-Germ Protein on Catalase Activity of Plasma and Erythocyte of Metabolic Syndrome Women

    Directory of Open Access Journals (Sweden)

    HERY WINARSI

    2015-01-01

    Full Text Available Oxidative stress always accompany patients with metabolic syndrome (MS. Several researchers reported that soy-protein is able to decrease oxidative stress level. However, there is no report so far about soy-germ protein in relation to its potential to the decrease oxidative stress level of MS patients. The aim of this study was to explore the potential of soy-germ protein on activity of catalase enzyme in blood's plasma as well as erythrocytes of MS patients. Double-blind randomized clinical trial was used as an experimental study. Thirty respondents were included in this study with MS, normal level blood sugar, low-HDL cholesterol but high in triglyceride, 40-65 years old, Body Mass Index > 25 kg/m2, live in Purwokerto and agreed to sign the informed consent. They were randomly grouped into 3 different groups, 10 each: Group I, was given special milk that contains soy-germ protein and Zn; Group II, soy-germ protein, while Group III was placebo; for two consecutive months. Data were taken from blood samples in 3 different periods i.e. 0, 1, and 2 months after treatment. Two months after treatment, there was an increase from 5.36 to 20.17 IU/mg (P = 0.028 in activity of catalase enzyme in blood's plasma respondents who consumed milk containing soy-germ protein with or without Zn. A similar trend of catalase activity, but at a lower level, was also noticed in erythrocyte; which increased from 88.31 to 201.11 IU/mg (P = 0.013. The increase in activity of catalase enzyme in blood's plasma was 2.2 times higher than that in erythrocytes.

  19. Ratio of dietary rumen degradable protein to rumen undegradable protein affects nitrogen partitioning but does not affect the bovine milk proteome produced by mid-lactation Holstein dairy cows.

    Science.gov (United States)

    Tacoma, R; Fields, J; Ebenstein, D B; Lam, Y-W; Greenwood, S L

    2017-09-01

    Little is known about the bovine milk proteome or whether it can be affected by diet. The objective of this study was to determine if the dietary rumen degradable protein (RDP):rumen undegradable protein (RUP) ratio could alter the bovine milk proteome. Six Holstein cows (parity: 2.5 ± 0.8) in mid lactation were blocked by days in milk (80 ± 43 d in milk) and milk yield (57.5 ± 6.0 kg) and randomly assigned to treatment groups. The experiment was conducted as a double-crossover design consisting of three 21-d periods. Within each period, treatment groups received diets with either (1) a high RDP:RUP ratio (RDP treatment: 62.4:37.6% of crude protein) or (2) a low RDP:RUP ratio (RUP treatment: 51.3:48.7% of crude protein). Both diets were isonitrogenous and isoenergetic (crude protein: 18.5%, net energy for lactation: 1.8 Mcal/kg of dry matter). To confirm N and energy status of cows, dry matter intake was determined daily, rumen fluid samples were collected for volatile fatty acid analysis, blood samples were collected for plasma glucose, β-hydroxybutyrate, urea nitrogen, and fatty acid analysis, and total 24-h urine and fecal samples were collected for N analysis. Milk samples were collected to determine the general milk composition and the protein profile. Milk samples collected for high-abundance protein analysis were subjected to HPLC analysis to determine the content of α-casein, β-casein, and κ-casein, as well as α-lactalbumin and β-lactoglobulin. Samples collected for low-abundance protein analysis were fractionated, enriched using ProteoMiner treatment, and separated using sodium dodecyl sulfate-PAGE. After excision and digestion, the peptides were analyzed using liquid chromatography (LC) tandem mass spectrometry (MS/MS). The LC-MS/MS data were analyzed using PROC GLIMMIX of SAS (version 9.4, SAS Institute Inc., Cary, NC) and adjusted using the MULTTEST procedure. All other parameters were analyzed using PROC MIXED of SAS. No treatment differences

  20. Evaluation of metabolism, plasma protein binding and other biological parameters after administration of (−)-[18 F]Flubatine in humans

    International Nuclear Information System (INIS)

    Patt, Marianne; Becker, Georg A.; Grossmann, Udo; Habermann, Bernd; Schildan, Andreas; Wilke, Stephan; Deuther-Conrad, Winnie; Graef, Susanne; Fischer, Steffen; Smits, René; Hoepping, Alexander; Wagenknecht, Gudrun; Steinbach, Jörg; Gertz, Hermann-Josef; Hesse, Swen; Schönknecht, Peter

    2014-01-01

    Introduction: (−)-[ 18 F]Flubatine is a PET tracer with high affinity and selectivity for the nicotinic acetylcholine α 4 β 2 receptor subtype. A clinical trial assessing the availability of this subtype of nAChRs was performed. From a total participant number of 21 Alzheimer’s disease (AD) patients and 20 healthy controls (HCs), the following parameters were determined: plasma protein binding, metabolism and activity distribution between plasma and whole blood. Methods: Plasma protein binding and fraction of unchanged parent compound were assessed by ultracentrifugation and HPLC, respectively. The distribution of radioactivity (parent compound + metabolites) between plasma and whole blood was determined ex vivo at different time-points after injection by gamma counting after separation of whole blood by centrifugation into the cellular and non-cellular components. In additional experiments in vitro, tracer distribution between these blood components was assessed for up to 90 min. Results: A fraction of 15% ± 2% of (−)-[ 18 F]Flubatine was found to be bound to plasma proteins. Metabolic degradation of (−)-[ 18 F]Flubatine was very low, resulting in almost 90% unchanged parent compound at 90 min p.i. with no significant difference between AD and HC. The radioactivity distribution between plasma and whole blood changed in vivo only slightly over time from 0.82 ± 0.03 at 3 min p.i. to 0.87 ± 0.03 at 270 min p.i. indicating the contribution of only a small amount of metabolites. In vitro studies revealed that (−)-[ 18 F]Flubatine was instantaneously distributed between cellular and non-cellular blood parts. Discussion: (−)-[ 18 F]Flubatine exhibits very favourable characteristics for a PET radiotracer such as slow metabolic degradation and moderate plasma protein binding. Equilibrium of radioactivity distribution between plasma and whole blood is reached instantaneously and remains almost constant over time allowing both convenient sample handling and

  1. Determination of protein carbonyls in plasma, cell extracts, tissue homogenates, isolated proteins: Focus on sample preparation and derivatization conditions.

    Science.gov (United States)

    Weber, Daniela; Davies, Michael J; Grune, Tilman

    2015-08-01

    Protein oxidation is involved in regulatory physiological events as well as in damage to tissues and is thought to play a key role in the pathophysiology of diseases and in the aging process. Protein-bound carbonyls represent a marker of global protein oxidation, as they are generated by multiple different reactive oxygen species in blood, tissues and cells. Sample preparation and stabilization are key steps in the accurate quantification of oxidation-related products and examination of physiological/pathological processes. This review therefore focuses on the sample preparation processes used in the most relevant methods to detect protein carbonyls after derivatization with 2,4-dinitrophenylhydrazine with an emphasis on measurement in plasma, cells, organ homogenates, isolated proteins and organelles. Sample preparation, derivatization conditions and protein handling are presented for the spectrophotometric and HPLC method as well as for immunoblotting and ELISA. An extensive overview covering these methods in previously published articles is given for researchers who plan to measure protein carbonyls in different samples. © 2015 Published by Elsevier Ltd.

  2. Platelet adhesion and plasma protein adsorption control of collagen surfaces by He+ ion implantation

    International Nuclear Information System (INIS)

    Kurotobi, K.; Suzuki, Y.; Nakajima, H.; Suzuki, H.; Iwaki, M.

    2003-01-01

    He + ion implanted collagen-coated tubes with a fluence of 1 x 10 14 ions/cm 2 were exhibited antithrombogenicity. To investigate the mechanisms of antithrombogenicity of these samples, plasma protein adsorption assay and platelet adhesion experiments were performed. The adsorption of fibrinogen (Fg) and von Willebrand factor (vWf) was minimum on the He + ion implanted collagen with a fluence of 1 x 10 14 ions/cm 2 . Platelet adhesion (using platelet rich plasma) was inhibited on the He + ion implanted collagen with a fluence of 1 x 10 14 ions/cm 2 and was accelerated on the untreated collagen and ion implanted collagen with fluences of 1 x 10 13 , 1 x 10 15 and 1 x 10 16 ions/cm 2 . Platelet activation with washed platelets was observed on untreated collagen and He + ion implanted collagen with a fluence of 1 x 10 14 ions/cm 2 and was inhibited with fluences of 1 x 10 13 , 1 x 10 15 and 1 x 10 16 ions/cm 2 . Generally, platelets can react with a specific ligand inside the collagen (GFOGER sequence). The results of platelets adhesion experiments using washed platelets indicated that there were no ligands such as GFOGER on the He + ion implanted collagen over a fluence of 1 x 10 13 ions/cm 2 . On the 1 x 10 14 ions/cm 2 implanted collagen, no platelet activation was observed due to the influence of plasma proteins. >From the above, it is concluded that the decrease of adsorbed Fg and vWf caused the antithrombogenicity of He + ion implanted collagen with a fluence of 1 x 10 14 ions/cm 2 and that plasma protein adsorption took an important role repairing the graft surface

  3. Multi-site assessment of the precision and reproducibility of multiple reaction monitoring–based measurements of proteins in plasma

    Science.gov (United States)

    Addona, Terri A; Abbatiello, Susan E; Schilling, Birgit; Skates, Steven J; Mani, D R; Bunk, David M; Spiegelman, Clifford H; Zimmerman, Lisa J; Ham, Amy-Joan L; Keshishian, Hasmik; Hall, Steven C; Allen, Simon; Blackman, Ronald K; Borchers, Christoph H; Buck, Charles; Cardasis, Helene L; Cusack, Michael P; Dodder, Nathan G; Gibson, Bradford W; Held, Jason M; Hiltke, Tara; Jackson, Angela; Johansen, Eric B; Kinsinger, Christopher R; Li, Jing; Mesri, Mehdi; Neubert, Thomas A; Niles, Richard K; Pulsipher, Trenton C; Ransohoff, David; Rodriguez, Henry; Rudnick, Paul A; Smith, Derek; Tabb, David L; Tegeler, Tony J; Variyath, Asokan M; Vega-Montoto, Lorenzo J; Wahlander, Åsa; Waldemarson, Sofia; Wang, Mu; Whiteaker, Jeffrey R; Zhao, Lei; Anderson, N Leigh; Fisher, Susan J; Liebler, Daniel C; Paulovich, Amanda G; Regnier, Fred E; Tempst, Paul; Carr, Steven A

    2010-01-01

    Verification of candidate biomarkers relies upon specific, quantitative assays optimized for selective detection of target proteins, and is increasingly viewed as a critical step in the discovery pipeline that bridges unbiased biomarker discovery to preclinical validation. Although individual laboratories have demonstrated that multiple reaction monitoring (MRM) coupled with isotope dilution mass spectrometry can quantify candidate protein biomarkers in plasma, reproducibility and transferability of these assays between laboratories have not been demonstrated. We describe a multilaboratory study to assess reproducibility, recovery, linear dynamic range and limits of detection and quantification of multiplexed, MRM-based assays, conducted by NCI-CPTAC. Using common materials and standardized protocols, we demonstrate that these assays can be highly reproducible within and across laboratories and instrument platforms, and are sensitive to low µg/ml protein concentrations in unfractionated plasma. We provide data and benchmarks against which individual laboratories can compare their performance and evaluate new technologies for biomarker verification in plasma. PMID:19561596

  4. Protein covalent immobilization via its scarce thiol versus abundant amine groups: Effect on orientation, cell binding domain exposure and conformational lability.

    Science.gov (United States)

    Ba, O M; Hindie, M; Marmey, P; Gallet, O; Anselme, K; Ponche, A; Duncan, A C

    2015-10-01

    Quantity, orientation, conformation and covalent linkage of naturally cell adhesive proteins adsorbed or covalently linked to a surface, are known to influence the preservation of their subsequent long term cell adhesion properties and bioactivity. In the present work, we explore two different strategies for the covalent linking of plasma fibronectin (pFN) - used as a cell adhesive model protein, onto a polystyrene (PS) surface. One is aimed at tethering the protein to the surface in a semi-oriented fashion (via one of the 4 free thiol reactive groups on the protein) with a heterofunctional coupling agent (SSMPB method). The other aims to immobilize the protein in a more random fashion by reaction between the abundant pendant primary amine bearing amino acids of the pFN and activated carboxylic surface functions obtained after glutaric anhydride surface treatment (GA method). The overall goal will be to verify the hypothesis of a correlation between covalent immobilization of a model cell adhesive protein to a PS surface in a semi-oriented configuration (versus randomly oriented) with promotion of enhanced exposure of the protein's cell binding domain. This in turn would lead to enhanced cell adhesion. Ideally the goal is to elaborate substrates exhibiting a long term stable protein monolayer with preserved cell adhesive properties and bioactivity for biomaterial and/or cell adhesion commercial plate applications. However, the initial restrictive objective of this paper is to first quantitatively and qualitatively investigate the reversibly (merely adsorbed) versus covalently irreversibly bound protein to the surface after the immobilization procedure. Although immobilized surface amounts were similar (close to the monolayer range) for all immobilization approaches, covalent grafting showed improved retention and stronger "tethering" of the pFN protein to the surface (roughly 40%) after SDS rinsing compared to that for mere adsorption (0%) suggesting an added value

  5. Thermolysin damages animal life through degradation of plasma proteins enhanced by rapid cleavage of serpins and activation of proteases.

    Science.gov (United States)

    Kong, Lulu; Lu, Anrui; Guan, Jingmin; Yang, Bing; Li, Muwang; Hillyer, Julián F; Ramarao, Nalini; Söderhäll, Kenneth; Liu, Chaoliang; Ling, Erjun

    2015-01-01

    Thermolysin, a metallopeptidase secreted by pathogenic microbes, is concluded as an important virulence factor due to cleaving purified host proteins in vitro. Using the silkworm Bombyx mori as a model system, we found that thermolysin injection into larvae induces the destruction of the coagulation response and the activation of hemolymph melanization, which results in larval death. Thermolysin triggers the rapid degradation of insect and mammalian plasma proteins at a level that is considerably greater than expected in vitro and/or in vivo. To more specifically explore the mechanism, thermolysin-induced changes to key proteins belonging to the insect melanization pathway were assessed as a window for observing plasma protein cleavage. The application of thermolysin induced the rapid cleavage of the melanization negative regulator serpin-3, but did not directly activate the melanization rate-limiting enzyme prophenoloxidase (PPO) or the terminal serine proteases responsible for PPO activation. Terminal serine proteases of melanization are activated indirectly after thermolysin exposure. We hypothesize that thermolysin induces the rapid degradation of serpins and the activation of proteases directly or indirectly, boosting uncontrolled plasma protein degradation in insects and mammalians. © 2014 Wiley Periodicals, Inc.

  6. Characterization of protein phosphatase 2A acting on phosphorylated plasma membrane aquaporin of tulip petals.

    Science.gov (United States)

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

    2004-05-01

    A protein phosphatase holo-type enzyme (38, 65, and 75 kDa) preparation and a free catalytic subunit (38 kDa) purified from tulip petals were characterized as protein phosphatase 2A (PP2A) by immunological and biochemical approaches. The plasma membrane containing the putative plasma membrane aquaporin (PM-AQP) was prepared from tulip petals, phosphorylated in vitro, and used as the substrate for both of the purified PP2A preparations. Although both preparations dephosphorylated the phosphorylated PM-AQP at 20 degrees C, only the holo-type enzyme preparation acted at 5 degrees C on the phosphorylated PM-AQP with higher substrate specificity, suggesting that regulatory subunits are required for low temperature-dependent dephosphorylation of PM-AQP in tulip petals.

  7. Plasma pro-surfactant protein B and lung function decline in smokers.

    Science.gov (United States)

    Leung, Janice M; Mayo, John; Tan, Wan; Tammemagi, C Martin; Liu, Geoffrey; Peacock, Stuart; Shepherd, Frances A; Goffin, John; Goss, Glenwood; Nicholas, Garth; Tremblay, Alain; Johnston, Michael; Martel, Simon; Laberge, Francis; Bhatia, Rick; Roberts, Heidi; Burrowes, Paul; Manos, Daria; Stewart, Lori; Seely, Jean M; Gingras, Michel; Pasian, Sergio; Tsao, Ming-Sound; Lam, Stephen; Sin, Don D

    2015-04-01

    Plasma pro-surfactant protein B (pro-SFTPB) levels have recently been shown to predict the development of lung cancer in current and ex-smokers, but the ability of pro-SFTPB to predict measures of chronic obstructive pulmonary disease (COPD) severity is unknown. We evaluated the performance characteristics of pro-SFTPB as a biomarker of lung function decline in a population of current and ex-smokers. Plasma pro-SFTPB levels were measured in 2503 current and ex-smokers enrolled in the Pan-Canadian Early Detection of Lung Cancer Study. Linear regression was performed to determine the relationship of pro-SFTPB levels to changes in forced expiratory volume in 1 s (FEV1) over a 2-year period as well as to baseline FEV1 and the burden of emphysema observed in computed tomography (CT) scans. Plasma pro-SFTPB levels were inversely related to both FEV1 % predicted (p=0.024) and FEV1/forced vital capacity (FVC) (p<0.001), and were positively related to the burden of emphysema on CT scans (p<0.001). Higher plasma pro-SFTPB levels were also associated with a more rapid decline in FEV1 at 1 year (p=0.024) and over 2 years of follow-up (p=0.004). Higher plasma pro-SFTPB levels are associated with increased severity of airflow limitation and accelerated decline in lung function. Pro-SFTPB is a promising biomarker for COPD severity and progression. Copyright ©ERS 2015.

  8. Identification of lipopolysaccharide-interacting plasma membrane-type proteins in Arabidopsis thaliana.

    Science.gov (United States)

    Vilakazi, Cornelius S; Dubery, Ian A; Piater, Lizelle A

    2017-02-01

    Lipopolysaccharide (LPS) is an amphiphatic bacterial glycoconjugate found on the external membrane of Gram-negative bacteria. This endotoxin is considered as a microbe-associated molecular pattern (MAMP) molecule and has been shown to elicit defense responses in plants. Here, LPS-interacting proteins from Arabidopsis thaliana plasma membrane (PM)-type fractions were captured and identified in order to investigate those involved in LPS perception and linked to triggering of innate immune responses. A novel proteomics-based affinity-capture strategy coupled to liquid chromatography-tandem mass spectrometry (LC-MS/MS) was employed for the enrichment and identification of LPS-interacting proteins. As such, LPS isolated from Burkholderia cepacia (LPS B.cep. ) was immobilized on three independent and distinct affinity-based matrices to serve as bait for interacting proteins from A. thaliana leaf and callus tissue. These were resolved by 1D electrophoresis and identified by mass spectrometry. Proteins specifically bound to LPS B.cep. have been implicated in membrane structure (e.g. COBRA-like and tubulin proteins), membrane trafficking and/or transport (e.g. soluble NSF attachment protein receptor (SNARE) proteins, patellin, aquaporin, PM instrinsic proteins (PIP) and H + -ATPase), signal transduction (receptor-like kinases and calcium-dependent protein kinases) as well as defense/stress responses (e.g. hypersensitive-induced response (HIR) proteins, jacalin-like lectin domain-containing protein and myrosinase-binding proteins). The novel affinity-capture strategy for the enrichment of LPS-interacting proteins proved to be effective, especially in the binding of proteins involved in plant defense responses, and can thus be used to elucidate LPS-mediated molecular recognition and disease mechanism(s). Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  9. Acute and chronic effects of a 24-hour intravenous triglyceride emulsion challenge on plasma lecithin : cholesterol acyltransferase, phospholipid transfer protein, and cholesteryl ester transfer protein activities

    NARCIS (Netherlands)

    Riemens, SC; Van Tol, A; Sluiter, WJ; Dullaart, RPF

    Lecithin:cholesterol acyltransferase (LCAT), phospholipid transfer protein (PLTP), and cholesteryl ester transfer protein (CETP) are key factors in remodeling of high density lipoproteins (HDL) and triglyceride-rich lipoproteins. We examined the effect of a large, 24 h intravenous fat load on plasma

  10. Biogenesis of the rat hepatocyte plasma membrane in vivo: comparison of the pathways taken by apical and basolateral proteins using subcellular fractionation

    International Nuclear Information System (INIS)

    Bartles, J.R.; Feracci, H.M.; Stieger, B.; Hubbard, A.L.

    1987-01-01

    We have used pulse-chase metabolic radiolabeling with L-[ 35 S]methionine in conjunction with subcellular fractionation and specific protein immunoprecipitation techniques to compare the posttranslational transport pathways taken by endogenous domain-specific integral proteins of the rat hepatocyte plasma membrane in vivo. Our results suggest that both apical (HA 4, dipeptidylpeptidase IV, and aminopeptidase N) and basolateral (CE 9 and the asialoglycoprotein receptor [ASGP-R]) proteins reach the hepatocyte plasma membrane with similar kinetics. The mature molecular mass form of each of these proteins reaches its maximum specific radioactivity in a purified hepatocyte plasma membrane fraction after only 45 min of chase. However, at this time, the mature radiolabeled apical proteins are not associated with vesicles derived from the apical domain of the hepatocyte plasma membrane, but instead are associated with vesicles which, by several criteria, appear to be basolateral plasma membrane. These vesicles: (a) fractionate like basolateral plasma membrane in sucrose density gradients and in free-flow electrophoresis; (b) can be separated from the bulk of the likely organellar contaminants, including membranes derived from the late Golgi cisternae, transtubular network, and endosomes; (c) contain the proven basolateral constituents CE 9 and the ASGP-R, as judged by vesicle immunoadsorption using fixed Staphylococcus aureus cells and anti-ASGP-R antibodies; and (d) are oriented with their ectoplasmic surfaces facing outward, based on the results of vesicle immunoadsorption experiments using antibodies specific for the ectoplasmic domain of the ASGP-R. Only at times of chase greater than 45 min do significant amounts of the mature radiolabeled apical proteins arrive at the apical domain, and they do so at different rates

  11. Proteomic Characterization of Differential Abundant Proteins Accumulated between Lower and Upper Epidermises of Fleshy Scales in Onion (Allium cepa L. Bulbs.

    Directory of Open Access Journals (Sweden)

    Si Wu

    Full Text Available The onion (Allium cepa L. is widely planted worldwide as a valuable vegetable crop. The scales of an onion bulb are a modified type of leaf. The one-layer-cell epidermis of onion scales is commonly used as a model experimental material in botany and molecular biology. The lower epidermis (LE and upper epidermis (UE of onion scales display obvious differences in microscopic structure, cell differentiation and pigment synthesis; however, associated proteomic differences are unclear. LE and UE can be easily sampled as single-layer-cell tissues for comparative proteomic analysis. In this study, a proteomic approach based on 2-DE and mass spectrometry (MS was applied to compare LE and UE of fleshy scales from yellow and red onions. We identified 47 differential abundant protein spots (representing 31 unique proteins between LE and UE in red and yellow onions. These proteins are mainly involved in pigment synthesis, stress response, and cell division. Particularly, the differentially accumulated chalcone-flavanone isomerase and flavone O-methyltransferase 1-like in LE may result in the differences in the onion scale color between red and yellow onions. Moreover, stress-related proteins abundantly accumulated in both LE and UE. In addition, the differential accumulation of UDP-arabinopyranose mutase 1-like protein and β-1,3-glucanase in the LE may be related to the different cell sizes between LE and UE of the two types of onion. The data derived from this study provides new insight into the differences in differentiation and developmental processes between onion epidermises. This study may also make a contribution to onion breeding, such as improving resistances and changing colors.

  12. G protein-coupled receptor 30 (GPR30) forms a plasma membrane complex with membrane-associated guanylate kinases (MAGUKs) and protein kinase A-anchoring protein 5 (AKAP5) that constitutively inhibits cAMP production.

    Science.gov (United States)

    Broselid, Stefan; Berg, Kelly A; Chavera, Teresa A; Kahn, Robin; Clarke, William P; Olde, Björn; Leeb-Lundberg, L M Fredrik

    2014-08-08

    GPR30, or G protein-coupled estrogen receptor, is a G protein-coupled receptor reported to bind 17β-estradiol (E2), couple to the G proteins Gs and Gi/o, and mediate non-genomic estrogenic responses. However, controversies exist regarding the receptor pharmacological profile, effector coupling, and subcellular localization. We addressed the role of the type I PDZ motif at the receptor C terminus in receptor trafficking and coupling to cAMP production in HEK293 cells and CHO cells ectopically expressing the receptor and in Madin-Darby canine kidney cells expressing the native receptor. GPR30 was localized both intracellularly and in the plasma membrane and subject to limited basal endocytosis. E2 and G-1, reported GPR30 agonists, neither stimulated nor inhibited cAMP production through GPR30, nor did they influence receptor localization. Instead, GPR30 constitutively inhibited cAMP production stimulated by a heterologous agonist independently of Gi/o. Moreover, siRNA knockdown of native GPR30 increased cAMP production. Deletion of the receptor PDZ motif interfered with inhibition of cAMP production and increased basal receptor endocytosis. GPR30 interacted with membrane-associated guanylate kinases, including SAP97 and PSD-95, and protein kinase A-anchoring protein (AKAP) 5 in the plasma membrane in a PDZ-dependent manner. Knockdown of AKAP5 or St-Ht31 treatment, to disrupt AKAP interaction with the PKA RIIβ regulatory subunit, decreased inhibition of cAMP production, and St-Ht31 increased basal receptor endocytosis. Therefore, GPR30 forms a plasma membrane complex with a membrane-associated guanylate kinase and AKAP5, which constitutively attenuates cAMP production in response to heterologous agonists independently of Gi/o and retains receptors in the plasma membrane. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  13. Protein Detection Using the Multiplexed Proximity Extension Assay (PEA) from Plasma and Vaginal Fluid Applied to the Indicating FTA Elute Micro Card™

    Science.gov (United States)

    Berggrund, Malin; Ekman, Daniel; Gustavsson, Inger; Sundfeldt, Karin; Olovsson, Matts; Enroth, Stefan; Gyllensten, Ulf

    2016-01-01

    The indicating FTA elute micro card™ has been developed to collect and stabilize the nucleic acid in biological samples and is widely used in human and veterinary medicine and other disciplines. This card is not recommended for protein analyses, since surface treatment may denature proteins. We studied the ability to analyse proteins in human plasma and vaginal fluid as applied to the indicating FTA elute micro card™ using the sensitive proximity extension assay (PEA). Among 92 proteins in the Proseek Multiplex Oncology Iv2 panel, 87 were above the limit of detection (LOD) in liquid plasma and 56 among 92 above LOD in plasma applied to FTA cards. Washing and protein elution protocols were compared to identify an optimal method. Liquid-based cytology samples showed a lower number of proteins above LOD than FTA cards with vaginal fluid samples applied. Our results demonstrate that samples applied to the indicating FTA elute micro card™ are amendable to protein analyses, given that a sensitive protein detection assay is used. The results imply that biological samples applied to FTA cards can be used for DNA, RNA and protein detection. PMID:28936257

  14. Protein Detection Using the Multiplexed Proximity Extension Assay (PEA from Plasma and Vaginal Fluid Applied to the Indicating FTA Elute Micro Card™

    Directory of Open Access Journals (Sweden)

    Malin Berggrund

    2016-01-01

    Full Text Available The indicating FTA elute micro card™ has been developed to collect and stabilize the nucleic acid in biological samples and is widely used in human and veterinary medicine and other disciplines. This card is not recommended for protein analyses, since surface treatment may denature proteins. We studied the ability to analyse proteins in human plasma and vaginal fluid as applied to the indicating FTA elute micro card™ using the sensitive proximity extension assay (PEA. Among 92 proteins in the Proseek Multiplex Oncology Iv2 panel, 87 were above the limit of detection (LOD in liquid plasma and 56 among 92 above LOD in plasma applied to FTA cards. Washing and protein elution protocols were compared to identify an optimal method. Liquid-based cytology samples showed a lower number of proteins above LOD than FTA cards with vaginal fluid samples applied. Our results demonstrate that samples applied to the indicating FTA elute micro card™ are amendable to protein analyses, given that a sensitive protein detection assay is used. The results imply that biological samples applied to FTA cards can be used for DNA, RNA and protein detection.

  15. Protein Detection Using the Multiplexed Proximity Extension Assay (PEA) from Plasma and Vaginal Fluid Applied to the Indicating FTA Elute Micro Card™.

    Science.gov (United States)

    Berggrund, Malin; Ekman, Daniel; Gustavsson, Inger; Sundfeldt, Karin; Olovsson, Matts; Enroth, Stefan; Gyllensten, Ulf

    2016-01-01

    The indicating FTA elute micro card™ has been developed to collect and stabilize the nucleic acid in biological samples and is widely used in human and veterinary medicine and other disciplines. This card is not recommended for protein analyses, since surface treatment may denature proteins. We studied the ability to analyse proteins in human plasma and vaginal fluid as applied to the indicating FTA elute micro card™ using the sensitive proximity extension assay (PEA). Among 92 proteins in the Proseek Multiplex Oncology Iv2 panel, 87 were above the limit of detection (LOD) in liquid plasma and 56 among 92 above LOD in plasma applied to FTA cards. Washing and protein elution protocols were compared to identify an optimal method. Liquid-based cytology samples showed a lower number of proteins above LOD than FTA cards with vaginal fluid samples applied. Our results demonstrate that samples applied to the indicating FTA elute micro card™ are amendable to protein analyses, given that a sensitive protein detection assay is used. The results imply that biological samples applied to FTA cards can be used for DNA, RNA and protein detection.

  16. Cellulose membranes are more effective in holding back vital proteins and exhibit less interaction with plasma proteins during hemodialysis.

    Science.gov (United States)

    Pešić, Ivana; Müller, Gerhard A; Baumann, Cosima; Dihazi, Gry H; Koziolek, Michael J; Eltoweissy, Marwa; Bramlage, Carsten; Asif, Abdul R; Dihazi, Hassan

    2013-04-01

    The vast majority of patients with end-stage renal disease are treated with intermittent hemodialysis as a form of renal replacement therapy. To investigate the impact of hemodialysis membrane material on vital protein removal, dialysates from 26 well-characterized hemodialysis patients were collected 5 min after beginning, during 5h of treatment, as well as 5 min before ending of the dialysis sessions. Dialysis sessions were performed using either modified cellulose (n=12) (low-flux and high flux) or synthetic Polyflux (n=14) (low-flux and high-flux) dialyzer. Protein removal during hemodialysis was quantified and the dialysate proteome patterns were analyzed by 2-DE, MS and Western blot. There was a clear correlation between the type of membrane material and the amount of protein removed. Synthetic Polyflux membranes exhibit strong interaction with plasma proteins resulting in a significantly higher protein loss compared to modified cellulosic membrane. Moreover, the proteomics analysis showed that the removed proteins represented different molecular weight range and different functional groups: transport proteins, protease inhibitors, proteins with role in immune response and regulations, constructive proteins and as a part of HLA immune complex. The effect of this protein removal on hemodialysis treatment outcome should be investigated in further studies. Copyright © 2013 Elsevier B.V. All rights reserved.

  17. The AP2 clathrin adaptor protein complex regulates the abundance of GLR-1 glutamate receptors in the ventral nerve cord of Caenorhabditis elegans.

    Science.gov (United States)

    Garafalo, Steven D; Luth, Eric S; Moss, Benjamin J; Monteiro, Michael I; Malkin, Emily; Juo, Peter

    2015-05-15

    Regulation of glutamate receptor (GluR) abundance at synapses by clathrin-mediated endocytosis can control synaptic strength and plasticity. We take advantage of viable, null mutations in subunits of the clathrin adaptor protein 2 (AP2) complex in Caenorhabditis elegans to characterize the in vivo role of AP2 in GluR trafficking. In contrast to our predictions for an endocytic adaptor, we found that levels of the GluR GLR-1 are decreased at synapses in the ventral nerve cord (VNC) of animals with mutations in the AP2 subunits APM-2/μ2, APA-2/α, or APS-2/σ2. Rescue experiments indicate that APM-2/μ2 functions in glr-1-expressing interneurons and the mature nervous system to promote GLR-1 levels in the VNC. Genetic analyses suggest that APM-2/μ2 acts upstream of GLR-1 endocytosis in the VNC. Consistent with this, GLR-1 accumulates in cell bodies of apm-2 mutants. However, GLR-1 does not appear to accumulate at the plasma membrane of the cell body as expected, but instead accumulates in intracellular compartments including Syntaxin-13- and RAB-14-labeled endosomes. This study reveals a novel role for the AP2 clathrin adaptor in promoting the abundance of GluRs at synapses in vivo, and implicates AP2 in the regulation of GluR trafficking at an early step in the secretory pathway. © 2015 Garafalo et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

  18. Analysis of protein profiles in diabetic rat blood plasma that induced by alloxan

    Science.gov (United States)

    Hidayati, Dewi; Abdulgani, Nurlita; Setiyawan, Hengki; Trisnawati, Indah; Ashuri, Nova Maulidina; Sa'adah, Noor Nailis

    2017-06-01

    Proteomics is the study to identify the proteins involved in physiological metabolic pathway. The protein profiles of blood plasma from alloxan-induced diabetic rats has investigated using Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE). Data were analyzed descriptively based on variations of the type and intensity of the protein. There were identified the similarity of protein variant between diabetic and control rats included ankyrin (200kDa), IgG (150kDa), nephrin (136 kDa), IDE (112 kDA), albumin (66 kDa), prealbumin (55 kDA), CICP (43 kDa), ApoA-V (39 kDa), GAPDH (35 kDa), C-RP (27,1 kDa), leptin (16 kDa) and apelin (13 kDa). However, the apelin profile at diabetic rats shows the higher intensity than control.

  19. Scavenger receptor-mediated recognition of maleyl bovine plasma albumin and the demaleylated protein in human monocyte macrophages

    International Nuclear Information System (INIS)

    Haberland, M.E.; Fogelman, A.M.

    1985-01-01

    Maleyl bovine plasma albumin competed on an equimolar basis with malondialdehyde low density lipoprotein (LDL) in suppressing the lysosomal hydrolysis of 125 I-labeled malondialdehyde LDL mediated by the scavenger receptor of human monocyte macrophages. Maleyl bovine plasma albumin, in which 94% of the amino groups were modified, exhibited an anodic mobility in agarose electrophoresis 1.7 times that of the native protein. Incubation of maleyl bovine plasma albumin at pH 3.5 regenerated the free amino groups and restored the protein to the same electrophoretic mobility as native albumin. Although ligands recognized by the scavenger receptor typically are anionic, the authors propose that addition of new negative charge achieved by maleylation, rather than directly forming the receptor binding site(s), induces conformational changes in albumin as a prerequisite to expression of the recognition domain(s). They conclude that the primary sequence of albumin, rather than addition of new negative charge, provides the recognition determinant(s) essential for interaction of maleyl bovine plasma albumin with the scavenger receptor

  20. Do 14-3-3 proteins and plasma membrane H+-ATPases interact in the barley epidermis in response to the barley powdery mildew fungus?

    DEFF Research Database (Denmark)

    Finnie, C.; Andersen, C.H.; Borch, J.

    2002-01-01

    14-3-3 proteins form a family of highly conserved proteins with central roles in many eukaryotic signalling networks. In plants, they bind to and activate the plasma membrane H+-ATPase, creating a binding site for the phytotoxin fusicoccin. Barley 14-3-3 transcripts accumulate in the epidermis upon...... inoculation with the powdery mildew fungus. We have isolated a cDNA encoding a plasma membrane H+-ATPase (HvHA1), which is also induced by powdery mildew attack. The C-terminal domain of this H+-ATPase interacts with 14-3-3 proteins in the yeast two-hybrid system. Inoculation with the powdery mildew fungus......, or treatment with fusicoccin, results in an increase in fusicoccin binding ability of barley leaf membranes. Overlay assays show a fungus-induced increase in binding of digoxygenin-labelled 14-3-3 protein to several proteins including a 100 kDa membrane protein, probably the plasma membrane H...

  1. Palmitoylation of Sindbis Virus TF Protein Regulates Its Plasma Membrane Localization and Subsequent Incorporation into Virions.

    Science.gov (United States)

    Ramsey, Jolene; Renzi, Emily C; Arnold, Randy J; Trinidad, Jonathan C; Mukhopadhyay, Suchetana

    2017-02-01

    Palmitoylation is a reversible, posttranslational modification that helps target proteins to cellular membranes. The alphavirus small membrane proteins 6K and TF have been reported to be palmitoylated and to positively regulate budding. 6K and TF are isoforms that are identical in their N termini but unique in their C termini due to a -1 ribosomal frameshift during translation. In this study, we used cysteine (Cys) mutants to test differential palmitoylation of the Sindbis virus 6K and TF proteins. We modularly mutated the five Cys residues in the identical N termini of 6K and TF, the four additional Cys residues in TF's unique C terminus, or all nine Cys residues in TF. Using these mutants, we determined that TF palmitoylation occurs primarily in the N terminus. In contrast, 6K is not palmitoylated, even on these shared residues. In the C-terminal Cys mutant, TF protein levels increase both in the cell and in the released virion compared to the wild type. In viruses with the N-terminal Cys residues mutated, TF is much less efficiently localized to the plasma membrane, and it is not incorporated into the virion. The three Cys mutants have minor defects in cell culture growth but a high incidence of abnormal particle morphologies compared to the wild-type virus as determined by transmission electron microscopy. We propose a model where the C terminus of TF modulates the palmitoylation of TF at the N terminus, and palmitoylated TF is preferentially trafficked to the plasma membrane for virus budding. Alphaviruses are a reemerging viral cause of arthritogenic disease. Recently, the small 6K and TF proteins of alphaviruses were shown to contribute to virulence in vivo Nevertheless, a clear understanding of the molecular mechanisms by which either protein acts to promote virus infection is missing. The TF protein is a component of budded virions, and optimal levels of TF correlate positively with wild-type-like particle morphology. In this study, we show that the

  2. A SOLAR SPECTROSCOPIC ABSOLUTE ABUNDANCE OF ARGON FROM RESIK

    International Nuclear Information System (INIS)

    Sylwester, J.; Sylwester, B.; Phillips, K. J. H.; Kuznetsov, V. D.

    2010-01-01

    Observations of He-like and H-like Ar (Ar XVII and Ar XVIII) lines at 3.949 A and 3.733 A, respectively, with the RESIK X-ray spectrometer on the CORONAS-F spacecraft, together with temperatures and emission measures from the two channels of GOES, have been analyzed to obtain the abundance of Ar in flare plasmas in the solar corona. The line fluxes per unit emission measure show a temperature dependence like that predicted from theory and lead to spectroscopically determined values for the absolute Ar abundance, A(Ar) = 6.44 ± 0.07 (Ar XVII) and 6.49 ± 0.16 (Ar XVIII), which are in agreement to within uncertainties. The weighted mean is 6.45 ± 0.06, which is between two recent compilations of the solar Ar abundance and suggests that the photospheric and coronal abundances of Ar are very similar.

  3. Up-Regulation of Antioxidant Proteins in the Plasma Proteome during Saturation Diving: Unique Coincidence under Hypobaric Hypoxia.

    Science.gov (United States)

    Domoto, Hideharu; Iwaya, Keiichi; Ikomi, Fumitaka; Matsuo, Hirotaka; Tadano, Yutaka; Fujii, Shigenori; Tachi, Kazuyoshi; Itoh, Yoshiyuki; Sato, Michiya; Inoue, Kimitoshi; Shinomiya, Nariyoshi

    2016-01-01

    Saturation diving (SD) is one of the safest techniques for tolerating hyperbaric conditions for long durations. However, the changes in the human plasma protein profile that occur during SD are unknown. To identify differential protein expression during or after SD, 65 blood samples from 15 healthy Japanese men trained in SD were analyzed by two-dimensional fluorescence difference gel electrophoresis. The expression of two proteins, one 32.4 kDa with an isoelectric point (pI) of 5.8 and the other 44.8 kDa with pI 4.0, were elevated during SD to 60, 100, and 200 meters sea water (msw). The expression of these proteins returned to pre-diving level when the SD training was completed. The two proteins were identified using in-gel digestion and mass spectrometric analysis; the 32.4 kDa protein was transthyretin and the 44.8 kDa protein was alpha-1-acid glycoprotein 1. Oxidation was detected at methionine 13 of transthyretin and at methionine 129 of alpha-1-acid glycoprotein 1 by tandem mass spectrometry. Moreover, haptoglobin was up-regulated during the decompression phase of 200 msw. These plasma proteins up-regulated during SD have a common function as anti-oxidants. This suggests that by coordinating their biological effects, these proteins activate a defense mechanism to counteract the effects of hyperbaric-hyperoxic conditions during SD.

  4. Up-Regulation of Antioxidant Proteins in the Plasma Proteome during Saturation Diving: Unique Coincidence under Hypobaric Hypoxia.

    Directory of Open Access Journals (Sweden)

    Hideharu Domoto

    Full Text Available Saturation diving (SD is one of the safest techniques for tolerating hyperbaric conditions for long durations. However, the changes in the human plasma protein profile that occur during SD are unknown. To identify differential protein expression during or after SD, 65 blood samples from 15 healthy Japanese men trained in SD were analyzed by two-dimensional fluorescence difference gel electrophoresis. The expression of two proteins, one 32.4 kDa with an isoelectric point (pI of 5.8 and the other 44.8 kDa with pI 4.0, were elevated during SD to 60, 100, and 200 meters sea water (msw. The expression of these proteins returned to pre-diving level when the SD training was completed. The two proteins were identified using in-gel digestion and mass spectrometric analysis; the 32.4 kDa protein was transthyretin and the 44.8 kDa protein was alpha-1-acid glycoprotein 1. Oxidation was detected at methionine 13 of transthyretin and at methionine 129 of alpha-1-acid glycoprotein 1 by tandem mass spectrometry. Moreover, haptoglobin was up-regulated during the decompression phase of 200 msw. These plasma proteins up-regulated during SD have a common function as anti-oxidants. This suggests that by coordinating their biological effects, these proteins activate a defense mechanism to counteract the effects of hyperbaric-hyperoxic conditions during SD.

  5. Isobaric Tags for Relative and Absolute Quantification (iTRAQ)-Based Untargeted Quantitative Proteomic Approach To Identify Change of the Plasma Proteins by Salbutamol Abuse in Beef Cattle.

    Science.gov (United States)

    Zhang, Kai; Tang, Chaohua; Liang, Xiaowei; Zhao, Qingyu; Zhang, Junmin

    2018-01-10

    Salbutamol, a selective β 2 -agonist, endangers the safety of animal products as a result of illegal use in food animals. In this study, an iTRAQ-based untargeted quantitative proteomic approach was applied to screen potential protein biomarkers in plasma of cattle before and after treatment with salbutamol for 21 days. A total of 62 plasma