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Sample records for adsorbed plasma proteins

  1. Quantitative proteomics analysis of adsorbed plasma proteins classifies nanoparticles with different surface properties and size

    SciTech Connect

    Zhang, Haizhen; Burnum, Kristin E.; Luna, Maria L.; Petritis, Brianne O.; Kim, Jong Seo; Qian, Weijun; Moore, Ronald J.; Heredia-Langner, Alejandro; Webb-Robertson, Bobbie-Jo M.; Thrall, Brian D.; Camp, David G.; Smith, Richard D.; Pounds, Joel G.; Liu, Tao

    2011-12-01

    In biofluids (e.g., blood plasma) nanoparticles are readily embedded in layers of proteins that can affect their biological activity and biocompatibility. Herein, we report a study on the interactions between human plasma proteins and nanoparticles with a controlled systematic variation of properties using stable isotope labeling and liquid chromatography-mass spectrometry (LC-MS) based quantitative proteomics. Novel protocol has been developed to simplify the isolation of nanoparticle bound proteins and improve the reproducibility. Plasma proteins associated with polystyrene nanoparticles with three different surface chemistries and two sizes as well as for four different exposure times (for a total of 24 different samples) were identified and quantified by LC-MS analysis. Quantitative comparison of relative protein abundances were achieved by spiking an 18 O-labeled 'universal reference' into each individually processed unlabeled sample as an internal standard, enabling simultaneous application of both label-free and isotopic labeling quantitation across the sample set. Clustering analysis of the quantitative proteomics data resulted in distinctive pattern that classifies the nanoparticles based on their surface properties and size. In addition, data on the temporal study indicated that the stable protein 'corona' that was isolated for the quantitative analysis appeared to be formed in less than 5 minutes. The comprehensive results obtained herein using quantitative proteomics have potential implications towards predicting nanoparticle biocompatibility.

  2. Volumetric Interpretation of Protein Adsorption: Capacity Scaling with Adsorbate Molecular Weight and Adsorbent Surface Energy

    PubMed Central

    Parhi, Purnendu; Golas, Avantika; Barnthip, Naris; Noh, Hyeran; Vogler, Erwin A.

    2009-01-01

    Silanized-glass-particle adsorbent capacities are extracted from adsorption isotherms of human serum albumin (HSA, 66 kDa), immunoglobulin G (IgG, 160 kDa), fibrinogen (Fib, 341 kDa), and immunoglobulin M (IgM, 1000 kDa) for adsorbent surface energies sampling the observable range of water wettability. Adsorbent capacity expressed as either mass-or-moles per-unit-adsorbent-area increases with protein molecular weight (MW) in a manner that is quantitatively inconsistent with the idea that proteins adsorb as a monolayer at the solution-material interface in any physically-realizable configuration or state of denaturation. Capacity decreases monotonically with increasing adsorbent hydrophilicity to the limit-of-detection (LOD) near τo = 30 dyne/cm (θ~65o) for all protein/surface combinations studied (where τo≡γlvocosθ is the water adhesion tension, γlvo is the interfacial tension of pure-buffer solution, and θ is the buffer advancing contact angle). Experimental evidence thus shows that adsorbent capacity depends on both adsorbent surface energy and adsorbate size. Comparison of theory to experiment implies that proteins do not adsorb onto a two-dimensional (2D) interfacial plane as frequently depicted in the literature but rather partition from solution into a three-dimensional (3D) interphase region that separates the physical surface from bulk solution. This interphase has a finite volume related to the dimensions of hydrated protein in the adsorbed state (defining “layer” thickness). The interphase can be comprised of a number of adsorbed-protein layers depending on the solution concentration in which adsorbent is immersed, molecular volume of the adsorbing protein (proportional to MW), and adsorbent hydrophilicity. Multilayer adsorption accounts for adsorbent capacity over-and-above monolayer and is inconsistent with the idea that protein adsorbs to surfaces primarily through protein/surface interactions because proteins within second (or higher

  3. In situ modification of chromatography adsorbents using cold atmospheric pressure plasmas

    NASA Astrophysics Data System (ADS)

    Olszewski, P.; Willett, T. C.; Theodosiou, E.; Thomas, O. R. T.; Walsh, J. L.

    2013-05-01

    Efficient manufacturing of increasingly sophisticated biopharmaceuticals requires the development of new breeds of chromatographic materials featuring two or more layers, with each layer affording different functions. This letter reports the in situ modification of a commercial beaded anion exchange adsorbent using atmospheric pressure plasma generated within gas bubbles. The results show that exposure to He-O2 plasma in this way yields significant reductions in the surface binding of plasmid DNA to the adsorbent exterior, with minimal loss of core protein binding capacity; thus, a bi-layered chromatography material exhibiting both size excluding and anion exchange functionalities within the same bead is produced.

  4. Experimental characterization of adsorbed protein orientation, conformation, and bioactivity

    PubMed Central

    Thyparambil, Aby A.; Wei, Yang; Latour, Robert A.

    2015-01-01

    Protein adsorption on material surfaces is a common phenomenon that is of critical importance in many biotechnological applications. The structure and function of adsorbed proteins are tightly interrelated and play a key role in the communication and interaction of the adsorbed proteins with the surrounding environment. Because the bioactive state of a protein on a surface is a function of the orientation, conformation, and accessibility of its bioactive site(s), the isolated determination of just one or two of these factors will typically not be sufficient to understand the structure–function relationships of the adsorbed layer. Rather a combination of methods is needed to address each of these factors in a synergistic manner to provide a complementary dataset to characterize and understand the bioactive state of adsorbed protein. Over the past several years, the authors have focused on the development of such a set of complementary methods to address this need. These methods include adsorbed-state circular dichroism spectropolarimetry to determine adsorption-induced changes in protein secondary structure, amino-acid labeling/mass spectrometry to assess adsorbed protein orientation and tertiary structure by monitoring adsorption-induced changes in residue solvent accessibility, and bioactivity assays to assess adsorption-induced changes in protein bioactivity. In this paper, the authors describe the methods that they have developed and/or adapted for each of these assays. The authors then provide an example of their application to characterize how adsorption-induced changes in protein structure influence the enzymatic activity of hen egg-white lysozyme on fused silica glass, high density polyethylene, and poly(methyl-methacrylate) as a set of model systems. PMID:25708632

  5. Experimental characterization of adsorbed protein orientation, conformation, and bioactivity.

    PubMed

    Thyparambil, Aby A; Wei, Yang; Latour, Robert A

    2015-01-01

    Protein adsorption on material surfaces is a common phenomenon that is of critical importance in many biotechnological applications. The structure and function of adsorbed proteins are tightly interrelated and play a key role in the communication and interaction of the adsorbed proteins with the surrounding environment. Because the bioactive state of a protein on a surface is a function of the orientation, conformation, and accessibility of its bioactive site(s), the isolated determination of just one or two of these factors will typically not be sufficient to understand the structure-function relationships of the adsorbed layer. Rather a combination of methods is needed to address each of these factors in a synergistic manner to provide a complementary dataset to characterize and understand the bioactive state of adsorbed protein. Over the past several years, the authors have focused on the development of such a set of complementary methods to address this need. These methods include adsorbed-state circular dichroism spectropolarimetry to determine adsorption-induced changes in protein secondary structure, amino-acid labeling/mass spectrometry to assess adsorbed protein orientation and tertiary structure by monitoring adsorption-induced changes in residue solvent accessibility, and bioactivity assays to assess adsorption-induced changes in protein bioactivity. In this paper, the authors describe the methods that they have developed and/or adapted for each of these assays. The authors then provide an example of their application to characterize how adsorption-induced changes in protein structure influence the enzymatic activity of hen egg-white lysozyme on fused silica glass, high density polyethylene, and poly(methyl-methacrylate) as a set of model systems. PMID:25708632

  6. Activity of lactoperoxidase when adsorbed on protein layers.

    PubMed

    Haberska, Karolina; Svensson, Olof; Shleev, Sergey; Lindh, Liselott; Arnebrant, Thomas; Ruzgas, Tautgirdas

    2008-09-15

    Lactoperoxidase (LPO) is an enzyme, which is used as an antimicrobial agent in a number of applications, e.g., food technology. In the majority of applications LPO is added to a homogeneous product phase or immobilised on product surface. In the latter case, however, the measurements of LPO activity are seldom reported. In this paper we have assessed LPO enzymatic activity on bare and protein modified gold surfaces by means of electrochemistry. It was found that LPO rapidly adsorbs to bare gold surfaces resulting in an amount of LPO adsorbed of 2.9mg/m(2). A lower amount of adsorbed LPO is obtained if the gold surface is exposed to bovine serum albumin, bovine or human mucin prior to LPO adsorption. The enzymatic activity of the adsorbed enzyme is in general preserved at the experimental conditions and varies only moderately when comparing bare gold and gold surface pretreated with the selected proteins. The measurement of LPO specific activity, however, indicate that it is about 1.5 times higher if LPO is adsorbed on gold surfaces containing a small amount of preadsorbed mucin in comparison to the LPO directly adsorbed on bare gold.

  7. Discharge Plasma Assisted Adsorbents for Exhaust Treatment: A Comparative Analysis on Enhancing NOx Removal

    NASA Astrophysics Data System (ADS)

    Rajanikanth, B. S.; Dipanwita, Sinha; Emmanuel, P.

    2008-06-01

    An analysis has been made on the discharge plasma coupled with an adsorbent system for NOx removal. The cascaded plasma-adsorbent system may be perceived as a better alternative for the existing adsorbent-based abatement system in the industry. In this study the exhaust is sourced from a diesel generator set. It was observed that better NO removal in a plasma reactor can be made possible by achieving higher average fields and subsequent NO2 removal can be improved using an adsorbent system connected in cascade with the plasma system. The paper describes various findings pertaining to these comparative analyses.

  8. Pattern Recognition of Adsorbing HP Lattice Proteins

    NASA Astrophysics Data System (ADS)

    Wilson, Matthew S.; Shi, Guangjie; Wüst, Thomas; Landau, David P.; Schmid, Friederike

    2015-03-01

    Protein adsorption is relevant in fields ranging from medicine to industry, and the qualitative behavior exhibited by course-grained models could shed insight for further research in such fields. Our study on the selective adsorption of lattice proteins utilizes the Wang-Landau algorithm to simulate the Hydrophobic-Polar (H-P) model with an efficient set of Monte Carlo moves. Each substrate is modeled as a square pattern of 9 lattice sites which attract either H or P monomers, and are located on an otherwise neutral surface. The fully enumerated set of 102 unique surfaces is simulated with each protein sequence. A collection of 27-monomer sequences is used- each of which is non-degenerate and protein-like. Thermodynamic quantities such as the specific heat and free energy are calculated from the density of states, and are used to investigate the adsorption of lattice proteins on patterned substrates. Research supported by NSF.

  9. Adsorbed Proteins Influence the Biological Activity and Molecular Targeting of Nanomaterials

    SciTech Connect

    Dutta, Debamitra; Sundaram, S. K.; Teeguarden, Justin G.; Riley, Brian J.; Fifield, Leonard S.; Jacobs, Jon M.; Addleman, Raymond S.; Kaysen, George A.; Moudgil, Brij M.; Weber, Thomas J.

    2007-11-01

    The possible combination of unique physicochemical properties operating at unique sites of action within cells and tissues has led to considerable uncertainty surrounding nanomaterial toxic potential. Here we have investigated the relative importance of proteins adsorbed onto nanomaterial surfaces in guiding uptake and toxicity to determine whether a priori identification of adsorbed proteins will contribute to nanomaterial toxicity assessment. Albumin was identified as the major protein adsorbed onto single walled carbon nanotubes (SWCNTs) following incubation with fetal bovine or human serum/plasma, but not when plasma from the Nagase Analbuminemic Rat (NAR) was used, and precoating SWCNTs with a non-ionic surfactant (Pluronic F127) inhibited albumin adsorption. Damaged or structurally altered albumin is rapidly cleared by scavenger receptors. In the RAW 264.7 macrophage-like model, we observed that SWCNTs inhibited the induction of cyclooxygenase-2 (Cox-2) by lipopolysaccharide (LPS; 1 ng/ml, 6 hr) and this anti-inflammatory response was inhibited by fucoidan (scavenger receptor antagonist) and by precoating SWCNTs with Pluronic F127. Fucoidan also reduced the uptake of fluorescent SWCNTs (Alexa647) in RAW 264.7 cells. Albumin-coated SWCNTs reduced LPS-mediated Cox-2 induction. SWCNTs did not appear to reduce binding of a fluorescent LPS (Alexa488) to RAW 264.7 cells. The profile of proteins adsorbed onto amorphous silica (50 – 1000 nm) was qualitatively different, relative to SWCNTs, and coating amorphous silica with Pluronic F127 dramatically reduced protein binding and toxicity. Collectively, these observations are consistent with an important role for adsorbed proteins in guiding nanomaterial disposition and toxicity.

  10. High capacity cryogel-type adsorbents for protein purification.

    PubMed

    Singh, Naveen Kumar; Dsouza, Roy N; Grasselli, Mariano; Fernández-Lahore, Marcelo

    2014-08-15

    Cryogel bodies were modified to obtain epoxy groups by graft-copolymerization using both chemical and gamma irradiation initiation techniques. The free epoxy adsorbents were reacted further to introduce diethylaminoethanol (DEAE) functionalities. The resulting weak anion-exchange cryogel adsorbents showed dynamic binding capacities of ca. 27±3mg/mL, which was significantly higher than previously reported for this type of adsorbent material. Gamma irradiated grafting initiation showed a 4-fold higher capacity for proteins than chemical grafting initiation procedures. The phosphate capacity for these DEAE cryogels was 119mmol/L and also showed similar column efficiency as compared to commercial adsorbents. The large pores in the cryogel structure ensure convective transport of the molecules to active binding sites located on the polymer-grafted surface of cryogels. However, as cryogels have relatively large pores (10-100μm), the BET area available for surface activation is low, and consequently, the capacity of the cryogels is relatively low for biomolecules, especially when compared to commercial beaded adsorbents. Nevertheless, we have shown that gamma ray mediated surface grafting of cryogel matrices greatly enhance their functional and adsorptive properties.

  11. High capacity cryogel-type adsorbents for protein purification.

    PubMed

    Singh, Naveen Kumar; Dsouza, Roy N; Grasselli, Mariano; Fernández-Lahore, Marcelo

    2014-08-15

    Cryogel bodies were modified to obtain epoxy groups by graft-copolymerization using both chemical and gamma irradiation initiation techniques. The free epoxy adsorbents were reacted further to introduce diethylaminoethanol (DEAE) functionalities. The resulting weak anion-exchange cryogel adsorbents showed dynamic binding capacities of ca. 27±3mg/mL, which was significantly higher than previously reported for this type of adsorbent material. Gamma irradiated grafting initiation showed a 4-fold higher capacity for proteins than chemical grafting initiation procedures. The phosphate capacity for these DEAE cryogels was 119mmol/L and also showed similar column efficiency as compared to commercial adsorbents. The large pores in the cryogel structure ensure convective transport of the molecules to active binding sites located on the polymer-grafted surface of cryogels. However, as cryogels have relatively large pores (10-100μm), the BET area available for surface activation is low, and consequently, the capacity of the cryogels is relatively low for biomolecules, especially when compared to commercial beaded adsorbents. Nevertheless, we have shown that gamma ray mediated surface grafting of cryogel matrices greatly enhance their functional and adsorptive properties. PMID:24980092

  12. Human Plasma Protein C

    PubMed Central

    Kisiel, Walter

    1979-01-01

    Protein C is a vitamin K-dependent protein, which exists in bovine plasma as a precursor of a serine protease. In this study, protein C was isolated to homogeneity from human plasma by barium citrate adsorption and elution, ammonium sulfate fractionation, DEAE-Sephadex chromatography, dextran sulfate agarose chromatography, and preparative polyacrylamide gel electrophoresis. Human protein C (Mr = 62,000) contains 23% carbohydrate and is composed of a light chain (Mr = 21,000) and a heavy chain (Mr = 41,000) held together by a disulfide bond(s). The light chain has an amino-terminal sequence of Ala-Asn-Ser-Phe-Leu- and the heavy chain has an aminoterminal sequence of Asp-Pro-Glu-Asp-Gln. The residues that are identical to bovine protein C are underlined. Incubation of human protein C with human α-thrombin at an enzyme to substrate weight ratio of 1:50 resulted in the formation of activated protein C, an enzyme with serine amidase activity. In the activation reaction, the apparent molecular weight of the heavy chain decreased from 41,000 to 40,000 as determined by gel electrophoresis in the presence of sodium dodecyl sulfate. No apparent change in the molecular weight of the light chain was observed in the activation process. The heavy chain of human activated protein C also contains the active-site serine residue as evidenced by its ability to react with radiolabeled diisopropyl fluorophosphate. Human activated protein C markedly prolongs the kaolin-cephalin clotting time of human plasma, but not that of bovine plasma. The amidolytic and anticoagulant activities of human activated protein C were completely obviated by prior incubation of the enzyme with diisopropyl fluorophosphate. These results indicate that human protein C, like its bovine counterpart, exists in plasma as a zymogen and is converted to a serine protease by limited proteolysis with attendant anticoagulant activity. Images PMID:468991

  13. Identification of polymer surface adsorbed proteins implicated in pluripotent human embryonic stem cell expansion.

    PubMed

    Hammad, Moamen; Rao, Wei; Smith, James G W; Anderson, Daniel G; Langer, Robert; Young, Lorraine E; Barrett, David A; Davies, Martyn C; Denning, Chris; Alexander, Morgan R

    2016-08-16

    Improved biomaterials are required for application in regenerative medicine, biosensing, and as medical devices. The response of cells to the chemistry of polymers cultured in media is generally regarded as being dominated by proteins adsorbed to the surface. Here we use mass spectrometry to identify proteins adsorbed from a complex mouse embryonic fibroblast (MEF) conditioned medium found to support pluripotent human embryonic stem cell (hESC) expansion on a plasma etched tissue culture polystyrene surface. A total of 71 proteins were identified, of which 14 uniquely correlated with the surface on which pluripotent stem cell expansion was achieved. We have developed a microarray combinatorial protein spotting approach to test the potential of these 14 proteins to support expansion of a hESC cell line (HUES-7) and a human induced pluripotent stem cell line (ReBl-PAT) on a novel polymer (N-(4-Hydroxyphenyl) methacrylamide). These proteins were spotted to form a primary array yielding several protein mixture 'hits' that enhanced cell attachment to the polymer. A second array was generated to test the function of a refined set of protein mixtures. We found that a combination of heat shock protein 90 and heat shock protein-1 encourage elevated adherence of pluripotent stem cells at a level comparable to fibronectin pre-treatment. PMID:27466628

  14. Differential analysis of "protein corona" profile adsorbed onto different nonviral gene delivery systems.

    PubMed

    Capriotti, Anna Laura; Caracciolo, Giulio; Caruso, Giuseppe; Foglia, Patrizia; Pozzi, Daniela; Samperi, Roberto; Laganà, Aldo

    2011-12-15

    A shotgun proteomics approach was used to characterize and compare the proteins that lead to the formation of a rich "protein corona" adsorbed onto the surfaces of cationic liposomes (CLs), lipoplexes, and lipid/polycation/DNA (LPD) complexes, when they come into contact with plasma. After separation of the nanoparticle-protein complex from plasma, the protein mixture was digested, and peptides were analyzed by nanoliquid chromatography-Orbitrap LTQ-XL mass spectrometry. The number of proteins bound to lipoplexes was double that of those identified in the corona of CLs (208 vs 105), while 77 proteins were common to both coronas. The number of proteins bound to the surface of the LPD complexes (158, 133 of which are common to lipoplexes) is intermediate between those found in the protein corona of both CLs and lipoplexes. About half of them were found in the protein corona of CLs. By overlapping the three formulations, it can be seen that only 12 proteins are peculiar to LPD complexes. These results may help in designing gene delivery systems capable of binding the minimum possible quantity of proteins that influence transfection negatively, binding selectively proteins capable of helping in steering in vivo the vector toward the target, and obtaining more efficient and effective gene therapy.

  15. The effect of gold nanoparticle structure on the conformation and function of adsorbed proteins

    NASA Astrophysics Data System (ADS)

    Gagner, Jennifer E.

    Many applications of nanobiomaterials rely on or are enhanced by specific, protein-mediated interactions with biological systems. These interactions can be engineered by chemically modifying the surface of the material to affect protein adsorption, or by altering the topography of the nanoscale surface. The attachment or adsorption of proteins onto materials can greatly affect the structure and subsequent function of those proteins, giving rise to unpredictable and potentially undesirable effects. Thus, it is essential to develop a detailed understanding of how nanostructured surface characteristics, such as atomic-scale topography, surface energy, and chemical structure may affect protein adsorption, structure, function, and stability. The presented work on gold nanoparticles (AuNP) in the forms of spheres (AuNS), rods (AuNR), cubes (AuNC) and octahedra (AuNO) will elucidate the effect of nanoparticle morphology on adsorbed model proteins lysozyme (Lyz) and α-chymotrypsin (ChT). It has been found that nanoparticle morphology does affect the structure of adsorbed proteins as well as the extent of the surface coverage; however, the final form of the nano-bio conjugate is protein specific. Lyz conjugates underwent loss of structure and rapid aggregation regardless of AuNP morphology; however, ChT conjugates exhibited no structure loss when immobilized on AuNS, and a significant, loading specific structure loss when adsorbed on AuNR. Further work will be presented on efforts to determine the role of crystal structure, surface energy, and ligand chemistry on adsorbed proteins. Wet chemical methods are used to synthesize AuNC with f100g facets and AuNO with f111g facets. Nanoparticles are characterized through electron microscopy, X-ray and electron diffraction, X-ray photoelectron spectroscopy and inductively coupled plasma mass spectroscopy. Protein conjugation and changes in protein structure are monitored through a variety of physical and spectroscopic techniques

  16. Shotgun proteomic analytical approach for studying proteins adsorbed onto liposome surface.

    PubMed

    Capriotti, Anna Laura; Caracciolo, Giulio; Cavaliere, Chiara; Crescenzi, Carlo; Pozzi, Daniela; Laganà, Aldo

    2011-09-01

    The knowledge about the interaction between plasma proteins and nanocarriers employed for in vivo delivery is fundamental to understand their biodistribution. Protein adsorption onto nanoparticle surface (protein corona) is strongly affected by vector surface characteristics. In general, the primary interaction is thought to be electrostatic, thus surface charge of carrier is supposed to play a central role in protein adsorption. Because protein corona composition can be critical in modifying the interactive surface that is recognized by cells, characterizing its formation onto lipid particles may serve as a fundamental predictive model for the in vivo efficiency of a lipidic vector. In the present work, protein coronas adsorbed onto three differently charged cationic liposome formulations were compared by a shotgun proteomic approach based on nano-liquid chromatography-high-resolution mass spectrometry. About 130 proteins were identified in each corona, with only small differences between the different cationic liposome formulations. However, this study could be useful for the 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 into body fluids.

  17. XPS and bioactivity study of the bisphosphonate pamidronate adsorbed onto plasma sprayed hydroxyapatite coatings

    NASA Astrophysics Data System (ADS)

    McLeod, Kate; Kumar, Sunil; Smart, Roger St. C.; Dutta, Naba; Voelcker, Nicolas H.; Anderson, Gail I.; Sekel, Ron

    2006-12-01

    This paper reports the use of X-ray photoelectron spectroscopy (XPS) to investigate bisphosphonate (BP) adsorption onto plasma sprayed hydroxyapatite (HA) coatings commonly used for orthopaedic implants. BPs exhibit high binding affinity for the calcium present in HA and hence can be adsorbed onto HA-coated implants to exploit their beneficial properties for improved bone growth at the implant interface. A rigorous XPS analysis of pamidronate, a commonly used nitrogenous BP, adsorbed onto plasma sprayed HA-coated cobalt-chromium substrates has been carried out, aimed at: (a) confirming the adsorption of this BP onto HA; (b) studying the BP diffusion profile in the HA coating by employing the technique of XPS depth profiling; (c) confirming the bioactivity of the adsorbed BP. XPS spectra of plasma sprayed HA-coated discs exposed to a 10 mM aqueous BP solution (pamidronate) for periods of 1, 2 and 24 h showed nitrogen and phosphorous photoelectron signals corresponding to the BP, confirming its adsorption onto the HA substrate. XPS depth profiling of the 2 h BP-exposed HA discs showed penetration of the BP into the HA matrix to depths of at least 260 nm. The bioactivity of the adsorbed BP was confirmed by the observed inhibition of osteoclast (bone resorbing) cell activity. In comparison to the HA sample, the HA sample with adsorbed BP exhibited a 25-fold decrease in primary osteoclast cells.

  18. Differential plasma protein binding to metal oxide nanoparticles

    NASA Astrophysics Data System (ADS)

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

    2009-11-01

    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 TiO2, 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 TiO2 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.

  19. Plasma-induced conversion of surface-adsorbed hydrocarbons

    SciTech Connect

    Sackinger, W.M.

    1992-07-01

    Experimental results are reported for an electrical device for direct conversion of methane into higher hydrocarbons. A microchannel plate is excited with electrons from a photoemissive source, and electron impact ionization of methane on the inner surfaces of the microchannels creates an ion feedback process. The resulting low-density plasma creates higher hydrocarbons when charged particles impact the surfaces at grazing incidence. The production Of C{sub 2} to C{sub 8}-containing gases was noted, with a selectivity for C{sub 2} of 39% in one case. The proportions of converted products and the conversion rates depend upon the electrical voltage, the microchannel geometry, and the operating pressure. Conversion rates increase with operating pressure.

  20. Plasma-induced conversion of surface-adsorbed hydrocarbons

    SciTech Connect

    Sackinger, W.M.

    1992-01-01

    Experimental results are reported for an electrical device for direct conversion of methane into higher hydrocarbons. A microchannel plate is excited with electrons from a photoemissive source, and electron impact ionization of methane on the inner surfaces of the microchannels creates an ion feedback process. The resulting low-density plasma creates higher hydrocarbons when charged particles impact the surfaces at grazing incidence. The production Of C{sub 2} to C{sub 8}-containing gases was noted, with a selectivity for C{sub 2} of 39% in one case. The proportions of converted products and the conversion rates depend upon the electrical voltage, the microchannel geometry, and the operating pressure. Conversion rates increase with operating pressure.

  1. Sorbent track: Quantitative monitoring of adsorbed VOCs under in-situ plasma exposure

    PubMed Central

    Jia, Zixian; Rousseau, Antoine

    2016-01-01

    Sorbent-TRACK is a new device developed to monitor adsorption and surface oxidation of pollutants under direct plasma exposure. It is based on direct transmitted Fourier Transformed Infrared (FTIR) spectroscopy. A pyrex reactor under controlled gas pressure and composition is inserted on the infrared beam of a commercially available Nicolet 5700 FTIR spectrometer. A substrate holder is located on the optical path of the infrared beam. A thin pellet of a dedicated catalyst (CeO2 in the present work) is inserted in a substrate holder and can be exposed to direct plasma treatment using a Dielectric Barrier Discharge. The time resolution of Sorbent-TRACK is limited by the time resolution of the Nicolet 5700 FTIR spectrometer and close to 30 s. The dynamic of the adsorption and plasma oxidation of acetone and isopropanol on CeO2 are studied and intermediates are monitored. Performances and sensitivity of Sorbent-TRACK are reported Adsorption and oxidation of acetone leads to production of adsorbed isobutene and acetic acid, where oxidation of isopropanol gives mainly to adsorbed acetone, mesityl oxide and acetate. An increase of the plasma power leads to an increase of the isopropanol and acetone oxidation rate and a related increase of the production of adsorbed intermediates. PMID:27555531

  2. Sorbent track: Quantitative monitoring of adsorbed VOCs under in-situ plasma exposure

    NASA Astrophysics Data System (ADS)

    Jia, Zixian; Rousseau, Antoine

    2016-08-01

    Sorbent-TRACK is a new device developed to monitor adsorption and surface oxidation of pollutants under direct plasma exposure. It is based on direct transmitted Fourier Transformed Infrared (FTIR) spectroscopy. A pyrex reactor under controlled gas pressure and composition is inserted on the infrared beam of a commercially available Nicolet 5700 FTIR spectrometer. A substrate holder is located on the optical path of the infrared beam. A thin pellet of a dedicated catalyst (CeO2 in the present work) is inserted in a substrate holder and can be exposed to direct plasma treatment using a Dielectric Barrier Discharge. The time resolution of Sorbent-TRACK is limited by the time resolution of the Nicolet 5700 FTIR spectrometer and close to 30 s. The dynamic of the adsorption and plasma oxidation of acetone and isopropanol on CeO2 are studied and intermediates are monitored. Performances and sensitivity of Sorbent-TRACK are reported Adsorption and oxidation of acetone leads to production of adsorbed isobutene and acetic acid, where oxidation of isopropanol gives mainly to adsorbed acetone, mesityl oxide and acetate. An increase of the plasma power leads to an increase of the isopropanol and acetone oxidation rate and a related increase of the production of adsorbed intermediates.

  3. Sorbent track: Quantitative monitoring of adsorbed VOCs under in-situ plasma exposure.

    PubMed

    Jia, Zixian; Rousseau, Antoine

    2016-01-01

    Sorbent-TRACK is a new device developed to monitor adsorption and surface oxidation of pollutants under direct plasma exposure. It is based on direct transmitted Fourier Transformed Infrared (FTIR) spectroscopy. A pyrex reactor under controlled gas pressure and composition is inserted on the infrared beam of a commercially available Nicolet 5700 FTIR spectrometer. A substrate holder is located on the optical path of the infrared beam. A thin pellet of a dedicated catalyst (CeO2 in the present work) is inserted in a substrate holder and can be exposed to direct plasma treatment using a Dielectric Barrier Discharge. The time resolution of Sorbent-TRACK is limited by the time resolution of the Nicolet 5700 FTIR spectrometer and close to 30 s. The dynamic of the adsorption and plasma oxidation of acetone and isopropanol on CeO2 are studied and intermediates are monitored. Performances and sensitivity of Sorbent-TRACK are reported Adsorption and oxidation of acetone leads to production of adsorbed isobutene and acetic acid, where oxidation of isopropanol gives mainly to adsorbed acetone, mesityl oxide and acetate. An increase of the plasma power leads to an increase of the isopropanol and acetone oxidation rate and a related increase of the production of adsorbed intermediates. PMID:27555531

  4. Protein immobilization in hollow nanostructures and investigation of the adsorbed protein behavior.

    PubMed

    Qian, Xi; Levenstein, Alex; Gagner, Jennifer E; Dordick, Jonathan S; Siegel, Richard W

    2014-02-11

    Understanding nanomaterial-biomolecule interactions is critical to develop broad applications in sensors, devices, and therapeutics. During the past decade, in-depth studies have been performed on the effect of nanoscale surface topography on adsorbed protein structure and function. However, a fundamental understanding of nanobio interactions at concave surfaces is limited; the greatest challenge is to create a nanostructure that allows such interactions to occur and to be characterized. We have synthesized hollow nanocages (AuNG) through careful control of morphology and surface chemistry. Lysozyme was used as a model to probe interactions between a protein and these nanostructures. Solid Au nanoparticles with a similar morphology and surface chemistry were also used as a reference. Through a series of quantitative analyses of protein adsorption profiles and enzymatic activity assays of both nanobioconjugates, we discovered that a significant amount of protein could be delivered into the core of AuNG, while maintaining a substantial fraction of native activity.

  5. Influence of particle size on the binding activity of proteins adsorbed onto gold nanoparticles.

    PubMed

    Kaur, Kanwarjeet; Forrest, James A

    2012-02-01

    We used optical extinction spectroscopy to study the structure of proteins adsorbed onto gold nanoparticles of sizes 5-60 nm and their resulting biological binding activity. For these studies, proteins differing in size and shape, with well-characterized and specific interactions-rabbit immunoglobulin G (IgG), goat anti-rabbit IgG (anti-IgG), Staphylococcal protein A, streptavidin, and biotin-were used as model systems. Protein interaction with gold nanoparticles was probed by optical extinction measurements of localized surface plasmon resonance (LSPR) of the gold nanoparticles. Binding of the ligands in solution to protein molecules already immobilized on the surface of gold causes a small but detectable shift in the LSPR peak of the gold nanoparticles. This shift can be used to probe the binding activity of the adsorbed protein. Within the context of Mie theory calculations, the thickness of the adsorbed protein layer as well as its apparent refractive index is shown to depend on the size of the gold nanoparticle. The results suggest that proteins can adopt different orientations that depend on the size of the gold nanospheres. These different orientations, in turn, can result in different levels of biological activity. For example, we find that IgG adsorbed on spheres with diameter ≥20 nm does not bind to protein A. This study illustrates the principle that the size of nanoparticles can strongly influence the binding activity of adsorbed proteins. In addition to the importance of this in cases of direct exposure of proteins to nanoparticles, the results have implications for proteins adsorbed to materials with nanometer scale surface roughness.

  6. Plant Plasma Membrane Proteins 1

    PubMed Central

    Grimes, Howard D.; Breidenbach, R. William

    1987-01-01

    A major 75 kD protein group from the tomato plasma membrane was semipurified on polyacrylamide gels and used to raise a rabbit antiserum. The resulting antiserum recognized a single 75 kilodalton band from phase partitioned tomato plasma membrane (from both suspension cells and mature, green fruit) after resolution on one-dimensional polyacrylamide gels. Two-dimensional polyacrylamide gel analysis of proteins from tomato plasma membrane showed that the 75 kilodalton antiserum recognized a group of proteins ranging from 63.1 to 88.2 kilodaltons (mean = 75.6 kilodaltons) and with isoelectric point values ranging from 5.7 to 6.3. No other spots were visible on the two-dimensional blots. This antiserum was shown to bind protoplast surface epitopes by indirect immunofluorescence. The presence of this protein group in both monocotyledonous and dicotyledonous plants was established by immunoblotting the tomato 75 kilodalton antiserum against proteins obtained from plasma membrane-enriched fractions from corn roots and soybean roots. The data suggest that this 75 kilodalton protein group is a major proteinaceous component of the plant plasma membrane. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 PMID:16665801

  7. Continuous reduction of cyclic adsorbed and desorbed NO(x) in diesel emission using nonthermal plasma.

    PubMed

    Kuwahara, Takuya; Nakaguchi, Harunobu; Kuroki, Tomoyuki; Okubo, Masaaki

    2016-05-01

    Considering the recent stringent regulations governing diesel NO(x) emission, an aftertreatment system for the reduction of NO(x) in the exhaust gas has been proposed and studied. The proposed system is a hybrid method combining nonthermal plasma and NOx adsorbent. The system does not require precious metal catalysts or harmful chemicals such as urea and ammonia. In the present system, NO(x) in diesel emission is treated by adsorption and desorption by adsorbent as well as nonthermal plasma reduction. In addition, the remaining NO(x) in the adsorbent is desorbed again in the supplied air by residual heat. The desorbed NO(x) in air recirculates into the intake of the engine, and this process, i.e., exhaust gas components' recirculation (EGCR) achieves NO(x) reduction. Alternate utilization of two adsorption chambers in the system can achieve high-efficiency NO(x) removal continuously. An experiment with a stationary diesel engine for electric power generation demonstrates an energy efficiency of 154 g(NO2)/kWh for NO(x) removal and continuous NO(x) reduction of 70.3%. Considering the regulation against diesel emission in Japan, i.e., the new regulation to be imposed on vehicles of 3.5-7.5 ton since 2016, the present aftertreatment system fulfills the requirement with only 1.0% of engine power.

  8. Continuous reduction of cyclic adsorbed and desorbed NO(x) in diesel emission using nonthermal plasma.

    PubMed

    Kuwahara, Takuya; Nakaguchi, Harunobu; Kuroki, Tomoyuki; Okubo, Masaaki

    2016-05-01

    Considering the recent stringent regulations governing diesel NO(x) emission, an aftertreatment system for the reduction of NO(x) in the exhaust gas has been proposed and studied. The proposed system is a hybrid method combining nonthermal plasma and NOx adsorbent. The system does not require precious metal catalysts or harmful chemicals such as urea and ammonia. In the present system, NO(x) in diesel emission is treated by adsorption and desorption by adsorbent as well as nonthermal plasma reduction. In addition, the remaining NO(x) in the adsorbent is desorbed again in the supplied air by residual heat. The desorbed NO(x) in air recirculates into the intake of the engine, and this process, i.e., exhaust gas components' recirculation (EGCR) achieves NO(x) reduction. Alternate utilization of two adsorption chambers in the system can achieve high-efficiency NO(x) removal continuously. An experiment with a stationary diesel engine for electric power generation demonstrates an energy efficiency of 154 g(NO2)/kWh for NO(x) removal and continuous NO(x) reduction of 70.3%. Considering the regulation against diesel emission in Japan, i.e., the new regulation to be imposed on vehicles of 3.5-7.5 ton since 2016, the present aftertreatment system fulfills the requirement with only 1.0% of engine power. PMID:26844402

  9. [Analysis of FT-IR-ATR spectra of serum proteins adsorbed on carbonaceous materials].

    PubMed

    Li, Bogang; Na, Juanjuan; Yin, Jie; Yin, Guangfu

    2006-10-01

    To clarify the reason causing difference of serum proteins adsorbability on different carbonaceous materials, FT-IR-ATR spectra of human serum albumin (HSA) and human serum fibrinogen(HFG) before and after adsorbing on diamond like carbon film (DLC),diamond film (DF) and graphite were analyzed. It has been shown that there are hydrogen bond because of -NH at the interfaces of HSA-DLC, HFG-DF and HFG-graphite. Based on the results, earlier research conclusion that the adsorbability of HSA on DLC higher than that on DF and graphite, but on DF and graphite the adsorption of HFG takes precedence can be explained rationally.

  10. Removal of formaldehyde by adsorption and plasma treatment of mineral adsorbent

    NASA Astrophysics Data System (ADS)

    Saulich, K.; Müller, S.

    2013-01-01

    Formaldehyde is a harmful ambient air pollutant which can be produced by incomplete combustion processes, e.g. in power plants or automobiles. In this work a cycled adsorption and discharge process using mineral granulate in a packed bed dielectric barrier discharge plasma reactor was applied for formaldehyde (99 ppm) removal from gas streams. The mineral granulate used consisted of 80% halloysite and showed a good adsorption capacity for formaldehyde. In the discharge step, the adsorbed formaldehyde molecules were decomposed to COx and hydrocarbons in a N2 plasma at a low input discharge power of 2.2 W. The decomposition performance on adsorbed formaldehyde molecules was studied depending on space-time, a specific oxygen fraction of the carrier gas and the influence of temperature. With rising N2 space times in the discharge area, the total amount of decomposed formaldehyde molecules increased and the decomposition reaction mechanism shifted to CO2 formation. An oxygen fraction in the carrier gas further raised the oxidized amount of formaldehyde to CO2. The mineral granulate showed satisfied regeneration ability during the cycled plasma process.

  11. Effect of the interplay between protein and surface on the properties of adsorbed protein layers.

    PubMed

    Ouberai, Myriam M; Xu, Kairuo; Welland, Mark E

    2014-08-01

    Although protein adsorption to surface is a common phenomenon, investigation of the process is challenging due to the complexity of the interplay between external factors, protein and surface properties. Therefore experimental approaches have to measure the properties of adsorbed protein layers with high accuracy in order to achieve a comprehensive description of the process. To this end, we used a combination of two biosensing techniques, dual polarization interferometry and quartz crystal microbalance with dissipation. From this, we are able to extract surface coverage values, layer structural parameters, water content and viscoelastic properties to examine the properties of protein layers formed at the liquid/solid interface. Layer parameters were examined upon adsorption of proteins of varying size and structural properties, on surfaces with opposite polarity. We show that "soft" proteins such as unfolded α-synuclein and high molecular weight albumin are highly influenced by the surface polarity, as they form a highly diffuse and hydrated layer on the hydrophilic silica surface as opposed to the denser, less hydrated layer formed on a hydrophobic methylated surface. These layer properties are a result of different orientations and packing of the proteins. By contrast, lysozyme is barely influenced by the surface polarity due to its intrinsic structural stability. Interestingly, we show that for a similar molecular weight, the unfolded α-synuclein forms a layer with the highest percentage of solvation not related to surface coverage but resulting from the highest water content trapped within the protein. Together, these data reveal a trend in layer properties highlighting the importance of the interplay between protein and surface for the design of biomaterials. PMID:24780165

  12. Volumetric Interpretation of Protein Adsorption: Interfacial Packing of Protein Adsorbed to Hydrophobic Surfaces from Surface-Saturating Solution Concentrations

    PubMed Central

    Kao, Ping; Parhi, Purnendu; Krishnan, Anandi; Noh, Hyeran; Haider, Waseem; Tadigadapa, Srinivas; Allara, David L.; Vogler, Erwin A.

    2010-01-01

    The maximum capacity of a hydrophobic adsorbent is interpreted in terms of square or hexagonal (cubic and face-centered-cubic, FCC) interfacial packing models of adsorbed blood proteins in a way that accommodates experimental measurements by the solution-depletion method and quartz-crystal-microbalance (QCM) for the human proteins serum albumin (HSA, 66 kDa), immunoglobulin G (IgG, 160 kDa), fibrinogen (Fib, 341 kDa), and immunoglobulin M (IgM, 1000 kDa). A simple analysis shows that adsorbent capacity is capped by a fixed mass/volume (e.g. mg/mL) surface-region (interphase) concentration and not molar concentration. Nearly analytical agreement between the packing models and experiment suggests that, at surface saturation, above-mentioned proteins assemble within the interphase in a manner that approximates a well-ordered array. HSA saturates a hydrophobic adsorbent with the equivalent of a single square-or-hexagonally-packed layer of hydrated molecules whereas the larger proteins occupy two-or-more layers, depending on the specific protein under consideration and analytical method used to measure adsorbate mass (solution depletion or QCM). Square-or-hexagonal (cubic and FCC) packing models cannot be clearly distinguished by comparison to experimental data. QCM measurement of adsorbent capacity is shown to be significantly different than that measured by solution depletion for similar hydrophobic adsorbents. The underlying reason is traced to the fact that QCM measures contribution of both core protein, water of hydration, and interphase water whereas solution depletion measures only the contribution of core protein. It is further shown that thickness of the interphase directly measured by QCM systematically exceeds that inferred from solution-depletion measurements, presumably because the static model used to interpret solution depletion does not accurately capture the complexities of the viscoelastic interfacial environment probed by QCM. PMID:21035180

  13. Volumetric Interpretation of Protein Adsorption: Ion-Exchange Adsorbent Capacity, Protein pI, and Interaction Energetics

    PubMed Central

    Noh, Hyeran; Yohe, Stefan; Vogler, Erwin A.

    2008-01-01

    Adsorption of lysozyme (Lys), human serum albumin (HSA), and immunoglobulin G (IgG) to anion- and cation-exchange resins is dominated by electrostatic interactions between protein and adsorbent. The solution-depletion method of measuring adsorption shows, however, that these proteins do not irreversibly adsorb to ion-exchange surfaces, even when the charge disparity between adsorbent and protein inferred from protein pI is large. Net-positively-charged Lys (pI = 11) and net-negatively-charged HSA (pI = 5.5) adsorb so strongly to sulfopropyl sepharose (SP; a negatively-charged, strong cation exchange resin, −0.22 mmol/mL exchange capacity) that both resist displacement by net-neutral IgG (pI = 7.0) in simultaneous adsorption-competition experiments. By contrast, IgG readily displaces both Lys and HSA adsorbed either to quarternary-ammonium sepharose (Q; a positively-charged, strong anion exchanger, + 0.22 mmol/mL exchange capacity) or octadecyl sepharose (ODS, a neutral hydrophobic resin, 0 mmol/mL exchange capacity). Thus it is concluded that adsorption results do not sensibly correlate with protein pI and that pI is actually a rather poor predictor of affinity for ion-exchange surfaces. Adsorption of Lys, HSA, and IgG to ion-exchange resins from stagnant solution leads to adsorbed multi-layers, into-or-onto which IgG adsorbs in adsorption-competition experiments. Comparison of adsorption to ion-exchange resins and neutral ODS leads to the conclusion that the apparent standard free-energy-of-adsorption ΔGadso of Lys, HSA, and IgG is not large in comparison to thermal energy due to energy-compensating interactions between water, protein, and ion-exchange surfaces that leaves a small net ΔGadso. Thus water is found to control protein adsorption to a full range of substratum types spanning hydrophobic (poorly water wettable) surfaces, hydrophilic surfaces bearing relatively-weak Lewis acid/base functionalities that wet with (hydrogen bond to) water but do not

  14. Depletion of Abundant Plasma Proteins and Limitations of Plasma Proteomics

    PubMed Central

    Tu, Chengjian; Rudnick, Paul A.; Martinez, Misti Y.; Cheek, Kristin L.; Stein, Stephen E.; Slebos, Robbert J. C.; Liebler, Daniel C.

    2010-01-01

    Immunoaffinity depletion with antibodies to the top 7 or top 14 high abundance plasma proteins is used to enhance detection of lower abundance proteins in both shotgun and targeted proteomic analyses. We evaluated the effects of top 7/top 14 immunodepletion on the shotgun proteomic analysis of human plasma. Our goal was to evaluate the impact of immunodepletion on detection of proteins across detectable ranges of abundance. The depletion columns afforded highly repeatable and efficient plasma protein fractionation. Relatively few nontargeted proteins were captured by the depletion columns. Analyses of unfractionated and immunodepleted plasma by peptide isoelectric focusing (IEF), followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) demonstrated enrichment of nontargeted plasma proteins by an average of 4-fold, as assessed by MS/MS spectral counting. Either top 7 or top 14 immunodepletion resulted in a 25% increase in identified proteins compared to unfractionated plasma. Although 23 low abundance (<10 ng mL−1) plasma proteins were detected, they accounted for only 5–6% of total protein identifications in immunodepleted plasma. In both unfractionated and immunodepleted plasma, the 50 most abundant plasma proteins accounted for 90% of cumulative spectral counts and precursor ion intensities, leaving little capacity to sample lower abundance proteins. Untargeted proteomic analyses using current LC-MS/MS platforms—even with immunodepletion—cannot be expected to efficiently discover low abundance, disease-specific biomarkers in plasma. PMID:20677825

  15. Multiscale simulations of protein G B1 adsorbed on charged self-assembled monolayers.

    PubMed

    Liu, Jie; Liao, Chenyi; Zhou, Jian

    2013-09-10

    The orientation of an antibody plays an important role in the development of immunosensors. Protein G is an antibody binding protein, which specifically targets the Fc fragment of an antibody. In this work, the orientation of prototypical and mutated protein G B1 adsorbed on positively and negatively charged self-assembled monolayers was studied by parallel tempering Monte Carlo and all-atom molecular dynamics simulations. Both methods present generally similar orientation distributions of protein G B1 for each kind of surface. The root-mean-square deviation, DSSP, gyration radius, eccentricity, dipole moment, and superimposed structures of protein G B1 were analyzed. Moreover, the orientation of binding antibody was also predicted in this work. Simulation results show that with the same orientation trends, the mutant exhibits narrower orientation distributions than does the prototype, which was mainly caused by the stronger dipole of the mutant. Both kinds of proteins adsorbed on charged surfaces were induced by the competition of electrostatic interaction and vdW interaction; the electrostatic interaction energy dominated the adsorption behavior. The protein adsorption was also largely affected by the distribution of charged residues within the proteins. Thus, the prototype could adsorb on a negatively charged surface, although it keeps a net charge of -4 e. The mutant has imperfect opposite orientation when it adsorbed on oppositely charged surfaces. For the mutant on a carboxyl-functionalized self-assembled monolayer (COOH-SAM), the orientation was the same as that inferred by experiments. While for the mutant on amine-functionalized self-assembled monolayer (NH2-SAM), the orientation was induced by the competition between attractive interactions (led by ASP40 and GLU56) and repulsive interactions (led by LYS10); thus, the perfect opposite orientation could not be obtained. On both surfaces, the adsorbed protein could retain its native conformation. The desired

  16. The special features of protein adsorption isotherms on silica adsorbents

    NASA Astrophysics Data System (ADS)

    Chukhrai, E. S.; Atyaksheva, L. F.; Pilipenko, O. S.

    2011-05-01

    The adsorption isotherms of hemoglobin, peroxidase, and β-galactosidase on silochrome and mesoporous and biporous silicas were comparatively studied. Adsorption developed in two stages, including fast "reversible" protein adsorption (equilibrium was reached in t ≤ 1-2 h) and a "slow stage" of irreversible binding in t ≫ 24 h (multipoint adsorption). The corresponding equilibrium constants were determined. The mechanism of unlimited linear association of peroxidase in the adsorption layer on the surface of silochrome was established.

  17. Spectrofluorimetric determination of glutathione in human plasma by solid-phase extraction using graphene as adsorbent

    NASA Astrophysics Data System (ADS)

    Huang, Ke-Jing; Jing, Qiang-Shan; Wei, Cai-Yun; Wu, Ying-Ying

    2011-09-01

    An efficient solid phase extraction-spectrofluorimetric method using graphene as adsorbent was developed to sensitively determine glutathione (GSH) in biological samples. Fluorescent probe N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-yl)methyl)iodoacetamide (BODIPY Fl-C 1-IA) was applied for the derivatization of GSH. The procedure was based on BODIPY Fl-C 1-IA selective reaction with GSH to form highly fluorescent product BODIPY Fl-C 1-IA-GSH, its extraction to the graphene-packed SPE cartridge and spectrofluorimetric determination. Some factors affecting the extraction efficiency, such as the type of the eluent and its volume, sample pH, extraction time, and sample volume were optimized. Comparative studies were also performed between graphene and other adsorbents including C 18 silica, graphitic carbon, and multi-walled carbon nanotubes for the extraction of analyte. The calibration graph using the pretreatment system for GSH was linear over the range of 0.5-200 nM. The limit of detection was 0.01 nM (signal-to-noise ratio = 3). Relative standard deviation for six replicate determinations of GSH at 80 nM concentration level was lower than 5.0%. The developed method was applied to the determination of GSH in human plasma with recoveries of 92-108%. This work revealed the great potentials of graphene as an excellent sorbent material in the analysis of biological samples.

  18. Spectrofluorimetric determination of glutathione in human plasma by solid-phase extraction using graphene as adsorbent.

    PubMed

    Huang, Ke-Jing; Jing, Qiang-Shan; Wei, Cai-Yun; Wu, Ying-Ying

    2011-09-01

    An efficient solid phase extraction-spectrofluorimetric method using graphene as adsorbent was developed to sensitively determine glutathione (GSH) in biological samples. Fluorescent probe N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-yl)methyl)iodoacetamide (BODIPY Fl-C1-IA) was applied for the derivatization of GSH. The procedure was based on BODIPY Fl-C1-IA selective reaction with GSH to form highly fluorescent product BODIPY Fl-C1-IA-GSH, its extraction to the graphene-packed SPE cartridge and spectrofluorimetric determination. Some factors affecting the extraction efficiency, such as the type of the eluent and its volume, sample pH, extraction time, and sample volume were optimized. Comparative studies were also performed between graphene and other adsorbents including C18 silica, graphitic carbon, and multi-walled carbon nanotubes for the extraction of analyte. The calibration graph using the pretreatment system for GSH was linear over the range of 0.5-200 nM. The limit of detection was 0.01 nM (signal-to-noise ratio=3). Relative standard deviation for six replicate determinations of GSH at 80 nM concentration level was lower than 5.0%. The developed method was applied to the determination of GSH in human plasma with recoveries of 92-108%. This work revealed the great potentials of graphene as an excellent sorbent material in the analysis of biological samples.

  19. Role of Structure and Glycosylation of Adsorbed Protein Films in Biolubrication

    PubMed Central

    Veeregowda, Deepak H.; Busscher, Henk J.; Vissink, Arjan; Jager, Derk-Jan; Sharma, Prashant K.; van der Mei, Henny C.

    2012-01-01

    Water forms the basis of lubrication in the human body, but is unable to provide sufficient lubrication without additives. The importance of biolubrication becomes evident upon aging and disease, particularly under conditions that affect secretion or composition of body fluids. Insufficient biolubrication, may impede proper speech, mastication and swallowing, underlie excessive friction and wear of articulating cartilage surfaces in hips and knees, cause vaginal dryness, and result in dry, irritated eyes. Currently, our understanding of biolubrication is insufficient to design effective therapeutics to restore biolubrication. Aim of this study was to establish the role of structure and glycosylation of adsorbed protein films in biolubrication, taking the oral cavity as a model and making use of its dynamics with daily perturbations due to different glandular secretions, speech, drinking and eating, and tooth brushing. Using different surface analytical techniques (a quartz crystal microbalance with dissipation monitoring, colloidal probe atomic force microscopy, contact angle measurements and X-ray photo-electron spectroscopy), we demonstrated that adsorbed salivary conditioning films in vitro are more lubricious when their hydrophilicity and degree of glycosylation increase, meanwhile decreasing their structural softness. High-molecular-weight, glycosylated proteins adsorbing in loops and trains, are described as necessary scaffolds impeding removal of water during loading of articulating surfaces. Comparing in vitro and in vivo water contact angles measured intra-orally, these findings were extrapolated to the in vivo situation. Accordingly, lubricating properties of teeth, as perceived in 20 volunteers comprising of equal numbers of male and female subjects, could be related with structural softness and glycosylation of adsorbed protein films on tooth surfaces. Summarizing, biolubrication is due to a combination of structure and glycosylation of adsorbed protein

  20. Determinants of protein elution rates from preparative ion-exchange adsorbents.

    PubMed

    Angelo, James M; Lenhoff, Abraham M

    2016-04-01

    The rate processes involved in elution in preparative chromatography can affect both peak resolution and hence selectivity as well as practical factors such as facility fit. These processes depend on the physical structure of the adsorbent particles, the amount of bound solute, the solution conditions for operation or some combination of these factors. Ion-exchange adsorbents modified with covalently attached or grafted polymer layers have become widely used in preparative chromatography. Their often easily accessible microstructures offer substantial binding capacities for biomolecules, but elution has sometimes been observed to be undesirably slow. In order to determine which physicochemical phenomena control elution behavior, commercially available cellulosic, dextran-grafted and unmodified agarose materials were characterized here by their elution profiles at various conditions, including different degrees of loading. Elution data were analyzed under the assumption of purely diffusion-limited control, including the role of pore structure properties such as porosity and tortuosity. In general, effective elution rates decreased with the reduction of accessible pore volume, but differences among different proteins indicated the roles of additional factors. Additional measurements and analysis, including the use of confocal laser scanning microscopy to observe elution within single chromatographic particles, indicated the importance of protein association within the particle during elution. The use of protein stabilizing agents was explored in systems presenting atypical elution behavior, and l-arginine and disaccharide excipients were shown to alleviate the effects for one protein, lysozyme, in the presence of sodium chloride. Incorporation of these excipients into eluent buffer gave rise to faster elution and significantly lower pool volumes in elution from polymer-modified adsorbents.

  1. The leucine rich amelogenin protein (LRAP) adsorbs as monomers or dimers onto surfaces

    SciTech Connect

    Tarasevich, Barbara J.; Lea, Alan S.; Shaw, Wendy J.

    2010-03-15

    Amelogenin and amelogenin splice variants are believed to be involved in controlling the formation of the highly anisotropic and ordered hydroxyapatite crystallites that form enamel. The adsorption behavior of amelogenin proteins onto substrates is very important because protein-surface interactions are critical to it’s function. We have studied the adsorption of LRAP, a splice variant of amelogenin which may also contribute to enamel function, onto model self-assembled monolayers on gold containing of COOH, CH3, and NH2 end groups. Dynamic light scattering (DLS) experiments indicated that LRAP in phosphate buffered saline (PBS) and solutions at saturation with calcium phosphate contained aggregates of nanospheres. Null ellipsometry and atomic force microscopy (AFM) were used to study protein adsorption amounts and structures. Relatively high amounts of adsorption occurred onto the CH3 and NH2 surfaces from both calcium phosphate and PBS solutions. Adsorption was also promoted onto COOH surfaces when calcium was present in the solutions suggesting an interaction that involves calcium bridging with the negatively charged C-terminus. The ellipsometry and AFM studies suggested that the protein adsorbed onto all surfaces as LRAP monomers. We propose that the monomers adsorb onto the surfaces by disassembling or “shedding” from the nanospheres that are present in solution. This work reveals the importance of small subnanosphere-sized structures of LRAP at interfaces, structures that may be important in the biomineralization of tooth enamel.

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

    PubMed

    Capriotti, Anna Laura; Caracciolo, Giulio; Cavaliere, Chiara; Foglia, Patrizia; Pozzi, Daniela; Samperi, Roberto; Laganà, Aldo

    2012-03-16

    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.

  3. Ultra-Thin Optically Transparent Carbon Electrodes Produced from Layers of Adsorbed Proteins

    PubMed Central

    Alharthi, Sarah A.; Benavidez, Tomas E.; Garcia, Carlos D.

    2013-01-01

    This work describes a simple, versatile, and inexpensive procedure to prepare optically transparent carbon electrodes, using proteins as precursors. Upon adsorption, the protein-coated substrates were pyrolyzed under reductive conditions (5% H2) to form ultra-thin, conductive electrodes. Because proteins spontaneously adsorb to interfaces forming uniform layers, the proposed method does not require a precise control of the preparation conditions, specialized instrumentation, or expensive precursors. The resulting electrodes were characterized by a combination of electrochemical, optical, and spectroscopic means. As a proof-of-concept, the optically-transparent electrodes were also used as substrate for the development of an electrochemical glucose biosensor. The proposed films represent a convenient alternative to more sophisticated, and less available, carbon-based nanomaterials. Furthermore, these films could be formed on a variety of substrates, without classical limitations of size or shape. PMID:23421732

  4. Redox regulation of protein damage in plasma

    PubMed Central

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

    2014-01-01

    The presence and concentrations of modified proteins circulating in plasma depend on rates of protein synthesis, modification and clearance. In early studies, the proteins most frequently analysed for damage were those which were more abundant in plasma (e.g. albumin and immunoglobulins) which exist at up to 10 orders of magnitude higher concentrations than other plasma proteins e.g. cytokines. However, advances in analytical techniques using mass spectrometry and immuno-affinity purification methods, have facilitated analysis of less abundant, modified proteins and the nature of modifications at specific sites is now being characterised. The damaging reactive species that cause protein modifications in plasma principally arise from reactive oxygen species (ROS) produced by NADPH oxidases (NOX), nitric oxide synthases (NOS) and oxygenase activities; reactive nitrogen species (RNS) from myeloperoxidase (MPO) and NOS activities; and hypochlorous acid from MPO. Secondary damage to proteins may be caused by oxidized lipids and glucose autooxidation. In this review, we focus on redox regulatory control of those enzymes and processes which control protein maturation during synthesis, produce reactive species, repair and remove damaged plasma proteins. We have highlighted the potential for alterations in the extracellular redox compartment to regulate intracellular redox state and, conversely, for intracellular oxidative stress to alter the cellular secretome and composition of extracellular vesicles. Through secreted, redox-active regulatory molecules, changes in redox state may be transmitted to distant sites. PMID:24624332

  5. Plasma protein denaturation with graded heat exposure.

    PubMed

    Vazquez, R; Larson, D F

    2013-11-01

    During cardiopulmonary bypass (CPB), perfusion at tepid temperatures (33-35 °C) is recommended to avoid high temperature cerebral hyperthermia during and after the operation. However, the ideal temperature for uncomplicated adult cardiac surgery is an unsettled question. Typically, the heat exchanger maximum temperature is monitored between 40-42 °C to prevent denaturation of plasma proteins, but studies have not been performed to make these conclusions. Therefore, our hypothesis was to determine the temperature in which blood plasma protein degradation occurs after 2 hours of heat exposure. As a result, blood plasma proteins were exposed to heat in the 37-50 °C range for 2 hours. Plasma protein samples were loaded onto an 8-12% gradient gel for SDS-PAGE and low molecular weight plasma protein degradation was detected with graded heat exposure. Protein degradation was first detected between 43-45 °C of heat exposure. This study supports the practice of monitoring the heat exchanger between 40-42 °C to prevent denaturation of plasma proteins.

  6. Characterization of cross-linked cellulosic ion-exchange adsorbents: 2. Protein sorption and transport.

    PubMed

    Angelo, James M; Cvetkovic, Aleksandar; Gantier, Rene; Lenhoff, Abraham M

    2016-03-18

    Adsorption behavior in the HyperCel family of cellulosic ion-exchange materials (Pall Corporation) was characterized using methods to assess, quantitatively and qualitatively, the dynamics of protein uptake as well as static adsorption as a function of ionic strength and protein concentration using several model proteins. The three exchangers studied all presented relatively high adsorptive capacities under low ionic strength conditions, comparable to commercially available resins containing polymer functionalization aimed at increasing that particular characteristic. The strong cation- and anion-exchange moieties showed higher sensitivity to increasing salt concentrations, but protein affinity on the salt-tolerant STAR AX HyperCel exchanger remained strong at ionic strengths normally used in downstream processing to elute material fully during ion-exchange chromatography. Very high uptake rates were observed in both batch kinetics experiments and time-series confocal laser scanning microscopy, suggesting low intraparticle transport resistances relative to external film resistance, even at higher bulk protein concentrations where the opposite is typically observed. Electron microscopy imaging of protein adsorbed phases provided additional insight into particle structure that could not be resolved in previous work on the bare resins.

  7. Influence of fluoride-detergent combinations on the visco-elasticity of adsorbed salivary protein films.

    PubMed

    Veeregowda, Deepak H; van der Mei, Henny C; Busscher, Henk J; Sharma, Prashant K

    2011-02-01

    The visco-elasticity of salivary-protein films is related to mouthfeel, lubrication, biofilm formation, and protection against erosion and is influenced by the adsorption of toothpaste components. The thickness and the visco-elasticity of hydrated films (determined using a quartz crystal microbalance) of 2-h-old in vitro-adsorbed salivary-protein films were 43.5 nm and 9.4 MHz, respectively, whereas the dehydrated thickness, measured using X-ray photoelectron spectroscopy, was 2.4 nm. Treatment with toothpaste slurries decreased the thickness of the film, depending on the fluoride-detergent combination involved. Secondary exposure to saliva resulted in a regained thickness of the film to a level similar to its original thickness; however, no association was found between the thickness of hydrated and dehydrated films, indicating differences in film structure. Treatment with stannous fluoride/sodium lauryl sulphate (SnF(2)/SLS)-containing toothpaste slurries yielded a strong, immediate two-fold increase in characteristic film frequency (f(c)) with respect to untreated films, indicating cross-linking in adsorbed salivary-protein films by Sn(2+) that was absent when SLS was replaced with sodium hexametaphosphate (NaHMP). Secondary exposure to saliva of films treated with SnF(2) caused a strong, six-fold increase in f(c) compared with primary salivary-protein films, regardless of whether SLS or NaHMP was the detergent. This suggests that ionized stannous is not directly available for cross-linking in combination with highly negatively charged NaHMP, but becomes slowly available after initial treatment to cause cross-linking during secondary exposure to saliva.

  8. Targeted Mutagenesis and Combinatorial Library Screening Enables Control of Protein Orientation on Surfaces and Increased Activity of Adsorbed Proteins.

    PubMed

    Cruz-Teran, Carlos A; Carlin, Kevin B; Efimenko, Kirill; Genzer, Jan; Rao, Balaji M

    2016-08-30

    While nonspecific adsorption is widely used for immobilizing proteins on solid surfaces, the random nature of protein adsorption may reduce the activity of immobilized proteins due to occlusion of the active site. We hypothesized that the orientation a protein assumes on a given surface can be controlled by systematically introducing mutations into a region distant from its active site, thereby retaining activity of the immobilized protein. To test this hypothesis, we generated a combinatorial protein library by randomizing six targeted residues in a binding protein derived from highly stable, nonimmunoglobulin Sso7d scaffold; mutations were targeted in a region that is distant from the binding site. This library was screened to isolate binders that retain binding to its cognate target (chicken immunoglobulin Y, cIgY) as well as exhibit adsorption on unmodified silica at pH 7.4 and high ionic strength conditions. A single mutant, Sso7d-2B5, was selected for further characterization. Sso7d-2B5 retained binding to cIgY with an apparent dissociation constant similar to that of the parent protein; both mutant and parent proteins saturated the surface of silica with similar densities. Strikingly, however, silica beads coated with Sso7d-2B5 could achieve up to 7-fold higher capture of cIgY than beads coated with the parent protein. These results strongly suggest that mutations introduced in Sso7d-2B5 alter its orientation relative to the parent protein, when adsorbed on silica surfaces. Our approach also provides a generalizable strategy for introducing mutations in proteins so as to improve their activity upon immobilization, and has direct relevance to development of protein-based biosensors and biocatalysts. PMID:27490089

  9. Human plasma protein adsorption onto dextranized surfaces: a two-dimensional electrophoresis and mass spectrometry study.

    PubMed

    Tsai, Irene Y; Tomczyk, Nancy; Eckmann, Joshua I; Composto, Russell J; Eckmann, David M

    2011-05-01

    Protein adsorption is fundamental to thrombosis and to the design of biocompatible materials. We report a two-dimensional electrophoresis and mass spectrometry study to characterize multiple human plasma proteins adsorbed onto four different types of model surfaces: silicon oxide, dextranized silicon, polyurethane and dextranized polyurethane. Dextran was grafted onto the surfaces of silicon and polyurethane to mimic the blood-contacting endothelial cell glycocalyx surface. Surface topography and hydrophobicity/hydrophilicity were determined and analyzed using atomic force microscopy and water contact angle measurements, respectively. Using two-dimensional electrophoresis, we show that, relative to the unmodified surfaces, dextranization significantly inhibits the adsorption of several human plasma proteins including IGHG1 protein, fibrinogen, haptoglobin, Apo A-IV, Apo A-I, immunoglobulin, serum retinal-binding protein and truncated serum albumin. We further demonstrate the selectivity of plasma protein adsorbed onto the different functionalized surfaces and the potential to control and manipulate proteins adsorption on the surfaces of medical devices, implants and microfluidic devices. This result shows that adsorption experiments using a single protein or a binary mixture of proteins are consistent with competitive protein adsorption studies. In summary, these studies indicate that coating blood-contacting biomedical applications with dextran is an effective route to reduce thrombo-inflammatory responses and to surface-direct biological activities. PMID:21277175

  10. Inhibition of Lipid Oxidation in Oil-in-Water Emulsions by Interface-Adsorbed Myofibrillar Protein.

    PubMed

    Yang, Jiayi; Xiong, Youling L

    2015-10-14

    This study investigated the role of interfacial myofibrillar protein (MFP) in the oxidative stabilization of meat emulsions. Emulsions with 10% oil were prepared using either 2% (w/v) Tween 20 or 0.25, 0.5, and 1% (w/v) MFP and then subjected to hydroxyl radical oxidation at 4 °C for 0, 2, and 24 h. MFP was more readily oxidized (intrinsic fluorescence quenching, sulfur losses, and carbonyl formation) than oil [conjugated dienes and 2-thiobarbituric acid-reactive substances (TBARS)]. However, oxidized MFP in the continuous phase stimulated lipid oxidation after 24 h, sharply contrasting with interface-adsorbed MFP that inhibited TBARS formation nearly 90% (p < 0.05). Interfacial MFP from 2 h oxidized samples exhibited greater losses of fluorescence and more extensive polymerization of myosin (detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) than MFP present in the continuous phase. Results indicated that, due to the physical localization, interface-adsorbed MFP in general and myosin in particular provided accentuated protection of emulsions against oxidation. PMID:26414649

  11. Exploring the interfacial structure of protein adsorbates and the kinetics of protein adsorption: an in situ high-energy X-ray reflectivity study.

    PubMed

    Evers, Florian; Shokuie, Kaveh; Paulus, Michael; Sternemann, Christian; Czeslik, Claus; Tolan, Metin

    2008-09-16

    The high energy X-ray reflectivity technique has been applied to study the interfacial structure of protein adsorbates and protein adsorption kinetics in situ. For this purpose, the adsorption of lysozyme at the hydrophilic silica-water interface has been chosen as a model system. The structure of adsorbed lysozyme layers was probed for various aqueous solution conditions. The effect of solution pH and lysozyme concentration on the interfacial structure was measured. Monolayer formation was observed for all cases except for the highest concentration. The adsorbed protein layers consist of adsorbed lysozyme molecules with side-on or end-on orientation. By means of time-dependent X-ray reflectivity scans, the time-evolution of adsorbed proteins was monitored as well. The results of this study demonstrate the capabilities of in situ X-ray reflectivity experiments on protein adsorbates. The great advantages of this method are the broad wave vector range available and the high time resolution.

  12. Exploring the interfacial structure of protein adsorbates and the kinetics of protein adsorption: an in situ high-energy X-ray reflectivity study.

    PubMed

    Evers, Florian; Shokuie, Kaveh; Paulus, Michael; Sternemann, Christian; Czeslik, Claus; Tolan, Metin

    2008-09-16

    The high energy X-ray reflectivity technique has been applied to study the interfacial structure of protein adsorbates and protein adsorption kinetics in situ. For this purpose, the adsorption of lysozyme at the hydrophilic silica-water interface has been chosen as a model system. The structure of adsorbed lysozyme layers was probed for various aqueous solution conditions. The effect of solution pH and lysozyme concentration on the interfacial structure was measured. Monolayer formation was observed for all cases except for the highest concentration. The adsorbed protein layers consist of adsorbed lysozyme molecules with side-on or end-on orientation. By means of time-dependent X-ray reflectivity scans, the time-evolution of adsorbed proteins was monitored as well. The results of this study demonstrate the capabilities of in situ X-ray reflectivity experiments on protein adsorbates. The great advantages of this method are the broad wave vector range available and the high time resolution. PMID:18715021

  13. Evaluation of the Effectiveness of Surfactants and Denaturants to Elute and Denature Adsorbed Protein on Different Surface Chemistries.

    PubMed

    Thyparambil, Aby A; Wei, Yang; Latour, Robert A

    2015-11-01

    The elution and/or denaturation of proteins from material surfaces by chemical excipients such as surfactants and denaturants is important for numerous applications including medical implant reprocessing, bioanalyses, and biodefense. The objective of this study was to develop and apply methods to quantitatively assess how surface chemistry and adsorption conditions influence the effectiveness of three commonly used surfactants (sodium dodecyl sulfate, n-octyl-β-d-glucoside, and 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) and two denaturants (guanidium hydrochloride and urea) to elute protein (hen egg white lysozyme and bovine pancreatic ribonuclease A) from three different surface chemistries (silica glass, poly(methyl methacrylate), and high-density polyethylene). The structure and bioactivity of residual protein on the surface following elution were characterized using circular dichroism spectropolarimetry and enzyme assays to assess the extent of protein denaturation. Our results indicate that the denaturants were generally more effective than the surfactants in removing the adsorbed proteins from each type of surface. Also, the denaturing capacity of these excipients on the residual proteins on the surfaces was distinctly different from their influence on the proteins in solution and was unique for each of the adsorption conditions. Taken altogether, these results reveal that the effectiveness of surfactants and denaturants to elute and denature adsorbed protein is significantly influenced by surface chemistry and the conditions from which the protein was adsorbed. These results provide a basis for the selection, design, and further development of chemical agents for protein elution and surface decontamination.

  14. Evaluation of the Effectiveness of Surfactants and Denaturants to Elute and Denature Adsorbed Protein on Different Surface Chemistries.

    PubMed

    Thyparambil, Aby A; Wei, Yang; Latour, Robert A

    2015-11-01

    The elution and/or denaturation of proteins from material surfaces by chemical excipients such as surfactants and denaturants is important for numerous applications including medical implant reprocessing, bioanalyses, and biodefense. The objective of this study was to develop and apply methods to quantitatively assess how surface chemistry and adsorption conditions influence the effectiveness of three commonly used surfactants (sodium dodecyl sulfate, n-octyl-β-d-glucoside, and 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) and two denaturants (guanidium hydrochloride and urea) to elute protein (hen egg white lysozyme and bovine pancreatic ribonuclease A) from three different surface chemistries (silica glass, poly(methyl methacrylate), and high-density polyethylene). The structure and bioactivity of residual protein on the surface following elution were characterized using circular dichroism spectropolarimetry and enzyme assays to assess the extent of protein denaturation. Our results indicate that the denaturants were generally more effective than the surfactants in removing the adsorbed proteins from each type of surface. Also, the denaturing capacity of these excipients on the residual proteins on the surfaces was distinctly different from their influence on the proteins in solution and was unique for each of the adsorption conditions. Taken altogether, these results reveal that the effectiveness of surfactants and denaturants to elute and denature adsorbed protein is significantly influenced by surface chemistry and the conditions from which the protein was adsorbed. These results provide a basis for the selection, design, and further development of chemical agents for protein elution and surface decontamination. PMID:26449787

  15. Preparation of new amphiphilic macroporous nonwoven polymeric adsorbents aimed for selective removal of low-density lipoprotein from plasma.

    PubMed

    Hou, Xiaodong; Zhang, Tao; Cao, Amin

    2015-01-01

    In the present work, new amphiphilic macroporous polymeric adsorbent (AMPA) membranes for LDL-apheresis were prepared by (60)Co γ-ray irradiation-induced grafting copolymerization of polypropylene (PP) nonwoven fabric with acrylic acid, followed by bonding cholesterol through linkers of different length. The new AMPA membranes were characterized by attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy, scanning electron microscope (SEM), and contact angle microscopy. Static adsorption and hemo-perfusion tests show these new adsorbents could efficiently remove LDL from human plasma. Meanwhile, the AMPA displayed good adsorption capacity for triglyceride (TG) as well. The static adsorption performance of the AMPA membranes depends on the length of linker. In addition, a balance between the amount of bonded cholesterol and remaining carboxyl group was found necessary to reach the optimal adsorption performance. The best result was achieved by the AMPA membrane PA15C6-3, by which 62.8 ± 3.8 μg of LDL-C, 16.5 ± 0.71 μg of HDL-C, 132.4 ± 3.0 μg of TG are removed from human plasma per square centimeter. PMID:24764047

  16. Changes in solvent exposure reveal the kinetics and equilibria of adsorbed protein unfolding in hydrophobic interaction chromatography.

    PubMed

    Deitcher, R W; O'Connell, J P; Fernandez, E J

    2010-08-27

    Hydrogen exchange has been a useful technique for studying the conformational state of proteins, both in bulk solution and at interfaces, for several decades. Here, we propose a physically based model of simultaneous protein adsorption, unfolding and hydrogen exchange in HIC. An accompanying experimental protocol, utilizing mass spectrometry to quantify deuterium labeling, enables the determination of both the equilibrium partitioning between conformational states and pseudo-first order rate constants for folding and unfolding of adsorbed protein. Unlike chromatographic techniques, which rely on the interpretation of bulk phase behavior, this methodology utilizes the measurement of a molecular property (solvent exposure) and provides insight into the nature of the unfolded conformation in the adsorbed phase. Three model proteins of varying conformational stability, alpha-chymotrypsinogen A, beta-lactoglobulin B, and holo alpha-lactalbumin, are studied on Sepharose HIC resins possessing assorted ligand chemistries and densities. alpha-Chymotrypsinogen, conformationally the most stable protein in the set, exhibits no change in solvent exposure at all the conditions studied, even when isocratic pulse-response chromatography suggests nearly irreversible adsorption. Apparent unfolding energies of adsorbed beta-lactoglobulin B and holo alpha-lactalbumin range from -4 to 3 kJ/mol and are dependent on resin properties and salt concentration. Characteristic pseudo-first order rate constants for surface-induced unfolding are 0.2-0.9 min(-1). While poor protein recovery in HIC is often associated with irreversible unfolding, this study documents that non-eluting behavior can occur when surface unfolding is reversible or does not occur at all. Further, this hydrogen exchange technique can be used to assess the conformation of adsorbed protein under conditions where the protein is non-eluting and chromatographic methods are not applicable.

  17. Zeta potential, contact angles, and AFM imaging of protein conformation adsorbed on hybrid nanocomposite surfaces.

    PubMed

    Pinho, Ana C; Piedade, Ana P

    2013-08-28

    The sputtering deposition of gold (Au) and poly(tetrafluoroethylene) (PTFE) was used to prepare a nanocomposite hybrid thin film suitable for protein adsorption while maintaining the native conformation of the biological material. The monolithic PTFE and the nanocomposite PTFE/Au thin films, with Au content up to 1 at %, were co-deposited by r.f. magnetron sputtering using argon as a discharge gas and deposited onto 316L stainless steel substrates, the most commonly used steel in biomaterials. The deposited thin films, before and after bovine serum albumin (BSA) adsorption, were thoroughly characterized with special emphasis on the surface properties/characteristics by atomic force microscopy (AFM), zeta potential, and static and dynamic contact angle measurements, in order to assess the relationship between structure and conformational changes. The influence of a pre-adsorbed peptide (RGD) was also evaluated. The nanotopographic and chemical changes induced by the presence of gold in the nanocomposite thin films enable RGD bonding, which is critical for the maintenance of the BSA native conformation after adsorption.

  18. In vitro studies of PBT Nonwoven Fabrics adsorbent for the removal of low density lipoprotein from hyperlipemia plasma

    NASA Astrophysics Data System (ADS)

    Cao, Ye; Wang, Hong; Yang, Chao; Zhong, Rui; Lei, Yu; Sun, Kang; Liu, Jiaxin

    2011-06-01

    Polyanion ligands such as acrylic acid (AA) and heparin were grafted on PBT Nonwoven Fabrics (PBTNF) to study their effect on the adsorption of low density lipoprotein (LDL). These modified PBTNFs were characterized by Horizontal Attenuated Total Reflectance Fourier Transform Infrared spectroscopy and X-ray Photoelectron spectroscopy. The blood compatibilities of the modified PBTNFs were examined using in vitro hemolysis rate (HR), platelet adhesion, total protein (TP) and activated partial thromboplastin time. The results showed that direct immobilized heparin could improve PBTNF-PAA's blood compatibility and decrease the adsorption capability of useful high density lipoprotein, but would possess so low bioactivity that could not further improve the absorption of LDL and TC. Since the PBTNF-PAA55-Heparin adsorbent had quite good adsorption selectivity for these proteins, it can be an excellent candidate for depletion of LDL with good blood compatibility.

  19. Plasma protein binding of zomepirac sodium.

    PubMed

    O'Neill, P J

    1981-07-01

    The plasma protein binding of zomepirac, a new nonnarcotic analgesic, was studied using equilibrium dialysis. Experiments were performed using human plasma and plasma from mice, rats, and rhesus monkeys, all species of pharmacological or toxicological interest. At concentrations approximating those achieved in vivo, the binding was fairly constant at 98-99% in all species except the rhesus monkey, where binding was decreased from 98 to approximately 96% at higher concentrations (greater then 50 microgram/ml). Zomepirac (10 microgram/ml) did not appear to displace or to be displaced by warfarin (10 microgram/ml) caused a concentration-dependent decrease in zomepirac (10 microgram/ml) binding. Zomepirac did not affect salicylate binding.

  20. 21 CFR 640.90 - Plasma Protein Fraction (Human).

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 7 2010-04-01 2010-04-01 false Plasma Protein Fraction (Human). 640.90 Section...) BIOLOGICS ADDITIONAL STANDARDS FOR HUMAN BLOOD AND BLOOD PRODUCTS Plasma Protein Fraction (Human) § 640.90 Plasma Protein Fraction (Human). (a) Proper name and definition. The proper name of the product shall...

  1. 21 CFR 640.90 - Plasma Protein Fraction (Human).

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 7 2011-04-01 2010-04-01 true Plasma Protein Fraction (Human). 640.90 Section 640...) BIOLOGICS ADDITIONAL STANDARDS FOR HUMAN BLOOD AND BLOOD PRODUCTS Plasma Protein Fraction (Human) § 640.90 Plasma Protein Fraction (Human). (a) Proper name and definition. The proper name of the product shall...

  2. 21 CFR 640.90 - Plasma Protein Fraction (Human).

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 7 2012-04-01 2012-04-01 false Plasma Protein Fraction (Human). 640.90 Section...) BIOLOGICS ADDITIONAL STANDARDS FOR HUMAN BLOOD AND BLOOD PRODUCTS Plasma Protein Fraction (Human) § 640.90 Plasma Protein Fraction (Human). (a) Proper name and definition. The proper name of the product shall...

  3. 21 CFR 640.90 - Plasma Protein Fraction (Human).

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 7 2013-04-01 2013-04-01 false Plasma Protein Fraction (Human). 640.90 Section...) BIOLOGICS ADDITIONAL STANDARDS FOR HUMAN BLOOD AND BLOOD PRODUCTS Plasma Protein Fraction (Human) § 640.90 Plasma Protein Fraction (Human). (a) Proper name and definition. The proper name of the product shall...

  4. 21 CFR 640.90 - Plasma Protein Fraction (Human).

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 7 2014-04-01 2014-04-01 false Plasma Protein Fraction (Human). 640.90 Section...) BIOLOGICS ADDITIONAL STANDARDS FOR HUMAN BLOOD AND BLOOD PRODUCTS Plasma Protein Fraction (Human) § 640.90 Plasma Protein Fraction (Human). (a) Proper name and definition. The proper name of the product shall...

  5. Plasma membrane sterols are involved in yeast's ability to adsorb polyphenolic compounds resulting from wine model solution browning.

    PubMed

    Márquez, Trinidad; Millán, Carmen; Salmon, Jean-Michel

    2009-09-01

    The aim of this work was to demonstrate the direct interaction between membrane sterols of yeast lees and some polymerized phenolic compounds resulting from wine model solution browning. For this purpose, we first demonstrated by measurement of steady-state fluorescence anisotropy of the cationic fluorescent TMA-DPH probe the effect of polymerized compounds from the model reactions of (+)-catechin/acetaldehyde and (+)-catechin/glyoxylic acid on the plasma membrane order of Saccharomyces cerevisiae yeast lees enriched with different sterols. In a second set of experiments, we used S. cerevisiae plasma membrane vesicles spiked with different sources of sterol (ergosterol, cholesterol or a mix of grape phytosterols) to assess the effect of the same polymerized compounds on both vesicle integrity and membrane leakiness to protons by ACMA fluorescence. All the obtained results prove that yeast membrane sterols are able to strongly interact with some polymerized compounds resulting from the browning of model solutions, likely explaining the yeast ability to adsorb polyphenolic compounds and mainly the colorless intermediate compounds of the browning reactions. PMID:19691282

  6. Human plasma protein N-glycosylation.

    PubMed

    Clerc, Florent; Reiding, Karli R; Jansen, Bas C; Kammeijer, Guinevere S M; Bondt, Albert; Wuhrer, Manfred

    2016-06-01

    Glycosylation is the most abundant and complex protein modification, and can have a profound structural and functional effect on the conjugate. The oligosaccharide fraction is recognized to be involved in multiple biological processes, and to affect proteins physical properties, and has consequentially been labeled a critical quality attribute of biopharmaceuticals. Additionally, due to recent advances in analytical methods and analysis software, glycosylation is targeted in the search for disease biomarkers for early diagnosis and patient stratification. Biofluids such as saliva, serum or plasma are of great use in this regard, as they are easily accessible and can provide relevant glycosylation information. Thus, as the assessment of protein glycosylation is becoming a major element in clinical and biopharmaceutical research, this review aims to convey the current state of knowledge on the N-glycosylation of the major plasma glycoproteins alpha-1-acid glycoprotein, alpha-1-antitrypsin, alpha-1B-glycoprotein, alpha-2-HS-glycoprotein, alpha-2-macroglobulin, antithrombin-III, apolipoprotein B-100, apolipoprotein D, apolipoprotein F, beta-2-glycoprotein 1, ceruloplasmin, fibrinogen, immunoglobulin (Ig) A, IgG, IgM, haptoglobin, hemopexin, histidine-rich glycoprotein, kininogen-1, serotransferrin, vitronectin, and zinc-alpha-2-glycoprotein. In addition, the less abundant immunoglobulins D and E are included because of their major relevance in immunology and biopharmaceutical research. Where available, the glycosylation is described in a site-specific manner. In the discussion, we put the glycosylation of individual proteins into perspective and speculate how the individual proteins may contribute to a total plasma N-glycosylation profile determined at the released glycan level. PMID:26555091

  7. Identification of polymer surface adsorbed proteins implicated in pluripotent human embryonic stem cell expansion† †Electronic supplementary information (ESI) available. See DOI: 10.1039/c6bm00214e Click here for additional data file.

    PubMed Central

    Hammad, Moamen; Rao, Wei; Smith, James G. W.; Anderson, Daniel G.; Langer, Robert; Young, Lorraine E.; Barrett, David A.; Davies, Martyn C.; Denning, Chris

    2016-01-01

    Improved biomaterials are required for application in regenerative medicine, biosensing, and as medical devices. The response of cells to the chemistry of polymers cultured in media is generally regarded as being dominated by proteins adsorbed to the surface. Here we use mass spectrometry to identify proteins adsorbed from a complex mouse embryonic fibroblast (MEF) conditioned medium found to support pluripotent human embryonic stem cell (hESC) expansion on a plasma etched tissue culture polystyrene surface. A total of 71 proteins were identified, of which 14 uniquely correlated with the surface on which pluripotent stem cell expansion was achieved. We have developed a microarray combinatorial protein spotting approach to test the potential of these 14 proteins to support expansion of a hESC cell line (HUES-7) and a human induced pluripotent stem cell line (ReBl-PAT) on a novel polymer (N-(4-Hydroxyphenyl) methacrylamide). These proteins were spotted to form a primary array yielding several protein mixture ‘hits’ that enhanced cell attachment to the polymer. A second array was generated to test the function of a refined set of protein mixtures. We found that a combination of heat shock protein 90 and heat shock protein-1 encourage elevated adherence of pluripotent stem cells at a level comparable to fibronectin pre-treatment. PMID:27466628

  8. PLASMA PROTEIN METABOLISM—NORMAL AND ASSOCIATED WITH SHOCK

    PubMed Central

    Fink, R. M.; Enns, T.; Kimball, C. P.; Silberstein, H. E.; Bale, W. F.; Madden, S. C.; Whipple, G. H.

    1944-01-01

    Labeled plasma proteins are produced by administering to dogs the amino acid lysine synthesized with heavy nitrogen. Such labeled proteins are apparently indistinguishable biologically from proteins of normal isotope concentration. Labeled plasma proteins, as plasma, injected into normal dogs pass out of the blood stream at an initially rapid but constantly decreasing non-logarithmic rate. This outflow is balanced by a simultaneous inflow of plasma proteins from the tissues. Fifty per cent of the labeled protein is out of the blood stream in about 24 hours; 75 per cent in about 6 days. Shock due to trauma of intestine or leg shows a dilution curve of labeled plasma protein not unlike that of the normal dog. If anything, dilution appears a little less rapid in shock. Since the usual shrinkage of plasma volume and plasma protein mass is present in these shocked dogs, these data are compatible with a decreased inflow of protein into the plasma during shock. Methods are described which are suitable for the use of heavy nitrogen incorporated in the epsilon group of lysine and its subsequent analysis in body fluids. These data may indicate that the plasma proteins are normally in constant and rapid exchange with a mobile pool of body protein. PMID:19871430

  9. Atmospheric pressure plasma polymers for tuned QCM detection of protein adhesion.

    PubMed

    Rusu, G B; Asandulesa, M; Topala, I; Pohoata, V; Dumitrascu, N; Barboiu, M

    2014-03-15

    Our efforts have been concentrated in preparing plasma polymeric thin layers at atmospheric pressure grown on Quartz Crystal Microbalance-QCM electrodes for which the non-specific absorption of proteins can be efficiently modulated, tuned and used for QCM biosensing and quantification. Plasma polymerization reaction at atmospheric pressure has been used as a simple and viable method for the preparation of QCM bioactive surfaces, featuring variable protein binding properties. Polyethyleneglycol (ppEG), polystyrene (ppST) and poly(ethyleneglycol-styrene) (ppST-EG) thin-layers have been grown on QCM electrodes. These layers were characterized by Atomic Force Microscopy (AFM), Contact angle measurements, Fourier transform infrared (FTIR) and X-ray photoelectron spectroscopy (XPS). The plasma ppST QCM electrodes present a higher adsorption of Concanavalin A (ConA) and Bovine Serum Albumin (BSA) proteins when compared with the commercial coated polystyrene (ppST) ones. The minimum adsorption was found for ppEG, surface, known by their protein anti-fouling properties. The amount of adsorbed proteins can be tuned by the introduction of PEG precursors in the plasma discharge during the preparation of ppST polymers.

  10. Fabrication of a biomimetic adsorbent imprinted with a common specificity determinant for the removal of α- and β-amanitin from plasma.

    PubMed

    Tan, Lei; He, Rong; Li, Yongxian; Liang, Yong; Li, He; Tang, Youwen

    2016-08-12

    α-Amanitin and β-amanitin are the main toxins of mushroom poisoning. The application of traditional non-selective adsorbents is not satisfactory in clinical treatment of amanita mushroom poisoning due to lack of specificity adsorption capability of these adsorbents toward amanitin toxins. In the current work, we introduce a novel molecularly imprinted biomimetic adsorbent based on a ligand specificity determinant through surface imprinted strategy. Owing to the expensive price of the amanitin sources, we selected a typical common moiety of α, β-amanitin as specificity determinant to synthesize a template necessary for the preparation of molecularly imprinted polymers (MIPs). Computer simulation was used to initially select acidic methacrylic acid (MAA) and basic 4-vinyl pyridine (4-VP) together as functional monomers. The experiments further demonstrated that the synergistic interaction of MAA and 4-VP played a primary role in the recognition of α, β-amanitin by MIPs. By means of batch and packed-bed column experiment and the hemocompatibility evaluation, the resultant biomimetic adsorbent has been proved to be capable of selectively removing α, β-amanitin and possess good hemocompatibility. This novel adsorbent has great potential to find application in human plasma purification. PMID:27394089

  11. Synthesis of adsorbents with dendronic structures for protein hydrophobic interaction chromatography.

    PubMed

    Mata-Gómez, Marco A; Yaman, Sena; Valencia-Gallegos, Jesus A; Tari, Canan; Rito-Palomares, Marco; González-Valdez, José

    2016-04-22

    Here, we introduced a new technology based on the incorporation of dendrons-branched chemical structures-onto supports for synthesis of HIC adsorbents. In doing so we studied the synthesis and performance of these novel HIC dendron-based adsorbents. The adsorbents were synthesized in a facile two-step reaction. First, Sepharose 4FF (R) was chemically modified with polyester dendrons of different branching degrees i.e. third (G3) or fifth (G5) generations. Then, butyl-end valeric acid ligands were coupled to dendrons via ester bond formation. UV-vis spectrophotometry and FTIR analyses of the modified resins confirmed the presence of the dendrons and their ligands on them. Inclusion of dendrons allowed the increment of ligand density, 82.5 ± 11 and 175.6 ± 5.7 μmol ligand/mL resin for RG3 and RG5, respectively. Static adsorption capacity of modified resins was found to be ∼ 60 mg BSA/mL resin. Interestingly, dynamic binding capacity was higher at high flow rates, 62.5 ± 0.8 and 58.0 ± 0.5mg/mL for RG3 and RG5, respectively. RG3 was able to separate lipase, β-lactoglobulin and α-chymotrypsin selectively as well as fractionating of a whole proteome from yeast. This innovative technology will improve the existing HIC resin synthesis methods. It will also allow the reduction of the amount of adsorbent used in a chromatographic procedure and thus permit the use of smaller columns resulting in faster processes. Furthermore, this method could potentially be considered as a green technology since both, dendrons and ligands, are formed by ester bonds that are more biodegradable allowing the disposal of used resin waste in a more ecofriendly manner when compared to other exiting resins.

  12. Synthesis of adsorbents with dendronic structures for protein hydrophobic interaction chromatography.

    PubMed

    Mata-Gómez, Marco A; Yaman, Sena; Valencia-Gallegos, Jesus A; Tari, Canan; Rito-Palomares, Marco; González-Valdez, José

    2016-04-22

    Here, we introduced a new technology based on the incorporation of dendrons-branched chemical structures-onto supports for synthesis of HIC adsorbents. In doing so we studied the synthesis and performance of these novel HIC dendron-based adsorbents. The adsorbents were synthesized in a facile two-step reaction. First, Sepharose 4FF (R) was chemically modified with polyester dendrons of different branching degrees i.e. third (G3) or fifth (G5) generations. Then, butyl-end valeric acid ligands were coupled to dendrons via ester bond formation. UV-vis spectrophotometry and FTIR analyses of the modified resins confirmed the presence of the dendrons and their ligands on them. Inclusion of dendrons allowed the increment of ligand density, 82.5 ± 11 and 175.6 ± 5.7 μmol ligand/mL resin for RG3 and RG5, respectively. Static adsorption capacity of modified resins was found to be ∼ 60 mg BSA/mL resin. Interestingly, dynamic binding capacity was higher at high flow rates, 62.5 ± 0.8 and 58.0 ± 0.5mg/mL for RG3 and RG5, respectively. RG3 was able to separate lipase, β-lactoglobulin and α-chymotrypsin selectively as well as fractionating of a whole proteome from yeast. This innovative technology will improve the existing HIC resin synthesis methods. It will also allow the reduction of the amount of adsorbent used in a chromatographic procedure and thus permit the use of smaller columns resulting in faster processes. Furthermore, this method could potentially be considered as a green technology since both, dendrons and ligands, are formed by ester bonds that are more biodegradable allowing the disposal of used resin waste in a more ecofriendly manner when compared to other exiting resins. PMID:27018188

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

    PubMed Central

    Tepel, Martin; Beck, Hans C.; Tan, Qihua; Borst, Christoffer; Rasmussen, Lars M.

    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 transplant recipients and quantified 359 plasma proteins simultaneously using nano-Liquid-Chromatography-Tandem Mass-Spectrometry in individual samples and plasma C-reactive protein on the index day and the next day. Next-day C-reactive protein increased in 59 patients whereas it decreased in 32 patients. The 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 < 0.0001). Multivariable logistic regression showed that 82-plex protein signature (P < 0.001) 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 signature increased net reclassification index with a clinical meaningful 10% increase of risk mainly by the improvement of reclassification of subjects in the event group. An 82-plex plasma protein signature predicts an increase of the inflammatory marker C-reactive protein. PMID:26445912

  14. Preliminary study on the effects of ageing cold oxygen plasma treated PET/PP with respect to protein adsorption.

    PubMed

    Chen, Rui; Bayon, Yves; Hunt, John A

    2012-08-01

    Surfaces of polyethylene terephthalate (PET) and polypropylene (PP) have been modified by oxygen plasma. The surface hydrophilicity and changes in topography during up to 90 days storage in water and in dry air in a desiccator were analysed by dynamic contact angle test and atomic force microscopy (AFM). Clear ageing effects on the plasma treated surface were observed as increases in contact angle and changes in roughness as functions of increasing storage time. However, the effect of oxygen plasma treatment to increase the hydrophilicity of surface was still evident on the treated surfaces even after 90 days storage either in dry air or in water. In protein adsorption experiments, human serum albumin (HSA) and fibrinogen (Fg) were adsorbed on untreated and oxygen plasma treated PET and PP surfaces. The quantified ATR-FTIR results showed that both HSA and Fg adsorption on PET and PP surfaces decreased after oxygen plasma treatment, with the effect most evident for HSA. Although for both proteins adsorption increased with ageing, the amount of adsorbed proteins was still lower than untreated surface at 30 days. This suggests the shelf life of oxygen plasma treated samples could be as long as 30 days. PMID:22521680

  15. Mammalian plasma membrane proteins as potential biomarkers and drug targets.

    PubMed

    Rucevic, Marijana; Hixson, Douglas; Josic, Djuro

    2011-06-01

    Defining the plasma membrane proteome is crucial to understand the role of plasma membrane in fundamental biological processes. Change in membrane proteins is one of the first events that take place under pathological conditions, making plasma membrane proteins a likely source of potential disease biomarkers with prognostic or diagnostic potential. Membrane proteins are also potential targets for monoclonal antibodies and other drugs that block receptors or inhibit enzymes essential to the disease progress. Despite several advanced methods recently developed for the analysis of hydrophobic proteins and proteins with posttranslational modifications, integral membrane proteins are still under-represented in plasma membrane proteome. Recent advances in proteomic investigation of plasma membrane proteins, defining their roles as diagnostic and prognostic disease biomarkers and as target molecules in disease treatment, are presented.

  16. Microwave plasma activation of a polyvinylidene fluoride surface for protein immobilization

    NASA Astrophysics Data System (ADS)

    Vasile, C.; Baican, M. C.; Tibirna, C. M.; Tuchilus, C.; Debarnot, D.; Pâslaru, E.; Poncin-Epaillard, F.

    2011-11-01

    Polyvinylidene fluoride (PVDF) was modified by CO2, N2 or N2/H2 plasmas, which permitted the attachment of short carboxyl or amino groups. A variation of the discharge parameters was performed, for their optimization, as well as for minimizing degradation in favour of acidic, amphiphilic or basic functionalization, respectively. The optimum parameters of discharge for CO2, N2 or N2/H2 plasmas were P = 50 W, gas flow rate Q = 16 × 10-8 m3 s-1, exposure time t = 30-60 s, d = 0.1 m, pressure 15 Pa. The new surfaces were characterized by wettability measurements, attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR), x-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM) methods. In a second step, the proteins (triglycine (TG) and protein A) were adsorbed or chemically grafted onto the carboxyl or amino functionalized surface, after EDC/NHS (1-ethyl-3-(-dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide) activation of proteins. ATR-FTIR, XPS and AFM investigations confirmed the presence of protein on the surface. The XPS C1s core levels at 286.3 eV (C-N), 288 eV (amide bond) and 298 eV (carboxylic acid), together with variation of the O1s and N1s signals, illustrated the immobilization of proteins. It was established that TG was better attached on surfaces activated with N2/H2 plasma, while protein A was more tightly anchored on CO2, N2 plasma-activated surfaces. The former procedure allowed higher surface densities, while the latter permitted a better chemical control. The results proved that plasma-treated PVDF is a good substrate for protein coating, which can be further used for microorganisms' detection, as evidenced by the immunoassay test.

  17. Affinity binding of antibodies to supermacroporous cryogel adsorbents with immobilized protein A for removal of anthrax toxin protective antigen.

    PubMed

    Ingavle, Ganesh C; Baillie, Les W J; Zheng, Yishan; Lis, Elzbieta K; Savina, Irina N; Howell, Carol A; Mikhalovsky, Sergey V; Sandeman, Susan R

    2015-05-01

    Polymeric cryogels are efficient carriers for the immobilization of biomolecules because of their unique macroporous structure, permeability, mechanical stability and different surface chemical functionalities. The aim of the study was to demonstrate the potential use of macroporous monolithic cryogels for biotoxin removal using anthrax toxin protective antigen (PA), the central cell-binding component of the anthrax exotoxins, and covalent immobilization of monoclonal antibodies. The affinity ligand (protein A) was chemically coupled to the reactive hydroxyl and epoxy-derivatized monolithic cryogels and the binding efficiencies of protein A, monoclonal antibodies to the cryogel column were determined. Our results show differences in the binding capacity of protein A as well as monoclonal antibodies to the cryogel adsorbents caused by ligand concentrations, physical properties and morphology of surface matrices. The cytotoxicity potential of the cryogels was determined by an in vitro viability assay using V79 lung fibroblast as a model cell and the results reveal that the cryogels are non-cytotoxic. Finally, the adsorptive capacities of PA from phosphate buffered saline (PBS) were evaluated towards a non-glycosylated, plant-derived human monoclonal antibody (PANG) and a glycosylated human monoclonal antibody (Valortim(®)), both of which were covalently attached via protein A immobilization. Optimal binding capacities of 108 and 117 mg/g of antibody to the adsorbent were observed for PANG attached poly(acrylamide-allyl glycidyl ether) [poly(AAm-AGE)] and Valortim(®) attached poly(AAm-AGE) cryogels, respectively, This indicated that glycosylation status of Valortim(®) antibody could significantly increase (8%) its binding capacity relative to the PANG antibody on poly(AAm-AGE)-protien-A column (p < 0.05). The amounts of PA which remained in the solution after passing PA spiked PBS through PANG or Valortim bound poly(AAm-AGE) cryogel were significantly (p < 0

  18. Affinity binding of antibodies to supermacroporous cryogel adsorbents with immobilized protein A for removal of anthrax toxin protective antigen.

    PubMed

    Ingavle, Ganesh C; Baillie, Les W J; Zheng, Yishan; Lis, Elzbieta K; Savina, Irina N; Howell, Carol A; Mikhalovsky, Sergey V; Sandeman, Susan R

    2015-05-01

    Polymeric cryogels are efficient carriers for the immobilization of biomolecules because of their unique macroporous structure, permeability, mechanical stability and different surface chemical functionalities. The aim of the study was to demonstrate the potential use of macroporous monolithic cryogels for biotoxin removal using anthrax toxin protective antigen (PA), the central cell-binding component of the anthrax exotoxins, and covalent immobilization of monoclonal antibodies. The affinity ligand (protein A) was chemically coupled to the reactive hydroxyl and epoxy-derivatized monolithic cryogels and the binding efficiencies of protein A, monoclonal antibodies to the cryogel column were determined. Our results show differences in the binding capacity of protein A as well as monoclonal antibodies to the cryogel adsorbents caused by ligand concentrations, physical properties and morphology of surface matrices. The cytotoxicity potential of the cryogels was determined by an in vitro viability assay using V79 lung fibroblast as a model cell and the results reveal that the cryogels are non-cytotoxic. Finally, the adsorptive capacities of PA from phosphate buffered saline (PBS) were evaluated towards a non-glycosylated, plant-derived human monoclonal antibody (PANG) and a glycosylated human monoclonal antibody (Valortim(®)), both of which were covalently attached via protein A immobilization. Optimal binding capacities of 108 and 117 mg/g of antibody to the adsorbent were observed for PANG attached poly(acrylamide-allyl glycidyl ether) [poly(AAm-AGE)] and Valortim(®) attached poly(AAm-AGE) cryogels, respectively, This indicated that glycosylation status of Valortim(®) antibody could significantly increase (8%) its binding capacity relative to the PANG antibody on poly(AAm-AGE)-protien-A column (p < 0.05). The amounts of PA which remained in the solution after passing PA spiked PBS through PANG or Valortim bound poly(AAm-AGE) cryogel were significantly (p < 0

  19. Adsorption of proteins at physiological concentrations on pegylated surfaces and the compatibilizing role of adsorbed albumin with respect to other proteins according to optical waveguide lightmode spectroscopy (OWLS).

    PubMed

    Leclercq, Laurent; Modena, Enrico; Vert, Michel

    2013-01-01

    In literature, contacts between pegylated compounds and blood proteins are generally discussed in terms of excluded volume-related repulsions although adsorption and compatibility have been reported for some of these proteins occasionally. The major problem to investigate the behavior of blood in contact with pegylated surfaces is the complexity of the medium and especially the presence of albumin in large excess. In a model approach, optical waveguide lightmode spectroscopy (OWLS) was used to monitor the fate of albumin, fibrinogen, and γ-globulins at physiological concentrations in pH = 7.4 isotonic HEPES buffer after contact with SiTiO2 chips coated with diblock poly(DL-lactic acid)-block-poly(ethylene oxide)s and triblock poly(DL-lactic acid)-block-poly(ethylene oxide)-block-poly(DL-lactic acid) copolymers. Corresponding homopolymers were used as controls. The three protein systems were investigated separately, as a mixture and when added successively according to different orders of addition. OWLS gave access to the mass and the thickness of adhering protein layers that resist washing with HEPES buffer. Protein depositions were detected regardless of the presence of poly(ethylene glycol) segments on surfaces. Adsorption depended on the protein, on the surface and also on the presence of the other proteins. Unexpectedly any surface coated with a layer of adsorbed albumin prevented deposition of other proteins, including albumin itself. This outstanding finding suggests that it was the presence of albumin adsorbed on a surface, pegylated or not, that made that surface compatible with other proteins. As a consequence, dipping a device to be in contact with the blood of a patient in a solution of albumin could be a very simple means to avoid further protein deposition and maybe platelets adhesion after in vivo implantation.

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

    PubMed

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

    2015-07-01

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

  1. Clinical relevance of drug binding to plasma proteins

    NASA Astrophysics Data System (ADS)

    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.

  2. Sum Frequency Generation Vibrational Spectroscopy of Adsorbed Amino Acids, Peptides and Proteins of Hydrophilic and Hydrophobic Solid-Water Interfaces

    SciTech Connect

    Holinga IV, George Joseph

    2010-09-01

    Sum frequency generation (SFG) vibrational spectroscopy was used to investigate the interfacial properties of several amino acids, peptides, and proteins adsorbed at the hydrophilic polystyrene solid-liquid and the hydrophobic silica solid-liquid interfaces. The influence of experimental geometry on the sensitivity and resolution of the SFG vibrational spectroscopy technique was investigated both theoretically and experimentally. SFG was implemented to investigate the adsorption and organization of eight individual amino acids at model hydrophilic and hydrophobic surfaces under physiological conditions. Biointerface studies were conducted using a combination of SFG and quartz crystal microbalance (QCM) comparing the interfacial structure and concentration of two amino acids and their corresponding homopeptides at two model liquid-solid interfaces as a function of their concentration in aqueous solutions. The influence of temperature, concentration, equilibration time, and electrical bias on the extent of adsorption and interfacial structure of biomolecules were explored at the liquid-solid interface via QCM and SFG. QCM was utilized to quantify the biological activity of heparin functionalized surfaces. A novel optical parametric amplifier was developed and utilized in SFG experiments to investigate the secondary structure of an adsorbed model peptide at the solid-liquid interface.

  3. Adsorption kinetics of plasma proteins on ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles.

    PubMed

    Jansch, M; Stumpf, P; Graf, C; Rühl, E; Müller, R H

    2012-05-30

    In this study the kinetics of plasma protein adsorption onto ultrasmall superparamagnetic iron oxide (USPIO) particles have been analyzed and compared to previously published kinetic studies on polystyrene particles (PS particles), oil-in-water nanoemulsions and solid lipid nanoparticles (SLNs). SPIO and USPIO nanoparticles are commonly used as magnetic resonance imaging (MRI) enhancers for tumor imaging as well as in drug delivery applications. Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) has been used to determine the plasma protein adsorption onto the citrate/triethylene glycol-stabilized iron oxide surface. The results indicate that the existence of a Vroman effect, a displacement of previously adsorbed abundant proteins, such as albumin or fibrinogen, respectively, on USPIO particles has to be denied. Previously, identical findings have been reported for oil-in-water nanoemulsions. Furthermore, the protein adsorption kinetics differs dramatically from that of other solid drug delivery systems (PS, SLN). More relevant for the in vivo fate of long circulating particles is the protein corona after several minutes or even hours. Interestingly, the patterns received after an incubation time of 0.5 min to 240 min are found to be qualitatively and quantitatively similar. This leads to the assumption of a long-lived ("hard") protein corona around the iron oxide nanoparticles. PMID:22342465

  4. Elastic response of a protein monolayer adsorbed at decorated water surface

    NASA Astrophysics Data System (ADS)

    Singh, Amarjeet; Konovalov, Oleg

    2015-05-01

    Under the in-plane isothermal compression the self-assembled protein monolayer expand in the direction perpendicular to the applied force as a function of applied compression. The structure finally buckle beyond a critical compression, which finally returns to the initial structure when the compression force was removed, behaving like an elastic body. We modelled the layer as homogeneous elastic medium and calculated elastic constants. Young's modulus of the protein layer is 2 orders of magnitude smaller than the bulk lysozyme crystals. It is of fundamental significance to be able to predict the elastic properties of the proteins at air-water interface since protein remains in their natural environment unlike protein crystals.

  5. Plasma PIVKA proteins in rabbits given warfarin.

    PubMed

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

    1996-06-01

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

  6. Porous ceramic/agarose composite adsorbents for fast protein liquid chromatography.

    PubMed

    Xia, Haifeng; Jin, Xionghua; Wu, Puqiang; Zheng, Zhiyong

    2012-02-01

    Porous ceramic/agarose composite adsorbents were designed and prepared with silica ceramic beads and 4% agarose gel, and then functionalized with a special ligand carboxymethyl. A novel method was introduced to fabricating of the porous silica ceramic beads. The morphology of SEM shows a spherical shape and a porous structure of the ceramic beads. Nitrogen adsorption-desorption analysis gives an average pore size of 287.5 Å, a BET surface area of 29.33 m²/g and a porosity of 41.8%, respectively. Additionally, X-ray diffraction pattern indicates that the amorphous silica has been transformed into two crystal phases of quartz and cristobalite, leading to a porous and rigid skeleton and ensuring the application of the composite beads at high flow velocities. Lysozyme of hen egg-white with the activity of 12,700 U/mg was purified by the composite ion-exchanger in one step and the recovery and purification factor reaches 95.2% and 7.9, respectively.

  7. X-ray Reflectivity Studies of Adsorbed Proteins on Langmuir Layers

    NASA Astrophysics Data System (ADS)

    Málková, Šárka; Pingali, Sai V.; Long, Fei; Cho, Wonhwa; Schlossman, Mark L.

    2002-03-01

    Synchrotron X-ray reflectivity is used to study the interaction of the C2 domain of cytosolic phospholipase A2 (cPLA_2-C2) with a phospholipid membrane. SOPC (1-steraoyl-2-oleoyl-sn-glycero-3-phosphocholine) monolayer has been chosen as our model membrane. SOPC monolayer is supported on a buffered, Ca^2+ containing aqueous solution. The original phospholipid layer, which can be described by a two layer model roughened by capillary wave theory, is modified and a three layer model is necessary to fit the data after the protein is injected. The data analysis indicates that a third layer attached to the phospholipid headgroup region is formed. This additional layer corresponds to proteins bound to the phospholipid. The mechanism of the protein binding to the lipid depends on the initial lipid pressure with adsorption being less pronounced at higher pressures. Two control experiments are performed. Buffer containing no Ca^2+ is used in the first and F35/L39A mutant of cPLA_2-C2 (the mutation is in the region containing ligands for multiple Ca^2+ ions) is used in the second. Reflectivity curves do not show any evidence of protein adsorption to the phospholipid monolayer in any of our two control experiments. Our results thus support the idea that the initial pressure of the lipid, Ca^2+ ions and Ca^2+ binding sites of cPLA_2-C2 are important factors for the membrane binding of this protein.

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

    PubMed Central

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

    2011-01-01

    Plasma proteome is widely used in studying changes occurring in human body during disease or other disturbances. Immunological methods are commonly used in such studies. In recent years, mass spectrometry has gained popularity in high-throughput analysis of plasma proteins. In this study, we tested whether mass spectrometry and iTRAQ-based protein quantification might be used in proteomic analysis of human plasma during liver transplantation surgery to characterize changes in protein abundances occurring during early graft reperfusion. We sampled blood from systemic circulation as well as blood entering and exiting the liver. After immunodepletion of six high-abundant plasma proteins, trypsin digestion, iTRAQ labeling, and cation-exchange fractionation, the peptides were analyzed by reverse phase nano-LC-MS/MS. In total, 72 proteins were identified of which 31 could be quantified in all patient specimens collected. Of these 31 proteins, ten, mostly medium-to-high abundance plasma proteins with a concentration range of 50–2000 mg/L, displayed relative abundance change of more than 10%. The changes in protein abundance observed in this study allow further research on the role of several proteins in ischemia-reperfusion injury during liver transplantation and possibly in other surgery. PMID:22187521

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

    PubMed

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

    2011-01-01

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

  10. Molecular interactions of graphene oxide with human blood plasma proteins

    NASA Astrophysics Data System (ADS)

    Kenry, Affa Affb Affc; Loh, Kian Ping; Lim, Chwee Teck

    2016-04-01

    We investigate the molecular interactions between graphene oxide (GO) and human blood plasma proteins. To gain an insight into the bio-physico-chemical activity of GO in biological and biomedical applications, we performed a series of biophysical assays to quantify the molecular interactions between GO with different lateral size distributions and the three essential human blood plasma proteins. We elucidate the various aspects of the GO-protein interactions, particularly, the adsorption, binding kinetics and equilibrium, and conformational stability, through determination of quantitative parameters, such as GO-protein association constants, binding cooperativity, and the binding-driven protein structural changes. We demonstrate that the molecular interactions between GO and plasma proteins are significantly dependent on the lateral size distribution and mean lateral sizes of the GO nanosheets and their subtle variations may markedly influence the GO-protein interactions. Consequently, we propose the existence of size-dependent molecular interactions between GO nanosheets and plasma proteins, and importantly, the presence of specific critical mean lateral sizes of GO nanosheets in achieving very high association and fluorescence quenching efficiency of the plasma proteins. We anticipate that this work will provide a basis for the design of graphene-based and other related nanomaterials for a plethora of biological and biomedical applications.

  11. Colloid-probe AFM studies of the interaction forces of proteins adsorbed on colloidal crystals.

    PubMed

    Singh, Gurvinder; Bremmell, Kristen E; Griesser, Hans J; Kingshott, Peter

    2015-04-28

    In recent years, colloid-probe AFM has been used to measure the direct interaction forces between colloidal particles of different size or surface functionality in aqueous media, as one can study different forces in symmerical systems (i.e., sphere-sphere geometry). The present study investigates the interaction between protein coatings on colloid probes and hydrophilic surfaces decorated with hexagonally close packed single particle layers that are either uncoated or coated with proteins. Controlled solvent evaporation from aqueous suspensions of colloidal particles (coated with or without lysozyme and albumin) produces single layers of close-packed colloidal crystals over large areas on a solid support. The measurements have been carried out in an aqueous medium at different salt concentrations and pH values. The results show changes in the interaction forces as the surface charge of the unmodified or modified particles, and ionic strength or pH of the solution is altered. At high ionic strength or pH, electrostatic interactions are screened, and a strong repulsive force at short separation below 5 nm dominates, suggesting structural changes in the absorbed protein layer on the particles. We also study the force of adhesion, which decreases with an increment in the salt concentration, and the interaction between two different proteins indicating a repulsive interaction on approach and adhesion on retraction. PMID:25758979

  12. The Effect of Plasma Treatment on Adsorbed Iodine as a Catalyst in Chemical Vapor Deposition of Copper and Application to Filling of Deep Trenches with High Aspect Ratios

    NASA Astrophysics Data System (ADS)

    Lee, Do-Seon; Lee, Won-Jong

    2009-07-01

    Plasma treatment was introduced in order to control the catalytic properties of iodine in catalyst-enhanced chemical vapor deposition (CECVD) of copper (Cu). The iodine adatoms are deactivated (i.e., lose their catalytic effect) by forming Cu-I bonds through reaction with Cu atoms by the bombardment of ions during the plasma treatment. The surface concentration of effective iodine adatoms that can act as catalysts decreases exponentially with an increasing of ion exposure which is the product of ion flux and plasma treatment time. The deactivated iodine can be reactivated by annealing above 200 °C. The enhancement factor, defined as the ratio of the enhanced deposition rate of Cu film by the adsorbed iodine to the deposition rate without the catalytic effect of iodine, is proportional to the surface concentration of effective iodine adatoms. The distribution of the surface concentration of effective iodine adatoms inside the trench can be controlled by the plasma treatment. CECVD coupled with plasma treatment enables void-free filling of deep trenches with an aspect ratio of 14.

  13. Expanded bed adsorption/desorption of proteins with Streamline Direct CST I adsorbent.

    PubMed

    Li, Ping; Xiu, Guohua; Mata, Vera G; Grande, Carlos A; Rodrigues, Alirio E

    2006-08-20

    Streamline Direct CST I is a new type of ion exchanger with multi-modal functional groups, specially designed for an expanded bed adsorption (EBA) process, which can capture directly the proteins from the high ionic strength feedstocks with a high binding capacity. In this study, an experimental study is carried out for two-component proteins (BSA and myoglobin) competitive adsorption and desorption in an expanded bed packed with Streamline Direct CST I. Based on the measurements of the single- and two-component bovine serum albumin (BSA)/myoglobin adsorption isotherm on Streamline Direct CST I, the binding and elution conditions for the whole EBA process are selected; and then frontal analysis for a longer timescale and column displacement experiments in a fixed bed (XK16/20 column) are carried out to evaluate the two-component proteins (BSA and myoglobin) competitive adsorption and displacement on Streamline Direct CST I. Finally, the feasibility of capturing both BSA and myoglobin by an expanded bed packed with Streamline Direct CST I is addressed in a Streamline 50 column packed with 300 mL Streamline Direct CST I. PMID:16572450

  14. Zeolite Nanoparticles for Selective Sorption of Plasma Proteins

    PubMed Central

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

    2015-01-01

    The affinity of zeolite nanoparticles (diameter of 8–12 nm) possessing high surface area and high pore volume towards human plasma proteins has been investigated. The protein composition (corona) of zeolite nanoparticles has been shown to be more dependent on the plasma protein concentrations and 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. PMID:26616161

  15. Zeolite Nanoparticles for Selective Sorption of Plasma Proteins.

    PubMed

    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

    2015-01-01

    The affinity of zeolite nanoparticles (diameter of 8-12 nm) possessing high surface area and high pore volume towards human plasma proteins has been investigated. The protein composition (corona) of zeolite nanoparticles has been shown to be more dependent on the plasma protein concentrations and 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. PMID:26616161

  16. Zeolite Nanoparticles for Selective Sorption of Plasma Proteins

    NASA Astrophysics Data System (ADS)

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

    2015-11-01

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

  17. [Chemistry and clinical significance of human plasma proteins].

    PubMed

    Haupt, H

    1990-10-01

    Enormous progress has been made in the course of the past few years in the various fields of plasma protein research. The primary and disulfide bridge structures are now known for almost all of the 120 proteins thus far isolated from human plasma, including trace and ultratrace proteins as well as a number of genetic variants. Genetic cloning and the derivation of the amino-acid sequence from the nucleotide sequence have played a decisive role here. However, we are only in possession of the exact three-dimensional structure for a small number of plasma proteins. The major problem in this respect is, at present, the lack of suitable protein crystals for X-ray structure analysis. We still do not know the physiological function of a large number of plasma proteins, despite the fact that they, in part, have been well characterised both physically and chemically and could be assigned to their respective protein families on the basis of their amino-acid sequence. The development of techniques for protein structure determination is relatively well advanced today, yet we lack methods of illuminating the structure-function relationship. There are more than 20 different highly purified protein preparations in virus-safe form available today for substitution therapy. To this effect new purification procedures have been developed which pay particular attention to virus elimination and inactivation. Should present indications be confirmed, one may assume that further plasma proteins (e. g. proteinase inhibitors, apolipoproteins, fibronectin) could be of significance in therapy and prophylaxis. Unlimited amounts of human blood are not available. Gene technology offers a promising alternative, at least for the production of plasma protein administered to patients in small amounts. Work is being done intensively on various blood coagulation factors and proteinase inhibitors at the moment, and factor VIII: C is already being successfully used for the treatment of patients with

  18. Transport proteins of the plant plasma membrane

    NASA Technical Reports Server (NTRS)

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

    1996-01-01

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

  19. Atomic force microscopy compatible device for stretching cells and adsorbed proteins

    NASA Astrophysics Data System (ADS)

    De Jong, K. L.; MacLeod, H. C.; Norton, P. R.; Petersen, N. O.; Jasnin, M. F.

    2006-02-01

    A device that we term a "microrack" was designed to provide a means to stretch cells and proteins and to permit the measurement of any changes in adhesion forces that might occur as a result of the strain, with an atomic force microscope. The device requires an elastic material that allows adsorption of proteins and attachment of cells. The elastomer polydimethylsiloxane (PDMS) was chosen, and its suitability for short-term cell studies was tested by comparing cell morphology and fiber distribution on PDMS with cells grown on glass, a conventional substrate for cell study. Atomic force microscopy (AFM) images and section analysis of beads and scrape marks on the PDMS surface before and after stretching indicate that the microrack can provide up to 21%-29% deformation of PDMS. AFM images of cells grown on PDMS show that material attached to the surface is also affected by stretching of the microrack. The rupture of the cell after stretching and rippling of the cell under compression can be attributed to the fixation treatment, but indicates that the cell morphology is significantly affected by the movement of the substrate on the microrack.

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

    PubMed

    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.

  1. Adsorbent phosphates

    NASA Technical Reports Server (NTRS)

    Watanabe, S.

    1983-01-01

    An adsorbent which uses as its primary ingredient phosphoric acid salts of zirconium or titanium is presented. Production methods are discussed and several examples are detailed. Measurements of separating characteristics of some gases using the salts are given.

  2. Green algae (Chlorella pyrenoidosa) adsorbs Bacillus thurigiensis (Bt) toxin, Cry1Ca insecticidal protein, without an effect on growth.

    PubMed

    Wang, Jiamei; Chen, Xiuping; Li, Yunhe; Su, Changqing; Ding, Jiatong; Peng, Yufa

    2014-08-01

    The effect of purified Cry1Ca insecticidal protein on the growth of Chlorella pyrenoidosa was studied in a three-generation toxicity test. The C. pyrenoidosa medium with a density of 5.4 × 10(5) cells/mL was subcultured for three generations with added Cry1Ca at 0, 10, 100, and 1000 µg/L, and cell numbers were determined daily. To explore the distribution of Cry1Ca in C. pyrenoidosa and the culture medium, Cry1Ca was added at 1000 µg/L to algae with a high density of 4.8 × 10(6) cells/mL, and Cry1Ca content was determined daily in C. pyrenoidosa and the culture medium by enzyme-linked immunosorbent assays. Our results showed that the growth curves of C. pyrenoidosa exposed to 10, 100, and 1000 µg/L of Cry1Ca almost overlapped with that of the blank control, and there were no statistically significant differences among the four treatments from day 0 to day 7, regardless of generation. Moreover, the Cry1Ca content in the culture medium and in C. pyrenoidosa sharply decreased under exposure of 1000 µg/L Cry1Ca with high initial C. pyrenoidosa cell density. The above results demonstrate that Cry1Ca in water can be rapidly adsorbed and degraded by C. pyrenoidosa, but it has no suppressive or stimulative effect on algae growth.

  3. Genetic polymorphism of six plasma proteins in the domestic cat.

    PubMed

    Juneja, R K; Niini, T; Larsson, H E; Sandberg, K

    1991-01-01

    Phenotypes of cat plasma apolipoprotein A4 (APOA4), antithrombin 3 (AT3), alpha 1B-glycoprotein (A1BG), transferrin (TF), vitamin D-binding protein (GC), and an unidentified pretransferrin (PTF) were determined by using simple methods of horizontal, nondenaturing gel electrophoresis followed by protein staining. The cat proteins were identified by immunoblotting using antisera for human plasma proteins. Three alleles were reported for each of TF and PTF, and two alleles were reported for each of GC, APOA4, AT3, and A1BG. The mongrels and Persians showed a high degree of polymorphism at most of the loci whereas the Birmans exhibited much less variation. Genetic evidence indicating the occurrence of a monomeric and a dimeric form of APOA4 in cat plasma was reported. PMID:2013693

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

    PubMed

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

    2011-10-01

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

  5. Intravenous delivery of hydrophobin-functionalized porous silicon nanoparticles: stability, plasma protein adsorption and biodistribution.

    PubMed

    Sarparanta, Mirkka; Bimbo, Luis M; Rytkönen, Jussi; Mäkilä, Ermei; Laaksonen, Timo J; Laaksonen, Päivi; Nyman, Markus; Salonen, Jarno; Linder, Markus B; Hirvonen, Jouni; Santos, Hélder A; Airaksinen, Anu J

    2012-03-01

    Rapid immune recognition and subsequent elimination from the circulation hampers the use of many nanomaterials as carriers to targeted drug delivery and controlled release in the intravenous route. Here, we report the effect of a functional self-assembled protein coating on the intravenous biodistribution of (18)F-labeled thermally hydrocarbonized porous silicon (THCPSi) nanoparticles in rats. (18)F-Radiolabeling enables the sensitive and easy quantification of nanoparticles in tissues using radiometric methods and allows imaging of the nanoparticle biodistribution with positron emission tomography. Coating with Trichoderma reesei HFBII altered the hydrophobicity of (18)F-THCPSi nanoparticles and resulted in a pronounced change in the degree of plasma protein adsorption to the nanoparticle surface in vitro. The HFBII-THCPSi nanoparticles were biocompatible in RAW 264.7 macrophages and HepG2 liver cells making their intravenous administration feasible. In vivo, the distribution of the nanoparticles between the liver and spleen, the major mononuclear phagocyte system organs in the body, was altered compared to that of uncoated (18)F-THCPSi. Identification of the adsorbed proteins revealed that certain opsonins and apolipoproteins are enriched in HFBII-functionalized nanoparticles, whereas the adsorption of abundant plasma components such as serum albumin and fibrinogen is decreased.

  6. Cardiovascular-related proteins identified in human plasma by the HUPO Plasma Proteome Project pilot phase.

    PubMed

    Berhane, Beniam T; Zong, Chenggong; Liem, David A; Huang, Aaron; Le, Steven; Edmondson, Ricky D; Jones, Richard C; Qiao, Xin; Whitelegge, Julian P; Ping, Peipei; Vondriska, Thomas M

    2005-08-01

    Proteomic profiling of accessible bodily fluids, such as plasma, has the potential to accelerate biomarker/biosignature development for human diseases. The HUPO Plasma Proteome Project pilot phase examined human plasma with distinct proteomic approaches across multiple laboratories worldwide. Through this effort, we confidently identified 3020 proteins, each requiring a minimum of two high-scoring MS/MS spectra. A critical step subsequent to protein identification is functional annotation, in particular with regard to organ systems and disease. Performing exhaustive literature searches, we have manually annotated a subset of these 3020 proteins that have cardiovascular-related functions on the basis of an existing body of published information. These cardiovascular-related proteins can be organized into eight groups: markers of inflammation and/or cardiovascular disease, vascular and coagulation, signaling, growth and differentiation, cytoskeletal, transcription factors, channels/receptors and heart failure and remodeling. In addition, analysis of the peptide per protein ratio for MS/MS identification reveals group-specific trends. These findings serve as a resource to interrogate the functions of plasma proteins, and moreover, the list of cardiovascular-related proteins in plasma constitutes a baseline proteomic blueprint for the future development of biosignatures for diseases such as myocardial ischemia and atherosclerosis. PMID:16052623

  7. Neutrophils Turn Plasma Proteins into Weapons against HIV-1

    PubMed Central

    Hagleitner, Magdalena; Rambach, Günter; Van Aken, Hugo; Dierich, Manfred; Kehrel, Beate E.

    2013-01-01

    As a consequence of innate immune activation granulocytes and macrophages produce hypochlorite/hypochlorous acid (HOCl) via secretion of myeloperoxidase (MPO) to the outside of the cells, where HOCl immediately reacts with proteins. Most proteins that become altered by this system do not belong to the invading microorganism but to the host. While there is no doubt that the myeloperoxidase system is capable of directly inactivating HIV-1, we hypothesized that it may have an additional indirect mode of action. We show in this article that HOCl is able to chemically alter proteins and thus turn them into Idea-Ps (Idea-P = immune defence-altered protein), potent amyloid-like and SH-groups capturing antiviral weapons against HIV-1. HOCl-altered plasma proteins (Idea-PP) have the capacity to bind efficiently and with high affinity to the HIV-1 envelope protein gp120, and to its receptor CD4 as well as to the protein disulfide isomerase (PDI). Idea-PP was able to inhibit viral infection and replication in a cell culture system as shown by reduced number of infected cells and of syncytia, resulting in reduction of viral capsid protein p24 in the culture supernatant. The unmodified plasma protein fraction had no effect. HOCl-altered isolated proteins antithrombin III and human serum albumin, taken as representative examples of the whole pool of plasma proteins, were both able to exert the same activity of binding to gp120 and inhibition of viral proliferation. These data offer an opportunity to improve the understanding of the intricacies of host-pathogen interactions and allow the generation of the following hypothetical scheme: natural immune defense mechanisms generate by posttranslational modification of plasma proteins a potent virucidal weapon that immobilizes the virus as well as inhibits viral fusion and thus entry into the host cells. Furthermore simulation of this mechanism in vitro might provide an interesting new therapeutic approach against microorganisms

  8. Glycan Moieties as Bait to Fish Plasma Membrane Proteins.

    PubMed

    Fang, Fei; Zhao, Qun; Sui, Zhigang; Liang, Yu; Jiang, Hao; Yang, Kaiguang; Liang, Zhen; Zhang, Lihua; Zhang, Yukui

    2016-05-17

    Plasma membrane proteome analysis is of significance for screening candidate biomarkers and drug targets. However, due to their low abundance and lack of specific groups that can enable their capture, the plasma membrane proteins (PMPs) are under-represented. On the basis of the fact that PMPs are embedded in or anchored to the phospholipid bilayer of the plasma membrane and the glycan moieties of proteins and lipids located on the plasma membrane are exposed outside of the cell surface, we proposed a strategy to capture PMPs, termed as glycan moieties-directed PMPs enrichment (GMDPE). With the glycan moieties exposed outside of the cells as bait to ensure the selectivity and the phospholipid bilayer as raft to provide the sensitivity, we applied this strategy into the plasma membrane proteome analysis of HeLa cells, and in total, 772 PMPs were identified, increased by 4.5 times compared to those identified by the reported cell surface biotinylation method. Notably, among them, 86 CD antigens and 16 ion channel proteins were confidently identified. All these results demonstrated that our proposed approach has great potential in the large scale plasma membrane proteome profiling.

  9. Bioactive glass 45S5 powders: effect of synthesis route and resultant surface chemistry and crystallinity on protein adsorption from human plasma.

    PubMed

    Bahniuk, Markian S; Pirayesh, Hamidreza; Singh, Harsh D; Nychka, John A; Unsworth, Larry D

    2012-12-01

    Despite its medical applications, the mechanisms responsible for the osseointegration of bioactive glass (45S5) have yet to be fully understood. Evidence suggests that the strongest predictor for osseointegration of bioactive glasses, and ceramics, with bone tissue as the formation of an apatitic calcium phosphate layer atop the implanted material, with osteoblasts being the main mediator for new bone formation. Most have tried to understand the formation of this apatitic calcium phosphate layer, and other bioresponses between the host and bioactive glass 45S5 using Simulated Body Fluid; a solution containing ion concentrations similar to that found in human plasma without the presence of proteins. However, it is likely that cell attachment is probably largely mediated via the adsorbed protein layer. Plasma protein adsorption at the tissue bioactive glass interface has been largely overlooked. Herein, we compare crystalline and amorphous bioactive glass 45S5, in both melt-derived as well as sol-gel forms. Thus, allowing for a detailed understanding of both the role of crystallinity and powder morphology on surface ions, and plasma protein adsorption. It was found that sol-gel 45S5 powders, regardless of crystallinity, adsorbed 3-5 times as much protein as the crystalline melt-derived counterpart, as well as a greater variety of plasma proteins. The devitrification of melt-cast 45S5 resulted in only small differences in the amount and variety of the adsorbed proteome. Surface properties, and not material crystallinity, play a role in directing protein adsorption phenomena for bioactive glasses given the differences found between crystalline melt-cast 45S5 and sol-gel derived 45S5.

  10. Drug-drug plasma protein binding interactions of ivacaftor.

    PubMed

    Schneider, Elena K; Huang, Johnny X; Carbone, Vincenzo; Baker, Mark; Azad, Mohammad A K; Cooper, Matthew A; Li, Jian; Velkov, Tony

    2015-06-01

    Ivacaftor is a novel cystic fibrosis (CF) transmembrane conductance regulator (CFTR) potentiator that improves the pulmonary function for patients with CF bearing a G551D CFTR-protein mutation. Because ivacaftor is highly bound (>97%) to plasma proteins, there is the strong possibility that co-administered CF drugs may compete for the same plasma protein binding sites and impact the free drug concentration. This, in turn, could lead to drastic changes in the in vivo efficacy of ivacaftor and therapeutic outcomes. This biochemical study compares the binding affinity of ivacaftor and co-administered CF drugs for human serum albumin (HSA) and α1 -acid glycoprotein (AGP) using surface plasmon resonance and fluorimetric binding assays that measure the displacement of site-selective probes. Because of their ability to strongly compete for the ivacaftor binding sites on HSA and AGP, drug-drug interactions between ivacaftor are to be expected with ducosate, montelukast, ibuprofen, dicloxacillin, omeprazole, and loratadine. The significance of these plasma protein drug-drug interactions is also interpreted in terms of molecular docking simulations. This in vitro study provides valuable insights into the plasma protein drug-drug interactions of ivacaftor with co-administered CF drugs. The data may prove useful in future clinical trials for a staggered treatment that aims to maximize the effective free drug concentration and clinical efficacy of ivacaftor. PMID:25707701

  11. Plasma proteins predict conversion to dementia from prodromal disease

    PubMed Central

    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

    2014-01-01

    Background 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. Methods 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. Results 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%). Conclusions 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. PMID:25012867

  12. Stereoselective binding of chiral drugs to plasma proteins

    PubMed Central

    Shen, Qi; Wang, Lu; Zhou, Hui; Jiang, Hui-di; Yu, Lu-shan; Zeng, Su

    2013-01-01

    Chiral drugs show distinct biochemical and pharmacological behaviors in the human body. The binding of chiral drugs to plasma proteins usually exhibits stereoselectivity, which has a far-reaching influence on their pharmacological activities and pharmacokinetic profiles. In this review, the stereoselective binding of chiral drugs to human serum albumin (HSA), α1-acid glycoprotein (AGP) and lipoprotein, three most important proteins in human plasma, are detailed. Furthermore, the application of AGP variants and recombinant fragments of HSA for studying enantiomer binding properties is also discussed. Apart from the stereoselectivity of enantiomer-protein binding, enantiomer-enantiomer interactions that may induce allosteric effects are also described. Additionally, the techniques and methods used to determine drug-protein binding parameters are briefly reviewed. PMID:23852086

  13. Supramolecular Structures with Blood Plasma Proteins, Sugars and Nanosilica

    NASA Astrophysics Data System (ADS)

    Turov, V. V.; Gun'ko, V. M.; Galagan, N. P.; Rugal, A. A.; Barvinchenko, V. M.; Gorbyk, P. P.

    Supramolecular structures with blood plasma proteins (albumin, immunoglobulin and fibrinogen (HPF)), protein/water/silica and protein/water/ silica/sugar (glucose, fructose and saccharose) were studied by NMR, adsorption, IR and UV spectroscopy methods. Hydration parameters, amounts of weakly and strongly bound waters and interfacial energy (γ S) were determined over a wide range of component concentrations. The γ S(C protein,C silica) graphs were used to estimate the energy of protein-protein, protein-surface and particle-particle interactions. It was shown that interfacial energy of self-association (γ as) of protein molecules depends on a type of proteins. A large fraction of water bound to proteins can be displaced by sugars, and the effect of disaccharide (saccharose) was greater than that of monosugars. Changes in the structural parameters of cavities in HPF molecules and complexes with HPF/silica nanoparticles filled by bound water were analysed using NMR-cryoporometry showing that interaction of proteins with silica leads to a significant decrease in the amounts of water bound to both protein and silica surfaces. Bionanocomposites with BSA/nanosilica/sugar can be used to influence states of living cells and tissues after cryopreservation or other treatments. It was shown that interaction of proteins with silica leads to strong decrease in the volume of all types of internal cavities filled by water.

  14. Lotus-leaf-like topography predominates over adsorbed ECM proteins in poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) surface/cell interactions.

    PubMed

    Zheng, Jun; Li, Dan; Yuan, Lin; Liu, Xiaoli; Chen, Hong

    2013-06-26

    It is well-known that extracellular matrix (ECM) proteins mediate cell/surface interactions. However, introduction of a specific surface topography may disturb the correlation between ECM proteins adsorption and cells adhesion on a given surface. In present study, lotus-leaf-like topography was introduced on the surface of a biodegradable material, poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx). Protein adsorption and cell interactions with this lotus-leaf-like surface (designated PHBHHx-L) were investigated. Water contact angle data indicated that the hydrophobicity of PHBHHx was enhanced by the introduction of lotus-leaf-like topography. The adsorption of extracellular matrix proteins (fibronectin and vitronectin) on PHBHHx-L was measured by enzyme linked immunosorbent assay (ELISA). Compared with flat PHBHHx, adsorption on the PHBHHx-L surface increased by ~260% for fibronectin and ~40% for vitronectin. In contrast, fibroblast and endothelial cell adhesion and proliferation were reduced on the PHBHHx-L compared to the flat polymer surface. These results suggest that the inhibition of cell adhesion and proliferation caused by the lotus-leaf-like topography dominates over the effect of the adsorbed adhesive proteins in promoting adhesion and proliferation. It can be concluded that the lotus-leaf-like topography plays a dominant role in cell/PHBHHx-L interactions. The present findings indicate the complexity of the interplay among surface topography, adsorbed proteins, and cell-surface interactions.

  15. Plasma protein carbonyl levels and breast cancer risk.

    PubMed

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

    2007-01-01

    To study the role of oxidative stress in breast cancer risk, we analysed plasma levels of protein carbonyls in 1050 cases and 1107 controls. We found a statistically significant trend in breast cancer risk in relation to increasing quartiles of plasma protein carbonyl levels (OR = 1.2, 95% CI = 0.9-1.5; OR = 1.5, 95% CI = 1.2-2.0; OR = 1.6, 95% CI = 1.2-2.1, for the 2(nd), 3(rd) and 4(th) quartile relative to the lowest quartile, respectively, P for trend = 0.0001). The increase in risk was similar for younger (<50 years) and older women, more pronounced among women with higher physical activity levels (0.7 hrs/week for 4(th) quartile versus lowest quartile OR = 2.0, 95% CI = 1.4-3.0), higher alcohol consumption (> or = 15 grams/day for 4(th) quartile versus lowest quartile OR = 2.3, 95% CI = 1.1-4.7), and hormone replacement therapy use (HRT, OR = 2.6, 95% CI = 1.6-4.4 for 4(th) quartile versus lowest quartile). The multiplicative interaction terms were statistically significant only for physical activity and HRT. The positive association between plasma protein carbonyl levels and breast cancer risk was also observed when the analysis was restricted to women who had not received chemotherapy or radiation therapy prior to blood collection. Among controls, oxidized protein levels significantly increased with cigarette smoking and higher fruit and vegetable consumption, and decreased with alcohol consumption >30 grams per day. Women with higher levels of plasma protein carbonyl and urinary 15F(2t)-isoprostane had an 80% increase in breast cancer risk (OR = 1.8, 95% CI = 1.2-2.6) compared to women with levels below the median for both markers of oxidative stress. In summary, our results suggest that increased plasma protein carbonyl levels may be associated with breast cancer risk.

  16. Viscoelastic properties of adsorbed and cross-linked polypeptide and protein layers at a solid-liquid interface.

    PubMed

    Dutta, Amit K; Nayak, Arpan; Belfort, Georges

    2008-08-01

    The real-time changes in viscoelasticity of adsorbed poly(L-lysine) (PLL) and adsorbed histone (lysine rich fraction) due to cross-linking by glutaraldehyde and corresponding release of associated water were investigated using a quartz crystal microbalance with dissipation monitoring (QCM-D) and attenuated total reflection Fourier transform infrared spectroscopy (ATR/FTIR). The kinetics of PLL and histone adsorption were measured through changes in mass adsorbed onto a gold-coated quartz surface from changes in frequency and dissipation and using the Voigt viscoelastic model. Prior to cross-linking, the shear viscosity and shear modulus of the adsorbed PLL layer were approximately 3.0 x 10(-3) Pas and approximately 2.5 x 10(5) Pa, respectively, while after cross-linking, they increased to approximately 17.5 x 10(-3) Pas and approximately 2.5 x 10(6) Pa, respectively. For the adsorbed histone layer, shear viscosity and shear modulus increased modestly from approximately 1.3 x 10(-3) to approximately 2.0 x 10(-3) Pas and from approximately 1.2 x 10(4) to approximately 1.6 x 10(4) Pa, respectively. The adsorbed mass estimated from the Sauerbrey equation (perfectly elastic) and the Voigt viscoelastic model differ appreciably prior to cross-linking whereas after cross-linking they converged. This is because trapped water molecules were released during cross-linking. This was confirmed experimentally via ATR/FTIR measurements. The variation in viscoelastic properties increased substantially after cross-linking presumably due to fluctuation of the randomly cross-linked network structure. An increase in fluctuation of the viscoelastic properties and the loss of imbibed water could be used as a signature of the formation of a cross-linked network and the amount of cross-linking, respectively. PMID:18508070

  17. A radioimmunoassay for bone Gla protein (BGP) in human plasma.

    PubMed

    Johansen, J S; Mølholm Hansen, J E; Christiansen, C

    1987-03-01

    To study the value of bone Gla protein (BGP) as a biochemical marker of normal bone physiology and metabolic bone disorders, we have developed a radioimmunoassay (RIA) for the detection of BGP in human plasma. Antibodies were generated in rabbits immunized with purified calf BGP conjugated to thyroglobulin. Human plasma BGP reacted identically with the calf BGP standard, thus demonstrating the suitability of the assay to measure plasma BGP levels in man. The RIA is sensitive, accurate, and technically simple. Plasma BGP levels were determined in normal subjects (N = 35) and in patients with hypothyroidism (N = 10), hyperthyroidism (N = 22) and chronic renal failure (N = 35). The mean (+/- 1 SEM) concentration of plasma BGP in normal subjects was 1.27 +/- 0.07 nmol/l. Plasma BGP was significantly increased in patients with hyperthyroidism, 4.04 +/- 0.78 nmol/l (P less than 0.001) and chronic renal failure, 10.17 +/- 2.47 nmol/l (P less than 0.001). Low concentrations were found in patients with hypothyroidism, 0.74 +/- 0.11 nmol/l (P less than 0.01). Our studies indicate that plasma BGP provides a useful technique in the diagnosis of patients with bone disease.

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

    PubMed

    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 <200 nm. Parallel advances in molecular simulations provide near-atomic-resolution models of the dynamics of the organization of 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.

  19. Integrated system for temperature-controlled fast protein liquid chromatography comprising improved copolymer modified beaded agarose adsorbents and a travelling cooling zone reactor arrangement.

    PubMed

    Müller, Tobias K H; Cao, Ping; Ewert, Stephanie; Wohlgemuth, Jonas; Liu, Haiyang; Willett, Thomas C; Theodosiou, Eirini; Thomas, Owen R T; Franzreb, Matthias

    2013-04-12

    An integrated approach to temperature-controlled chromatography, involving copolymer modified agarose adsorbents and a novel travelling cooling zone reactor (TCZR) arrangement, is described. Sepharose CL6B was transformed into a thermoresponsive cation exchange adsorbent (thermoCEX) in four synthetic steps: (i) epichlorohydrin activation; (ii) amine capping; (iii) 4,4'-azobis(4-cyanovaleric acid) immobilization; and 'graft from' polymerization of poly(N-isopropylacrylamide-co-N-tert-butylacrylamide-co-acrylic acid-co-N,N'-methylenebisacrylamide). FT-IR, (1)H NMR, gravimetry and chemical assays allowed precise determination of the adsorbent's copolymer composition and loading, and identified the initial epoxy activation step as a critical determinant of 'on-support' copolymer loading, and in turn, protein binding performance. In batch binding studies with lactoferrin, thermoCEX's binding affinity and maximum adsorption capacity rose smoothly with temperature increase from 20 to 50 °C. In temperature shifting chromatography experiments employing thermoCEX in thermally jacketed columns, 44-51% of the lactoferrin adsorbed at 42 °C could be desorbed under binding conditions by cooling the column to 22 °C, but the elution peaks exhibited strong tailing. To more fully exploit the potential of thermoresponsive chromatography adsorbents, a new column arrangement, the TCZR, was developed. In TCZR chromatography, a narrow discrete cooling zone (special assembly of copper blocks and Peltier elements) is moved along a bespoke fixed-bed separation columnfilled with stationary phase. In tests with thermoCEX, it was possible to recover 65% of the lactoferrin bound at 35 °C using 8 successive movements of the cooling zone at a velocity of 0.1mm/s; over half of the recovered protein was eluted in the first peak in more concentrated form than in the feed. Intra-particle diffusion of desorbed protein out of the support pores, and the ratio between the velocities of the cooling

  20. Development of high-productivity, strong cation-exchange adsorbers for protein capture by graft polymerization from membranes with different pore sizes

    PubMed Central

    Chenette, Heather C.S.; Robinson, Julie R.; Hobley, Eboni; Husson, Scott M.

    2012-01-01

    This paper describes the surface modification of macroporous membranes using ATRP (atom transfer radical polymerization) to create cation-exchange adsorbers with high protein binding capacity at high product throughput. The work is motivated by the need for a more economical and rapid capture step in downstream processing of protein therapeutics. Membranes with three reported nominal pore sizes (0.2, 0.45, 1.0 μm) were modified with poly(3-sulfopropyl methacrylate, potassium salt) tentacles, to create a high density of protein binding sites. A special formulation was used in which the monomer was protected by a crown ether to enable surface-initiated ATRP of this cationic polyelectrolyte. Success with modification was supported by chemical analysis using Fourier-transform infrared spectroscopy and indirectly by measurement of pure water flux as a function of polymerization time. Uniformity of modification within the membranes was visualized with confocal laser scanning microscopy. Static and dynamic binding capacities were measured using lysozyme protein to allow comparisons with reported performance data for commercial cation-exchange materials. Dynamic binding capacities were measured for flow rates ranging from 13 to 109 column volumes (CV)/min. Results show that this unique ATRP formulation can be used to fabricate cation-exchange membrane adsorbers with dynamic binding capacities as high as 70 mg/mL at a throughput of 100 CV/min and unprecedented productivity of 300 mg/mL/min. PMID:23175597

  1. A candidate plasma protein classifier to identify Alzheimer's disease.

    PubMed

    Zhao, Xuemei; Lejnine, Serguei; Spond, Jeffrey; Zhang, Chunsheng; Ramaraj, T C; Holder, Daniel J; Dai, Hongyue; Weiner, Russell; Laterza, Omar F

    2015-01-01

    Biomarkers currently used in the aid for the diagnosis of Alzheimer's disease (AD) are cerebrospinal fluid (CSF) protein markers and brain neuroimaging markers. These biomarkers, however, either involve semi-invasive procedures or are costly to measure. Thus, AD biomarkers from more easily accessible body fluids, such as plasma, are very enticing. Using an aptamer-based proteomic technology, we profiled 1,129 plasma proteins of AD patients and non-demented control individuals. A 5-protein classifier for AD identification was constructed in the discovery study with excellent 10-fold cross-validation performance (90.1% sensitivity, 84.2% specificity, 87.9% accuracy, and AUC as 0.94). In an independent validation study, the classifier was applied and correctly predicted AD with 100.0% sensitivity, 80.0% specificity, and 90.0% accuracy, matching or outperforming the CSF Aβ42 and tau biomarkers whose performance were assessed in individual-matched CSF samples obtained at the same visit as plasma sample collection. Moreover, the classifier also correctly predicted mild cognitive impairment, an early pre-dementia state of the disease, with 96.7% sensitivity, 80.0% specificity, and 92.5% accuracy. These studies demonstrate that plasma proteins could be used effectively and accurately to contribute to the clinical diagnosis of AD. Although additional and more diverse cohorts are needed for further validation of the robustness, including the support of postmortem diagnosis, the 5-protein classifier appears to be a promising blood test to contribute diagnosis of AD. PMID:25114072

  2. A candidate plasma protein classifier to identify Alzheimer's disease.

    PubMed

    Zhao, Xuemei; Lejnine, Serguei; Spond, Jeffrey; Zhang, Chunsheng; Ramaraj, T C; Holder, Daniel J; Dai, Hongyue; Weiner, Russell; Laterza, Omar F

    2015-01-01

    Biomarkers currently used in the aid for the diagnosis of Alzheimer's disease (AD) are cerebrospinal fluid (CSF) protein markers and brain neuroimaging markers. These biomarkers, however, either involve semi-invasive procedures or are costly to measure. Thus, AD biomarkers from more easily accessible body fluids, such as plasma, are very enticing. Using an aptamer-based proteomic technology, we profiled 1,129 plasma proteins of AD patients and non-demented control individuals. A 5-protein classifier for AD identification was constructed in the discovery study with excellent 10-fold cross-validation performance (90.1% sensitivity, 84.2% specificity, 87.9% accuracy, and AUC as 0.94). In an independent validation study, the classifier was applied and correctly predicted AD with 100.0% sensitivity, 80.0% specificity, and 90.0% accuracy, matching or outperforming the CSF Aβ42 and tau biomarkers whose performance were assessed in individual-matched CSF samples obtained at the same visit as plasma sample collection. Moreover, the classifier also correctly predicted mild cognitive impairment, an early pre-dementia state of the disease, with 96.7% sensitivity, 80.0% specificity, and 92.5% accuracy. These studies demonstrate that plasma proteins could be used effectively and accurately to contribute to the clinical diagnosis of AD. Although additional and more diverse cohorts are needed for further validation of the robustness, including the support of postmortem diagnosis, the 5-protein classifier appears to be a promising blood test to contribute diagnosis of AD.

  3. Graft copolymer composed of cationic backbone and bottle brush-like side chains as a physically adsorbed coating for protein separation by capillary electrophoresis.

    PubMed

    Zhou, Dan; Xiang, Lina; Zeng, Rongju; Cao, Fuhu; Zhu, Xiaoxi; Wang, Yanmei

    2011-12-01

    To stabilize electroosmotic flow (EOF) and suppress protein adsorption onto the silica capillary inner wall, a cationic hydroxyethylcellulose-graft-poly (poly(ethylene glycol) methyl ether methacrylate) (cat-HEC-g-PPEGMA) graft copolymer composed of cationic backbone and bottle brush-like side chains was synthesized for the first time and used as a novel physically adsorbed coating for protein separation by capillary electrophoresis. Reversed (anodal) and very stable EOF was obtained in cat-HEC-g-PPEGMA-coated capillary at pH 2.2-7.8. The effects of degree of cationization, PEGMA grafting ratio, PEGMA molecular mass, and buffer pH on the separation of basic proteins were investigated. A systematic comparative study of protein separation in bare and HEC-coated capillaries and in cat-HEC-g-PPEGMA-coated capillary was also performed. The basic proteins can be well separated in cat-HEC-g-PPEGMA-coated capillary over the pH range of 2.8-6.8 with good repeatability and high separation efficiency, because the coating combines good protein-resistant property of bottle brush-like PPEGMA side chains with excellent coating ability of cat-HEC backbone. Besides its success in separation of basic proteins, the cat-HEC-g-PPEGMA coating was also superior in the fast separation of other protein samples, such as protein mixture, egg white, and saliva, which indicates that it is a promising coating for further proteomics analysis. PMID:22038787

  4. Analysis of plasma protein adsorption onto DC-Chol-DOPE cationic liposomes by HPLC-CHIP coupled to a Q-TOF mass spectrometer.

    PubMed

    Capriotti, Anna Laura; Caracciolo, Giulio; Caruso, Giuseppe; Cavaliere, Chiara; Pozzi, Daniela; Samperi, Roberto; Laganà, Aldo

    2010-12-01

    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.

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

    PubMed Central

    Wickwire, Kathie; Ho, Emily; Chung, Carolyn S.; King, Janet

    2014-01-01

    Zinc (Zn) deficiency is a problem worldwide. Current methods for assessing Zn status are limited to measuring plasma or serum Zn within populations suspected of deficiency. Despite the high prevalence of Zn deficiency in the human population there are no methods currently available for sensitively assessing Zn status among individuals. The purpose of this research was to utilize a proteomic approach using two-dimensional gel electrophoresis (2DE) and mass spectrometry to identify protein biomarkers that were sensitive to changes in dietary Zn levels in humans. Proteomic analysis was performed in human plasma samples (n = 6) obtained from healthy adult male subjects that completed a dietary Zn depletion/repletion protocol, current dietary zinc intake has a greater effect on fractional zinc absorption than does longer term zinc consumption in healthy adult men. Chung et al. (Am J Clin Nutr 87 (5):1224–1229, 2008). After a 13 day Zn acclimatization period where subjects consumed a Zn-adequate diet, the male subjects consumed a marginal Zn-depleted diet for 42 days followed by consumption of a Zn-repleted diet for 28 days. The samples at baseline, end of depletion and end of repletion were pre-fractionated through immuno-affinity columns to remove 14 highly abundant proteins, and each fraction separated by 2DE. Following staining by colloidal Coomassie blue and densitometric analysis, three proteins were identified by mass spectrometry as affected by changes in dietary Zn. Fibrin β and chain E, fragment double D were observed in the plasma protein fraction that remained bound to the immuno-affinity column. An unnamed protein that was related to immunoglobulins was observed in the immunode-pleted plasma fraction. Fibrin β increased two-fold following the Zn depletion period and decreased to baseline values following the Zn repletion period; this protein may serve as a viable biomarker for Zn status in the future. PMID:23255060

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

    PubMed

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

    2013-02-01

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

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

    PubMed

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

    2013-02-01

    Zinc (Zn) deficiency is a problem world-wide. Current methods for assessing Zn status are limited to measuring plasma or serum Zn within populations suspected of deficiency. Despite the high prevalence of Zn deficiency in the human population there are no methods currently available for sensitively assessing Zn status among individuals. The purpose of this research was to utilize a proteomic approach using two-dimensional gel electrophoresis (2DE) and mass spectrometry to identify protein biomarkers that were sensitive to changes in dietary Zn levels in humans. Proteomic analysis was performed in human plasma samples (n = 6) obtained from healthy adult male subjects that completed a dietary Zn depletion/repletion protocol, current dietary zinc intake has a greater effect on fractional zinc absorption than does longer term zinc consumption in healthy adult men. Chung et al. (Am J Clin Nutr 87 (5):1224-1229, 2008). After a 13 day Zn acclimatization period where subjects consumed a Zn-adequate diet, the male subjects consumed a marginal Zn-depleted diet for 42 days followed by consumption of a Zn-repleted diet for 28 days. The samples at baseline, end of depletion and end of repletion were pre-fractionated through immuno-affinity columns to remove 14 highly abundant proteins, and each fraction separated by 2DE. Following staining by colloidal Coomassie blue and densitometric analysis, three proteins were identified by mass spectrometry as affected by changes in dietary Zn. Fibrin β and chain E, fragment double D were observed in the plasma protein fraction that remained bound to the immunoaffinity column. An unnamed protein that was related to immunoglobulins was observed in the immunodepleted plasma fraction. Fibrin β increased two-fold following the Zn depletion period and decreased to baseline values following the Zn repletion period; this protein may serve as a viable biomarker for Zn status in the future. PMID:23255060

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

    PubMed

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

    1992-05-01

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

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

    PubMed Central

    Lysne, Vegard; Bjørndal, Bodil; Vik, Rita; Nordrehaug, Jan Erik; Skorve, Jon; Nygård, Ottar; Berge, Rolf K.

    2015-01-01

    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. PMID:26053618

  10. Liver takes up retinol-binding protein from plasma

    SciTech Connect

    Gjoen, T.; Bjerkelund, T.; Blomhoff, H.K.; Norum, K.R.; Berg, T.; Blomhoff, R.

    1987-08-15

    Retinol is transported in plasma bound to a specific transport protein, retinol-binding protein. We prepared /sup 125/I-tyramine cellobiose-labeled rat retinol-binding protein and studied its tissue uptake 1, 5, and 24 h after intravenous injection into rats. The liver was the organ containing most radioactivity at all time points studied. After 5 and 24 h, 30 and 22% of the injected dose were recovered in liver, respectively. After separating the liver into parenchymal and nonparenchymal cells in the 5-h group, we found that both cell fractions contained approximately the same amount of radioactivity (per gram of liver). Most of the retinol-binding protein radioactivity in the nonparenchymal cell fraction was in the stellate cells. The implication of these results for a possible transfer mechanism for retinol between parenchymal and stellate cells is discussed.

  11. Smoking, COPD and 3-Nitrotyrosine Levels of Plasma Proteins

    SciTech Connect

    Jin, Hongjun; Webb-Robertson, Bobbie-Jo M.; Peterson, Elena S.; Tan, Ruimin; Bigelow, Diana J.; Scholand, Mary Beth; Hoidal, John R.; Pounds, Joel G.; Zangar, Richard C.

    2011-09-01

    BACKGROUND: Nitric oxide is a physiologically regulator of endothelial function and hemodynamics. Oxidized products of nitric oxide can form nitrotyrosine, which is a marker of nitrative stress. Cigarette smoking decreases exhaled nitric oxide, and the underlying mechanism may be important in the cardiovascular toxicity of cigarette smoke, although it is not clear if this effect results from decreased nitric oxide production or oxidation of nitric oxide to reactive, nitrating, species. These processes would be expected to have opposite effects on nitrotyrosine levels, a marker of nitrative stress. OBJECTIVE: In this study, we determine the effects of smoking and chronic obstructive pulmonary disease (COPD) on circulating levels of nitrotyrosine, and thereby gain insight into the processes regulating nitrotyrosine formation. METHODS: A custom antibody microarray platform was used to analyze the levels of 3-nitrotyrosine modifications on 24 proteins in plasma. Plasma samples from 458 individuals were analyzed. RESULTS: Nitrotyrosine levels in circulating proteins were uniformly reduced in smokers but increased in COPD patients. We also observed a persistent suppression of nitrotyrosine in former smokers. CONCLUSIONS: Smoking broadly suppresses the levels of 3-nitrotyrosine in plasma proteins, suggesting that cigarette smoke suppresses endothelial nitric oxide production. In contrast, the increase in nitrotyrosine levels in COPD patients most likely results from inflammatory processes. This study provides the first evidence that smoking has irreversible effects on endothelial production of nitric oxide, and provides insight into how smoking could induce a loss of elasticity in the vasculature and a long-term increase in the risk of cardiovascular disease.

  12. No effect of cigarette smoking dose on oxidized plasma proteins

    PubMed Central

    Yeh, Chih-Ching; Barr, R. Graham; Powell, Charles A.; Mesia-Vela, Sonia; Wang, Yuanjia; Hamade, Nada K.; Austin, John H.M.; Santella, Regina M.

    2008-01-01

    Cigarette smoking is a major source of oxidative stress. Protein carbonyls have been used as a biomarker of oxidative stress because of the relative stability of carbonylated proteins and the high protein concentration in blood. Increased levels of carbonyl groups have been found in serum proteins of smokers compared to nonsmokers. However, neither the dose effect of current cigarette smoke nor other predictors of oxidative stress have been studied. Hence, we used an ELISA (Enzyme-Linked Immunosorbent Assay) to evaluate plasma protein carbonyls in smokers recruited in the Early Lung Cancer Action Project (ELCAP) program. The lung cancer screening program enrolled current and former smokers age 60 years and over without a prior cancer diagnosis. A total of 542 participants (282 men and 260 women) completed a baseline questionnaire and provided blood samples for the biomarker study. Protein oxidation was measured by derivatization of the carbonyl groups with 2,4-dinitrophenylhydrazine (DNPH) and ELISA quantitation of the DNPH group. Current smoking status was confirmed with urinary cotinine. The mean (± SD) protein carbonyl level was 17.9 ± 2.9 nmol carbonyls/ml plasma. Protein carbonyls did not differ significantly by gender. Carbonyl levels were higher among current than former smokers, but these differences did not attain statistical significance, nor did differences by urine cotinine levels, pack-years, pack/day among current smokers, and smoking duration. In a multiple regression analysis, higher protein carbonyl levels were independently associated with increasing age (0.59 nmol/ml increase per 10 years, 95% CI 0.14, 1.05, p = 0.01), African-American vs. white race/ethnicity, (1.30 nmol/ml, 95% CI 0.4, 2.19, p =0.008), and lower educational attainment (0.75 nmol/ml, 95% CI 0.12, 1.38, p = 0.02). Although we found no significant difference between current versus past cigarette smoking and protein carbonyls in this older group of smokers, associations were

  13. No effect of cigarette smoking dose on oxidized plasma proteins.

    PubMed

    Yeh, Chih-Ching; Barr, R Graham; Powell, Charles A; Mesia-Vela, Sonia; Wang, Yuanjia; Hamade, Nada K; Austin, John H M; Santella, Regina M

    2008-02-01

    Cigarette smoking is a major source of oxidative stress. Protein carbonyls have been used as a biomarker of oxidative stress because of the relative stability of carbonylated proteins and the high protein concentration in blood. Increased levels of carbonyl groups have been found in serum proteins of smokers compared to nonsmokers. However, neither the dose effect of current cigarette smoke nor other predictors of oxidative stress have been studied. Hence, we used an Enzyme-Linked Immunosorbent Assay (ELISA) to evaluate plasma protein carbonyls in smokers recruited in the Early Lung Cancer Action Project (ELCAP) program. The lung cancer screening program enrolled current and former smokers age 60 years and over without a prior cancer diagnosis. A total of 542 participants (282 men and 260 women) completed a baseline questionnaire and provided blood samples for the biomarker study. Protein oxidation was measured by derivatization of the carbonyl groups with 2,4-dinitrophenylhydrazine (DNPH) and ELISA quantitation of the DNPH group. Current smoking status was confirmed with urinary cotinine. The mean (+/-S.D.) protein carbonyl level was 17.9+/-2.9 nmol carbonyl/ml plasma. Protein carbonyls did not differ significantly by gender. Carbonyl levels were higher among current than former smokers, but these differences did not attain statistical significance, nor did differences by urine cotinine levels, pack-years, pack/day among current smokers, and smoking duration. In a multiple regression analysis, higher protein carbonyl levels were independently associated with increasing age (0.59 nmol/ml increase per 10 years, 95% CI 0.14, 1.05, p=0.01), African-American vs. white race/ethnicity, (1.30 nmol/ml, 95% CI 0.4, 2.19, p=0.008), and lower educational attainment (0.75 nmol/ml, 95% CI 0.12, 1.38, p=0.02). Although we found no significant difference between current vs. past cigarette smoking and protein carbonyls in this older group of smokers, associations were found for

  14. Interactions between plasma proteins and naturally occurring polyphenols.

    PubMed

    Li, Min; Hagerman, Ann E

    2013-05-01

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

  15. Biomarkers of Adiponectin: Plasma Protein Variation and Genomic DNA Polymorphisms

    PubMed Central

    Gu, Harvest F.

    2009-01-01

    Adiponectin is secreted by white adipose tissue and exists as the most abundant adipokine in the human plasma. Recent research has indicated that plasma adiponectin levels are inversely correlated with body mass index (BMI) and insulin resistance. Reduction of plasma adiponectin levels is commonly observed in the patients with type 2 diabetes (T2D) and/or in those who are obese in comparison with healthy control individuals. The adiponectin (AdipoQ) gene has a moderate linkage disequilibrium (LD), but two small LD blocks are observed, respectively, in the promoter region and the boundary of exon 2-intron 2. Genetic association studies have demonstrated that single nucleotide polymorphisms (SNPs) +45G15G(T/G) in exon 2 and +276G/T in intron 2 of the AdipoQ gene confer the risk susceptibility to the development of T2D, obesity and diabetic nephropathy (DN). The SNPs in the promoter region, including −11426A/G, −11377C/G and −11391G/A, are found to be associated with T2D and DN. Recent research has indicated that the promoter polymorphisms interfere with the AdipoQ promoter activity. The haplotypes constructed by the promoter polymorphisms and SNP +276G/T in intron 2 are associated with circulating adiponectin levels. This review summarises genetic and pathophysiological relevancies of adiponectin and discusses about the biomarkers of adiponectin plasma protein variation and genomic DNA polymorphisms. PMID:20029651

  16. Altering adsorbed proteins or cellular gene expression in bone-metastatic cancer cells affects PTHrP and Gli2 without altering cell growth.

    PubMed

    Page, Jonathan M; Merkel, Alyssa R; Ruppender, Nazanin S; Guo, Ruijing; Dadwal, Ushashi C; Cannonier, Shellese; Basu, Sandip; Guelcher, Scott A; Sterling, Julie A

    2015-09-01

    The contents of this data in brief are related to the article titled "Matrix Rigidity Regulates the Transition of Tumor Cells to a Bone-Destructive Phenotype through Integrin β3 and TGF-β Receptor Type II". In this DIB we will present our supplemental data investigating Integrin expression, attachment of cells to various adhesion molecules, and changes in gene expression in multiple cancer cell lines. Since the interactions of Integrins with adsorbed matrix proteins are thought to affect the ability of cancer cells to interact with their underlying substrates, we examined the expression of Integrin β1, β3, and β5 in response to matrix rigidity. We found that only Iβ3 increased with increasing substrate modulus. While it was shown that fibronectin greatly affects the expression of tumor-produced factors associated with bone destruction (parathyroid hormone-related protein, PTHrP, and Gli2), poly-l-lysine, vitronectin and type I collagen were also analyzed as potential matrix proteins. Each of the proteins was independently adsorbed on both rigid and compliant polyurethane films which were subsequently used to culture cancer cells. Poly-l-lysine, vitronectin and type I collagen all had negligible effects on PTHrP or Gli2 expression, but fibronectin was shown to have a dose dependent effect. Finally, altering the expression of Iβ3 demonstrated that it is required for tumor cells to respond to the rigidity of the matrix, but does not affect other cell growth or viability. Together these data support the data presented in our manuscript to show that the rigidity of bone drives Integrinβ3/TGF-β crosstalk, leading to increased expression of Gli2 and PTHrP.

  17. [Surface-enhanced Raman spectroscopy of biopolymers: membrane proteins, bacteriorhodopsin and rhodopsin adsorbed on silver electrodes and silver hydrosols].

    PubMed

    Nabiev, I R; Efremov, R G; Chumanov, G D

    1986-01-01

    Surface-enhanced Raman (SER) spectra of purple membranes of Halobacterium halobium and photoreceptor disks of the rod outer segments adsorbed on silver hydrosols were analysed. It has been shown that the intensity of SER spectra of bacterial and visual rhodopsins increases 5 X 10(4) times at adsorption. Concentration relationship of the signal intensity of SER spectra has the maximum at bacteriorhodopsin concentration about 2 X 10(-7) M. It has been shown that adsorption on silver hydrosol leads to fixation of light-induced photochemical transformations in bacterial and visual rhodopsins. Adsorption on the "smooth" electrodes at the potential of the zero charge of silver does not affect the photocycle of bacteriorhodopsin. An increase or decrease of the electrode potential relative to the zero charge point of silver leads to the accumulation of kinetic intermediate K610 and a decrease of the concentration of the form BRh570. It has been shown that on the "smooth" electrode primarily the long-range component of the SER mechanism is realized. Bands corresponding to the vibrations of the atom groups directly contacting with the metal are mainly intensified after redox cycle which increases the concentration of chemosorption centres. A conclusion is drawn that the method of SER spectroscopy of biomolecules adsorbed on "smooth" electrodes, permits obtaining information similar to that obtained from the analysis of Raman spectra of unadsorbed molecules, but at concentrations by two orders less. Adsorption on the electrodes treated with the help of redox cycle permits to obtain highly oriented preparations and to study topography of biopolymers in water solutions and suspensions.

  18. A differential protein solubility approach for the depletion of highly abundant proteins in plasma using ammonium sulfate.

    PubMed

    Bollineni, Ravi Chand; Guldvik, Ingrid J; Grönberg, Henrik; Wiklund, Fredrik; Mills, Ian G; Thiede, Bernd

    2015-12-21

    Depletion of highly abundant proteins is an approved step in blood plasma analysis by mass spectrometry (MS). In this study, we explored a precipitation and differential protein solubility approach as a fractionation strategy for abundant protein removal from plasma. Total proteins from plasma were precipitated with 90% saturated ammonium sulfate, followed by differential solubilization in 55% and 35% saturated ammonium sulfate solutions. Using a four hour liquid chromatography (LC) gradient and an LTQ-Orbitrap XL mass spectrometer, a total of 167 and 224 proteins were identified from the 55% and 35% ammonium sulfate fractions, whereas 235 proteins were found in the remaining protein fractions with at least two unique peptides. SDS-PAGE and exclusive total spectrum counts from LC-MS/MS analyses clearly showed that majority of the abundant plasma proteins were solubilized in 55% and 35% ammonium sulfate solutions, indicating that the remaining protein fraction is of potential interest for identification of less abundant plasma proteins. Serum albumin, serotransferrin, alpha-1-antitrypsin and transthyretin were the abundant proteins that were highly enriched in 55% ammonium sulfate fractions. Immunoglobulins, complement system proteins, and apolipoproteins were among other abundant plasma proteins that were enriched in 35% ammonium sulfate fractions. In the remaining protein fractions a total of 40 unique proteins were identified of which, 32 proteins were identified with at least 10 exclusive spectrum counts. According to PeptideAtlas, 9 of these 32 proteins were estimated to be present at low μg ml(-1) (0.12-1.9 μg ml(-1)) concentrations in the plasma, and 17 at low ng ml(-1) (0.1-55 ng ml(-1)) range.

  19. Determination of chicken and turkey plasma and serum protein concentrations by refractometry and the biuret method.

    PubMed

    Andreasen, C B; Latimer, K S; Kircher, I M; Brown, J

    1989-01-01

    Plasma and serum protein concentrations were determined in chickens and turkeys by refractometry (with human and veterinary refractometers) and by the biuret method. Chicken and turkey serum protein values were significantly lower than respective plasma protein values according to both methods. Refractometer readings for both plasma and serum correlated closely with the results of the biuret test (r2 = 0.72 to 0.97). These findings indicate that plasma and serum protein values may be determined accurately in chickens and turkeys with a handheld refractometer.

  20. Specific Interactions of Neutral Side Chains of an Adsorbed Protein with the Surface of α-Quartz and Silica Gel.

    PubMed

    Odinokov, Alexey V; Bagaturyants, Alexander A

    2015-07-16

    Many key features of the protein adsorption on the silica surfaces still remain unraveled. One of the open questions is the interaction of nonpolar side chains with siloxane cavities. Here, we use nonequilibrium molecular dynamics simulations for the detailed investigation of the binding of several hydrophobic and amphiphilic protein side chains with silica surface. These interactions were found to be a possible driving force for protein adsorption. The free energy gain was larger for the disordered surface of amorphous silica gel as compared to α-quartz, but the impact depended on the type of amino acid. The dependence was analyzed from the structural point of view. For every amino acid an enthalpy-entropy compensation behavior was observed. These results confirm a hypothesis of an essential role of hydrophobic interactions in protein unfolding and irreversible adsorption on the silica surface.

  1. Properties of proteins binding plasma progesterone in pregnant Cape porcupines (Hystrix africaeaustralis).

    PubMed

    Louw, A I; van Wyk, V; van Aarde, R J

    1992-09-01

    The properties of progesterone-binding proteins in plasma of pregnant Cape porcupines were investigated using radiolabelled progesterone and either progesterone or cortisol as competing ligands as well as native plasma and heated (60 degrees C for 30 min) plasma. The results demonstrated that plasma from pregnant porcupines contains corticosteroid-binding globulin, but that it constitutes a significant portion of plasma progesterone-binding proteins only during the early stages of pregnancy. Corticosteroid-binding globulin of porcupines appears to be as heat labile as that of guinea-pigs. Concentrations of progesterone-binding proteins in plasma increased during pregnancy to reach concentrations at the eleventh week that were 25 times higher than those of progesterone; concentrations increased significantly (r2 = 0.88) with the increase in progesterone concentration. The results indicate that plasma progesterone-binding proteins in Cape porcupines (Old World hystricomorph) are similar in composition to those in guinea-pigs (New World hystricomorph).

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

    PubMed Central

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

    2014-01-01

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

  3. Protein-Centric N-Glycoproteomics Analysis of Membrane and Plasma Membrane Proteins

    PubMed Central

    2015-01-01

    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. PMID:24754784

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

    PubMed

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

    2015-06-01

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

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

    NASA Technical Reports Server (NTRS)

    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

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

    SciTech Connect

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

    1988-01-01

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

  7. Influence of surface chemistry on the structural organization of monomolecular protein layers adsorbed to functionalized aqueous interfaces.

    PubMed Central

    Lösche, M; Piepenstock, M; Diederich, A; Grünewald, T; Kjaer, K; Vaknin, D

    1993-01-01

    The molecular organization of streptavidin (SA) bound to aqueous surface monolayers of biotin-functionalized lipids and binary lipid mixtures has been investigated with neutron reflectivity and electron and fluorescence microscopy. The substitution of deuterons (2H) for protons (1H), both in subphase water molecules and in the alkyl chains of the lipid surface monolayer, was utilized to determine the interface structure on the molecular length scale. In all cases studied, the protein forms monomolecular layers underneath the interface with thickness values of approximately 40 A. A systematic dependence of the structural properties of such self-assembled SA monolayers on the surface chemistry was observed: the lateral protein density depends on the length of the spacer connecting the biotin moiety and its hydrophobic anchor. The hydration of the lipid head groups in the protein-bound state depends on the dipole moment density at the interface. Images FIGURE 1 FIGURE 2 FIGURE 3 FIGURE 5 FIGURE 11 FIGURE 12 FIGURE A1 PMID:8298041

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

    NASA Astrophysics Data System (ADS)

    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.

  9. Adsorption of peptides and small proteins with control access polymer permeation to affinity binding sites. Part I: Polymer permeation-immobilized metal ion affinity chromatography separation adsorbents with polyethylene glycol and immobilized metal ions.

    PubMed

    González-Ortega, Omar; Porath, Jerker; Guzmán, Roberto

    2012-03-01

    Despite the many efforts to develop efficient protein purification techniques, the isolation of peptides and small proteins on a larger than analytical scale remains a significant challenge. Recovery of small biomolecules from diluted complex biological mixtures, such as human serum, employing porous adsorbents is a difficult task mainly due to the presence of concentrated large biomolecules that can add undesired effects in the system such as blocking of adsorbent pores, impairing diffusion of small molecules, or competition for adsorption sites. Adsorption and size exclusion chromatography (AdSEC) controlled access media, using polyethylene glycol (PEG) as a semi-permeable barrier on a polysaccharide matrix, have been developed and explored in this work to overcome such effects and to preferentially adsorb small molecules while rejecting large ones. In the first part of this work, adsorption studies were performed with small peptides and proteins from synthetic mixtures using controlled access polymer permeation adsorption (CAPPA) media created by effectively grafting PEG on an immobilized metal affinity chromatography (IMAC) agarose resin, where chelating agents and immobilized metal ions were used as the primary affinity binding sites. Synthetic mixtures consisted of bovine serum albumin (BSA) with small proteins, peptides, amino acids (such as histidine or Val⁴-Angiotensin III), and small molecules-spiked human serum. The synthesized hybrid adsorbent consisted of agarose beads modified with iminodiacetic (IDA) groups, loaded with immobilized Cu(II) ions, and PEG. These CAPPA media with grafted PEG on the interior and exterior surfaces of the agarose matrix were effective in rejecting high molecular weight proteins. Different PEG grafting densities and PEG of different molecular weight were tested to determine their effect in rejecting and controlling adsorbent permeation properties. Low grafting density of high molecular weight PEG was found to be as

  10. Analyte induced water adsorbability in gas phase biosensors: the influence of ethinylestradiol on the water binding protein capacity.

    PubMed

    Snopok, Borys; Kruglenko, Ivanna

    2015-05-01

    An ultra-sensitive gas phase biosensor/tracer/bio-sniffer is an emerging technology platform designed to provide real-time information on air-borne analytes, or those in liquids, through classical headspace analysis. The desired bio-sniffer measures gaseous 17α- ethinylestradiol (ETED) as frequency changes on a quartz crystal microbalance (QCM), which is a result of the interactions of liquid sample components in the headspace (ETED and water) with a biorecognition layer. The latter was constructed by immobilization of polyclonal antiserum against a phenolic A-ring of estrogenic receptors through protein A. The QCM response exhibited stretched exponential kinetics of negative frequency shifts with reversible and "irreversible" components of mass uptake onto the sensor surface in static headspace conditions when exposed to water solutions of ETED over the sensor working range, from 10(-10) to 10(-17) g L(-1). It was shown that the variations in the QCM response characteristics are due to the change of the water-binding capacity of the sensing layer induced by protein transformations initiated by the binding of ETED molecules. This result is well correlated with the natural physiological function of estrogens in controlling the homeostasis of body fluids in living beings. PMID:25763411

  11. Plasma binding proteins as mediators of corticosteroid action in vertebrates.

    PubMed

    Breuner, C W; Orchinik, M

    2002-10-01

    Stressors elicit a complex but variable suite of endocrine events. Comparative studies of the stress response have focused primarily on the adrenocortical response to stress, in particular the measurement of plasma levels of glucocorticoids. However, a number of other factors contribute to and modify cellular and organismal responses to glucocorticoids. Notably, plasma corticosteroid binding globulins (CBGs) can regulate the general availability of steroid to tissues, and/or direct the delivery of hormones to specific sites. In this paper, we discuss possible functions of CBG and mechanisms of CBG action, review CBG characteristics among vertebrates, and discuss our recent studies indicating that CBG may indeed modulate responses to stressors. For example, in house sparrows, we found that basal and stress-induced concentrations of total corticosteroid (cortisol or corticosterone) (CORT) vary seasonally, but CBG concentrations change proportionally, so that free CORT concentrations appear static year-round. In contrast, in white-crowned sparrows and tree lizards, CBG concentrations change under conditions when total CORT levels do not, resulting in significant changes in circulating free CORT. These differences in free CORT are masked if CBG is not accounted for. We have also found that the binding properties of CBG vary considerably between species and need to be determined empirically. Such studies led to the observation that CBG in several species may also serve as a functional androgen binding protein; this is especially important for birds, because previous studies had concluded that birds lack androgen binding globulins. We propose that consideration of CBG is paramount to understanding the role of glucocorticoids in mediating behavioral and physiological responses to stress.

  12. Protein binding of prilocaine in human plasma: influence of concentration, pH and temperature.

    PubMed

    Bachmann, B; Biscoping, J; Sinning, E; Hempelmann, G

    1990-05-01

    Protein binding of prilocaine was investigated in vitro under various conditions of changing pH, temperature and total plasma concentration by means of HPLC with UV-detection and ultrafiltration. Whereas changes in temperature (25 degrees C-40 degrees C) and pH (pH 5-pH 10) influenced protein binding markedly, rising plasma concentrations up to 16 micrograms/ml did not affect plasma protein binding significantly. This may be a possible explanation for clinical evidence of low toxicity associated with the use of prilocaine. Discussions concerning protein binding of local anaesthetics should always be based on defined ambient conditions (pH, temperature, concentration).

  13. Treatment Characteristics of Second Order Structure of Proteins Using Low-Pressure Oxygen RF Plasma

    NASA Astrophysics Data System (ADS)

    Hayashi, Nobuya; Nakahigashi, Akari; Kawaguchi, Ryutaro; Goto, Masaaki

    2010-10-01

    Removal of proteins from the surface of medical equipments is attempted using oxygen plasma produced by RF discharge. FTIR spectra indicate that the bonding of C-H and N-H in the casein protein is reduced after irradiation of oxygen plasma. Also, the second order structure of a protein such as α-helix and β-sheet are modified by the oxygen plasma. Complete removal of casein protein with the concentration of 0.016 mg/cm2 that is equivalent to remnants on the medical equipment requires two hours avoiding the damage to medical equipments.

  14. Treatment Characteristics of Second Order Structure of Proteins Using Low-Pressure Oxygen RF Plasma

    SciTech Connect

    Hayashi, Nobuya; Nakahigashi, Akari; Kawaguchi, Ryutaro; Goto, Masaaki

    2010-10-13

    Removal of proteins from the surface of medical equipments is attempted using oxygen plasma produced by RF discharge. FTIR spectra indicate that the bonding of C-H and N-H in the casein protein is reduced after irradiation of oxygen plasma. Also, the second order structure of a protein such as {alpha}-helix and {beta}-sheet are modified by the oxygen plasma. Complete removal of casein protein with the concentration of 0.016 mg/cm{sup 2} that is equivalent to remnants on the medical equipment requires two hours avoiding the damage to medical equipments.

  15. Extraction of methocarbamol from human plasma with a polypyrrole/multiwalled carbon nanotubes composite decorated with magnetic nanoparticles as an adsorbent followed by electrospray ionization ion mobility spectrometry detection.

    PubMed

    Saraji, Mohammad; Khayamian, Taghi; Hashemian, Zahra

    2014-12-01

    In this work, a polypyrrole/multiwalled carbon nanotubes composite decorated with Fe3 O4 nanoparticles was chemically synthesized and applied as a novel adsorbent for the extraction of methocarbamol from human plasma. Electrospray ionization ion mobility spectrometry was used for the determination of the analyte. The properties of the magnetic-modified adsorbent were characterized by scanning electron microscopy, transmission electron microscopy, Fourier transform IR spectroscopy, and X-ray diffraction. The effects of experimental parameters on the extraction efficiency of the sorbent were investigated. Under the optimized conditions, the linear dynamic range was found to be 2-150 ng/mL with the detection limit of 0.9 ng/mL. The relative standard deviation was 5.3% for three replicate measurements of methocarbamol in plasma sample. The extraction efficiency of the sorbent for the determination of different drugs with various polarities was also compared to that of Fe3 O4 -polypyrrole and Fe3 O4 -multiwalled carbon nanotubes sorbents. Finally, the method was used for the determination of methocarbamol in blood samples. PMID:25243817

  16. Extraction of methocarbamol from human plasma with a polypyrrole/multiwalled carbon nanotubes composite decorated with magnetic nanoparticles as an adsorbent followed by electrospray ionization ion mobility spectrometry detection.

    PubMed

    Saraji, Mohammad; Khayamian, Taghi; Hashemian, Zahra

    2014-12-01

    In this work, a polypyrrole/multiwalled carbon nanotubes composite decorated with Fe3 O4 nanoparticles was chemically synthesized and applied as a novel adsorbent for the extraction of methocarbamol from human plasma. Electrospray ionization ion mobility spectrometry was used for the determination of the analyte. The properties of the magnetic-modified adsorbent were characterized by scanning electron microscopy, transmission electron microscopy, Fourier transform IR spectroscopy, and X-ray diffraction. The effects of experimental parameters on the extraction efficiency of the sorbent were investigated. Under the optimized conditions, the linear dynamic range was found to be 2-150 ng/mL with the detection limit of 0.9 ng/mL. The relative standard deviation was 5.3% for three replicate measurements of methocarbamol in plasma sample. The extraction efficiency of the sorbent for the determination of different drugs with various polarities was also compared to that of Fe3 O4 -polypyrrole and Fe3 O4 -multiwalled carbon nanotubes sorbents. Finally, the method was used for the determination of methocarbamol in blood samples.

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

    PubMed

    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.

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

    NASA Astrophysics Data System (ADS)

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

    2014-06-01

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

  19. Analysis of the induction of the myelin basic protein binding to the plasma membrane phospholipid monolayer

    NASA Astrophysics Data System (ADS)

    Zhang, Lei; Hao, Changchun; Feng, Ying; Gao, Feng; Lu, Xiaolong; Li, Junhua; Sun, Runguang

    2016-09-01

    Myelin basic protein (MBP) is an essential structure involved in the generation of central nervous system (CNS) myelin. Myelin shape has been described as liquid crystal structure of biological membrane. The interactions of MBP with monolayers of different lipid compositions are responsible for the multi-lamellar structure and stability of myelin. In this paper, we have designed MBP-incorporated model lipid monolayers and studied the phase behavior of MBP adsorbed on the plasma membrane at the air/water interface by thermodynamic method and atomic force microscopy (AFM). By analyzing the pressure-area (π-A) and pressure-time (π-T) isotherms, univariate linear regression equation was obtained. In addition, the elastic modulus, surface pressure increase, maximal insertion pressure, and synergy factor of monolayers were detected. These parameters can be used to modulate the monolayers binding of protein, and the results show that MBP has the strongest affinity for 1,2-dipalmitoyl-sn-glycero-3- phosphoserine (DPPS) monolayer, followed by DPPC/DPPS mixed and 1,2-dipalmitoyl-sn-glycero-3-phospho-choline (DPPC) monolayers via electrostatic and hydrophobic interactions. AFM images of DPPS and DPPC/DPPS mixed monolayers in the presence of MBP (5 nM) show a phase separation texture at the surface pressure of 20 mN/m and the incorporation of MBP put into the DPPC monolayers has exerted a significant effect on the domain structure. MBP is not an integral membrane protein but, due to its positive charge, interacts with the lipid head groups and stabilizes the membranes. The interaction between MBP and phospholipid membrane to determine the nervous system of the disease has a good biophysical significance and medical value. Project supported by the National Natural Science Foundation of China (Grant Nos. 21402114 and 11544009), the Natural Science Basic Research Plan in Shaanxi Province of China (Grant No. 2016JM2010), the Fundamental Research Funds for the Central

  20. Analysis of the induction of the myelin basic protein binding to the plasma membrane phospholipid monolayer

    NASA Astrophysics Data System (ADS)

    Zhang, Lei; Hao, Changchun; Feng, Ying; Gao, Feng; Lu, Xiaolong; Li, Junhua; Sun, Runguang

    2016-09-01

    Myelin basic protein (MBP) is an essential structure involved in the generation of central nervous system (CNS) myelin. Myelin shape has been described as liquid crystal structure of biological membrane. The interactions of MBP with monolayers of different lipid compositions are responsible for the multi-lamellar structure and stability of myelin. In this paper, we have designed MBP-incorporated model lipid monolayers and studied the phase behavior of MBP adsorbed on the plasma membrane at the air/water interface by thermodynamic method and atomic force microscopy (AFM). By analyzing the pressure–area (π–A) and pressure–time (π–T) isotherms, univariate linear regression equation was obtained. In addition, the elastic modulus, surface pressure increase, maximal insertion pressure, and synergy factor of monolayers were detected. These parameters can be used to modulate the monolayers binding of protein, and the results show that MBP has the strongest affinity for 1,2-dipalmitoyl-sn-glycero-3- phosphoserine (DPPS) monolayer, followed by DPPC/DPPS mixed and 1,2-dipalmitoyl-sn-glycero-3-phospho-choline (DPPC) monolayers via electrostatic and hydrophobic interactions. AFM images of DPPS and DPPC/DPPS mixed monolayers in the presence of MBP (5 nM) show a phase separation texture at the surface pressure of 20 mN/m and the incorporation of MBP put into the DPPC monolayers has exerted a significant effect on the domain structure. MBP is not an integral membrane protein but, due to its positive charge, interacts with the lipid head groups and stabilizes the membranes. The interaction between MBP and phospholipid membrane to determine the nervous system of the disease has a good biophysical significance and medical value. Project supported by the National Natural Science Foundation of China (Grant Nos. 21402114 and 11544009), the Natural Science Basic Research Plan in Shaanxi Province of China (Grant No. 2016JM2010), the Fundamental Research Funds for the Central

  1. Enrichment of plasma membrane proteins using nanoparticle pellicles: comparison between silica and higher density nanoparticles.

    PubMed

    Choksawangkarn, Waeowalee; Kim, Sung-Kyoung; Cannon, Joe R; Edwards, Nathan J; Lee, Sang Bok; Fenselau, Catherine

    2013-03-01

    Proteomic and other characterization of plasma membrane proteins is made difficult by their low abundance, hydrophobicity, frequent carboxylation, and dynamic population. We and others have proposed that underrepresentation in LC-MS/MS analysis can be partially compensated by enriching the plasma membrane and its proteins using cationic nanoparticle pellicles. The nanoparticles increase the density of plasma membrane sheets and thus enhance separation by centrifugation from other lysed cellular components. Herein, we test the hypothesis that the use of nanoparticles with increased densities can provide enhanced enrichment of plasma membrane proteins for proteomic analysis. Multiple myeloma cells were grown and coated in suspension with three different pellicles of three different densities and both pellicle coated and uncoated suspensions analyzed by high-throughput LC-MS/MS. Enrichment was evaluated by the total number and the spectral counts of identified plasma membrane proteins.

  2. Continuous Reduction of Protein-Bound Uraemic Toxins with Improved Oxidative Stress by Using the Oral Charcoal Adsorbent AST-120 in Haemodialysis Patients.

    PubMed

    Yamamoto, Suguru; Kazama, Junichiro J; Omori, Kentaro; Matsuo, Koji; Takahashi, Yoshimitsu; Kawamura, Kazuko; Matsuto, Takayuki; Watanabe, Hiroshi; Maruyama, Toru; Narita, Ichiei

    2015-09-23

    Accumulation of protein-bound uraemic toxins (PBUTs) is one of the reasons for the development of uraemia-related complications including cardiovascular disease; however, conventional haemodialysis is limited in its ability to remove PBUTs. We aimed to examine whether the oral charcoal adsorbent AST-120 has an additive effect on PBUT removal in haemodialysis patients. During the 4-week study, anuric patients undergoing haemodialysis received AST-120 (6 g/day) in the last 2 weeks (n = 10) or the first 2 weeks (n = 10). Serum levels of total and free PBUTs such as indoxyl sulfate, p-cresyl sulfate, and phenyl sulfate at the pre- and postdialysis sessions were measured before and after AST-120 use and after discontinuation. Levels of the oxidative stress markers oxidized albumin and 8-isoprostane were also measured. AST-120 use induced dramatic reduction of indoxyl sulfate (total, 45.7% [33.2-50.5%]; free, 70.4% [44.8-79.8%]), p-cresyl sulfate (total, 31.1% [25.0-48.0%]; free, 63.5% [49.3-70.9%]), and phenyl sulfate (free, 50.6% [32.3-71.2%]) levels; however, this effect disappeared after the discontinuation of AST-120. AST-120 use also induced substantial reduction of the oxidized albumin and 8-isoprostane levels. In conclusion, oral administration of AST-120 had additive effects on the continuous reduction of some PBUTs in anuric patients undergoing haemodialysis.

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

    NASA Technical Reports Server (NTRS)

    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

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

    NASA Astrophysics Data System (ADS)

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

    2015-09-01

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

  5. The uranium from seawater program at PNNL: Overview of marine testing, adsorbent characterization, adsorbent durability, adsorbent toxicity, and deployment studies

    DOE PAGES

    Gill, Gary A.; Kuo, Li -Jung; Janke, Christopher James; Park, Jiyeon; Jeters, Robert T.; Bonheyo, George T.; Pan, Horng -Bin; Wai, Chien; Khangaonkar, Tarang P.; Bianucci, Laura; et al

    2016-02-07

    The Pacific Northwest National Laboratory's (PNNL) Marine Science Laboratory (MSL) located along the coast of Washington State is evaluating the performance of uranium adsorption materials being developed for seawater extraction under realistic marine conditions with natural seawater. Two types of exposure systems were employed in this program: flow-through columns for testing of fixed beds of individual fibers and pellets and a recirculating water flume for testing of braided adsorbent material. Testing consists of measurements of the adsorption of uranium and other elements from seawater as a function of time, typically 42 to 56 day exposures, to determine the adsorbent capacitymore » and adsorption rate (kinetics). Analysis of uranium and other trace elements collected by the adsorbents was conducted following strong acid digestion of the adsorbent with 50% aqua regia using either Inductively Coupled Plasma Optical Emission Spectrometry (ICP-OES) or Inductively Coupled Plasma Mass Spectrometer (ICP-MS). The ORNL 38H adsorbent had a 56 day adsorption capacity of 3.30 ± 0.68 g U/ kg adsorbent (normalized to a salinity of 35 psu), a saturation adsorption capacity of 4.89 ± 0.83 g U/kg of adsorbent material (normalized to a salinity of 35 psu) and a half-saturation time of 28 10 days. The AF1 adsorbent material had a 56 day adsorption capacity of 3.9 ± 0.2 g U/kg adsorbent material (normalized to a salinity of 35 psu), a saturation capacity of 5.4 ± 0.2 g U/kg adsorbent material (normalized to a salinity of 35 psu) and a half saturation time of 23 2 days. The ORNL amidoxime-based adsorbent materials are not specific for uranium, but also adsorb other elements from seawater. The major doubly charged cations in seawater (Ca and Mg) account for a majority of the cations adsorbed (61% by mass and 74% by molar percent). For the ORNL AF1 adsorbent material, U is the 4th most abundant element adsorbed by mass and 7th most abundant by molar percentage. Marine testing

  6. Plasmolysis, red blood cell partitioning, and plasma protein binding of etofibrate, clofibrate, and their degradation products.

    PubMed

    Altmayer, P; Garrett, E R

    1983-11-01

    Etofibrate (I), the ethylene glycol diester of clofibric and nicotinic acids, degrades almost equally through both half-esters with half-lives of approximately 10 and 1 min in fresh dog and human plasma, respectively. The nicotinate V degrades with half-lives of approximately 12 hr and 50 min in fresh dog and human plasma, respectively. Ester III and clofibrate VI degrade by saturable Michaelis-Menten kinetics in fresh human plasma, with similar maximum initial rates and respective terminal first-order half-lives of 12 and 26 min. Tetraethyl pyrophosphate at 100 micrograms/ml inhibited human plasma and red blood cell esterases permitting plasma protein binding and red blood cell partitioning studies. The red blood cell-plasma water partition coefficient was 5.4 for 0.2-80 micrograms/ml of I. Clofibrate (VI) showed a saturable erythrocyte partitioning that decreased from 7.8 (10 micrograms/ml) to 1 (50 micrograms/ml). The strong binding of I and VI to ultrafiltration membranes necessitated the determination of their plasma protein binding by the method of variable plasma concentrations of erythrocyte suspensions to give 96.6% (0.2-80 micrograms/ml) and 98.2% (13.6-108.4 micrograms/ml) binding, respectively. Methods for the determination of the parameters of saturable and nonsaturable plasma protein binding for unstable and membrane-binding drugs by the method of variable plasma concentrations in partitioning erythrocyte suspensions are presented.

  7. A Multi-technique Characterization of Adsorbed Protein Films: Orientation and Structure by ToF-SIMS, NEXAFS, SFG, and XPS

    NASA Astrophysics Data System (ADS)

    Baio, Joseph E.

    immobilization schemes. This protein contained both a hexahistidine tag and a cysteine residue, introduced at opposite ends of the HuLys Fv, for immobilization onto nitrilotriacetic acid (NTA) and maleimide oligo- (ethylene glycol) (MEG)-terminated substrates. The thiol group on the cysteine residue selectively binds to the MEG groups, while the his-tag selectively binds to the Ni-loaded NTA groups. XPS was used to monitor protein coverage on both surfaces by following the change in the nitrogen atomic %. The ToF-SIMS data provided a clear differentiation between the two samples due to the intensity differences of secondary ions originating from asymmetrically located amino acids in HuLys Fv. Indicating that the HuLys Fv fragment when adsorbed into the NTA and MEG substrates will be induced into two different orientations. On the NTA substrate the protein's binding site is accessible, while on the MEG substrate the binding site is oriented towards the surface. By taking advantage of the electron pathway through the heme group in cytochrome c (CytoC) electrochemists have built sensors based upon CytoC immobilized onto functionalized metal electrodes. When immobilized onto a charged surface, CytoC, with its distribution of lysine and glutamate residues around its surface, should orient and form a well-ordered protein film. Here a detailed examination of CytoC orientation when electrostatically immobilized onto both amine (NH 3+) and carboxyl (COO-) functionalized gold is presented. Again, protein coverage, on both surfaces, was monitored by the change in the atomic % N, as determined by XPS. ToF-SIMS data demonstrated a clear separation between the two samples based on the intensity differences of secondary ions stemming from amino acids located asymmetrically within CytoC, indicating opposite orientations of the protein on the two different surfaces. Spectral features within the in situ sum frequency generation vibrational spectra, acquired for the protein interacting with

  8. Modeling adsorption: Investigating adsorbate and adsorbent properties

    NASA Astrophysics Data System (ADS)

    Webster, Charles Edwin

    1999-12-01

    Surface catalyzed reactions play a major role in current chemical production technology. Currently, 90% of all chemicals are produced by heterogeneously catalyzed reactions. Most of these catalyzed reactions involve adsorption, concentrating the substrate(s) (the adsorbate) on the surface of the solid (the adsorbent). Pore volumes, accessible surface areas, and the thermodynamics of adsorption are essential in the understanding of solid surface characteristics fundamental to catalyst and adsorbent screening and selection. Molecular properties such as molecular volumes and projected molecular areas are needed in order to convert moles adsorbed to surface volumes and areas. Generally, these molecular properties have been estimated from bulk properties, but many assumptions are required. As a result, different literature values are employed for these essential molecular properties. Calculated molar volumes and excluded molecular areas are determined and tabulated for a variety of molecules. Molecular dimensions of molecules are important in the understanding of molecular exclusion as well as size and shape selectivity, diffusion, and adsorbent selection. Molecular dimensions can also be used in the determination of the effective catalytic pore size of a catalyst. Adsorption isotherms, on zeolites, (crystalline mineral oxides) and amorphous solids, can be analyzed with the Multiple Equilibrium Analysis (MEA) description of adsorption. The MEA produces equilibrium constants (Ki), capacities (ni), and thermodynamic parameters (enthalpies, ΔHi, and entropies, ΔSi) of adsorption for each process. Pore volumes and accessible surface areas are calculated from the process capacities. Adsorption isotherms can also be predicted for existing and new adsorbate-adsorbent systems with the MEA. The results show that MEA has the potential of becoming a standard characterization method for microporous solids that will lead to an increased understanding of their behavior in gas

  9. Increased levels of hyper-stable protein aggregates in plasma of older adults.

    PubMed

    Xia, Ke; Trasatti, Hannah; Wymer, James P; Colón, Wilfredo

    2016-06-01

    Proteins that misfold into hyper-stable/degradation-resistant species during aging may accumulate and disrupt protein homeostasis (i.e., proteostasis), thereby posing a survival risk to any organism. Using the method diagonal two-dimensional (D2D) SDS-PAGE, which separates hyper-stable SDS-resistant proteins at a proteomics level, we analyzed the plasma of healthy young (<30 years) and older (60-80 years) adults. We discovered the presence of soluble SDS-resistant protein aggregates in the plasma of older adults, but found significantly lower levels in the plasma of young adults. We identified the inflammation-related chaperone protein haptoglobin as the main component of the hyper-stable aggregates. This observation is consistent with the growing link between accumulations of protein aggregates and aging across many organisms. It is plausible higher amounts of SDS-resistant protein aggregates in the plasma of older adults may reflect a compromise in proteostasis that may potentially indicate cellular aging and/or disease risk. The results of this study have implications for further understanding the link between aging and the accumulation of protein aggregates, as well as potential for the development of aging-related biomarkers. More broadly, this novel application of D2D SDS-PAGE may be used to identify, quantify, and characterize the degradation-resistant protein aggregates in human plasma or any biological system. PMID:27179971

  10. The impact of the concentration of drug binding plasma proteins on drug distribution according to Øie-Tozer's model.

    PubMed

    Svennebring, Andreas Mats

    2016-01-01

    1. New equations have been developed from an updated version of Øie-Tozer's model expressing how the free concentration and volume of distribution change in relation to changes in the concentration of drug binding plasma proteins. This updated model accommodates more than one drug binding plasma protein to contribute to the plasma protein binding. 2. Demonstrations of the model show that variability in the concentration of one plasma protein has considerably less impact on the free drug concentration and volume of distribution if other plasma proteins contribute to binding, than if they don't.

  11. Synthesis and secretion of plasma proteins by embryonic chick hepatocytes: changing patterns during the first three days of culture

    PubMed Central

    1978-01-01

    A simple model system is described for studying synthesis of plasma proteins. The system is based on chick embryo hepatocytes in primary monolayer culture which synthesize a broad spectrum of plasma proteins and secrete them into the culture medium. The secreted proteins are stable and consist almost exclusively of plasma proteins. The cultured cells are nonproliferating hepatic parenchymal cells whose cell mass remains constant in culture. By a modification of Laurell's rocket immunoelectrophoresis, the secreted plasma proteins can be detected in nanogram amounts in 3 microliter of unconcentrated culture medium. Kinetics of secretion are obtained by sequential assay of proteins accumulating in the medium. In this system it is demonstrated that: (a) intracellular plasma protein levels are equivalent to less than 5% of the daily secretion; (b) synthesis and secretion are continuous; and (c) the overall half-time for plasma protein movement along the secretory pathway is less than 10 min. From these results, it follows that the rate at which the plasma proteins are secreted gives a valid estimate of their rate of synthesis. This feature of the culture and the sensitivity of the assay allow routine measurements of plasma protein synthesis without disruption of the cells and without the use of radioisotopes. It is shown, furthermore, that the overall rate of plasma protein synthesis in cultured hepatocytes is constant over a 3- day period and is similar to that of the intact liver. 3,000,000 cells, containing 1 mg cell protein, synthesize 0.2 mg of plasma proteins daily, amounting to one-fifth of hepatocellular protein synthesis. Under the conditions used, albumin synthesis steadily decreases with culture time whereas the synthesis of many other plasma proteins increases. The observed phenotypic changes and reorganization of plasma protein synthesis illustrate how the system may be exploited for studying the regulatory processes governing plasma protein synthesis. PMID

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  13. Prednisolone-induced predisposition to femoral head separation and the accompanying plasma protein changes in chickens

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Femoral head separation (FHS) is an idiopathic bone problem that causes lameness and production losses in commercial poultry. In a model of prednisolone induced susceptibility to FHS, the changes in plasma proteins and peptides were analyzed to find possible biomarkers. Plasma from control and FHS-s...

  14. The Relationship of Novel Plasma Proteins in the Early Neonatal Period With Retinopathy of Prematurity

    PubMed Central

    Lynch, Anne M.; Wagner, Brandie D.; Mandava, Naresh; Palestine, Alan G.; Mourani, Peter M.; McCourt, Emily A.; Oliver, Scott C. N.; Abman, Steven H.

    2016-01-01

    Purpose Retinopathy of prematurity (ROP) is a vision-threatening disease associated with abnormal retinal vascular development. Proteins from the insulin-like growth factor pathway are related to ROP. However, there is a paucity of research on the role of other proteins in ROP. The aim of this study was to identify plasma proteins related to clinically significant ROP. Methods We measured 1121 plasma proteins in the early neonatal period in infants at risk for ROP using an aptamer-based proteomic technology. The primary aim of the study was to compare plasma protein concentrations in infants who did (n = 12) and did not (n = 23) subsequently develop clinically significant ROP using logistic regression. As a secondary aim, we examined patterns in the proteins across categories of clinically significant, low-grade, and no ROP groups. Results Lower levels of 16 proteins were associated with an increased risk of clinically significant ROP. In this group, superoxide dismutase (Mn), mitochondrial (MnSOD), and chordin-like protein 1 (CRDL1) were highly ranked. Other proteins in this group included: C-C motif chemokine 14 (HCC-1), prolactin, insulin-like growth factor-binding protein 7 (IGFBP-7), and eotaxin. Higher levels of 12 proteins were associated with a higher risk for ROP. Fibroblast growth factor 19 (FGF-19) was the top-ranked protein target followed by hepatocyte growth factor-like protein (MSP), luteinizing hormone (LH), cystatin M, plasminogen, and proprotein convertase subtilisin/kexin type 9 (PCSK9). We also noted different patterns in the trend of concentrations of proteins across the clinically significant, low-grade, and no ROP groups. Conclusions We discovered plasma proteins with novel associations with clinically significant ROP (MnSOD, CRDL1, PCSK9), proteins with links to established ROP signaling pathways (IGFBP-7), and proteins such as MnSOD that may be a target for future therapeutic interventions. PMID:27679852

  15. Immunoprecipitation of Plasma Membrane Receptor-Like Kinases for Identification of Phosphorylation Sites and Associated Proteins.

    PubMed

    Kadota, Yasuhiro; Macho, Alberto P; Zipfel, Cyril

    2016-01-01

    Membrane proteins are difficult to study for numerous reasons. The surface of membrane proteins is relatively hydrophobic and sometimes very unstable, additionally requiring detergents for their extraction from the membrane. This leads to challenges at all levels, including expression, solubilization, purification, identification of associated proteins, and the identification of post-translational modifications. However, recent advances in immunoprecipitation technology allow to isolate membrane proteins efficiently, facilitating the study of protein-protein interactions, the identification of novel associated proteins, and to identify post-translational modifications, such as phosphorylation. Here, we describe an optimized immunoprecipitation protocol for plant plasma membrane receptor-like kinases. PMID:26577786

  16. Blimp-1 controls plasma cell function through regulation of immunoglobulin secretion and the unfolded protein response

    PubMed Central

    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

  17. Controlling protein adsorption on graphene for cryo-EM using low-energy hydrogen plasmas.

    PubMed

    Russo, Christopher J; Passmore, Lori A

    2014-06-01

    Despite its many favorable properties as a sample support for biological electron microscopy, graphene is not widely used because its hydrophobicity precludes reliable protein deposition. We describe a method to modify graphene with a low-energy hydrogen plasma, which reduces hydrophobicity without degrading the graphene lattice. Use of plasma-treated graphene enables better control of protein distribution in ice for electron cryo-microscopy and improves image quality by reducing radiation-induced sample motion.

  18. Controlling protein adsorption on graphene for cryo-EM using low-energy hydrogen plasmas.

    PubMed

    Russo, Christopher J; Passmore, Lori A

    2014-06-01

    Despite its many favorable properties as a sample support for biological electron microscopy, graphene is not widely used because its hydrophobicity precludes reliable protein deposition. We describe a method to modify graphene with a low-energy hydrogen plasma, which reduces hydrophobicity without degrading the graphene lattice. Use of plasma-treated graphene enables better control of protein distribution in ice for electron cryo-microscopy and improves image quality by reducing radiation-induced sample motion. PMID:24747813

  19. Identification of Trypanosome Proteins in Plasma from African Sleeping Sickness Patients Infected with T. b. rhodesiense

    PubMed Central

    Enyaru, John C.; Carr, Steven A.; Pearson, Terry W.

    2013-01-01

    Control of human African sleeping sickness, caused by subspecies of the protozoan parasite Trypanosoma brucei, is based on preventing transmission by elimination of the tsetse vector and by active diagnostic screening and treatment of infected patients. To identify trypanosome proteins that have potential as biomarkers for detection and monitoring of African sleeping sickness, we have used a ‘deep-mining” proteomics approach to identify trypanosome proteins in human plasma. Abundant human plasma proteins were removed by immunodepletion. Depleted plasma samples were then digested to peptides with trypsin, fractionated by basic reversed phase and each fraction analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). This sample processing and analysis method enabled identification of low levels of trypanosome proteins in pooled plasma from late stage sleeping sickness patients infected with Trypanosoma brucei rhodesiense. A total of 254 trypanosome proteins were confidently identified. Many of the parasite proteins identified were of unknown function, although metabolic enzymes, chaperones, proteases and ubiquitin-related/acting proteins were found. This approach to the identification of conserved, soluble trypanosome proteins in human plasma offers a possible route to improved disease diagnosis and monitoring, since these molecules are potential biomarkers for the development of a new generation of antigen-detection assays. The combined immuno-depletion/mass spectrometric approach can be applied to a variety of infectious diseases for unbiased biomarker identification. PMID:23951171

  20. Plasma surface modification of poly(D,L-lactic acid) as a tool to enhance protein adsorption and the attachment of different cell types.

    PubMed

    Alves, C M; Yang, Y; Marton, D; Carnes, D L; Ong, J L; Sylvia, V L; Dean, D D; Reis, R L; Agrawal, C M

    2008-10-01

    We have studied the influence of oxygen radio frequency glow discharge (RfGD) on the surface and bulk properties of poly(D,L-lactic acid) (PDLLA) and the effect of this surface modification on both protein adsorption and bone cell behavior. PDLLA films were characterized before and after plasma surface modification by water contact angle, surface energy, and adhesion tension of water as well as by scanning electron microscopy (SEM), X-ray electron spectroscopy (XPS), and Fourier transform infra-red (FTIR) spectroscopy. RfGD-films showed an increase in hydrophilicity and surface energy when compared with untreated films. Surface morphological changes were observed by SEM. Chemical analysis indicated significant differences in both atomic percentages and oxygen functional group. Protein adsorption was evaluated by combining solute depletion and spectroscopic techniques. Bovine serum albumin (BSA), fibronectin (FN), vitronectin (VN), and fetal bovine serum (FBS) were used in this study. RfGD-treated surfaces adsorbed more BSA and FN from single specie solutions than FBS that is a more complex, multi-specie solution. MG63 osteoblast-like cells and primary cultures of fetal rat calvarial (FRC) cells were used to assess both the effect of RfGD treatment and protein adsorption on cell attachment and proliferation. In the absence of preadsorbed proteins, cells could not distinguish between treated and untreated surfaces, with the exception of MG63 cells cultured for longer periods of time. In contrast, the adsorption of proteins increased the cells' preference for treated surfaces, thus indicating a crucial role for adsorbed proteins in mediating the response of osteogenic cells to the RfGD-treated PDLLA surface. PMID:18360882

  1. Desialylation of plasma proteins in severe dengue infection: possible role of oxidative stress.

    PubMed

    Rajendiran, Soundravally; Lakshamanappa, Hoti Sugeerappa; Zachariah, Bobby; Nambiar, Selvaraj

    2008-09-01

    Oxidative stress in dengue infection has been suggested. This study was carried out to explore the plasma protein oxidation and its sialic acid content in dengue infection. Thirty-two dengue hemorrhagic fever (DHF), 25 dengue shock syndrome (DSS), 29 dengue fever (DF), and 63 healthy controls were included in this study. The extent of carbonylation, sulphydryl content, and desialylation of plasma protein was estimated in acute phase sample. Significantly higher levels of protein carbonyls and lower levels of sialic acid and sulphydryl groups were found in DHF and DSS compared with DF using one-way analysis of variance. Regression analysis showed that desialylation is dependent on protein carbonyls in DHF/DSS. This study indicates that, in dengue infection, plasma proteins undergo increased levels of desialylation, which can be attributed to the oxidative stress. Future studies on sialylation status of endothelium and platelets can show light into the pathogenesis of the dengue infection.

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

    PubMed

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

    2014-09-01

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

  3. Adsorption of HSA, IgG and laminin-1 on model titania surfaces--effects of glow discharge treatment on competitively adsorbed film composition.

    PubMed

    Santos, Olga; Svendsen, Ida E; Lindh, Liselott; Arnebrant, Thomas

    2011-10-01

    This study investigated the effect of glow discharge treatment of titania surfaces on plasma protein adsorption, by means of ellipsometry and mechanically assisted SDS elution. The adsorption and film elution of three plasma proteins, viz. human serum albumin (HSA), human immunoglobulin G (IgG) and laminin-1, as well as competitive adsorption from a mixture of the three proteins, showed that the adsorbed amount of the individual proteins after 1 h increased in the order HSA protein mixture. Film elutability showed that 30 min of SDS interaction resulted in almost complete removal of adsorbed films. No difference in the total adsorbed amounts of individual proteins, or from the mixture, was observed between untreated and glow discharge treated titania surfaces. However, the composition of the adsorbed films from the mixture differed between the untreated and glow discharge treated substrata. On glow discharge-treated titania the fraction of HSA increased, the fraction of laminin-1 decreased and the fraction of IgG was unchanged compared to the adsorption on the untreated titania, which was attributed to protein-protein interactions and competitive/associative adsorption behaviour.

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

    SciTech Connect

    Sakamoto, Hikaru; Sakata, Keiko; Kusumi, Kensuke; Kojima, Mikiko; Sakakibara, Hitoshi; Iba, Koh

    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.

  5. Biochemical studies on proteins from cheese whey and blood plasma by-products.

    PubMed

    el-Sayed, M M; Abdel Hamid, F F; Ahmed, Y M; Ali, S H; Mansour, O Y; Abdallah, N M

    1998-02-01

    Efforts have been done to recover proteins from waste liquors rich in protein in a soluble form. Cheese whey and animal bloods are by-products from the manufacture of cheese and meat. It contains a variety of proteins which can be reclaimed. The efficiency of protein precipitation from the sweet-cheese whey by the use of hydroxyethyl cellulose (HEC) was similar to that precipitated by the use of carboxymethyl cellulose (CMC). Both are greater than that precipitated by trichloro acetic acid. The same results of the efficiency of precipitation were attained when the plasma was precipitated. It was found that cheese-whey protein-HEC-complex and plasma protein-HEC-complex contain a large amount of essential amino acids. Electrophoretic separation of whey protein complex showed that beta-Lactoglobulin forms the major fraction while in case of plasma protein complex albumin forms the major fraction. The fractionation patterns of different complexes with HEC, CMC or TCA gave the same components and about the same ratio. It appears from these results that HEC-protein complexes are preferable than CMC-protein complexes or proteins precipitated by TCA. Chemical analysis of whey protein complexes revealed that lactose content of whey protein-HEC-complex was higher than that of CMC-complex or protein precipitated by TCA. Elemental analysis of protein complexes showed that the level of sodium, phosphorus, and potassium was increased while that of copper or zinc decreased. Cellulose derivative protein complexes showed no significant effects on the liver or kidney function of albino rat and these results indicted that no toxic effect was observed from the uses of these protein complexes in feeding.

  6. A comprehensive analysis of the Streptococcus pyogenes and human plasma protein interaction network.

    PubMed

    Sjöholm, Kristoffer; Karlsson, Christofer; Linder, Adam; Malmström, Johan

    2014-07-01

    Streptococcus pyogenes is a major human bacterial pathogen responsible for severe and invasive disease associated with high mortality rates. The bacterium interacts with several human blood plasma proteins and clarifying these interactions and their biological consequences will help to explain the progression from mild to severe infections. In this study, we used a combination of mass spectrometry (MS) based techniques to comprehensively quantify the components of the S. pyogenes-plasma protein interaction network. From an initial list of 181 interacting human plasma proteins defined using liquid chromatography (LC)-MS/MS analysis we further subdivided the interacting protein list using selected reaction monitoring (SRM) depending on the level of enrichment and protein concentration on the bacterial surface. The combination of MS methods revealed several previously characterized interactions between the S. pyogenes surface and human plasma along with many more, so far uncharacterised, possible plasma protein interactions with S. pyogenes. In follow-up experiments, the combination of MS techniques was applied to study differences in protein binding to a S. pyogenes wild type strain and an isogenic mutant lacking several important virulence factors, and a unique pair of invasive and non-invasive S. pyogenes isolates from the same patient. Comparing the plasma protein-binding properties of the wild type and the mutant and the invasive and non-invasive S. pyogenes bacteria revealed considerable differences, underlining the significance of these protein interactions. The results also demonstrate the power of the developed mass spectrometry method to investigate host-microbial relationships with a large proteomics depth and high quantitative accuracy.

  7. Nedocromil sodium in obstructive airways disease: effect on symptoms and plasma protein leakage in sputum.

    PubMed

    Schoonbrood, D F; Out, T A; Hart, A A; Habets, F J; Roos, C M; Jansen, H M

    1997-07-01

    In patients with asthma or chronic obstructive pulmonary disease, there is chronic airway inflammation with increased leakage of plasma proteins into the airway lumen, which can be reduced by inhaled glucocorticosteroids. Nedocromil sodium is an anti-inflammatory drug, and we questioned whether it also affects the leakage of plasma proteins. In a double-blind placebo-controlled study we investigated the effect of 12 weeks of treatment with nedocromil on forced expiratory volume in one second (FEV1), provocative concentration of histamine causing a 20% fall in FEV1 (PC20), peak flow, symptom scores, and plasma protein leakage in sputum, in 31 patients with obstructive airways disease and sputum production (mean (range) FEV1 61% of predicted (42-87%); geometric mean (range) PC20 0.39 (0.04-2.9) mg x mL(-1)). As a measure for plasma protein leakage we calculated the relative coefficients of excretion (RCE) of proteins from serum to the soluble phase of sputum. There was a small increase in morning and evening peak flow (p<0.05) and a decrease in night-time bronchodilator use (p<0.02) in favour of nedocromil. The RCE of alpha2-macroglobulin to albumin significantly decreased after treatment with nedocromil (p=0.03). The results show limited clinical efficacy of nedocromil in our study group. They further suggest that the anti-inflammatory properties of nedocromil extend to inhibition of plasma protein leakage into the airways.

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

    PubMed Central

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

    2014-01-01

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

  9. Comparative proteomic analysis of plasma proteins in patients with age-related macular degeneration

    PubMed Central

    Xu, Xin-Rong; Zhong, Lu; Huang, Bing-Lin; Wei, Yuan-Hua; Zhou, Xin; Wang, Ling; Wang, Fu-Qiang

    2014-01-01

    AIM To find the significant altered proteins in age-related macular degeneration (AMD) patients as potential biomarkers of AMD. METHODS A comparative analysis of the protein pattern of AMD patients versus healthy controls was performed by means of proteomic analysis using two-dimensional gel electrophoresis followed by protein identification with MALDI TOF/TOF mass spectrometry. RESULTS We identified 28 proteins that were significantly altered with clinical relevance in AMD patients. These proteins were involved in a wide range of biological functions including immune responses, growth cytokines, cell fate determination, wound healing, metabolism, and anti-oxidance. CONCLUSION These results demonstrate the capacity of proteomic analysis of AMD patient plasma. In addition to the utility of this approach for biomarker discovery, identification of alterations in endogenous proteins in the plasma of AMD patient could improve our understanding of the disease pathogenesis. PMID:24790867

  10. Direct Capture of Functional Proteins from Mammalian Plasma Membranes into Nanodiscs.

    PubMed

    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.

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

    SciTech Connect

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

    2008-05-01

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

  12. Proteomic Identification of Novel Differentiation Plasma Protein Markers in Hypobaric Hypoxia-Induced Rat Model

    PubMed Central

    Ahmad, Mohammad Faiz; Sharma, Manish; Garg, Iti; Bhargava, Kalpana

    2014-01-01

    Background Hypobaric hypoxia causes complex changes in the expression of genes, including stress related genes and corresponding proteins that are necessary to maintain homeostasis. Whereas most prior studies focused on single proteins, newer methods allowing the simultaneous study of many proteins could lead to a better understanding of complex and dynamic changes that occur during the hypobaric hypoxia. Methods In this study we investigated the temporal plasma protein alterations of rat induced by hypobaric hypoxia at a simulated altitude of 7620 m (25,000 ft, 282 mm Hg) in a hypobaric chamber. Total plasma proteins collected at different time points (0, 6, 12 and 24 h), separated by two-dimensional electrophoresis (2-DE) and identified using matrix assisted laser desorption ionization time of flight (MALDI-TOF/TOF). Biological processes that were enriched in the plasma proteins during hypobaric hypoxia were identified using Gene Ontology (GO) analysis. According to their properties and obvious alterations during hypobaric hypoxia, changes of plasma concentrations of Ttr, Prdx-2, Gpx -3, Apo A-I, Hp, Apo-E, Fetub and Nme were selected to be validated by Western blot analysis. Results Bioinformatics analysis of 25 differentially expressed proteins showed that 23 had corresponding candidates in the database. The expression patterns of the eight selected proteins observed by Western blot were in agreement with 2-DE results, thus confirming the reliability of the proteomic analysis. Most of the proteins identified are related to cellular defense mechanisms involving anti-inflammatory and antioxidant activity. Their presence reflects the consequence of serial cascades initiated by hypobaric hypoxia. Conclusion/Significance This study provides information about the plasma proteome changes induced in response to hypobaric hypoxia and thus identification of the candidate proteins which can act as novel biomarkers. PMID:24842778

  13. Glycoproteomic Study Reveals Altered Plasma Proteins Associated with HIV Elite Suppressors

    PubMed Central

    Yang, Weiming; Laeyendecker, Oliver; Wendel, Sarah K.; Zhang, Bai; Sun, Shisheng; Zhou, Jian-Ying; Ao, Minghui; Moore, Richard D.; Jackson, J. Brooks; Zhang, Hui

    2014-01-01

    HIV elite suppressors (ES) or controllers are individuals achieving control of viremia by their natural immunological mechanisms without highly active antiretroviral therapy (HAART). Study of the mechanisms responsible for the immunological suppression of viremia in ES may lead to the detection of individuals with ES and the effective control of HIV infection. We hypothesize that plasma glycoproteins play essential roles in the immune system of ES since plasma proteins are critical and highly relevant in anti-viral immunity and most plasma proteins are glycoproteins. To examine glycoproteins associated with ES, plasma samples from ES individuals (n=20), and from individuals on HAART (n=20), with AIDS (n=20), and no HIV infection (n=10) were analyzed by quantitative glycoproteomics. We found that a number of glycoproteins changed between ES versus HAART, AIDS and HIV- individuals. In sharp contrast, the level of plasma glycoproteins in the HAART cohort showed fewer changes compared with AIDS and HIV- individuals. These results showed that although both ES and HAART effectively suppress viremia, ES appeared to profoundly affect immunologically relevant glycoproteins in plasma as consequence of or support for anti-viral immunity. Bioinformatic analysis revealed that altered proteins in ES plasma were mainly associated with inflammation. This analysis suggests that overlapping, while distinguishable, glycoprotein profiles for inflammation and immune activation appeared to be present between ES and non-ES (HAART+AIDS) cohorts, indicating different triggers for inflammation and immune activation between natural and treatment-related viral suppression. PMID:25285165

  14. Quantitative plasma proteome analysis reveals aberrant level of blood coagulation-related proteins in nasopharyngeal carcinoma.

    PubMed

    Peng, Pei-Hua; Wu, Chih-Ching; Liu, Shu-Chen; Chang, Kai-Ping; Chen, Chi-De; Chang, Ya-Ting; Hsu, Chia-Wei; Chang, Yu-Sun; Yu, Jau-Song

    2011-05-01

    Nasopharyngeal carcinoma (NPC), one of the most common cancers in Southeast Asia, is not easily diagnosed until advanced stages. To discover potential biomarkers for improving NPC diagnosis, we herein identified the aberrant plasma proteins in NPC patients. We first removed the top-seven abundant proteins from plasma samples of healthy controls and NPC patients, and then labeled the samples with different fluorescent cyanine dyes. The labeled samples were then mixed equally and fractionated with ion-exchange chromatography followed by SDS-PAGE. Proteins showing altered levels in NPC patients were identified by in-gel tryptic digestion and LC-MS/MS. When the biological roles of the 45 identified proteins were assessed via MetaCore™ analysis, the blood coagulation pathway emerged as the most significantly altered pathway in NPC plasma. Plasma kallikrein (KLKB1) and thrombin-antithrombin III complex (TAT) were chosen for evaluation as the candidate NPC biomarkers because of their involvement in blood coagulation. ELISAs confirmed the elevation of their plasma levels in NPC patients versus healthy controls. Western blot and activity assays further showed that the KLKB1 active form was significantly increased in NPC plasma. Collectively, our results identified the significant alteration of blood coagulation pathway in NPC patients, and KLKB1 and TAT may represent the potential NPC biomarkers.

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

    NASA Astrophysics Data System (ADS)

    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.

  16. With or without you - Proteomics with or without major plasma/serum proteins.

    PubMed

    Gianazza, Elisabetta; Miller, Ingrid; Palazzolo, Luca; Parravicini, Chiara; Eberini, Ivano

    2016-05-17

    The first sections of this review compile and discuss strategies and protocols for managing plasma/serum as a source of biomarkers relevant to human disease. In many such cases, depletion of abundant protein(s) is a crucial preliminary step to the procedure; specific conceptual and technical approaches, however, make it possible to effectively use to this purpose whole plasma/serum. The final sections focus instead on the complexity associated with each of the major serum/plasma proteins in terms of both, multiple molecular structures (existence of a number of protein species) and of multiple molecular functions (behavior as multifunctional/multitasking/moonlighting proteins). Reviewing evidence in these and some related fields (regulation of the synthetic pattern by proteins and non-protein compounds and its connection with health and disease) prompts the suggestion/recommendation that information on the abundant components of plasma/serum proteome is routinely obtained and processed/mined as a valuable contribution to the characterization of any non-physiological condition and to the understanding of its mechanisms and of its implications/sequels. PMID:27072114

  17. Multiple cellular proteins modulate the dynamics of K-ras association with the plasma membrane.

    PubMed

    Bhagatji, Pinkesh; Leventis, Rania; Rich, Rebecca; Lin, Chen-ju; Silvius, John R

    2010-11-17

    Although specific proteins have been identified that regulate the membrane association and facilitate intracellular transport of prenylated Rho- and Rab-family proteins, it is not known whether cellular proteins fulfill similar roles for other prenylated species, such as Ras-family proteins. We used a previously described method to evaluate how several cellular proteins, previously identified as potential binding partners (but not effectors) of K-ras4B, influence the dynamics of K-ras association with the plasma membrane. Overexpression of either PDEδ or PRA1 enhances, whereas knockdown of either protein reduces, the rate of dissociation of K-ras from the plasma membrane. Inhibition of calmodulin likewise reduces the rate of K-ras dissociation from the plasma membrane, in this case in a manner specific for the activated form of K-ras. By contrast, galectin-3 specifically reduces the rate of plasma membrane dissociation of activated K-ras, an effect that is blocked by the K-ras antagonist farnesylthiosalicylic acid (salirasib). Multiple cellular proteins thus control the dynamics of membrane association and intercompartmental movement of K-ras to an important degree even under basal cellular conditions.

  18. With or without you - Proteomics with or without major plasma/serum proteins.

    PubMed

    Gianazza, Elisabetta; Miller, Ingrid; Palazzolo, Luca; Parravicini, Chiara; Eberini, Ivano

    2016-05-17

    The first sections of this review compile and discuss strategies and protocols for managing plasma/serum as a source of biomarkers relevant to human disease. In many such cases, depletion of abundant protein(s) is a crucial preliminary step to the procedure; specific conceptual and technical approaches, however, make it possible to effectively use to this purpose whole plasma/serum. The final sections focus instead on the complexity associated with each of the major serum/plasma proteins in terms of both, multiple molecular structures (existence of a number of protein species) and of multiple molecular functions (behavior as multifunctional/multitasking/moonlighting proteins). Reviewing evidence in these and some related fields (regulation of the synthetic pattern by proteins and non-protein compounds and its connection with health and disease) prompts the suggestion/recommendation that information on the abundant components of plasma/serum proteome is routinely obtained and processed/mined as a valuable contribution to the characterization of any non-physiological condition and to the understanding of its mechanisms and of its implications/sequels.

  19. Plasma-surface modification vs air oxidation on carbon obtained from peach stone: Textural and chemical changes and the efficiency as adsorbents

    NASA Astrophysics Data System (ADS)

    De Velasco Maldonado, Paola S.; Hernández-Montoya, Virginia; Montes-Morán, Miguel A.

    2016-10-01

    Carbons were prepared from peach stones (Prunus persica) using different carbonization temperatures (600, 800 and 1000 °C). A selected sample was modified by oxidation using conventional oxidation techniques (thermal treatment in air atmosphere) and with cold oxygen plasma oxidation, under different conditions. Samples were characterized using elemental analysis, FT-IR spectroscopy, nitrogen adsorption isotherms at -196 °C, SEM/EDX analysis, potentiometric titration and XPS analysis. Carbons with and without oxidation were employed in the adsorption of Pb2+ in aqueous solution. Results obtained indicated that the materials with high contents of acidic oxygen groups were more efficient in the removal of Pb2+, values as high as approx. 40 mg g-1 being obtained for the best performing carbon. Textural properties of the original, un-oxidized carbon were significantly altered only after oxidation under air atmosphere at 450 °C. On the other hand, the samples oxidized with plasma show little changes in the textural parameters and a slight increase in the specific surface was observed for the sample treated at high RF power (100 W). Additionally, a significant increment of the oxygen content was observed for the plasma oxidized samples, as measured by XPS.

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

    PubMed

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

    2015-10-14

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

  1. Plasma proteomics, the Human Proteome Project, and cancer-associated alternative splice variant proteins.

    PubMed

    Omenn, Gilbert S

    2014-05-01

    This article addresses three inter-related subjects: the development of the Human Plasma Proteome Peptide Atlas, the launch of the Human Proteome Project, and the emergence of alternative splice variant transcripts and proteins as important features of evolution and pathogenesis. The current Plasma Peptide Atlas provides evidence on which peptides have been detected for every protein confidently identified in plasma; there are links to their spectra and their estimated abundance, facilitating the planning of targeted proteomics for biomarker studies. The Human Proteome Project (HPP) combines a chromosome-centric C-HPP with a biology and disease-driven B/D-HPP, upon a foundation of mass spectrometry, antibody, and knowledgebase resource pillars. The HPP aims to identify the approximately 7000 "missing proteins" and to characterize all proteins and their many isoforms. Success will enable the larger research community to utilize newly-available peptides, spectra, informative MS transitions, and databases for targeted analyses of priority proteins for each organ and disease. Among the isoforms of proteins, splice variants have the special feature of greatly enlarging protein diversity without enlarging the genome; evidence is accumulating of striking differential expression of splice variants in cancers. In this era of RNA-sequencing and advanced mass spectrometry, it is no longer sufficient to speak simply of increased or decreased expression of genes or proteins without carefully examining the splice variants in the protein mixture produced from each multi-exon gene. This article is part of a Special Issue entitled: Biomarkers: A Proteomic Challenge.

  2. Molecular characterization of a cold-induced plasma membrane protein gene from wheat.

    PubMed

    Koike, Michiya; Sutoh, Keita; Kawakami, Akira; Torada, Atsushi; Oono, Kiyoharu; Imai, Ryozo

    2005-12-01

    As a means to study the function of plasma membrane proteins during cold acclimation, we have isolated a cDNA clone for wpi6 which encodes a putative plasma membrane protein from cold-acclimated winter wheat. The wpi6 gene encodes a putative 5.9 kDa polypeptide with two predicted membrane-spanning domains, the sequence of which shows high sequence similarity with BLT101-family proteins from plants and yeast. Strong induction of wpi6 mRNA was observed during an early stage of cold acclimation in root and shoot tissues of both winter and spring wheat cultivars. In contrast to blt101 in barley, wpi6 mRNA was also induced by drought and salinity stresses, and exogenous application of ABA. Expression of wpi6 in a Deltapmp3 mutant of Saccharomyces cerevisiae, which is disturbed in plasma membrane potential due to the lack of a BLT101-family protein, partially complemented NaCl sensitivity of the mutant. Transient expression analysis of a WPI6::GFP fusion protein in onion epidermal cells revealed that WPI6 is localized in the plasma membrane. Taken together, these data suggested that WPI6 may have a protective role in maintaining plasma membrane function during cold acclimation in wheat.

  3. THE INFLUENCE OF NITROGEN RETENTION UPON THE REGENERATION OF PLASMA PROTEINS

    PubMed Central

    Holman, Russell L.; Mebane, J. Gilmer

    1940-01-01

    1. Because of the clinical observation that the capacity to form new plasma proteins is sometimes impaired in cases of nephritis (2), experiments were performed to determine whether the impaired function in the nephritic is related to nitrogen retention. 2. These experiments consisted of producing renal injury by injecting uranium nitrate into standard hypoproteinemic dogs and comparing the rate of blood plasma protein formation under these conditions of nitrogen retention with that in the uninjured dog. 3. Despite elevations in blood N.P.N. to more than ten times normal, no interference with plasma protein formation was observed. These elevations in N.P.N. affected principally the urea and undetermined fractions. 4. Neither elevation in N.P.N. nor proteinuria per se appears to have any effect upon plasma protein production. Possibly the deficient production of plasma proteins in the nephritic is related to a more general disturbance in metabolism in which the elevation in N.P.N. is secondary. PMID:19870964

  4. Quantitative variability of 342 plasma proteins in a human twin population

    PubMed Central

    Liu, Yansheng; Buil, Alfonso; Collins, Ben C; Gillet, Ludovic CJ; Blum, Lorenz C; Cheng, Lin-Yang; Vitek, Olga; Mouritsen, Jeppe; Lachance, Genevieve; Spector, Tim D; Dermitzakis, Emmanouil T; Aebersold, Ruedi

    2015-01-01

    The degree and the origins of quantitative variability of most human plasma proteins are largely unknown. Because the twin study design provides a natural opportunity to estimate the relative contribution of heritability and environment to different traits in human population, we applied here the highly accurate and reproducible SWATH mass spectrometry technique to quantify 1,904 peptides defining 342 unique plasma proteins in 232 plasma samples collected longitudinally from pairs of monozygotic and dizygotic twins at intervals of 2–7 years, and proportioned the observed total quantitative variability to its root causes, genes, and environmental and longitudinal factors. The data indicate that different proteins show vastly different patterns of abundance variability among humans and that genetic control and longitudinal variation affect protein levels and biological processes to different degrees. The data further strongly suggest that the plasma concentrations of clinical biomarkers need to be calibrated against genetic and temporal factors. Moreover, we identified 13 cis-SNPs significantly influencing the level of specific plasma proteins. These results therefore have immediate implications for the effective design of blood-based biomarker studies. PMID:25652787

  5. Large-scale inference of protein tissue origin in gram-positive sepsis plasma using quantitative targeted proteomics.

    PubMed

    Malmström, Erik; Kilsgård, Ola; Hauri, Simon; Smeds, Emanuel; Herwald, Heiko; Malmström, Lars; Malmström, Johan

    2016-01-06

    The plasma proteome is highly dynamic and variable, composed of proteins derived from surrounding tissues and cells. To investigate the complex processes that control the composition of the plasma proteome, we developed a mass spectrometry-based proteomics strategy to infer the origin of proteins detected in murine plasma. The strategy relies on the construction of a comprehensive protein tissue atlas from cells and highly vascularized organs using shotgun mass spectrometry. The protein tissue atlas was transformed to a spectral library for highly reproducible quantification of tissue-specific proteins directly in plasma using SWATH-like data-independent mass spectrometry analysis. We show that the method can determine drastic changes of tissue-specific protein profiles in blood plasma from mouse animal models with sepsis. The strategy can be extended to several other species advancing our understanding of the complex processes that contribute to the plasma proteome dynamics.

  6. Age-related variations of protein carbonyls in human saliva and plasma: is saliva protein carbonyls an alternative biomarker of aging?

    PubMed

    Wang, Zhihui; Wang, Yanyi; Liu, Hongchen; Che, Yuwei; Xu, Yingying; E, Lingling

    2015-06-01

    Free radical hypothesis which is one of the most acknowledged aging theories was developed into oxidative stress hypothesis. Protein carbonylation is by far one of the most widely used markers of protein oxidation. We studied the role of age and gender in protein carbonyl content of saliva and plasma among 273 Chinese healthy subjects (137 females and 136 males aged between 20 and 79) and discussed the correlation between protein carbonyl content of saliva and plasma. Protein carbonyl content of saliva and plasma were, respectively, 2.391 ± 0.639 and 0.838 ± 0.274 nmol/mg. Variations of saliva and plasma different age groups all reached significant differences in both male and female (all p < 0.05) while both saliva and plasma protein carbonyls were found to be significantly correlated with age (r = 0.6582 and r = 0.5176, all p < 0.001). Gender was discovered to be unrelated to saliva and plasma protein carbonyl levels (all p > 0.05). Saliva and plasma protein carbonyls were positively related (r = 0.4405, p < 0.001). Surprisingly, saliva and plasma protein carbonyls/ferric reducing ability of plasma (FRAP) ratios were proved to be significantly correlated with age (r = 0.7796 and r = 0.6938, all p < 0.001) while saliva protein carbonyls/FRAP ratio and plasma protein carbonyls/FRAP ratio were also correlated (r = 0.5573, p < 0.001). We concluded that saliva protein carbonyls seem to be an alternative biomarker of aging while the mechanisms of protein carbonylation and oxidative stress and the relationship between saliva protein carbonyls and diseases need to be further investigated.

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

    PubMed Central

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

    2006-01-01

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

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

    SciTech Connect

    Springer, David L.; Auberry, Deanna L.; Ahram, Mamoun; Adkins, Joshua N.; Feldhaus, Jane M.; Wahl, Jon H.; Wunsch, David M.; Rodland, Karin D.

    2003-01-01

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

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

    DOE PAGES

    Springer, David L.; Auberry, Deanna L.; Ahram, Mamoun; Adkins, Joshua N.; Feldhaus, Jane M.; Wahl, Jon H.; Wunschel, David S.; Rodland, Karin D.

    2004-01-01

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

  10. Protein retention on plasma-treated hierarchical nanoscale gold-silver platform

    PubMed Central

    Fang, Jinghua; Levchenko, Igor; Mai-Prochnow, Anne; Keidar, Michael; Cvelbar, Uros; Filipic, Gregor; Han, Zhao Jun; Ostrikov, Kostya (Ken)

    2015-01-01

    Dense arrays of gold-supported silver nanowires of about 100 nm in diameter grown directly in the channels of nanoporous aluminium oxide membrane were fabricated and tested as a novel platform for the immobilization and retention of BSA proteins in the microbial-protective environments. Additional treatment of the silver nanowires using low-temperature plasmas in the inductively-coupled plasma reactor and an atmospheric-pressure plasma jet have demonstrated that the morphology of the nanowire array can be controlled and the amount of the retained protein may be increased due to the plasma effect. A combination of the neutral gold sublayer with the antimicrobial properties of silver nanowires could significantly enhance the efficiency of the platforms used in various biotechnological processes. PMID:26307515

  11. Plasma-induced surface modification of polydimethylsiloxane aimed at reducing salt and protein deposition.

    PubMed

    De Smet, Nele; Rymarczyk-Machal, Monika; Schacht, Etienne

    2011-01-01

    Polydimethylsiloxane (PDMS) is an elastomer that is widely used in construction and for biological and biomedical applications. The biocompatibility of PDMS was improved by different surface treatment methods, i.e., plasma treatment or a combination of plasma treatment with UV-irradiation or redox initiator, to minimize the effects of deposition of salts and proteins. In this work we used the vinyl monomers sulfobetaine and AMPS which have good biocompatible properties. PMID:21176391

  12. Identification of DNA-binding proteins on human umbilical vein endothelial cell plasma membrane.

    PubMed Central

    Chan, T M; Frampton, G; Cameron, J S

    1993-01-01

    The binding of anti-DNA antibodies to the endothelial cell is mediated through DNA, which forms a bridge between the immunoglobulin and the plasma membrane. We have shown that 32P-labelled DNA bound to the plasma membrane of human umbilical vein endothelial cells (HUVEC) by a saturable process, which could be competitively inhibited by non-radiolabelled DNA. In addition, DNA-binding was enhanced in HUVEC that had been treated with IL-1 alpha or tumour necrosis factor-alpha (TNF-alpha). DNA-binding proteins of mol. wt 46,000, 92,000, and 84,000 were identified by the binding of 32P-labelled DNA to plasma membrane proteins separated on SDS-PAGE. DNA-binding proteins of mol. wt 46,000 and 84,000 were also present in the cytosol and nucleus. Murine anti-DNA MoAb410 bound to a single band, at mol. wt 46,000, of plasma membrane protein, in the presence of DNA. Our results showed that DNA-binding proteins are present in different cellular fractions of endothelial cells. DNA-binding proteins on the cell membrane could participate in the in situ formation of immune deposits; and their presence in the cell nucleus suggests a potential role in the modulation of cell function. Images Fig. 3 Fig. 4 PMID:8419070

  13. The effect of occupational lead exposure on lipid peroxidation, protein carbonylation, and plasma viscosity.

    PubMed

    Kasperczyk, Sławomir; Słowińska-Łożyńska, Ludmiła; Kasperczyk, Aleksandra; Wielkoszyński, Tomasz; Birkner, Ewa

    2015-12-01

    The aim of the study was to investigate the influence of occupational lead (Pb) exposure on lipid peroxidation, protein carbonylation, and plasma viscosity in workers. The examined group included 283 healthy male employees of manufacturing facilities using zinc and Pb. The mean blood concentrations of Pb and zinc protoporphyrin as well as the mean urine δ-aminolevulinic acid levels were used as markers of exposure for the examined group. Taking into account the obtained mean values of blood lead level, the examined group was divided into three subgroups. When comparing the control group with the subgroups, Pb exposure markers were significantly elevated in all the three subgroups. Concentrations of conjugated dienes (CD), lipid hydroperoxides, malondialdehyde (MDA), and protein carbonyl groups were also significantly increased. Conversely, the levels of total protein and protein sulfhydryls were significantly decreased in the subgroups compared with the controls. The plasma viscosity was significantly elevated in the subgroups. A dose-response between Pb levels and plasma viscosity was not observed. Pb supposedly elevates MDA and CD in a dose-dependent manner. In conclusion, occupational Pb exposure induces oxidative stress that results in lipid and protein damage. Moreover, Pb-induced oxidative stress is likely the primary factor that elevates plasma viscosity, despite decreased protein levels.

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

    PubMed

    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. PMID:27145233

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

    PubMed Central

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

    2013-01-01

    Sample displacement chromatography (SDC) in reversed-phase and ion-exchange modes was introduced approximately twenty years ago. This method was first used for the preparative purification of peptides and proteins. Recently, SDC in ion-exchange mode was also successfully used for enrichment of low abundance proteins from human plasma. In this paper, the use of SDC for the separation of plasma proteins in hydrophobic interaction mode is demonstrated. By use of two or more columns coupled in series during sample application, and subsequent elution of detached columns in parallel, additional separation of bound proteins was achieved. Further low-abundance, physiologically active proteins could be highly enriched and detected by ESI-MS/MS. PMID:22740472

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

    PubMed

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

    2012-07-01

    Sample displacement chromatography (SDC) in reversed-phase and ion-exchange modes was introduced approximately 20 years ago. This method was first used for the preparative purification of peptides and proteins. Recently, SDC in ion-exchange mode was also successfully used for enrichment of low-abundance proteins from human plasma. In this paper, the use of SDC for the separation of plasma proteins in hydrophobic interaction mode is demonstrated. By use of two or more columns coupled in series during sample application, and subsequent elution of detached columns in parallel, additional separation of bound proteins was achieved. Further low-abundance, physiologically active proteins could be highly enriched and detected by ESI-MS/MS.

  17. Whey protein supplementation increases methionine intake but not homocysteine plasma concentration in rats.

    PubMed

    Deminice, Rafael; Comparotto, Hugo; Jordao, Alceu Afonso

    2015-01-01

    The purpose of this study was to examine the effects of whey protein supplementation on homocysteine (Hcy) metabolism and liver oxidative stress in rats. Twenty-four rats were divided into 3 groups (n = 8) to receive one of the following diets for 4 weeks: control diet (C), whey protein-composed diet (WP), and whey protein-supplemented diet (WPS). The C and WP diets consisted of AIN-93 with 20% casein and 20% whey protein as protein source, respectively. WPS was AIN-93 (20% casein) supplemented by the addition of 20% (w/w) whey protein. Four weeks of ingesting a WPS diet resulted in a significantly higher (P < 0.05) total protein and methionine intakes. Although a significant increase (P < 0.05) in the hepatic S-adenosylmethionine and S-adenosylhomocysteine levels occurred in WPS group compared with C and WP, no significant change was observed in plasma Hcy concentration between groups. Furthermore, the levels of lipid hydroperoxides and advanced oxidation protein products, known liver oxidative stress markers, were increased in the WPS group compared with the C group. In addition, no change in glutathione liver concentration was observed in any of the groups studied. In conclusion, whey protein supplementation increases methionine intake substantially; however, it does not change plasma Hcy concentrations. On the other hand, increased hepatic oxidative stress markers were observed in whey protein supplemented rats were probably due to high protein intake.

  18. Analysis of Twenty-three plasma proteins in ascites. The depletion of fibrinogen and plasminogen.

    PubMed

    Henderson, J M; Stein, S F; Kutner, M; Wiles, M B; Ansley, J D; Rudman, D

    1980-12-01

    The concentrations of 23 plasma proteins were measured by radial immunodiffusion in the plasma and ascites of 17 patients with cirrhosis and four patients with intraperitoneal malignancies, to learn whether there is a selectivity in the movement of proteins from plasma into ascites, analogous to that of proteinuria. Additionally, since some of the proteins are involved in coagulation, we hoped to clarify the coagulopathy frequently seen following peritoneovenous shunting of ascites. Analysis was by groups: group 1 consisted of nine patients with cirrhosis with an ascites-total protein content less than 2.5 g/dl; group 2 consisted of eight patients with cirrhosis with ascites-total protein content greater than or equal to 2.5 g/dl; and group 3 consisted of four patients with malignant ascites. The ratio of the plasma concentration/ascites concentration ([P]/[A]) for each protein was calculated for each patient. In each group the median [P]/[A] for each protein was plotted against the natural logarithm of its molecular weight (In MW). For 21 of the 23 proteins, [P]/[A] showed a close linear relationship to In MW. Fibrogen and plasminogen showed significant (p < 0.0002) elevation above the regression line relating [P]/[A] to In MW. This indicates depletion of fibrinogen and plasminogen in ascites. The ascites in group 1 showed moderate selectivity, defined as the slope of the regression line (1.59), while groups 2 and 3 were essentialy nonselective (0.35 and 0.50). Fibrin-split products were elevated in all ascites but not in plasma, indicating either fibrinolysis or fibrinogenolysis within the ascites. A normal ratio for prothrombin suggests fibrinogenolysis may be the dominant mechanism. Thus the coagulopathy induced by LeVeen valve insertion may be in part secondary to the infusion of plasmin or a plasminogen activator into the circulation.

  19. Molecular Adsorber Coating

    NASA Technical Reports Server (NTRS)

    Straka, Sharon; Peters, Wanda; Hasegawa, Mark; Hedgeland, Randy; Petro, John; Novo-Gradac, Kevin; Wong, Alfred; Triolo, Jack; Miller, Cory

    2011-01-01

    A document discusses a zeolite-based sprayable molecular adsorber coating that has been developed to alleviate the size and weight issues of current ceramic puck-based technology, while providing a configuration that more projects can use to protect against degradation from outgassed materials within a spacecraft, particularly contamination-sensitive instruments. This coating system demonstrates five times the adsorption capacity of previously developed adsorber coating slurries. The molecular adsorber formulation was developed and refined, and a procedure for spray application was developed. Samples were spray-coated and tested for capacity, thermal optical/radiative properties, coating adhesion, and thermal cycling. Work performed during this study indicates that the molecular adsorber formulation can be applied to aluminum, stainless steel, or other metal substrates that can accept silicate-based coatings. The coating can also function as a thermal- control coating. This adsorber will dramatically reduce the mass and volume restrictions, and is less expensive than the currently used molecular adsorber puck design.

  20. Valproic acid: in vitro plasma protein binding and interaction with phenytoin.

    PubMed

    Cramer, J A; Mattson, R H

    1979-01-01

    Because valproic acid (VPA) is highly bound to plasma protein, several variables affecting binding will significantly alter the quantity of free drug which is pharmacologically active. Therefore, total VPA plasma concentrations do not reflect the therapeutic strength of the drug in tissue. We have performed equilibrium dialysis and ultrafiltration studies of VPA binding to plasma protein. The converging data in these in vitro studies indicate a clinically significant alteration in the percent of free VPA when total drug concentration exceeds 80 micrograms/ml. Saturation of drug binding sites probably occurs in this range. At 20--60 micrograms/ml VPA there is 5% free drug, with a significant increase to 8% free at 80 micrograms/ml; free drug increases to over 20% at 145 micrograms/ml total VPA. Human plasma, which is low in albumin, has twice the quantity of free VPA as normal plasma (10 versus 5% free). The clinical evidence of interaction between VPA and phenytoin is confirmed in vitro by the increase in the free fraction of both drugs. VPA binding decreases by 3--6%, while phenytoin binding decreases 5--6% as both drugs reach high plasma concentrations. When appropriate, laboratory reports should be available defining concentration of free drug in plasma for optimal interpretation of drug concetrations relative to clinical effects.

  1. Comparative Proteome Analysis of Cryopreserved Flagella and Head Plasma Membrane Proteins from Sea Bream Spermatozoa: Effect of Antifreeze Proteins

    PubMed Central

    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

  2. Plasma protein binding of an antisense oligonucleotide targeting human ICAM-1 (ISIS 2302).

    PubMed

    Watanabe, Tanya A; Geary, Richard S; Levin, Arthur A

    2006-01-01

    In vitro ultrafiltration was used to determine the plasma protein-binding characteristics of phosphorothioate oligonucleotides (PS ODNs). Although there are binding data on multiple PS ODNs presented here, the focus of this research is on the protein-binding characteristics of ISIS 2302, a PS ODN targeting human intercellular adhesion molecule-1 (ICAM-1) mRNA, which is currently in clinical trials for the treatment of ulcerative colitis. ISIS 2302 was shown to be highly bound (> 97%) across species (mouse, rat, monkey, human), with the mouse having the least degree of binding. ISIS 2302 was highly bound to albumin and, to a lesser, extent alpha2-macroglobulin and had negligible binding to alpha1-acid glycoprotein. Ten shortened ODN metabolites (8, 10, and 12-19 nucleotides [nt] in length, truncated from the 3' end) were evaluated in human plasma. The degree of binding was reduced as the ODN metabolite length decreased. Three additional 20-nt (20-mer) PS ODNs (ISIS 3521, ISIS 2503, and ISIS 5132) of varying sequence but similar chemistry were evaluated. Although the tested PS ODNs were highly bound to plasma proteins, suggesting a commonality within the chemical class, these results suggested that the protein-binding characteristics in human plasma may be sequence dependent. Lastly, drug displacement studies with ISIS 2302 and other concomitant drugs with known protein-binding properties were conducted to provide information on potential drug interactions. Coadministered ISIS 2302 and other high-binding drugs evaluated in this study did not displace one another at supraclinical plasma concentrations and, thus, are not anticipated to cause any pharmacokinetic interaction in the clinic as a result of the displacement of binding to plasma proteins.

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

    PubMed

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

    2006-01-01

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

  4. Optimization of hydrolysis conditions for bovine plasma protein using response surface methodology.

    PubMed

    Seo, Hyun-Woo; Jung, Eun-Young; Go, Gwang-Woong; Kim, Gap-Don; Joo, Seon-Tea; Yang, Han-Sul

    2015-10-15

    The purpose of this study was to establish optimal conditions for the hydrolysis of bovine plasma protein. Response surface methodology was used to model and optimize responses [degree of hydrolysis (DH), 2,2-diphenyl-1-picrydrazyl (DPPH) radical-scavenging activity and Fe(2+)-chelating activity]. Hydrolysis conditions, such as hydrolysis temperature (46.6-63.4 °C), hydrolysis time (98-502 min), and hydrolysis pH (6.32-9.68) were selected as the main processing conditions in the hydrolysis of bovine plasma protein. Optimal conditions for maximum DH (%), DPPH radical-scavenging activity (%) and Fe(2+)-chelating activity (%) of the hydrolyzed bovine plasma protein, were respectively established. We discovered the following three conditions for optimal hydrolysis of bovine plasma: pH of 7.82-8.32, temperature of 54.1 °C, and time of 338.4-398.4 min. We consequently succeeded in hydrolyzing bovine plasma protein under these conditions and confirmed the various desirable properties of optimal hydrolysis.

  5. Protein and energy intake during weaning. III. Effects on plasma amino acids.

    PubMed

    Axelsson, I; Borulf, S; Abildskov, K; Heird, W; Räihä, N

    1988-01-01

    Preprandial plasma amino acid concentrations were measured at 5 and 6 months of age in 30 healthy term infants who were either breast-fed ad libitum or fed one of two different formulas (1.9 g of protein per 100 ml with a whey:casein ratio of 50:50; 2.9 g of protein per 100 ml with a whey:casein ratio of 20:80) ad libitum, plus the same supplementary food regimen. The mean plasma concentrations of total amino acids and especially total essential amino acids were higher in the formula-fed infants. Those fed formula also had plasma concentrations of methionine, isoleucine, phenylalanine, leucine, valine, threonine, aspartate, proline, lysine, tyrosine, histidine that exceeded plasma concentrations of breast-fed infants by 2 or more standard deviations. Concentrations of arginine, glutamic acid, glutamine, ornithine, serine, cystine did not differ and taurine was higher in the breast-fed infants. The data indicate that formulas in common use today during weaning (4-6 months) provide excessive protein intakes when compared to the breast-fed control infants. A lowering of protein concentration and a further manipulation of the whey:casein ratio is necessary if plasma amino acid patterns similar to those found in breast-fed infants is to be achieved with artificial feeding.

  6. N-terminally myristoylated Ras proteins require palmitoylation or a polybasic domain for plasma membrane localization.

    PubMed

    Cadwallader, K A; Paterson, H; Macdonald, S G; Hancock, J F

    1994-07-01

    Plasma membrane targeting of Ras requires CAAX motif modifications together with a second signal from an adjacent polybasic domain or nearby cysteine palmitoylation sites. N-terminal myristoylation is known to restore membrane binding to H-ras C186S (C-186 is changed to S), a mutant protein in which all CAAX processing is abolished. We show here that myristoylated H-ras C186S is a substrate for palmitoyltransferase, despite the absence of C-terminal farnesylation, and that palmitoylation is absolutely required for plasma membrane targeting of myristoylated H-ras. Similarly, the polybasic domain is required for specific plasma membrane targeting of myristoylated K-ras. In contrast, the combination of myristoylation plus farnesylation results in the mislocalization of Ras to numerous intracellular membranes. Ras that is only myristoylated does not bind with a high affinity to any membrane. The specific targeting of Ras to the plasma membrane is therefore critically dependent on signals that are contained in the hypervariable domain but can be supported by N-terminal myristoylation or C-terminal prenylation. Interestingly, oncogenic Ras G12V that is localized correctly to the plasma membrane leads to mitogen-activated protein kinase activation irrespective of the combination of targeting signals used for localization, whereas Ras G12V that is mislocalized to the cytosol or to other membranes activates mitogen-activated protein kinase only if the Ras protein is farnesylated.

  7. Total Plasma Protein in Very Preterm Babies: Prognostic Value and Comparison with Illness Severity Scores

    PubMed Central

    Iacobelli, Silvia; Bonsante, Francesco; Quantin, Catherine; Robillard, Pierre-Yves; Binquet, Christine; Gouyon, Jean-Bernard

    2013-01-01

    Objective We aimed to investigate the predictive value for severe adverse outcome of plasma protein measurements on day one of life in very preterm infants and to compare total plasma protein levels with the validated illness severity scores CRIB, CRIB-II, SNAP-II and SNAPPE-II, regarding their predictive ability for severe adverse outcome. Methods We analyzed a cohort of infants born at 24–31 weeks gestation, admitted to the tertiary intensive care unit of a university hospital over 10.5 years. The outcome measure was “severe adverse outcome” defined as death before discharge or severe neurological injury on cranial ultrasound. The adjusted odd ratio (aOR) and 95% confidence interval (95% CI) of severe adverse outcome for hypoproteinemia (total plasma protein level <40 g/L) was calculated by univariate and multivariate analyses. Calibration (Hosmer-Lemeshow goodness-of-fit) was performed and the predictive ability for severe adverse outcome was assessed for total plasma protein and compared with CRIB, CRIB-II, SNAP-II and SNAPPE-II, by calculating receiver operating characteristic (ROC) curves and their associated area under the curve (AUC). Results 761 infants were studied: 14.4% died and 4.1% survived with severe cerebral ultrasound findings. The aOR of severe adverse outcome for hypoproteinemia was 6.1 (95% CI 3.8–9.9). The rank order for variables, as assessed by AUCs and 95% CIs, in predicting outcome was: total plasma protein [0.849 (0.821–0.873)], SNAPPE-II [0.822 (0.792–0.848)], CRIB [0.821 (0.792–0.848)], SNAP-II [0.810 (0.780–0.837)] and CRIB-II [0.803 (0.772–0.830)]. Total plasma protein predicted severe adverse outcome significantly better than CRIB-II and SNAP-II (both p<0.05). Calibration for total plasma protein was very good. Conclusions Early hypoproteinemia has prognostic value for severe adverse outcome in very preterm, sick infants. Total plasma protein has a predictive performance comparable with CRIB and SNAPPE-II and

  8. Determination of total protein in human seminal plasma.

    PubMed

    Hernvann, A; Gonzales, J; Diemert, M C; Galli, J

    1987-06-01

    Methodological problems are encountered in determinating human seminal protein content since results are influenced especially by techniques which are used, and by modifications due to liquefaction process. In order to test these two points, three methods (Folin, Biuret and Meulemans's reactions) were applied to the same seminal samples and results were compared. The differences seem principally due to the high level of glycoproteins. To determine the modifications of total protein content with time after semen collection, each sample was separated in aliquot fractions and proteolysis was blocked at different times for each fraction. There is an increase of the protein concentration during the first fifteen minutes that follows semen collection. The cause of this phenomenon is discussed.

  9. Isolation of human complex-forming glycoprotein, heterogeneous in charge (protein HC), and its IgA complex from plasma. Physiochemical and immunochemical properties, normal plasma concentration.

    PubMed

    Grubb, A O; López, C; Tejler, L; Mendez, E

    1983-12-10

    Human complex-forming glycoprotein, heterogeneous in charge (protein HC) has previously been isolated from urine and immunochemically shown to be present in low and high molecular weight forms in blood plasma (Tejler, L., and Grubb, A. O. (1976) Biochim. Biophys. Acta 439, 82-94). In the present work, the major low and high molecular weight forms of the protein were isolated from plasma by immunosorption followed by gel chromatography. The plasma low molecular weight protein HC and the urinary protein had similar, if not identical, molecular weight, amino acid composition, NH2-terminal and carboxyl-terminal amino acid sequences and electrophoretic mobility. The low molecular weight plasma protein HC carried a yellow chromophore like the urinary protein, but its molar extinction coefficient at 280 nm was lower and its charge heterogeneity less pronounced than that of urinary protein HC. The plasma high molecular weight protein HC had a hydrodynamic volume which was greater than that of monomeric IgA but smaller than that of dimeric IgA. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the isolated high molecular weight protein followed by electrophoretic blotting and immunochemical analysis demonstrated that the protein contained four polypeptide chains: two light immunoglobulin chains (Mr = 23,000), one IgA alpha-chain (Mr = 54,000), and one chain with Mr approximately 90,000 which carried both alpha-chain and protein HC antigenic determinants. Whether the protein HC X IgA complex is a functionally significant part of the humoral immune system cannot be decided without further experimentation, but the complex was found to be completely absent from the blood plasma of patients with a selective deficiency of IgA-secreting immunocytes. The isolated low and high molecular weight plasma protein HC components were used as standard proteins in the construction of a quantitative crossed immunoelectrophoretic assay for the simultaneous quantitation of the two

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

    PubMed

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

    2014-06-01

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

  11. Isolation and characterization of a new zinc-binding protein from albacore tuna plasma

    SciTech Connect

    Dyke, B.; Hegenauer, J.; Saltman, P.; Laurs, R.M.

    1987-06-02

    The protein responsible for sequestering high levels of zinc in the plasma of the albacore tuna (Thunnus alalunga) has been isolated by sequential chromatography. The glycoprotein has a molecular weight of 66,000. Approximately 8.2% of its amino acid residues are histidines. Equilibrium dialysis experiments show it to bind 3 mol of zinc/mol of protein. The stoichiometric constant for the association of zinc with a binding site containing three histidines was determined to be 10/sup 9.4/. This protein is different from albumin and represents a previously uncharacterized zinc transport protein.

  12. Maternal Low Quality Protein Diet Alters Plasma Amino Acid Concentrations of Weaning Rats.

    PubMed

    Kabasakal Cetin, Arzu; Dasgin, Halil; Gülec, Atila; Onbasilar, İlyas; Akyol, Asli

    2015-12-01

    Several studies have indicated the influence of a maternal low protein diet on the fetus. However, the effect of a maternal low quality protein diet on fetal growth and development is largely unknown. Wistar rats (11 weeks old) were mated and maintained on either a chow diet with 20% casein (n = 6) as the control group (C), or a low quality protein diet with 20% wheat gluten (n = 7) as the experimental group (WG) through gestation and lactation. Maternal body weights were similar in both groups throughout the study. Birth weights were not influenced by maternal diet and offspring body weights during lactation were similar between the groups. Offspring's plasma amino acid profiles showed that plasma methionine, glutamine and lysine were significantly lower and aspartic acid, ornithine and glycine-proline were significantly higher in the WG. Plant based protein comprises an important part of protein intake in developing countries. It is well-known that these diets can be inadequate in terms of essential amino acids. The current study shows differential effects of a maternal low quality protein diet on the offspring's plasma amino acids. Future studies will examine further aspects of the influence of maternal low quality protein diets on fetal growth and development.

  13. Maternal Low Quality Protein Diet Alters Plasma Amino Acid Concentrations of Weaning Rats

    PubMed Central

    Kabasakal Cetin, Arzu; Dasgin, Halil; Gülec, Atila; Onbasilar, İlyas; Akyol, Asli

    2015-01-01

    Several studies have indicated the influence of a maternal low protein diet on the fetus. However, the effect of a maternal low quality protein diet on fetal growth and development is largely unknown. Wistar rats (11 weeks old) were mated and maintained on either a chow diet with 20% casein (n = 6) as the control group (C), or a low quality protein diet with 20% wheat gluten (n = 7) as the experimental group (WG) through gestation and lactation. Maternal body weights were similar in both groups throughout the study. Birth weights were not influenced by maternal diet and offspring body weights during lactation were similar between the groups. Offspring’s plasma amino acid profiles showed that plasma methionine, glutamine and lysine were significantly lower and aspartic acid, ornithine and glycine-proline were significantly higher in the WG. Plant based protein comprises an important part of protein intake in developing countries. It is well-known that these diets can be inadequate in terms of essential amino acids. The current study shows differential effects of a maternal low quality protein diet on the offspring’s plasma amino acids. Future studies will examine further aspects of the influence of maternal low quality protein diets on fetal growth and development. PMID:26633475

  14. Mass spectrometric approach for identifying putative plasma membrane proteins of Arabidopsis leaves associated with cold acclimation.

    PubMed

    Kawamura, Yukio; Uemura, Matsuo

    2003-10-01

    Although enhancement of freezing tolerance in plants during cold acclimation is closely associated with an increase in the cryostability of plasma membrane, the molecular mechanism for the increased cryostability of plasma membrane is still to be elucidated. In Arabidopsis, enhanced freezing tolerance was detectable after cold acclimation at 2 degrees C for as short as 1 day, and maximum freezing tolerance was attained after 1 week. To identify the plasma membrane proteins that change in quantity in response to cold acclimation, a highly purified plasma membrane fraction was isolated from leaves before and during cold acclimation, and the proteins in the fraction were separated with gel electrophoresis. We found that there were substantial changes in the protein profiles after as short as 1 day of cold acclimation. Subsequently, using matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS), we identified 38 proteins that changed in quantity during cold acclimation. The proteins that changed in quantity during the first day of cold acclimation include those that are associated with membrane repair by membrane fusion, protection of the membrane against osmotic stress, enhancement of CO2 fixation, and proteolysis.

  15. A study of the interactions between carboplatin and blood plasma proteins using size exclusion chromatography coupled to inductively coupled plasma mass spectrometry.

    PubMed

    Xie, Ruimin; Johnson, Willie; Rodriguez, Lorna; Gounder, Murugesan; Hall, Gene S; Buckley, Brian

    2007-04-01

    To study the carboplatin-protein interaction, a sensitive method using size exclusion chromatography coupled to inductively coupled plasma mass spectrometry (SEC-ICP-MS) was developed. The complexes formed between plasma proteins and carboplatin were monitored and identified with this method. Composite blood plasma samples from patients who were undergoing chemotherapy were analyzed, and carboplatin was found to bind plasma proteins. In addition, blank plasma samples were spiked with carboplatin and were analyzed as a time course study, and the results confirmed that carboplatin formed complexes with plasma proteins, primarily albumin and gamma-globulin. To further substantiate the study, these two proteins were incubated with carboplatin. The binding between carboplatin and these proteins was then characterized qualitatively and quantitatively. In addition to a one-to-one binding of Pt to protein, protein aggregation was observed. The kinetics of the binding process of carboplatin to albumin and gamma-globulin was also studied. The initial reaction rate constant of carboplatin binding to albumin was determined to be 0.74 M(-1) min(-1), while that for gamma-globulin was 1.01 M(-1) min(-1), which are both lower than the rate constant of the cisplatin-albumin reaction previously reported.

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

    PubMed

    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.

  17. Using specialized adsorbents for remediation

    SciTech Connect

    Hochmuth, D.P.; Grant, A.

    1995-11-01

    This paper describes two remediation case studies in which specialized adsorbents were used. In one case, the adsorbents were used to treat effluent from a soil vapor extraction system. In the other case, the adsorbents were used to treat air from a groundwater air stripper. The specialized adsorbents effectively removed volatile organic compounds from each air stream.

  18. Garbage on, garbage off: new insights into plasma membrane protein quality control.

    PubMed

    MacGurn, Jason A

    2014-08-01

    Maintenance of cellular protein quality - by restoring misfolded proteins to their native state and by targeting terminally misfolded or damaged proteins for degradation - is a critical function of all cells. To ensure protein quality, cells have evolved various organelle-specific quality control mechanisms responsible for recognizing and responding to misfolded proteins at different subcellular locations of the cell. Recently, several publications have begun to elucidate mechanisms of quality control that operate at the plasma membrane (PM), recognizing misfolded PM proteins and targeting their endocytic trafficking and lysosomal degradation. Here, I discuss these recent developments in our understanding of PM quality control mechanisms and how they relate to global protein quality control strategies in the cell.

  19. Garbage on, Garbage off: New Insights into Plasma Membrane Protein Quality Control

    PubMed Central

    MacGurn, Jason A.

    2014-01-01

    Maintenance of cellular protein quality – by restoring misfolded proteins to their native state and by targeting terminally misfolded or damaged proteins for degradation – is a critical function of all cells. To ensure protein quality, cells have evolved various organelle-specific quality control mechanisms responsible recognizing and responding to misfolded proteins at different subcellular locations of the cell. Recently, several publications have begun to elucidate mechanisms of quality control that operate at the plasma membrane (PM), recognizing misfolded PM proteins and targeting their endocytic trafficking and lysosomal degradation. Here, I discuss these recent developments in our understanding of PM quality control mechanisms and how they relate to global protein quality control strategies in the cell. PMID:24908345

  20. Garbage on, garbage off: new insights into plasma membrane protein quality control.

    PubMed

    MacGurn, Jason A

    2014-08-01

    Maintenance of cellular protein quality - by restoring misfolded proteins to their native state and by targeting terminally misfolded or damaged proteins for degradation - is a critical function of all cells. To ensure protein quality, cells have evolved various organelle-specific quality control mechanisms responsible for recognizing and responding to misfolded proteins at different subcellular locations of the cell. Recently, several publications have begun to elucidate mechanisms of quality control that operate at the plasma membrane (PM), recognizing misfolded PM proteins and targeting their endocytic trafficking and lysosomal degradation. Here, I discuss these recent developments in our understanding of PM quality control mechanisms and how they relate to global protein quality control strategies in the cell. PMID:24908345

  1. Ethionine-dependent inhibition of acute-phase plasma protein synthesis in the rat.

    PubMed Central

    Kasperczyk, H.; Koj, A.

    1983-01-01

    Ethionine administered intraperitoneally to rats suffering from turpentine-induced inflammation preferentially reduced incorporation of 14C-leucine into fibrinogen, haptoglobin and other acute-phase proteins. The inhibitory effect was observed both in vivo and in liver slices obtained from ethionine-treated donors, while addition of ethionine to liver slices in vitro led to general reduction of synthesis of all liver and plasma proteins, including albumin. For comparison, the effects of galactosamine and actinomycin D on plasma protein synthesis in injured rats were also examined. It has been concluded that ethionine acts in the early phases of the acute-phase response, probably by inhibition of trauma-induced transcription of liver mRNA specific for acute-phase proteins. PMID:6882676

  2. Cell wall constrains lateral diffusion of plant plasma-membrane proteins

    PubMed Central

    Martinière, Alexandre; Lavagi, Irene; Nageswaran, Gayathri; Rolfe, Daniel J.; Maneta-Peyret, Lilly; Luu, Doan-Trung; Botchway, Stanley W.; Webb, Stephen E. D.; Mongrand, Sebastien; Maurel, Christophe; Martin-Fernandez, Marisa L.; Kleine-Vehn, Jürgen; Friml, Jirí; Moreau, Patrick; Runions, John

    2012-01-01

    A cell membrane can be considered a liquid-phase plane in which lipids and proteins theoretically are free to diffuse. Numerous reports, however, describe retarded diffusion of membrane proteins in animal cells. This anomalous diffusion results from a combination of structuring factors including protein–protein interactions, cytoskeleton corralling, and lipid organization into microdomains. In plant cells, plasma-membrane (PM) proteins have been described as relatively immobile, but the control mechanisms that structure the PM have not been studied. Here, we use fluorescence recovery after photobleaching to estimate mobility of a set of minimal PM proteins. These proteins consist only of a PM-anchoring domain fused to a fluorescent protein, but their mobilities remained limited, as is the case for many full-length proteins. Neither the cytoskeleton nor membrane microdomain structure was involved in constraining the diffusion of these proteins. The cell wall, however, was shown to have a crucial role in immobilizing PM proteins. In addition, by single-molecule fluorescence imaging we confirmed that the pattern of cellulose deposition in the cell wall affects the trajectory and speed of PM protein diffusion. Regulation of PM protein dynamics by the plant cell wall can be interpreted as a mechanism for regulating protein interactions in processes such as trafficking and signal transduction. PMID:22689944

  3. Membrane potential governs lateral segregation of plasma membrane proteins and lipids in yeast.

    PubMed

    Grossmann, Guido; Opekarová, Miroslava; Malinsky, Jan; Weig-Meckl, Ina; Tanner, Widmar

    2007-01-10

    The plasma membrane potential is mainly considered as the driving force for ion and nutrient translocation. Using the yeast Saccharomyces cerevisiae as a model organism, we have discovered a novel role of the membrane potential in the organization of the plasma membrane. Within the yeast plasma membrane, two non-overlapping sub-compartments can be visualized. The first one, represented by a network-like structure, is occupied by the proton ATPase, Pma1, and the second one, forming 300-nm patches, houses a number of proton symporters (Can1, Fur4, Tat2 and HUP1) and Sur7, a component of the recently described eisosomes. Evidence is presented that sterols, the main lipid constituent of the plasma membrane, also accumulate within the patchy compartment. It is documented that this compartmentation is highly dependent on the energization of the membrane. Plasma membrane depolarization causes reversible dispersion of the H(+)-symporters, not however of the Sur7 protein. Mitochondrial mutants, affected in plasma membrane energization, show a significantly lower degree of membrane protein segregation. In accordance with these observations, depolarized membranes also considerably change their physical properties (detergent sensitivity).

  4. Adsorption of protein streptavidin to the plasma treated surface of polystyrene

    NASA Astrophysics Data System (ADS)

    Vesel, Alenka; Elersic, Kristina

    2012-05-01

    Immobilization of protein streptavidin to the surface of polystyrene (PS) polymer was studied by X-ray photoelectron spectroscopy (XPS). Two different protocols were used to attach streptavidin to the PS surface: physical adsorption and chemical coupling. In both cases the surface properties of PS samples were modified by exposure to cold oxygen plasma for 10 s. Plasma was created in oxygen at 75 Pa by en electrode-less RF discharge. The RF generator operated at 27.12 MHz and the nominal power was about 120 W. The electron temperature was about 3 eV, the plasma density was about 3 × 1015 m-3 and the neutral oxygen atom density was about 3 × 1021 m-3. Oxygen plasma treatment caused formation of O-rich functional groups on the surface of PS. The concentration of oxygen was determined by XPS and was about 28 at.%. A thin film of streptavidin was deposited by physical adsorption and chemical bonding. The appearance of streptavidin on the surface was determined from XPS spectra measuring the ratio between N and C peaks. The plasma treatment caused poor adsorption and but strong chemisorption of streptavidin. The results were explained by specific interaction of protein with polar functional groups on the surface of PS after plasma treatment.

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

    SciTech Connect

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

    1985-10-01

    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.

  6. In Situ Synthesis of Porous Carbons by Using Room-Temperature, Atmospheric-Pressure Dielectric Barrier Discharge Plasma as High-Performance Adsorbents for Solid-Phase Microextraction.

    PubMed

    Lin, Yao; Wu, Li; Xu, Kailai; Tian, Yunfei; Hou, Xiandeng; Zheng, Chengbin

    2015-09-21

    A one-step, template-free method is described to synthesize porous carbons (PCs) in situ on a metal surface by using a room-temperature, atmospheric-pressure dielectric barrier discharge (DBD) plasma. This method not only features high efficiency, environmentally friendliness, and low cost and simple equipment, but also can conveniently realize large-area synthesis of PCs by only changing the design of the DBD reactor. The synthesized PCs have a regulated nestlike morphology, and thus, provide a high specific surface area and high pore volume, which result in excellent adsorption properties. Its applicability was demonstrated by using a PC-coated stainless-steel fiber as a solid-phase microextraction (SPME) fiber to preconcentrate polycyclic aromatic hydrocarbons (PAHs) prior to analysis by gas chromatography with flame ionization detection (GC-FID). The results showed that the fiber exhibited excellent enrichment factors (4.1×10(4) to 3.1×10(5)) toward all tested PAHs. Thus, the PC-based SPME-GC-FID provides low limits of detection (2 to 20 ng L(-1)), good precision (<7.8%), and good recoveries (80-115%) for ultra-sensitive determination of PAHs in real water samples. In addition, the PC-coated fiber could be stable enough for more than 500 replicate extraction cycles. PMID:26267394

  7. Stimulus-dependent secretion of plasma proteins from human neutrophils.

    PubMed Central

    Borregaard, N; Kjeldsen, L; Rygaard, K; Bastholm, L; Nielsen, M H; Sengeløv, H; Bjerrum, O W; Johnsen, A H

    1992-01-01

    In search for matrix proteins released from secretory vesicles of human neutrophils, a prominent 67-kD protein was identified in the extracellular medium of neutrophils stimulated by the chemotactic peptide, FMLP. The protein was purified to apparent homogeneity and partially sequenced. The sequence of the first 32 NH2-terminal amino acids was identical to the sequence of albumin. mRNA for human albumin could not be detected in bone marrow cells, nor could biosynthetic labeling of albumin be demonstrated in bone marrow cells during incubation with [14C]leucine. Immunofluorescence studies on single cells demonstrated the presence of intracellular albumin in fixed permeabilized neutrophils. Light microscopy of immunogold-silver-stained cryosections visualized albumin in cytoplasmic "granules." The morphology of these was determined by immunoelectron microscopy as vesicles of varying form and size. Subcellular fractionation studies on unstimulated neutrophils demonstrated the presence of albumin in the low density pre-gamma and gamma-regions that contain secretory vesicles, but are devoid of specific granules and azurophil granules. Albumin was readily released from these structures during activation of neutrophils with inflammatory mediators. Immunoblotting demonstrated the presence of immunoglobulin and transferrin along with albumin in exocytosed material from stimulated neutrophils. This indicates that secretory vesicles are unique endocytic vesicles that can be triggered to exocytose by inflammatory stimuli. Images PMID:1378856

  8. Adsorbed serum albumin is permissive to macrophage attachment to perfluorocarbon polymer surfaces in culture

    PubMed Central

    Godek, M.L.; Michel, R.; Chamberlain, L. M.; Castner, D. G.; Grainger, D.W.

    2013-01-01

    Monocyte/macrophage adhesion to biomaterials, correlated with foreign body response, occurs through protein-mediated surface interactions. Albumin-selective perfluorocarbon (FC) biomaterials are generally poorly cell-conducive due to insufficient receptor-mediated surface interactions, but macrophages bind to albumin-coated substrates and also preferentially to highly hydrophobic fluorinated surfaces. Bone marrow macrophages (BMMO) and IC-21, RAW 264.7 and J774A.1 monocyte/macrophage cells were cultured on FC surfaces. Protein deposition onto two distinct FC surfaces from complex and single-component solutions was tracked using fluorescence and time-of-flight secondary ion mass spectrometry (ToF-SIMS) methods. Cell adhesion and growth on protein pre-treated substrates were compared by light microscopy. Flow cytometry and integrin-directed antibody receptor blocking assessed integrins critical for monocyte/macrophage adhesion in vitro. Albumin predominantly adsorbs onto both FC surfaces from 10% serum. In cultures pre-adsorbed with albumin or serum-dilutions, BMMO responded similar to IC-21 at early time points. Compared to Teflon® AF, plasma-polymerized FC was less permissive to extended cell proliferation. The β2 integrins play major roles in macrophage adhesion to FC surfaces: antibody blocking significantly disrupted cell adhesion. Albumin-mediated cell adhesion mechanisms to FC surfaces could not be clarified. Primary BMMO and secondary IC-21 macrophages behave similarly on FC surfaces, regardless of pre-adsorbed protein biasing, with respect to adhesion, cell morphology, motility and proliferation. PMID:18306309

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

    PubMed

    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.

  10. Proteomic profiling of human plasma exosomes identifies PPAR{gamma} as an exosome-associated protein

    SciTech Connect

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

    2009-01-16

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

  11. Circulating plasma cholesteryl ester transfer protein activity and blood pressure tracking in the community

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Clinical trials using cholesteryl ester transfer protein (CETP) inhibitors to raise high-density lipoprotein cholesterol (HDL-C) concentrations reported an 'off-target' blood pressure (BP) raising effect. We evaluated the relations of baseline plasma CETP activity and longitudinal BP change. One tho...

  12. PLASMA PROTEIN PROFILING AS A HIGH THROUGHPUT TOOL FOR CHEMICAL SCREENING USING A SMALL FISH MODEL

    EPA Science Inventory

    Hudson, R. Tod, Michael J. Hemmer, Kimberly A. Salinas, Sherry S. Wilkinson, James Watts, James T. Winstead, Peggy S. Harris, Amy Kirkpatrick and Calvin C. Walker. In press. Plasma Protein Profiling as a High Throughput Tool for Chemical Screening Using a Small Fish Model (Abstra...

  13. Heterogeneity of Arabinogalactan-Proteins on the Plasma Membrane of Rose Cells.

    PubMed Central

    Serpe, M. D.; Nothnagel, E. A.

    1996-01-01

    Arabinogalactan-proteins (AGPs) have been purified from the plasma membrane of suspension-cultured Paul's Scarlet rose (Rosa sp.) cells. The two most abundant and homogeneous plasma membrane AGP fractions were named plasma membrane AGP1 (PM-AGP1) and plasma membrane AGP2 (PM-AGP2) and had apparent molecular masses of 140 and 217 kD, respectively. Both PM-AGP1 and PM-AGP2 had [beta]-(1-3)-, [beta]-(1,6)-, and [beta]-(1,3,6)-galactopyranosyl residues, predominantly terminal [alpha]-arabinofuranosyl residues, and (1,4)- and terminal glucuronopyranosyl residues. The protein moieties of PM-AGP1 and PM-AGP2 were both rich in hydroxyproline, alanine, and serine, but differed in the abundance of hydroxyproline, which was 1.6 times higher in PM-AGP2 than in PM-AGP1. Another difference was the overall protein content, which was 3.7% (w/w) in PM-AGP1 and 15% in PM-AGP2. As judged by their behavior on reverse-phase chromatography, PM-AGP1 and PM-AGP2 were not more hydrophobic than AGPs from the cell wall or culture medium. In contrast, a minor plasma membrane AGP fraction eluted later on reverse-phase chromatography and was more negatively charged at pH 5 than either PM-AGP1 or PM-AGP2. The more negatively charged fraction contained molecules with a glycosyl composition characteristic of AGPs and included at least two different macromolecules. The results of this investigation indicate that Rosa plasma membrane contains at least four distinct AGPs or AGP-like molecules. These molecules differed from each other in size, charge, hydrophobicity, amino-acyl composition, and/or protein content. PMID:12226444

  14. Regenerative adsorbent heat pump

    NASA Technical Reports Server (NTRS)

    Jones, Jack A. (Inventor)

    1991-01-01

    A regenerative adsorbent heat pump process and system is provided which can regenerate a high percentage of the sensible heat of the system and at least a portion of the heat of adsorption. A series of at least four compressors containing an adsorbent is provided. A large amount of heat is transferred from compressor to compressor so that heat is regenerated. The process and system are useful for air conditioning rooms, providing room heat in the winter or for hot water heating throughout the year, and, in general, for pumping heat from a lower temperature to a higher temperature.

  15. Malondialdehyde and 4-hydroxynonenal protein adducts in plasma and liver of rats with iron overload.

    PubMed Central

    Houglum, K; Filip, M; Witztum, J L; Chojkier, M

    1990-01-01

    In hepatic iron overload, iron-catalyzed lipid peroxidation has been implicated in the mechanisms of hepatocellular injury. Lipid peroxidation may produce reactive aldehydes such as malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE), which may form aldehyde-protein adducts. We investigated whether lipid peroxidation occurred in rats fed a diet containing 3% carbonyl iron for 5-13 wk, and if this resulted in the formation of MDA- and 4-HNE- protein adducts. Chronic iron feeding resulted in hepatic iron overload (greater than 10-fold) and concomitantly induced a 2-fold increase in hepatic lipid peroxidation. Using an antiserum specific for MDA-lysine protein adducts, we demonstrated by immunohistochemistry the presence of aldehyde-protein adducts in the cytosol of periportal hepatocytes, which co-localized with iron. In addition, MDA- and 4-HNE-lysine adducts were found in plasma proteins of animals with iron overload. Only MDA adducts were detected in albumin, while other plasma proteins including a approximately 120-kD protein had both MDA and 4-HNE adducts. In this animal model of hepatic iron overload, injury occurs primarily in periportal hepatocytes, where MDA-lysine protein adducts and excess iron co-localized. Images PMID:2123889

  16. Plasma Membrane Intrinsic Proteins from Maize Cluster in Two Sequence Subgroups with Differential Aquaporin Activity1

    PubMed Central

    Chaumont, François; Barrieu, François; Jung, Rudolf; Chrispeels, Maarten J.

    2000-01-01

    The transport of water through membranes is regulated in part by aquaporins or water channel proteins. These proteins are members of the larger family of major intrinsic proteins (MIPs). Plant aquaporins are categorized as either tonoplast intrinsic proteins (TIPs) or plasma membrane intrinsic proteins (PIPs). Sequence analysis shows that PIPs form several subclasses. We report on the characterization of three maize (Zea mays) PIPs belonging to the PIP1 and PIP2 subfamilies (ZmPIP1a, ZmPIP1b, and ZmPIP2a). The ZmPIP2a clone has normal aquaporin activity in Xenopus laevis oocytes. ZmPIP1a and ZmPIP1b have no activity, and a review of the literature shows that most PIP1 proteins identified in other plants have no or very low activity in oocytes. Arabidopsis PIP1 proteins are the only exception. Control experiments show that this lack of activity of maize PIP1 proteins is not caused by their failure to arrive at the plasma membrane of the oocytes. ZmPIP1b also does not appear to facilitate the transport of any of the small solutes tried (glycerol, choline, ethanol, urea, and amino acids). These results are discussed in relationship to the function and regulation of the PIP family of aquaporins. PMID:10759498

  17. Label-free quantitative analysis for studying the interactions between nanoparticles and plasma proteins.

    PubMed

    Capriotti, Anna Laura; Caracciolo, Giulio; Caruso, Giuseppe; Cavaliere, Chiara; Pozzi, Daniela; Samperi, Roberto; Laganà, Aldo

    2013-01-01

    A shotgun proteomics approach was used to compare human plasma protein binding capability with cationic liposomes, DNA-cationic lipid complexes (lipoplexes), and lipid-polycation-DNA (LPD) complexes. Nano-high-performance liquid chromatography coupled with a high-resolution LTQ Orbitrap XL mass spectrometer was used to characterize and compare their protein corona. Spectral counting and area under curve methods were used to perform label-free quantification. Substantial qualitative and quantitative differences were found among proteins bound to the three different systems investigated. Protein variety found on lipoplexes and LPD complexes was richer than that found on cationic liposomes. There were also significant differences between the amounts of protein. Such results could help in the design of gene-delivery systems, because some proteins could be more selectively bound rather than others, and their bio-distribution could be driven in vivo for more efficient and effective gene therapy.

  18. Presence of lunasin in plasma of men after soy protein consumption.

    PubMed

    Dia, Vermont P; Torres, Sofia; De Lumen, Ben O; Erdman, John W; De Mejia, Elvira Gonzalez

    2009-02-25

    Lunasin is a 43-amino acid bioactive peptide from soybean and other plant sources which is reported to possess anti-inflammatory and anticancer properties. The objective of this study was to assess the presence and concentration of lunasin in blood of men fed soy protein products. Five healthy male subjects who were 18-25 years old consumed 50 g of soy protein for 5 days, and blood was taken 30 min and 1 h after soy protein ingestion on day 5. Lunasin was isolated from plasma using strong anion exchange beads in a magnetic particle concentrator and eluted with 20 mM triethanolamine at pH 8.0 with 0.20 M NaCl. The concentration of lunasin in plasma as determined by an enzyme-linked immunosorbent assay ranged in the various subjects from 50.2 to 110.6 ng/mL of plasma (average +/- standard deviation, 66.0 +/- 25.4 ng/mL) for blood taken at 30 min and from 33.5 to 122.7 ng/mL of plasma (71.0 +/- 32.8 ng/mL) for blood withdrawn 1 h after ingestion on day 5. We estimated an average of 4.5% absorption (range of 2.2-7.8%) of lunasin from the total lunasin ingested from 50 g of soy protein. Matrix-assisted laser desorption ionization time-of-flight peptide mass mapping showed that a 5 kDa peptide similar to synthetic lunasin was present in plasma samples of people who consumed soy protein while absent at the baseline plasma samples from the same individuals. Liquid chromatography-tandem mass spectrometry analysis showed the presence of amino acid sequences from lunasin in plasma samples after soy intake for 30 min and 1 h. No peptides from lunasin were present in plasma samples without soy intake. The results of this study suggest that lunasin is bioavailable in humans, an important requirement for its anticancer potential.

  19. GPI-anchored proteins do not reside in ordered domains in the live cell plasma membrane

    PubMed Central

    Sevcsik, Eva; Brameshuber, Mario; Fölser, Martin; Weghuber, Julian; Honigmann, Alf; Schütz, Gerhard J.

    2015-01-01

    The organization of proteins and lipids in the plasma membrane has been subject of a long-lasting debate. Membrane rafts of higher lipid chain order were proposed to mediate protein interactions, but have thus far not been directly observed. Here, we use protein micropatterning combined with single-molecule tracking to put current models to the test: we rearranged lipid-anchored raft proteins (glycosylphosphatidylinositol(GPI)-anchored mGFP) directly in the live cell plasma membrane and measured the effect on the local membrane environment. Intriguingly, this treatment does neither nucleate the formation of an ordered membrane phase, nor result in any enrichment of nanoscopic ordered domains within the micropatterned regions. In contrast, we find that immobilized mGFP-GPIs behave as inert obstacles to the diffusion of other membrane constituents without influencing their membrane environment over distances beyond their physical size. Our results indicate that phase partitioning is not a fundamental element of protein organization in the plasma membrane. PMID:25897971

  20. GPI-anchored proteins do not reside in ordered domains in the live cell plasma membrane

    NASA Astrophysics Data System (ADS)

    Sevcsik, Eva; Brameshuber, Mario; Fölser, Martin; Weghuber, Julian; Honigmann, Alf; Schütz, Gerhard J.

    2015-04-01

    The organization of proteins and lipids in the plasma membrane has been the subject of a long-lasting debate. Membrane rafts of higher lipid chain order were proposed to mediate protein interactions, but have thus far not been directly observed. Here we use protein micropatterning combined with single-molecule tracking to put current models to the test: we rearranged lipid-anchored raft proteins (glycosylphosphatidylinositol(GPI)-anchored-mGFP) directly in the live cell plasma membrane and measured the effect on the local membrane environment. Intriguingly, this treatment does neither nucleate the formation of an ordered membrane phase nor result in any enrichment of nanoscopic-ordered domains within the micropatterned regions. In contrast, we find that immobilized mGFP-GPIs behave as inert obstacles to the diffusion of other membrane constituents without influencing their membrane environment over distances beyond their physical size. Our results indicate that phase partitioning is not a fundamental element of protein organization in the plasma membrane.

  1. Moesin, ezrin, and p205 are actin-binding proteins associated with neutrophil plasma membranes.

    PubMed Central

    Pestonjamasp, K; Amieva, M R; Strassel, C P; Nauseef, W M; Furthmayr, H; Luna, E J

    1995-01-01

    Actin-binding proteins in bovine neutrophil plasma membranes were identified using blot overlays with 125I-labeled F-actin. Along with surface-biotinylated proteins, membranes were enriched in major actin-binding polypeptides of 78, 81, and 205 kDa. Binding was specific for F-actin because G-actin did not bind. Further, unlabeled F-actin blocked the binding of 125I-labeled F-actin whereas other acidic biopolymers were relatively ineffective. Binding also was specifically inhibited by myosin subfragment 1, but not by CapZ or plasma gelsolin, suggesting that the membrane proteins, like myosin, bind along the sides of the actin filaments. The 78- and 81-kDa polypeptides were identified as moesin and ezrin, respectively, by co-migration on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoprecipitation with antibodies specific for moesin and ezrin. Although not present in detectable amounts in bovine neutrophils, radixin (a third and closely related member of this gene family) also bound 125I-labeled F-actin on blot overlays. Experiments with full-length and truncated bacterial fusion proteins localized the actin-binding site in moesin to the extreme carboxy terminus, a highly conserved sequence. Immunofluorescence micrographs of permeabilized cells and cell "footprints" showed moesin co-localization with actin at the cytoplasmic surface of the plasma membrane, consistent with a role as a membrane-actin-linking protein. Images PMID:7612961

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

    NASA Astrophysics Data System (ADS)

    Kylián, O.; Rauscher, H.; Gilliland, D.; Brétagnol, F.; Rossi, F.

    2008-05-01

    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.

  3. Extensive dataset of boar seminal plasma proteome displaying putative reproductive functions of identified proteins.

    PubMed

    Perez-Patiño, Cristina; Barranco, Isabel; Parrilla, Inmaculada; Martinez, Emilio A; Rodriguez-Martinez, Heriberto; Roca, Jordi

    2016-09-01

    A complete proteomic profile of seminal plasma (SP) remains challenging, particularly in porcine. The data reports on the analysis of boar SP-proteins by using a combination of SEC, 1-D SDS PAGE and NanoLC-ESI-MS/MS from 33 pooled SP-samples (11 boars, 3 ejaculates/boar). A complete dataset of the 536 SP-proteins identified and validated with confidence ≥95% (Unused Score >1.3) and a false discovery rate (FDR) ≤1%, is provided. In addition, the relative abundance of 432 of them is also shown. Gene ontology annotation of the complete SP-proteome complemented by an extensive description of the putative reproductive role of SP-proteins, providing a valuable source for a better understanding of SP role in the reproductive success. This data article refers to the article entitled "Characterization of the porcine seminal plasma proteome comparing ejaculate portions" (Perez-Patiño et al., 2016) [1]. PMID:27583342

  4. Proteomic analysis of mouse liver plasma membrane: use of differential extraction to enrich hydrophobic membrane proteins.

    PubMed

    Zhang, Lijun; Xie, Jinyun; Wang, Xi'e; Liu, Xiaohui; Tang, Xinke; Cao, Rui; Hu, Weijun; Nie, Song; Fan, Chunming; Liang, Songping

    2005-11-01

    To comprehensively identify proteins of liver plasma membrane (PM), we isolated PMs from mouse liver by sucrose density gradient centrifugation. An optimized extraction method for whole PM proteins and several methods of differential extraction expected to enrich hydrophobic membrane proteins were tested. The extracted PM proteins were separated by 2-DE, and were identified by MALDI-TOF-MS, and ESI-quadrupole-TOF MS. As the complementary method, 1-DE-MS/MS was also used to identify PM proteins. The optimized lysis buffer containing urea, thiourea, CHAPS and NP-40 was able to extract more PM proteins, and treatment of PM samples with chloroform/methanol and sodium carbonate led to enrichment of more hydrophobic PM proteins. From the mouse liver PM fraction, 175 non-redundant gene products were identified, of which 88 (about 50%) were integral membrane proteins with one to seven transmembrane domains. The remaining products were probably membrane-associated and cytosolic proteins. The function distribution of all the identified liver PM proteins was analyzed; 40% represented enzymes, 12% receptors and 9% proteins with unknown function.

  5. Electrochemical characterization of albumin protein on Ti-6AL-4V alloy immersed in a simulated plasma solution.

    PubMed

    Padilla, Norma; Bronson, Arturo

    2007-06-01

    The effect of oxygen and albumin on the electrochemical behavior of a Ti-6Al-4V alloy immersed in a simulated inorganic plasma (SIP) solution was studied with a rotating-cylindrical electrode configuration to focus on the surface/electrolyte reactions. Potentiokinetic scans and electrochemical impedance spectroscopy have been used to characterize the interface by determining the passive current density and capacitance. For the polarization scans, an albumin addition of 37.7 mg/cm(3) to the SIP solution (oxygenated and unoxygenated) decreased the passive current density, indicating a lowering of the corrosive rate. The surface capacitance for the Ti-6Al-4V alloy immersed in a SIP solution averaged 13 microF/cm(2), which transformed after albumin addition (37.7 mg/cm(3)) from a potential independent behavior to the capacitance ranging from 23 to 6 microF/cm(2) with increasing potentials from -800 to 1500 mV(SCE), respectively, indicative of albumin adsorption. Within the same potential range and albumin addition to oxygenated solutions, the capacitances expanded slightly with a similar decreasing trend from 31 to 6 microF/cm(2), although the capacitance depicts an interaction between the hydrated passive film and the adsorbed albumin from -550 to 500 mV(SCE) in which the capacitance plateaued at 15 microF/cm(2). The hydrated porous oxide film results from the porous rutile layer reacting with H(2)O(2) formed as an intermediary component of oxygen reduction at the Ti-6Al-4V surface. The passive film-albumin interaction would affect the processing of titanium alloys in their surface preparation for biocompatibility, as well as determining the reactivity of titanium alloys to proteins.

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

    SciTech Connect

    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.

  7. Characterization of Differential Protein Tethering at the Plasma Membrane in Response to Epidermal Growth Factor Signaling

    PubMed Central

    Looyenga, Brendan D.; MacKeigan, Jeffrey P.

    2013-01-01

    Physical tethering of membrane proteins to the cortical actin cytoskeleton provides functional organization to the plasma membrane and contributes to diverse cellular processes including cell signaling, vesicular trafficking, endocytosis, and migration. For these processes to occur, membrane protein tethering must be dynamically regulated in response to environmental cues. In this study, we describe a novel biochemical scheme for isolating the complement of plasma membrane proteins that are physically tethered to the actin cytoskeleton. We utilized this method in combination with tandem liquid chromatography/mass spectrometry (LC–MS/MS) to demonstrate that cytoskeletal tethering of membrane proteins is acutely regulated by epidermal growth factor (EGF) in normal human kidney (HK2) cells. Our results indicate that several proteins known to be involved in EGF signaling, as well as other proteins not traditionally associated with this pathway, are tethered to the cytoskeleton in dynamic fashion. Further analysis of one hit from our proteomic survey, the receptor phosphotyrosine phosphatase PTPRS, revealed a correlation between cytoskeletal tethering and endosomal trafficking in response to EGF. This finding parallels previous indications that PTPRS is involved in the desensitization of EGFR and provides a potential mechanism to coordinate localization of these two membrane proteins in the same compartment upon EGFR activation. PMID:22559174

  8. Determination of protein carbonyls in plasma, cell extracts, tissue homogenates, isolated proteins: Focus on sample preparation and derivatization conditions

    PubMed Central

    Weber, Daniela; Davies, Michael J.; Grune, Tilman

    2015-01-01

    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. PMID:26141921

  9. Adsorbent and adsorbent bed for materials capture and separation processes

    SciTech Connect

    Liu, Wei

    2011-01-25

    A method device and material for performing adsorption wherein a fluid mixture is passed through a channel in a structured adsorbent bed having a solid adsorbent comprised of adsorbent particles having a general diameter less than 100 um, loaded in a porous support matrix defining at least one straight flow channel. The adsorbent bed is configured to allow passage of a fluid through said channel and diffusion of a target material into said adsorbent under a pressure gradient driving force. The targeted molecular species in the fluid mixture diffuses across the porous support retaining layer, contacts the adsorbent, and adsorbs on the adsorbent, while the remaining species in the fluid mixture flows out of the channel.

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

    PubMed

    Jessen, Flemming; Wulff, Tune

    2015-09-15

    A simple and reproducible procedure for enrichment of a plasma protein subfraction suitable for two-dimensional polyacrylamide gel electrophoresis (2DE) was developed, using a Triton X-114-based cloud point extraction (CPE). Appropriate conditions for such a CPE procedure were found by SDS-PAGE to be a plasma protein concentration of about 10mg/ml in 3% (w/v) Triton X-114. 2DE of proteins obtained by CPE of 400 μl of human plasma revealed about 200 spots constituting a spot pattern very different from the pattern of total plasma. The CPE procedure only had a limited contribution to the technical variation. Identification of about 60 spots, representing only 22 proteins, revealed that several proteins in the obtained subfraction were present in more isoforms or modifications. Among these were apolipoproteins (A-1, D, E, L1, and M), haptoglobin-related protein, phosphatidylcholine-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 and their isoforms or modifications.

  11. Protein composition in human plasma after long-term orbital missions and in rodent plasma after spaceflights on biosatellites "Cosmos-1887" and "Cosmos-2044".

    PubMed

    Larina, O N

    1991-02-01

    The two-dimensional plasma protein map of crewmembers of long-duration "Mir" expeditions obtained the day after the recovery shows a manifold increase in the content of several proteins normally seen in trace amounts. The emergence of several unusual protein spots occurs as well, some of them probably due to charge shifts provided by the events influencing posttranslational modification processes. By the 8 postflight day these phenomena were disappeared. In the "Cosmos-1887" biosatellite experiment, the plasma samples obtained two days after the landing as well as plasma of synchronous animals exhibited the higher fibrinogen levels when compared to those of vivarium animals. The protein consisting of a number of fractions with molecular weight of 50 to 60 kD and pI 5 to 6 had protein spots of similar size in flight and synchronous animals while in vivarium rats one of the spots was larger in size as opposed to the others. The plasma protein spectrum of flight and synchronous groups of animals in "Cosmos-1887" experiment where plasma samples were prepared in the period of time from 5 to 10 hours after spaceflight coincided with the pattern of vivarium animals. The data suggest that the protein changes described above develop during postflight period and accelerations, vibrations, readaptation to 1 G gravity, emotional stress could be the cause of these alterations.

  12. Does smoking, age or gender affect the protein phenotype of extracellular vesicles in plasma?

    PubMed

    Bæk, R; Varming, K; Jørgensen, M M

    2016-08-01

    Extracellular vesicles (EVs) are involved in several diseases, which have formed the basis for the potential use of EV analyses in a clinical setting. The protein phenotype of EVs can provide information on the functionality of the vesicles and may be used for identification of disease-related biomarkers. With this extensive study of 161 healthy individuals it was elucidated that certain markers of plasma EVs are influenced by demographic variations such as gender, age and smoking status. When the purpose is to use EVs as a diagnostic tool, it should be emphasized how important it is to choose the correct demographic group when comparing marker levels of plasma EVs. PMID:27470710

  13. Plasma membrane protein trafficking in plant–microbe interactions: a plant cell point of view

    PubMed Central

    Nathalie Leborgne-Castel; Bouhidel, Karim

    2014-01-01

    In order to ensure their physiological and cellular functions, plasma membrane (PM) proteins must be properly conveyed from their site of synthesis, i.e., the endoplasmic reticulum, to their final destination, the PM, through the secretory pathway. PM protein homeostasis also relies on recycling and/or degradation, two processes that are initiated by endocytosis. Vesicular membrane trafficking events to and from the PM have been shown to be altered when plant cells are exposed to mutualistic or pathogenic microbes. In this review, we will describe the fine-tune regulation of such alterations, and their consequence in PM protein activity. We will consider the formation of intracellular perimicrobial compartments, the PM protein trafficking machinery of the host, and the delivery or retrieval of signaling and transport proteins such as pattern-recognition receptors, producers of reactive oxygen species, and sugar transporters. PMID:25566303

  14. Treatment of Second-Order Structures of Proteins Using Oxygen Radio Frequency Plasma

    NASA Astrophysics Data System (ADS)

    Hayashi, Nobuya; Nakahigashi, Akari; Liu, Hao; Goto, Masaaki

    2010-08-01

    Decomposition characteristics of second-order structures of proteins are determined using an oxygen radio frequency (RF) plasma sterilizer in order to prevent infectious proteins from contaminating medical equipment in hospitals. The removal of casein protein as a test protein with a concentration of 50 mg/cm2 on the plane substrate requires approximately 8 h when singlet atomic oxygen is irradiated. The peak intensity of Fourier transform infrared spectroscopy (FTIR) spectra of the β-sheet structures decreases at approximately the same rate as those of the α-helix and first-order structures of proteins. Active oxygen has a sufficient oxidation energy to dissociate hydrogen bonds within the β-sheet structure.

  15. A proteomic study reveals the diversified distribution of plasma membrane-associated proteins in rat hepatocytes.

    PubMed

    Li, Xuanwen; Cao, Jia; Jin, Qihui; Xie, Chunliang; He, Quanyuan; Cao, Rui; Xiong, Jixian; Chen, Ping; Wang, Xianchun; Liang, Songping

    2008-06-01

    To investigate the heterogeneous protein composition of highly polarized hepatocyte plasma membrane (PM), three PM-associated subfractions were obtained from freshly isolated rat hepatocytes using density gradient centrifugation. The origins of the three subfractions were determined by morphological analysis and western blotting. The proteins were subjected to either one-dimensional (1-D) SDS-PAGE or two-dimensional (2-D) benzyldimethyl-n-hexadecylammonium chloride (BAC)/SDS-PAGE before nano-Liquid Chromatography-Electrospray Ionization--tandem mass spectrometry analysis (LC-ESI-MS/MS). A total of 613 non-redundant proteins were identified, among which 371 (60.5%) proteins were classified as PM or membrane-associated proteins according to GO annotations and the literatures and 32.4% had transmembrane domains. PM proteins from microsomal portion possessed the highest percentage of transmembrane domain, about 46.5% of them containing at least one transmembrane domain. In addition to proteins known to be located at polarized liver PM regions, such as asialoglycoprotein receptor 2, desmoplakin and bile salt export pump, several proteins which had the potential to become novel subfraction-specific proteins were also identified, such as annexin a6, pannexin and radixin. Our analysis also evaluated the application of 1-D SDS-PAGE and 2-D 16-BAC/SDS-PAGE on the separation of integral membrane proteins.

  16. Plasma volumes, blood volumes, and plasma protein concentrations after moderate haemodilution with fluosol-DA or normal saline in the rat.

    PubMed

    Shrewsbury, R P

    1991-05-01

    Plasma volumes, blood volumes, and plasma total protein, albumin, and bilirubin concentrations have been determined in rats for 72 h following 20 or 40 mL kg-1 haemodilution with Fluosol-DA or 0.9% NaCl. Haemodilution with 20 mL kg-1 of either haemodiluent had no influence on the measured values. Plasma and blood volumes did not change after Fluosol-DA haemodilution at 40 mL kg-1, but albumin and bilirubin concentrations were decreased for 72 h. Only bilirubin concentrations were decreased for 72 h following haemodilution with 40 mL kg-1 of 0.9% NaCl. It was concluded that changes in a drug's plasma protein binding, and not the plasma or blood volume, are responsible for the reported alterations in a drug's apparent volume of distribution after haemodilution.

  17. Oral supplementation with whey proteins increases plasma glutathione levels of HIV-infected patients.

    PubMed

    Micke, P; Beeh, K M; Schlaak, J F; Buhl, R

    2001-02-01

    HIV infection is characterized by an enhanced oxidant burden and a systemic deficiency of the tripeptide glutathione (GSH), a major antioxidant. The semi-essential amino acid cysteine is the main source of the free sulfhydryl group of GSH and limits its synthesis. Therefore, different strategies to supplement cysteine supply have been suggested to increase glutathione levels in HIV-infected individuals. The aim of this study was to evaluate the effect of oral supplementation with two different cysteine-rich whey protein formulas on plasma GSH levels and parameters of oxidative stress and immune status in HIV-infected patients. In a prospective double blind clinical trial, 30 patients (25 male, 5 female; mean age (+/- SD) 42 +/- 9.8 years) with stable HIV infection (221 +/- 102 CD4 + lymphocytes L-1) were randomized to a supplemental diet with a daily dose of 45 g whey proteins of either Protectamin (Fresenius Kabi, Bad Hamburg, Germany) or Immunocal (Immunotec, Vandreuil, Canada) for two weeks. Plasma concentrations of total, reduced and oxidized GSH, superoxide anion (O2-) release by blood mononuclear cells, plasma levels of TNF-alpha and interleukins 2 and 12 were quantified with standard methods at baseline and after therapy. Pre-therapy, plasma GSH levels (Protectamin: 1.92 +/- 0.6 microM; Immunocal: 1.98 +/- 0.9 microM) were less than normal (2.64 +/- 0.7 microM, P = 0.03). Following two weeks of oral supplementation with whey proteins, plasma GSH levels increased in the Protectamin group by 44 +/- 56% (2.79 +/- 1.2 microM, P = 0.004) while the difference in the Immunocal group did not reach significance (+ 24.5 +/- 59%, 2.51 +/- 1.48 microM, P = 0.43). Spontaneous O2- release by blood mononuclear cells was stable (20.1 +/- 14.2 vs. 22.6 +/- 16.1 nmol h-1 10-6 cells, P = 0.52) whereas PMA-induced O2- release decreased in the Protectamin group (53.7 +/- 19 vs. 39.8 +/- 18 nmol h-1 10-6 cells, P = 0.04). Plasma concentrations of TNF-alpha and interleukins 2 and

  18. Quantitative and epitope-specific antigenicity analysis of the human papillomavirus 6 capsid protein in aqueous solution or when adsorbed on particulate adjuvants.

    PubMed

    Li, Min; Wang, Xin; Cao, Lu; Lin, Zhijie; Wei, Minxi; Fang, Mujin; Li, Shaowei; Zhang, Jun; Xia, Ningshao; Zhao, Qinjian

    2016-08-17

    Human papillomavirus (HPV) 6 is a human pathogen which causes genital warts. Recombinant virus-like particle (VLP) based antigens are the active components in prophylactic vaccines to elicit functional antibodies. The binding and functional characteristics of a panel of 15 murine monoclonal antibodies (mAbs) against HPV6 was quantitatively assessed. Elite conformational indicators, recognizing the conformational epitopes, are also elite viral neutralizers as demonstrated with their viral neutralization efficiency (5 mAbs with neutralization titer below 4ng/mL) in a pseudovirion (PsV)-based system. The functionality of a given mAb is closely related to the nature of the corresponding epitope, rather than the apparent binding affinity to antigen. The epitope-specific antigenicity assays can be used to assess the binding activity of PsV or VLP preparations to neutralizing mAbs. These mAb-based assays can be used for process monitoring and for product release and characterization to confirm the existence of functional epitopes in purified antigen preparations. Due to the particulate nature of the alum adjuvants, the vaccine antigen adsorbed on adjuvants was considered largely as "a black box" due to the difficulty in analysis and visualization. Here, a novel method with fluorescence-based high content imaging for visualization and quantitating the immunoreactivity of adjuvant-adsorbed VLPs with neutralizing mAbs was developed, in which antigen desorption was not needed. The facile and quantitative in situ antigenicity analysis was amendable for automation. The integrity of a given epitope or two non-overlapping epitopes on the recombinant VLPs in their adjuvanted form can be assessed in a quantitative manner for cross-lot or cross-product comparative analysis with minimal manipulation of samples. PMID:27426626

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

    PubMed Central

    2011-01-01

    Background During the peri-implantation period, the embryo adheres to an adequately prepared or receptive endometrial surface epithelium. Abnormal embryo adhesion to the endometrium results in embryo implantation failure and infertility. Endometrial epithelial cell plasma membrane proteins critical in regulating adhesion may potentially be infertility biomarkers or targets for treating infertility. Interleukin (IL) 11 regulates human endometrial epithelial cells (hEEC) adhesion. Its production is abnormal in women with infertility. The objective of the study was to identify IL11 regulated plasma membrane proteins in hEEC in vitro using a proteomic approach. Methods Using a 2D-differential in-gel electrophoresis (DIGE) electrophoresis combined with LCMS/MS mass spectrometry approach, we identified 20 unique plasma membrane proteins differentially regulated by IL11 in ECC-1 cells, a hEEC derived cell line. Two IL11 regulated proteins with known roles in cell adhesion, annexin A2 (ANXA2) and flotillin-1 (FLOT1), were validated by Western blot and immunocytochemistry in hEEC lines (ECC-1 and an additional cell line, Ishikawa) and primary hEEC. Flotilin-1 was further validated by immunohistochemistry in human endometrium throughout the menstrual cycle (n = 6-8/cycle). Results 2D-DIGE analysis identified 4 spots that were significantly different between control and IL11 treated group. Of these 4 spots, there were 20 proteins that were identified with LCMS/MS. Two proteins; ANXA2 and FLOT1 were chosen for further analyses and have found to be significantly up-regulated following IL11 treatment. Western blot analysis showed a 2-fold and a 2.5-fold increase of ANXA2 in hEEC membrane fraction of ECC-1 and Ishikawa cells respectively. Similarly, a 1.8-fold and a 2.3/2.4-fold increase was also observed for FLOT1 in hEEC membrane fraction of ECC-1 and Ishikawa cells respectively. In vitro, IL11 induced stronger ANXA2 expression on cell surface of primary hEEC and ECC-1 whilst

  20. HIV-1 RNA genome dimerizes on the plasma membrane in the presence of Gag protein.

    PubMed

    Chen, Jianbo; Rahman, Sheikh Abdul; Nikolaitchik, Olga A; Grunwald, David; Sardo, Luca; Burdick, Ryan C; Plisov, Sergey; Liang, Edward; Tai, Sheldon; Pathak, Vinay K; Hu, Wei-Shau

    2016-01-12

    Retroviruses package a dimeric genome comprising two copies of the viral RNA. Each RNA contains all of the genetic information for viral replication. Packaging a dimeric genome allows the recovery of genetic information from damaged RNA genomes during DNA synthesis and promotes frequent recombination to increase diversity in the viral population. Therefore, the strategy of packaging dimeric RNA affects viral replication and viral evolution. Although its biological importance is appreciated, very little is known about the genome dimerization process. HIV-1 RNA genomes dimerize before packaging into virions, and RNA interacts with the viral structural protein Gag in the cytoplasm. Thus, it is often hypothesized that RNAs dimerize in the cytoplasm and the RNA-Gag complex is transported to the plasma membrane for virus assembly. In this report, we tagged HIV-1 RNAs with fluorescent proteins, via interactions of RNA-binding proteins and motifs in the RNA genomes, and studied their behavior at the plasma membrane by using total internal reflection fluorescence microscopy. We showed that HIV-1 RNAs dimerize not in the cytoplasm but on the plasma membrane. Dynamic interactions occur among HIV-1 RNAs, and stabilization of the RNA dimer requires Gag protein. Dimerization often occurs at an early stage of the virus assembly process. Furthermore, the dimerization process is probably mediated by the interactions of two RNA-Gag complexes, rather than two RNAs. These findings advance the current understanding of HIV-1 assembly and reveal important insights into viral replication mechanisms.

  1. Seminal plasma protein profiles of ejaculates obtained by internal artificial vagina and electroejaculation in Brahman bulls.

    PubMed

    Rego, J P A; Moura, A A; Nouwens, A S; McGowan, M R; Boe-Hansen, G B

    2015-09-01

    The present study was conducted to investigate if differences exist in the seminal plasma protein profile from mature Brahman bulls using two methods of semen collection: internal artificial vagina (IAV) and electroejaculation (EEJ). Semen was collected four times from three bulls on the same day and parameters were assessed immediately post-collection. Seminal plasma proteins were evaluated by 2-D fluorescence difference gel electrophoresis and identified by mass spectrometry. Semen volume was greater (P < 0.05) for EEJ (4.6 ± 0.35 mL) than for IAV (1.86 ± 0.24 mL) but sperm concentration was greater in IAV (1505 ± 189 × 10(6) sperm/mL) than in EEJ samples (344 ± 87 × 10(6) sperm/mL). Sperm motility and the percentage of normal sperm were not different between treatments. Total concentration of seminal plasma proteins was greater for samples collected by IAV as compared to EEJ (19.3 ± 0.9 compared with 13.0 ± 1.8 mg/mL, P < 0.05; respectively). Based on 2-D gels, 22 spots had a greater volume (P < 0.05) in gels derived from IAV samples, corresponding to 21 proteins identified as transferrin, albumin, epididymal secretory glutathione peroxidase, among others. Thirty-three spots, corresponding to 26 proteins, had a greater volume (P < 0.05) in gels derived from EEJ samples. These proteins were identified as spermadhesin-1, Bovine Sperm Protin 1, 3 and 5 isoforms, angiogenin-1, alpha-1B-glycoprotein, clusterin, nucleobindin-1, cathepsins, spermadhesin Z13, annexins, among others. Thus, proteins in greater amounts in samples obtained by IAV and EEJ were mainly of epididymal origin and accessory sex glands, respectively. PMID:26282524

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

    PubMed

    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

  3. Ellipsometric studies of synthetic albumin-binding chitosan-derivatives and selected blood plasma proteins

    NASA Astrophysics Data System (ADS)

    Sarkar, Sabyasachi

    This dissertation summarizes work on the synthesis of chitosan-derivatives and the development of ellipsometric methods to characterize materials of biological origin. Albumin-binding chitosan-derivatives were synthesized via addition reactions that involve amine groups naturally present in chitosan. These surfaces were shown to have an affinity towards human serum albumin via ELISA, UV spectroscopy and SDS PAGE. Modified surfaces were characterized with IR ellipsometry at various stages of their synthesis using appropriate optical models. It was found that spin cast chitosan films were anisotropic in nature. All optical models used for characterizing chitosan-derivatives were thus anisotropic. Chemical signal dependence on molecular structure and composition was illustrated via IR spectroscopic ellipsometry (IRSE). An anisotropic optical model of an ensemble of Lorentz oscillators were used to approximate material behavior. The presence of acetic acid in spin-cast non-neutralized chitosan samples was thus shown. IRSE application to biomaterials was also demonstrated by performing a step-wise chemical characterizations during synthesis stages. Protein adsorbed from single protein solutions on these modified surfaces was monitored by visible in-situ variable wavelength ellipsometry. Based on adsorption profiles obtained from single protein adsorption onto silicon surfaces, lumped parameter kinetic models were developed. These models were used to fit experimental data of immunoglobulin-G of different concentrations and approximate conformational changes in fibrinogen adsorption. Biomaterial characterization by ellipsometry was further extended to include characterization of individual protein solutions in the IR range. Proteins in an aqueous environment were characterized by attenuated total internal reflection (ATR) IR ellipsometry using a ZnSe prism. Parameterized dielectric functions were created for individual proteins using Lorentz oscillators. These

  4. Hypochlorite-induced oxidation of proteins in plasma: formation of chloramines and nitrogen-centred radicals and their role in protein fragmentation.

    PubMed Central

    Hawkins, C L; Davies, M J

    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 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 cases chloramine formation accounts for approx. 20-30% of the added HOCl. These chloramines decompose in a time-dependent manner when incubated at 20 or 37 degrees C but not at 4 degrees C. Ascorbate and urate remove these chloramines in a time- and concentration-dependent manner, with the former being 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. Radical formation was inhibited by excess methionine, implicating protein-derived chloramines (probably from lysine side chains) as the radical source. Plasma protein fragmentation occurs in a time- and HOCl-concentration-dependent manner, as evidenced by the increased mobility of the EPR spin adducts, the detection of further radical species believed to be intermediates in protein degradation and the loss of the parent protein bands on SDS/PAGE. Fragmentation can be inhibited by methionine and other agents (ascorbate, urate, Trolox C or GSH) capable of removing chloramines and reactive radicals. These results are consistent with protein-derived chloramines, and the radicals derived from them, as contributing agents in HOCl-induced plasma protein oxidation. PMID:10333500

  5. Phosphorylation-dependent trafficking of plasma membrane proteins in animal and plant cells.

    PubMed

    Offringa, Remko; Huang, Fang

    2013-09-01

    In both unicellular and multicellular organisms, transmembrane (TM) proteins are sorted to and retained at specific membrane domains by endomembrane trafficking mechanisms that recognize sorting signals in the these proteins. The trafficking and distribution of plasma membrane (PM)-localized TM proteins (PM proteins), especially of those PM proteins that show an asymmetric distribution over the PM, has received much attention, as their proper PM localization is crucial for elementary signaling and transport processes, and defects in their localization often lead to severe disease symptoms or developmental defects. The subcellular localization of PM proteins is dynamically regulated by post-translational modifications, such as phosphorylation and ubiquitination. These modificaitons mostly occur on sorting signals that are located in the larger cytosolic domains of the cargo proteins. Here we review the effects of phosphorylation of PM proteins on their trafficking, and present the key examples from the animal field that have been subject to studies for already several decades, such as that of aquaporin 2 and the epidermal growth factor receptor. Our knowledge on cargo trafficking in plants is largely based on studies of the family of PIN FORMED (PIN) carriers that mediate the efflux of the plant hormone auxin. We will review what is known on the subcellular distribution and trafficking of PIN proteins, with a focus on how this is modulated by phosphorylation, and identify and discuss analogies and differences in trafficking with the well-studied animal examples. PMID:23945267

  6. Simulated gastrointestinal digestion, intestinal permeation and plasma protein interaction of white, green, and black tea polyphenols.

    PubMed

    Tenore, Gian Carlo; Campiglia, Pietro; Giannetti, Daniela; Novellino, Ettore

    2015-02-15

    The gastrointestinal digestion, intestinal permeation, and plasma protein interaction of polyphenols from a single tea cultivar at different stages of processing (white, green, and black teas) were simulated. The salivary phase contained 74.8-99.5% of native polyphenols, suggesting potential bioavailability of significant amounts of antioxidants through the oral mucosal epithelium that might be gastric sensitive and/or poorly absorbed in the intestine. White tea had the highest content and provided the best intestinal bioaccessibility and bioavailability for catechins. Since most of native catechins were not absorbed, they were expected to accumulate in the intestinal lumen where a potential inhibition capacity of cellular glucose and cholesterol uptake was assumed. The permeated catechins (approximately, 2-15% of intestinal levels) significantly bound (about 37%) to plasma HDLs, suggesting a major role in cholesterol metabolism. White tea and its potential nutraceuticals could be effective in the regulation of plasma glucose and cholesterol levels. PMID:25236233

  7. Simulated gastrointestinal digestion, intestinal permeation and plasma protein interaction of white, green, and black tea polyphenols.

    PubMed

    Tenore, Gian Carlo; Campiglia, Pietro; Giannetti, Daniela; Novellino, Ettore

    2015-02-15

    The gastrointestinal digestion, intestinal permeation, and plasma protein interaction of polyphenols from a single tea cultivar at different stages of processing (white, green, and black teas) were simulated. The salivary phase contained 74.8-99.5% of native polyphenols, suggesting potential bioavailability of significant amounts of antioxidants through the oral mucosal epithelium that might be gastric sensitive and/or poorly absorbed in the intestine. White tea had the highest content and provided the best intestinal bioaccessibility and bioavailability for catechins. Since most of native catechins were not absorbed, they were expected to accumulate in the intestinal lumen where a potential inhibition capacity of cellular glucose and cholesterol uptake was assumed. The permeated catechins (approximately, 2-15% of intestinal levels) significantly bound (about 37%) to plasma HDLs, suggesting a major role in cholesterol metabolism. White tea and its potential nutraceuticals could be effective in the regulation of plasma glucose and cholesterol levels.

  8. Fetuin-B, a liver-derived plasma protein is essential for fertilization.

    PubMed

    Dietzel, Eileen; Wessling, Jennifer; Floehr, Julia; Schäfer, Cora; Ensslen, Silke; Denecke, Bernd; Rösing, Benjamin; Neulen, Joseph; Veitinger, Thomas; Spehr, Marc; Tropartz, Tanja; Tolba, René; Renné, Thomas; Egert, Angela; Schorle, Hubert; Gottenbusch, Yuliya; Hildebrand, André; Yiallouros, Irene; Stöcker, Walter; Weiskirchen, Ralf; Jahnen-Dechent, Willi

    2013-04-15

    The zona pellucida (ZP) is a glycoprotein matrix surrounding mammalian oocytes. Upon fertilization, ZP hardening prevents sperm from binding to and penetrating the ZP. Here, we report that targeted gene deletion of the liver-derived plasma protein fetuin-B causes premature ZP hardening and, consequently, female infertility. Transplanting fetuin-B-deficient ovaries into wild-type recipients restores fertility, indicating that plasma fetuin-B is necessary and sufficient for fertilization. In vitro fertilization of oocytes from fetuin-B-deficient mice only worked after rendering the ZP penetrable by laser perforation. Mechanistically, fetuin-B sustains fertility by inhibiting ovastacin, a cortical granula protease known to trigger ZP hardening. Thus, plasma fetuin-B is necessary to restrain protease activity and thereby maintain ZP permeability until after gamete fusion. These results also show that premature ZP hardening can cause infertility in mice.

  9. Wettability Effect of PECVD-SiOx Films on Poly(lactic acid) Induced by Oxygen Plasma on Protein Adsorption and Cell Attachment

    NASA Astrophysics Data System (ADS)

    Sarapirom, S.; Lee, J. S.; Jin, S. B.; Song, D. H.; Yu, L. D.; Han, J. G.; Chaiwong, C.

    2013-04-01

    Surface wettability is an important property of biomaterials. Silicon oxide films have a wide range of applications due to a range of the properties such as the mechanical strength and surface wettability. This paper reports effect of the surface wettability of silicon oxide (SiOx) films on protein adsorption and cell attachment and proliferation. SiOx films were deposited onto poly(lactic acid) (PLA) substrate using plasma enhanced chemical vapor deposition (PECVD). Octamethylcyclotetrasiloxane (OMCTS:Si4O4C8H24) was used as a precursor with O2 as a carrier gas. After deposition, the films were treated with O2-plasma to adapt wettability. It was found that O2-plasma enhanced the wettability of the films without changing the film thickness, while made the surface morphology slightly smoother. The polar component increased after O2-plasma treatment as observed in the contact angle measurements. The surface energy of the films was calculated by means of the Owens-Wendt method to resolve the contributions of polar and dispersive components. The chemical structure was characterized using attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy. The films were dense with a high Si-network structure. The reduced carbon content (-CHn, Si-CH3) and increased hydrogen content (-OH) of the O2-plasma treated SiOx films led to the polar components enhancing the SiOx wettability. Adsorption of bovine serum albumin (BSA) on the films was investigated by using x-ray photoelectron spectroscopy (XPS). More BSA was adsorbed onto the O2-plasma treated SiOx films. Attachment and proliferation of MC3T3-E1 mouse pre-osteoblasts and L929 mouse fibroblasts cells on the SiOx films were evaluated via MTT assay. The cells were attached more to the untreated SiOx films but proliferated more on the surface of the O2-plasma treated SiOx films depending on the cell types.

  10. Identification of differentially expressed plasma proteins in atherosclerotic patients with type 2 diabetes.

    PubMed

    Lepedda, Antonio Junior; Lobina, Omar; Rocchiccioli, Silvia; Nieddu, Gabriele; Ucciferri, Nadia; De Muro, Pierina; Idini, Michela; Nguyen, Hai Quy Tram; Guarino, Anna; Spirito, Rita; Formato, Marilena

    2016-07-01

    Besides hyperglycaemia and insulin resistance, several factors are associated with a higher cardiovascular risk in type 2 diabetes mellitus (T2DM), many of them being closely related to each other owing to common origins or pathways. The pathophysiological mechanisms underlying vascular dysfunctions in diabetes include reduced bioavailability of nitric oxide, increased ROS and prothrombotic factors production, as well as activation of receptors for advanced glycation end-products. These alterations contribute to create a pro-inflammatory/thrombotic state that ultimately leads to plaque formation and complication. This study aimed at identifying differentially expressed plasma proteins between T2DM and non-diabetic patients undergoing carotid endarterectomy, by means of two-dimensional electrophoresis coupled with LC-MS/MS. Before analysis, plasma samples were enriched in low-expression proteins through combinatorial hexapeptide ligand libraries. Both mono- and two-dimensional western blotting were performed for data validation. Differentially expressed proteins were mapped onto STRING v10 to build a protein-protein interaction network. Sixteen differentially expressed spots were identified with a high score. Among them, there were fibrinogen beta and gamma chains, complement C1r, C3 and C4-B subcomponents, alpha-1-antitrypsin (AAT), vitronectin and CD5 antigen-like. Protein-Protein interaction analysis evidenced a network among differentially expressed proteins in which vitronectin seems to represent a potentially pivotal node among fibrinolysis, complement dependent immune responses and inflammation in accordance with a number of in vitro and in vivo evidences for a contributory role of these proteins to the development of diabetic atherosclerosis. PMID:27037039

  11. Quantitative analysis of aberrant protein glycosylation in liver cancer plasma by AAL-enrichment and MRM mass spectrometry.

    PubMed

    Ahn, Yeong Hee; Shin, Park Min; Kim, Yong-Sam; Oh, Na Ree; Ji, Eun Sun; Kim, Kwang Hoe; Lee, Yeon Jung; Kim, Sung Ho; Yoo, Jong Shin

    2013-11-01

    A lectin-coupled mass spectrometry (MS) approach was employed to quantitatively monitor aberrant protein glycosylation in liver cancer plasma. To do this, we compared the difference in the total protein abundance of a target glycoprotein between hepatocellular carcinoma (HCC) plasmas and hepatitis B virus (HBV) plasmas, as well as the difference in lectin-specific protein glycoform abundance of the target glycoprotein. Capturing the lectin-specific protein glycoforms from a plasma sample was accomplished by using a fucose-specific aleuria aurantia lectin (AAL) immobilized onto magnetic beads via a biotin-streptavidin conjugate. Following tryptic digestion of both the total plasma and its AAL-captured fraction of each HCC and HBV sample, targeted proteomic mass spectrometry was conducted quantitatively by a multiple reaction monitoring (MRM) technique. From the MRM-based analysis of the total plasmas and AAL-captured fractions, differences between HCC and HBV plasma groups in fucosylated glycoform levels of target glycoproteins were confirmed to arise from both the change in the total protein abundance of the target proteins and the change incurred by aberrant fucosylation on target glycoproteins in HCC plasma, even when no significant change occurs in the total protein abundance level. Combining the MRM-based analysis method with the lectin-capturing technique proved to be a successful means of quantitatively investigating aberrant protein glycosylation in cancer plasma samples. Additionally, it was elucidated that the differences between HCC and control groups in fucosylated biomarker candidates A1AT and FETUA mainly originated from an increase in fucosylation levels on these target glycoproteins, rather than an increase in the total protein abundance of the target glycoproteins.

  12. Surface Enhanced Raman Scattering studies of L-amino acids adsorbed on silver nanoclusters

    NASA Astrophysics Data System (ADS)

    Botta, Raju; Rajanikanth, A.; Bansal, C.

    2015-01-01

    Silver nanocluster films were prepared using plasma inert gas phase condensation technique. These were used as Raman active substrates for Surface Enhanced Raman Scattering (SERS) studies of 19 standard L-amino acids adsorbed on the surface of Ag nanoclusters via Agsbnd N bonds. A detailed study of two essential aromatic amino acids viz. L-Phenylalanine and L-Tryptophan showed a correlation between the Raman intensity of the characteristic lines of phenol and indole side chains and their molar concentrations in the range 1 μM-1 mM. This indicates that Raman studies can be used for quantitative determination of the amino acids in proteins.

  13. Detection of cellular prion protein in exosomes derived from ovine plasma.

    PubMed

    Berrone, Elena; Corona, Cristiano; Mazza, Maria; Vallino Costassa, Elena; Faro, Monica Lo; Properzi, Francesca; Guglielmetti, Chiara; Maurella, Cristiana; Caramelli, Maria; Deregibus, Maria Chiara; Camussi, Giovanni; Casalone, Cristina

    2015-12-01

    Prion protein (PrP) is present at extremely low levels in the blood of animals and its detection is complicated by the poor sensitivity of current standard methodologies. Interesting results have been obtained with recent advanced technologies that are able to detect minute amounts of the pathological PrP (PrPSc), but their efficiency is reduced by various factors present in blood. In this study, we were able to extract cellular PrP (PrPC) from plasma-derived exosomes by a simple, fast method without the use of differential ultracentrifugation and to visualize it by Western blotting, reducing the presence of most plasma proteins. This result confirms that blood is capable of releasing PrP in association with exosomes and could be useful to better study its role in the pathogenesis of transmissible spongiform encephalopathies. PMID:26399471

  14. Characterization of protein phosphatase 2A acting on phosphorylated plasma membrane aquaporin of tulip petals.

    PubMed

    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.

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

    PubMed

    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.

  16. The Ebola virus matrix protein deeply penetrates the plasma membrane: an important step in viral egress.

    PubMed

    Soni, Smita P; Adu-Gyamfi, Emmanuel; Yong, Sylvia S; Jee, Clara S; Stahelin, Robert V

    2013-05-01

    Ebola virus, from the Filoviridae family has a high fatality rate in humans and nonhuman primates and to date, to the best of our knowledge, has no FDA approved vaccines or therapeutics. Viral protein 40 (VP40) is the major Ebola virus matrix protein that regulates assembly and egress of infectious Ebola virus particles. It is well established that VP40 assembles on the inner leaflet of the plasma membrane; however, the mechanistic details of VP40 membrane binding that are important for viral release remain to be elucidated. In this study, we used fluorescence quenching of a tryptophan on the membrane-binding interface with brominated lipids along with mutagenesis of VP40 to understand the depth of membrane penetration into lipid bilayers. Experimental results indicate that VP40 penetrates 8.1 Å into the hydrocarbon core of the plasma membrane bilayer. VP40 also induces substantial changes to membrane curvature as it tubulates liposomes and induces vesiculation into giant unilamellar vesicles, effects that are abrogated by hydrophobic mutations. This is a critical step in viral egress as cellular assays demonstrate that hydrophobic residues that penetrate deeply into the plasma membrane are essential for plasma membrane localization and virus-like particle formation and release from cells.

  17. Plasma steroid-binding proteins: primary gatekeepers of steroid hormone action

    PubMed Central

    2016-01-01

    Biologically active steroids are transported in the blood by albumin, sex hormone-binding globulin (SHBG), and corticosteroid-binding globulin (CBG). These plasma proteins also regulate the non-protein-bound or ‘free’ fractions of circulating steroid hormones that are considered to be biologically active; as such, they can be viewed as the ‘primary gatekeepers of steroid action’. Albumin binds steroids with limited specificity and low affinity, but its high concentration in blood buffers major fluctuations in steroid concentrations and their free fractions. By contrast, SHBG and CBG play much more dynamic roles in controlling steroid access to target tissues and cells. They bind steroids with high (~nM) affinity and specificity, with SHBG binding androgens and estrogens and CBG binding glucocorticoids and progesterone. Both are glycoproteins that are structurally unrelated, and they function in different ways that extend beyond their transportation or buffering functions in the blood. Plasma SHBG and CBG production by the liver varies during development and different physiological or pathophysiological conditions, and abnormalities in the plasma levels of SHBG and CBG or their abilities to bind steroids are associated with a variety of pathologies. Understanding how the unique structures of SHBG and CBG determine their specialized functions, how changes in their plasma levels are controlled, and how they function outside the blood circulation provides insight into how they control the freedom of steroids to act in health and disease. PMID:27113851

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

    PubMed

    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.

  19. The importance of selecting a proper biological milieu for protein corona analysis in vitro: Human plasma versus human serum.

    PubMed

    Mirshafiee, Vahid; Kim, Raehyun; Mahmoudi, Morteza; Kraft, Mary L

    2016-06-01

    Nanoparticle (NP) exposure to biological fluids in the body results in protein binding to the NP surface, which forms a protein coating that is called the "protein corona". To simplify studies of protein-NP interactions and protein corona formation, NPs are incubated with biological solutions, such as human serum or human plasma, and the effects of this exposure are characterized in vitro. Yet, how NP exposure to these two different biological milieus affects protein corona composition and cell response has not been investigated. Here, we explore the differences between the protein coronas that form when NPs are incubated in human serum versus human plasma. NP characterization indicated that NPs that were exposed to human plasma had higher amounts of proteins bound to their surfaces, and were slightly larger in size than those exposed to human serum. In addition, significant differences in corona composition were also detected with gel electrophoresis and liquid chromatography-mass spectrometry/mass spectrometry, where a higher fraction of coagulation proteins and complement factors were found on the plasma-exposed NPs. Flow cytometry and confocal microscopy showed that the uptake of plasma-exposed NPs was higher than that of serum-exposed NPs by RAW 264.7 macrophage immune cells, but not by NIH 3T3 fibroblast cells. This difference is likely due to the elevated amounts of opsonins, such as fibrinogen, on the surfaces of the NPs exposed to plasma, but not serum, because these components trigger NP internalization by immune cells. As the human plasma better mimics the composition of the in vivo environment, namely blood, in vitro protein corona studies should employ human plasma, and not human serum, so the biological phenomena that is observed is more similar to that occurring in vivo.

  20. Modulating bone cells response onto starch-based biomaterials by surface plasma treatment and protein adsorption.

    PubMed

    Alves, Catarina M; Yang, Y; Carnes, D L; Ong, J L; Sylvia, V L; Dean, D D; Agrawal, C M; Reis, R L

    2007-01-01

    The effect of oxygen-based radio frequency glow discharge (rfGD) on the surface of different starch-based biomaterials (SBB) and the influence of proteins adsorption on modulating bone-cells behavior was studied. Bovine serum albumin, fibronectin and vitronectin were used in single and complex protein systems. RfGD-treated surfaces showed to increase in hydrophilicity and surface energy when compared to non-modified SBB. Biodegradable polymeric blends of cornstarch with cellulose acetate (SCA; 50/50wt%), ethylene vinyl alcohol (SEVA-C; 50/50wt%) and polycaprolactone (SPCL; 30/70wt%) were studied. SCA and SCA reinforced with 10% hydroxyapatite (HA) showed the highest degree of modification as result of the rfGD treatment. Protein and control solutions were used to incubate with the characterized SBB and, following this, MG63 osteoblast-like osteosarcoma cells were seeded over the surfaces. Cell adhesion and proliferation onto SCA was found to be enhanced for non-treated surfaces and on SCA+10%HA no alteration was brought up by the plasma modification. Onto SCA surfaces, BSA, FN and VN single solutions improved cell adhesion, and this same effect was found upscaled for ternary systems. In addition, plasma treated SEVA-C directed an increase in both adhesion and proliferation comparing to non-treated surfaces. Even though adhesion onto treated and untreated SPCL was quite similar, plasma modification clearly promoted MG63 cells proliferation. Regarding MG63 cells morphology it was shown that onto SEVA-C surfaces the variation of cell shape was primarily defined by the protein system, while onto SPCL it was mainly affected by the plasma treatment. PMID:17011619

  1. Sex steroid binding proteins in the plasma of hatchling Chelonia mydas.

    PubMed

    Ikonomopoulou, M P; Ibrahim, K; Bradley, A J

    2008-09-01

    Sex steroid binding proteins were identified in hatchling female and male Chelonia mydas by dialysis and steady-state gel electrophoresis when examined at 4 degrees C. A testosterone binding protein with high binding affinity (K (a) = 0.98 +/- 0.5 x 10(8) M(-1)) and low to moderate binding capacity (B (max) = 7.58 +/- 4.2 x 10(-5) M) was observed in male hatchlings. An oestradiol binding protein with high affinity (K (a) = 0.35 +/- 1.8 x 10(8) M(-1)) and low to moderate binding capacity (B (max) = 0.16 +/- 0.5 x 10(-4) M) was identified in female hatchlings. This study confirmed that sex steroid binding proteins (SSBPs) become inactivate in both sexes at 36 degrees C, the maximum body temperature of sea turtle hatchlings at emergence. The inactivation of SSBPs at this temperature indicates that sex steroid hormones circulate freely in the body of the green turtles and are biologically available in the blood plasma. This observation is consistent with female and male hatchling C. mydas having different physiological (hormonal) and developmental requirements around the time of emergence. Moreover, concurrently conducted competition studies showed that sex steroids including testosterone and oestradiol do compete for binding sites in both male and female C. mydas hatchling plasma. Competition also occurred between testosterone and dihydrotestosterone for binding sites in the male C. mydas plasma. However, competition studies in the plasma of female hatchling C. mydas demonstrate that oestrone does not compete with oestradiol for binding sites.

  2. Modulating bone cells response onto starch-based biomaterials by surface plasma treatment and protein adsorption.

    PubMed

    Alves, Catarina M; Yang, Y; Carnes, D L; Ong, J L; Sylvia, V L; Dean, D D; Agrawal, C M; Reis, R L

    2007-01-01

    The effect of oxygen-based radio frequency glow discharge (rfGD) on the surface of different starch-based biomaterials (SBB) and the influence of proteins adsorption on modulating bone-cells behavior was studied. Bovine serum albumin, fibronectin and vitronectin were used in single and complex protein systems. RfGD-treated surfaces showed to increase in hydrophilicity and surface energy when compared to non-modified SBB. Biodegradable polymeric blends of cornstarch with cellulose acetate (SCA; 50/50wt%), ethylene vinyl alcohol (SEVA-C; 50/50wt%) and polycaprolactone (SPCL; 30/70wt%) were studied. SCA and SCA reinforced with 10% hydroxyapatite (HA) showed the highest degree of modification as result of the rfGD treatment. Protein and control solutions were used to incubate with the characterized SBB and, following this, MG63 osteoblast-like osteosarcoma cells were seeded over the surfaces. Cell adhesion and proliferation onto SCA was found to be enhanced for non-treated surfaces and on SCA+10%HA no alteration was brought up by the plasma modification. Onto SCA surfaces, BSA, FN and VN single solutions improved cell adhesion, and this same effect was found upscaled for ternary systems. In addition, plasma treated SEVA-C directed an increase in both adhesion and proliferation comparing to non-treated surfaces. Even though adhesion onto treated and untreated SPCL was quite similar, plasma modification clearly promoted MG63 cells proliferation. Regarding MG63 cells morphology it was shown that onto SEVA-C surfaces the variation of cell shape was primarily defined by the protein system, while onto SPCL it was mainly affected by the plasma treatment.

  3. Optical tweezers study of red blood cell aggregation and disaggregation in plasma and protein solutions

    NASA Astrophysics Data System (ADS)

    Lee, Kisung; Kinnunen, Matti; Khokhlova, Maria D.; Lyubin, Evgeny V.; Priezzhev, Alexander V.; Meglinski, Igor; Fedyanin, Andrey A.

    2016-03-01

    Kinetics of optical tweezers (OT)-induced spontaneous aggregation and disaggregation of red blood cells (RBCs) were studied at the level of cell doublets to assess RBC interaction mechanics. Measurements were performed under in vitro conditions in plasma and fibrinogen and fibrinogen + albumin solutions. The RBC spontaneous aggregation kinetics was found to exhibit different behavior depending on the cell environment. In contrast, the RBC disaggregation kinetics was similar in all solutions qualitatively and quantitatively, demonstrating a significant contribution of the studied proteins to the process. The impact of the study on assessing RBC interaction mechanics and the protein contribution to the reversible RBC aggregation process is discussed.

  4. Exploring the stochastic dynamics of correlated movement of receptor proteins in plasma membranes in vivo

    SciTech Connect

    Huang, Jung Y.; Lin, Chien Y.

    2015-12-14

    Ligand-induced receptor dimerization plays a crucial role in the signaling process of living cells. In this study, we developed a theoretical model and performed single-molecule tracking to explore the correlated diffusion processes of liganded epidermal growth factor receptors prior to dimer formation. We disclosed that both an attractive potential between liganded receptor proteins in proximity and correlated fluctuations in the local environments of the proteins play an important role to produce the observed correlated movement of the receptors. This result can serve as the foundation to shed light on the way in which receptor functions are regulated in plasma membranes in vivo.

  5. Oral plasma zinc tolerance test in patients with protein energy malnutrition.

    PubMed Central

    Atalay, Y; Arcasoy, A; Kürkçüoğlu, M

    1989-01-01

    Zinc absorption was measured in 37 children with malnutrition using the oral zinc tolerance test (22.5 mg elementary zinc) and the results compared with those of a group of healthy control subjects. The increase in plasma zinc was significantly lower in patients with marasmic kwashiorkor than in the control group. The zinc tolerance test was, however, normal in marasmic patients. We conclude that zinc deficiency occurs in some types of protein energy malnutrition, and that malabsorption may aggravate zinc deficiency. It is reasonable to give higher doses of zinc than are usually recommended during oral zinc supplementation in patients with protein energy malnutrition. PMID:2513780

  6. The blood volume and plasma protein levels before and after gastrectomy 1

    PubMed Central

    Swan, Alexander; Allen, Geoffrey T.; Tanner, Norman C.

    1962-01-01

    This paper is a survey of a series of 185 plasma volume estimations carried out on 75 gastric surgical patients before and after operation at St. James's Hospital in 1958-59. In the majority of cases serum proteins were also measured. The purpose of this work was to study the effects of gastric operations, and especially of partial gastrectomy, on patients' blood volume, the oxygen-carrying capacity of the blood as measured by the total circulating red cell volume, and serum protein content. PMID:13918772

  7. Development of reduced fat minced meats using inulin and bovine plasma proteins as fat replacers.

    PubMed

    Rodriguez Furlán, Laura T; Padilla, Antonio Pérez; Campderrós, Mercedes E

    2014-02-01

    This work deals with the effect of the addition of inulin and bovine plasma proteins as fat replacers, on the quality of minced meat. The proteins are obtained by ultrafiltration and freeze-drying. The following determinations were carried out: chemical composition, sensorial analysis (color, flavor, taste and consistency), emulsion stability and instrumental texture analysis of samples. The resulting formulations were compared with full-fat minced meat, as control. The results showed an increase of protein contents after fat replacement, while a fat reduction of 20-35% produced light products enriched with proteins and inulin as the functional ingredient. No change was observed in color, flavor, or taste among the samples. However, the sensory analysis showed that the combination of plasma protein (2.5%w/w) and inulin (2%w/w) had the best acceptability with respect to consistency, and had a lower fat drain from the emulsion. Texture profile analysis revealed that this formulation assimilated the control texture properties, being that this result is required for adequate consumer acceptance.

  8. Development of reduced fat minced meats using inulin and bovine plasma proteins as fat replacers.

    PubMed

    Rodriguez Furlán, Laura T; Padilla, Antonio Pérez; Campderrós, Mercedes E

    2014-02-01

    This work deals with the effect of the addition of inulin and bovine plasma proteins as fat replacers, on the quality of minced meat. The proteins are obtained by ultrafiltration and freeze-drying. The following determinations were carried out: chemical composition, sensorial analysis (color, flavor, taste and consistency), emulsion stability and instrumental texture analysis of samples. The resulting formulations were compared with full-fat minced meat, as control. The results showed an increase of protein contents after fat replacement, while a fat reduction of 20-35% produced light products enriched with proteins and inulin as the functional ingredient. No change was observed in color, flavor, or taste among the samples. However, the sensory analysis showed that the combination of plasma protein (2.5%w/w) and inulin (2%w/w) had the best acceptability with respect to consistency, and had a lower fat drain from the emulsion. Texture profile analysis revealed that this formulation assimilated the control texture properties, being that this result is required for adequate consumer acceptance. PMID:24200568

  9. A radioiodinated, intracellularly trapped ligand for determining the sites of plasma protein degradation in vivo.

    PubMed Central

    Pittman, R C; Carew, T E; Glass, C K; Green, S R; Taylor, C A; Attie, A D

    1983-01-01

    We recently developed a general method for determining tissue sites of degradation of plasma proteins in vivo that made use of covalently attached radioactive sucrose. On degradation of the protein, the sucrose remained trapped in the cells as a cumulative marker of protein degradation. The method described here depends on the same principles, but uses an adduct of cellobiose and tyramine that is radioiodinated to high specific radioactivity and then covalently attached to protein. Use of the radioiodinated ligand increases the sensitivity of the method at least 100-fold and allows simplified tissue analysis. Proteins derivatized with the radioiodinated ligand were recognized as underivatized proteins both in vitro and in vivo. On degradation of derivatized low-density lipoprotein, the rate of leakage from cultured fibroblasts was only 5% during 24 h. Similarly, on injection of labelled proteins into rats and rabbits, urinary excretion of the label was in all cases less than 10% of total labelled catabolic products recovered 24 h after injection. Examination of the tissue contents of label at two times after injection of labelled asialofetuin or apolipoprotein A1 in rats, and asialotransferrin in rabbits showed that the label did not detectably redistribute between tissues after initial uptake and catabolism; a significant leakage from liver was quantitatively accounted for by label appearing in gut contents and faeces. A simple double-label method was devised to provide a correction for intact protein in trapped plasma, the extravascular spaces, and within cells. By using this method it becomes unnecessary to fractionate tissue samples. PMID:6882394

  10. Combining Ultracentrifugation and Peptide Termini Group-specific Immunoprecipitation for Multiplex Plasma Protein Analysis

    PubMed Central

    Volk, Sonja; Schreiber, Thomas D.; Eisen, David; Wiese, Calvin; Planatscher, Hannes; Pynn, Christopher J.; Stoll, Dieter; Templin, Markus F.; Joos, Thomas O.; Pötz, Oliver

    2012-01-01

    Blood plasma is a valuable source of potential biomarkers. However, its complexity and the huge dynamic concentration range of its constituents complicate its analysis. To tackle this problem, an immunoprecipitation strategy was employed using antibodies directed against short terminal epitope tags (triple X proteomics antibodies), which allow the enrichment of groups of signature peptides derived from trypsin-digested plasma. Isolated signature peptides are subsequently detected using MALDI-TOF/TOF mass spectrometry. Sensitivity of the immunoaffinity approach was, however, compromised by the presence of contaminant peaks derived from the peptides of nontargeted high abundant proteins. A closer analysis of the enrichment strategy revealed nonspecific peptide binding to the solid phase affinity matrix as the major source of the contaminating peptides. We therefore implemented a sucrose density gradient ultracentrifugation separation step into the procedure. This yielded a 99% depletion of contaminating peptides from a sucrose fraction containing 70% of the peptide-antibody complexes and enabled the detection of the previously undetected low abundance protein filamin-A. Assessment of this novel approach using 15 different triple X proteomics antibodies demonstrated a more consistent detection of a greater number of targeted peptides and a significant reduction in the intensity of nonspecific peptides. Ultracentrifugation coupled with immunoaffinity MS approaches presents a powerful tool for multiplexed plasma protein analysis without the requirement for demanding liquid chromatography separation techniques. PMID:22527512

  11. Remodeling of the plasma membrane in preparation for sperm-egg recognition: roles of acrosomal proteins.

    PubMed

    Tanphaichitr, Nongnuj; Kongmanas, Kessiri; Kruevaisayawan, Hathairat; Saewu, Arpornrad; Sugeng, Clarissa; Fernandes, Jason; Souda, Puneet; Angel, Jonathan B; Faull, Kym F; Aitken, R John; Whitelegge, Julian; Hardy, Daniel; Berger, Trish; Baker, Mark

    2015-01-01

    The interaction of sperm with the egg's extracellular matrix, the zona pellucida (ZP) is the first step of the union between male and female gametes. The molecular mechanisms of this process have been studied for the past six decades with the results obtained being both interesting and confusing. In this article, we describe our recent work, which attempts to address two lines of questions from previous studies. First, because there are numerous ZP binding proteins reported by various researchers, how do these proteins act together in sperm-ZP interaction? Second, why do a number of acrosomal proteins have ZP affinity? Are they involved mainly in the initial sperm-ZP binding or rather in anchoring acrosome reacting/reacted spermatozoa to the ZP? Our studies reveal that a number of ZP binding proteins and chaperones, extracted from the anterior sperm head plasma membrane, coexist as high molecular weight (HMW) complexes, and that these complexes in capacitated spermatozoa have preferential ability to bind to the ZP. Zonadhesin (ZAN), known as an acrosomal protein with ZP affinity, is one of these proteins in the HMW complexes. Immunoprecipitation indicates that ZAN interacts with other acrosomal proteins, proacrosin/acrosin and sp32 (ACRBP), also present in the HMW complexes. Immunodetection of ZAN and proacrosin/acrosin on spermatozoa further indicates that both proteins traffic to the sperm head surface during capacitation where the sperm acrosomal matrix is still intact, and therefore they are likely involved in the initial sperm-ZP binding step.

  12. Determining the N-terminal orientations of recombinant transmembrane proteins in the Escherichia coli plasma membrane

    PubMed Central

    Lee, Chien-Hsien; Chou, Chia-Cheng; Hsu, Min-Feng; Wang, Andrew H.-J.

    2015-01-01

    In silico algorithms have been the common approach for transmembrane (TM) protein topology prediction. However, computational tools may produce questionable results and experimental validation has proven difficult. Although biochemical strategies are available to determine the C-terminal orientation of TM proteins, experimental strategies to determine the N-terminal orientation are still limited but needed because the N-terminal end is essential for membrane targeting. Here, we describe a new and easy method to effectively determine the N-terminal orientation of the target TM proteins in Escherichia coli plasma membrane environment. D94N, the mutant of bacteriorhodopsin from Haloarcula marismortui, can be a fusion partner to increase the production of the target TM proteins if their N-termini are in cytoplasm (Nin orientation). To create a suitable linker for orientating the target TM proteins with the periplasmic N-termini (Nout orientation) correctly, we designed a three-TM-helix linker fused at the C-terminus of D94N fusion partner (termed D94N-3TM) and found that D94N-3TM can specifically improve the production of the Nout target TM proteins. In conclusion, D94N and D94N-3TM fusion partners can be applied to determine the N-terminal end of the target TM proteins oriented either Nin or Nout by evaluating the net expression of the fusion proteins. PMID:26462555

  13. Multifunctional Transmembrane Protein Ligands for Cell-Specific Targeting of Plasma Membrane-Derived Vesicles.

    PubMed

    Zhao, Chi; Busch, David J; Vershel, Connor P; Stachowiak, Jeanne C

    2016-07-01

    Liposomes and nanoparticles that bind selectively to cell-surface receptors can target specific populations of cells. However, chemical conjugation of ligands to these particles is difficult to control, frequently limiting ligand uniformity and complexity. In contrast, the surfaces of living cells are decorated with highly uniform populations of sophisticated transmembrane proteins. Toward harnessing cellular capabilities, here it is demonstrated that plasma membrane vesicles (PMVs) derived from donor cells can display engineered transmembrane protein ligands that precisely target cells on the basis of receptor expression. These multifunctional targeting proteins incorporate (i) a protein ligand, (ii) an intrinsically disordered protein spacer to make the ligand sterically accessible, and (iii) a fluorescent protein domain that enables quantification of the ligand density on the PMV surface. PMVs that display targeting proteins with affinity for the epidermal growth factor receptor (EGFR) bind at increasing concentrations to breast cancer cells that express increasing levels of EGFR. Further, as an example of the generality of this approach, PMVs expressing a single-domain antibody against green fluorescence protein (eGFP) bind to cells expressing eGFP-tagged receptors with a selectivity of ≈50:1. The results demonstrate the versatility of PMVs as cell targeting systems, suggesting diverse applications from drug delivery to tissue engineering. PMID:27294846

  14. Comprehensive and quantitative proteomic analyses of zebrafish plasma reveals conserved protein profiles between genders and between zebrafish and human

    PubMed Central

    Li, Caixia; Tan, Xing Fei; Lim, Teck Kwang; Lin, Qingsong; Gong, Zhiyuan

    2016-01-01

    Omic approaches have been increasingly used in the zebrafish model for holistic understanding of molecular events and mechanisms of tissue functions. However, plasma is rarely used for omic profiling because of the technical challenges in collecting sufficient blood. In this study, we employed two mass spectrometric (MS) approaches for a comprehensive characterization of zebrafish plasma proteome, i.e. conventional shotgun liquid chromatography-tandem mass spectrometry (LC-MS/MS) for an overview study and quantitative SWATH (Sequential Window Acquisition of all THeoretical fragment-ion spectra) for comparison between genders. 959 proteins were identified in the shotgun profiling with estimated concentrations spanning almost five orders of magnitudes. Other than the presence of a few highly abundant female egg yolk precursor proteins (vitellogenins), the proteomic profiles of male and female plasmas were very similar in both number and abundance and there were basically no other highly gender-biased proteins. The types of plasma proteins based on IPA (Ingenuity Pathway Analysis) classification and tissue sources of production were also very similar. Furthermore, the zebrafish plasma proteome shares significant similarities with human plasma proteome, in particular in top abundant proteins including apolipoproteins and complements. Thus, the current study provided a valuable dataset for future evaluation of plasma proteins in zebrafish. PMID:27071722

  15. Comprehensive and quantitative proteomic analyses of zebrafish plasma reveals conserved protein profiles between genders and between zebrafish and human.

    PubMed

    Li, Caixia; Tan, Xing Fei; Lim, Teck Kwang; Lin, Qingsong; Gong, Zhiyuan

    2016-01-01

    Omic approaches have been increasingly used in the zebrafish model for holistic understanding of molecular events and mechanisms of tissue functions. However, plasma is rarely used for omic profiling because of the technical challenges in collecting sufficient blood. In this study, we employed two mass spectrometric (MS) approaches for a comprehensive characterization of zebrafish plasma proteome, i.e. conventional shotgun liquid chromatography-tandem mass spectrometry (LC-MS/MS) for an overview study and quantitative SWATH (Sequential Window Acquisition of all THeoretical fragment-ion spectra) for comparison between genders. 959 proteins were identified in the shotgun profiling with estimated concentrations spanning almost five orders of magnitudes. Other than the presence of a few highly abundant female egg yolk precursor proteins (vitellogenins), the proteomic profiles of male and female plasmas were very similar in both number and abundance and there were basically no other highly gender-biased proteins. The types of plasma proteins based on IPA (Ingenuity Pathway Analysis) classification and tissue sources of production were also very similar. Furthermore, the zebrafish plasma proteome shares significant similarities with human plasma proteome, in particular in top abundant proteins including apolipoproteins and complements. Thus, the current study provided a valuable dataset for future evaluation of plasma proteins in zebrafish. PMID:27071722

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

    PubMed Central

    2010-01-01

    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 < 5 kDa. In Experiment 2, female llamas (n = 7 per group) were given an i.m. dose of seminal plasma treated previously by: 1) enzymatic digestion with proteinase-K, 2) incubation with charcoal-dextran, 3) heating to 65°C, or 4) untreated (control). In Experiment 3, female llamas (n = 10 per group) were given an i.m. dose of pronase-treated or non-treated (control) seminal plasma. In all experiments, llamas were examined by transrectal ultrasonography to detect ovulation and CL formation. Ovulation rate was compared among groups by Fisher's exact test and follicle and CL diameters were compared among groups by analyses of variance or student's t-tests. 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 < 0.001). In Experiment 2, ovulations were detected in all llamas in each treatment group; i.e., respective treatments of seminal plasma failed to inactivate the ovulation-inducing factor. In Experiment 3, ovulations were detected in 0/10 llamas given pronase-treated seminal plasma and in 9/10 controls (P < 0.01). 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

  17. Evaluation of Multi-Protein Immunoaffinity Subtraction for Plasma Proteomics and Candidate Biomarker Discovery Using Mass Spectrometry

    SciTech Connect

    Liu, Tao; Qian, Weijun; Mottaz, Heather M.; Gritsenko, Marina A.; Norbeck, Angela D.; Moore, Ronald J.; Purvine, Samuel O.; Camp, David G.; Smith, Richard D.

    2006-11-01

    The detection of low-abundance protein disease biomarkers from human blood poses significant challenges due to the high dynamic range of protein concentrations that span more than 10 orders of magnitude, as well as the extreme complexity of the serum/plasma proteome. Therefore, experimental strategies that include the removal of high-abundance proteins have been increasingly utilized in proteomic studies of serum, plasma, and other body fluids to enhance detection of low-abundance proteins and achieve broader proteome coverage. However, both the specificity and reproducibility of the high-abundance protein depletion process represent common concerns. Here, we report a detailed evaluation of the performance of two commercially available immunoaffinity subtraction systems commonly used in human serum/plasma proteome characterization by high resolution LC-MS/MS. One system uses mammalian IgG antibodies to remove six of the most abundant plasma proteins, and the other uses chicken immunoglobulin yolk (IgY) antibodies to remove twelve of the most abundant plasma proteins. Plasma samples were repeatedly processed using these two systems, and the resulting flow-through fractions and bound fractions were individually analyzed for comparison. Removal of target proteins by both immunoaffinity subtraction systems proved reproducible and efficient. Nontarget proteins, including spiked protein standards, were also observed to bind to the columns, but in a fairly reproducible manner. The results suggest that these multi-protein immunoaffinity subtraction systems are both highly effective and reproducible for removing high-abundance proteins and therefore, can be readily integrated into quantitative strategies to enhance detection of low-abundance proteins in biomarker discovery studies.

  18. In silicio identification of glycosyl-phosphatidylinositol-anchored plasma-membrane and cell wall proteins of Saccharomyces cerevisiae.

    PubMed

    Caro, L H; Tettelin, H; Vossen, J H; Ram, A F; van den Ende, H; Klis, F M

    1997-12-01

    Use of the Von Heijne algorithm allowed the identification of 686 open reading frames (ORFs) in the genome of Saccharomyces cerevisiae that encode proteins with a potential N-terminal signal sequence for entering the secretory pathway. On further analysis, 51 of these proteins contain a potential glycosyl-phosphatidylinositol (GPI)-attachment signal. Seven additional ORFs were found to belong to this group. Upon examination of the possible GPI-attachment sites, it was found that in yeast the most probable amino acids for GPI-attachment as asparagine and glycine. In yeast, GPI-proteins are found at the cell surface, either attached to the plasma-membrane or as an intrinsic part of the cell wall. It was noted that plasma-membrane GPI-proteins possess a dibasic residue motif just before their predicted GPI-attachment site. Based on this, and on homologies between proteins, families of plasma-membrane and cell wall proteins were assigned, revealing 20 potential plasma-membrane and 38 potential cell wall proteins. For members of three plasma-membrane protein families, a function has been described. On the other hand, most of the cell wall proteins seem to be structural components of the wall, responsive to different growth conditions. The GPI-attachment site of yeast slightly differs from mammalian cells. This might be of use in the development of anti-fungal drugs.

  19. Plasma protein thiols, ceruloplasmin, C-reactive protein and red blood cell acetylcholinesterase in patients undergoing intrauterine insemination

    PubMed Central

    Prabhu, Krishnananda; Kumar, Pratap; Adiga, Satish Kumar; Rao, Anjali; Lanka, Anupama; Singh, Jaipal

    2009-01-01

    OBJECTIVE: To estimate acetylcholinesterase (AChE), protein thiols (PT), ceruloplasmin (CP) and C-reactive proteins (CRPs) to assess any change in their levels following intrauterine insemination (IUI). MATERIALS AND METHODS: Forty-two patients aged 31 ± 4.65 years (mean ± SD) with primary infertility selected for IUI. All of them had induced ovulation with clomiphene citrate 50 mg from day 2 to day 6. After taking the consent, 2 ml of blood was withdrawn before and after 24 h of IUI for biochemical estimations. RESULTS: We observed a significant decrease in plasma CP, PT and RBC AChE (P < 0.001) following IUI compared with the respective pre-procedure levels. Highly sensitive CRP showed a marginal increase after IUI. CONCLUSION: Fluctuations in levels of the above parameters point to their role in the female reproductive system and in the outcome of the IUI. PMID:19562071

  20. Irreversible binding of zomepirac to plasma protein in vitro and in vivo.

    PubMed Central

    Smith, P C; McDonagh, A F; Benet, L Z

    1986-01-01

    Zomepirac is a nonsteroidal anti-inflammatory drug recently withdrawn from use because of an unexplained high incidence of immunological reactions. It is metabolized in humans to a reactive, unstable acyl glucuronide which accumulates in plasma. Because of the similarity of zomepirac glucuronide to bilirubin glucuronide in structure and stability and the documented irreversible binding of bilirubin to albumin through its acyl glucuronide, we studied the reaction of zomepirac acyl glucuronide with albumin in vitro from pH 5 to 9 and in vivo in six healthy human volunteers who had received a single 100-mg oral dose of zomepirac. Irreversible binding of zomepirac to protein was determined by exhaustive washing of protein, followed by hydrolysis of bound zomepirac-protein adduct with base, extraction of the liberated drug, and chromatographic measurement. Irreversible binding was observed both in vitro and in vivo. The extent of binding in vitro was time- and pH-dependent. In vitro drug binding was also observed for the isomers of zomepirac glucuronide which were formed by intramolecular acyl migration. Irreversible binding in vivo correlated with overall exposure to zomepirac glucuronide when exposure was expressed as the area under the plasma concentration vs. time curve. When probenecid (500 mg, twice daily), which decreases the plasma clearance of zomepirac glucuronide, was administered concurrently with zomepirac, irreversible binding of zomepirac was increased. The nature of the zomepirac protein binding is probably covalent. Formation of irreversibly protein-bound zomepirac occurs via the acyl glucuronide as previously shown for bilirubin glucuronide, and the reaction may be general for other drugs that are metabolized to acyl glucuronides. PMID:3949982

  1. Analyzing Protein Clusters on the Plasma Membrane: Application of Spatial Statistical Analysis Methods on Super-Resolution Microscopy Images.

    PubMed

    Paparelli, Laura; Corthout, Nikky; Pavie, Benjamin; Annaert, Wim; Munck, Sebastian

    2016-01-01

    The spatial distribution of proteins within the cell affects their capability to interact with other molecules and directly influences cellular processes and signaling. At the plasma membrane, multiple factors drive protein compartmentalization into specialized functional domains, leading to the formation of clusters in which intermolecule interactions are facilitated. Therefore, quantifying protein distributions is a necessity for understanding their regulation and function. The recent advent of super-resolution microscopy has opened up the possibility of imaging protein distributions at the nanometer scale. In parallel, new spatial analysis methods have been developed to quantify distribution patterns in super-resolution images. In this chapter, we provide an overview of super-resolution microscopy and summarize the factors influencing protein arrangements on the plasma membrane. Finally, we highlight methods for analyzing clusterization of plasma membrane proteins, including examples of their applications. PMID:27207364

  2. Analyzing Protein Clusters on the Plasma Membrane: Application of Spatial Statistical Analysis Methods on Super-Resolution Microscopy Images.

    PubMed

    Paparelli, Laura; Corthout, Nikky; Pavie, Benjamin; Annaert, Wim; Munck, Sebastian

    2016-01-01

    The spatial distribution of proteins within the cell affects their capability to interact with other molecules and directly influences cellular processes and signaling. At the plasma membrane, multiple factors drive protein compartmentalization into specialized functional domains, leading to the formation of clusters in which intermolecule interactions are facilitated. Therefore, quantifying protein distributions is a necessity for understanding their regulation and function. The recent advent of super-resolution microscopy has opened up the possibility of imaging protein distributions at the nanometer scale. In parallel, new spatial analysis methods have been developed to quantify distribution patterns in super-resolution images. In this chapter, we provide an overview of super-resolution microscopy and summarize the factors influencing protein arrangements on the plasma membrane. Finally, we highlight methods for analyzing clusterization of plasma membrane proteins, including examples of their applications.

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

    NASA Astrophysics Data System (ADS)

    Gupta, P.; Ferrer, J. F.

    1982-01-01

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

  4. Toxicity challenges in environmental chemicals: Prediction of human plasma protein binding through quantitative structure-activity relationship (QSAR) models

    EPA Science Inventory

    The present study explores the merit of utilizing available pharmaceutical data to construct a quantitative structure-activity relationship (QSAR) for prediction of the fraction of a chemical unbound to plasma protein (Fub) in environmentally relevant compounds. Independent model...

  5. Effects of sugar-sweetened beverages on plasma acylation stimulating protein, leptin, and adiponectin: Relationships with metabolic outcomes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    OBJECTIVE: The effects of fructose and glucose consumption on plasma acylation stimulating protein (ASP), adiponectin, and leptin concentrations relative to energy intake, body weight, adiposity, circulating triglycerides, and insulin sensitivity were determined. DESIGN AND METHODS: Thirty two over...

  6. EVALUATION OF A WASTEWATER DISCHARGE USING VITELLOGENIN GENE EXPRESSION AND PLASMA PROTEIN LEVELS IN MALE FATHEAD MINNOWS

    EPA Science Inventory

    Liver vitellogenin gene expression and plasma vitellogenin protein presence, indicators of exposure of fish to estrogens, were measured in male fathead minnows (Pimephales promelas) caged at two locations in a constructed wetland below a sewage treatment plant effluent outfall in...

  7. Integrin-like proteins are localized to plasma membrane fractions, not plastids, in Arabidopsis

    NASA Technical Reports Server (NTRS)

    Swatzell, L. J.; Edelmann, R. E.; Makaroff, C. A.; Kiss, J. Z.

    1999-01-01

    Integrins are a large family of integral membrane proteins that function in signal transduction in animal systems. These proteins are conserved in vertebrates, invertebrates, and fungi. Evidence from previous research suggests that integrin-like proteins may be present in plants as well, and that these proteins may function in signal transduction during gravitropism. In past studies, researchers have used monoclonal and polyclonal antibodies to localize beta 1 integrin-like proteins in plants. However, there is a disparity between data collected from these studies, especially since molecular weights obtained from these investigations range from 55-120 kDa for integrin-like proteins. To date, a complete investigation which employs all three basic immunolabeling procedures, immunoblotting, immunofluorescence microscopy, and immunogold labeling, in addition to extensive fractionation and exhaustive controls, has been lacking. In this paper, we demonstrate that use of a polyclonal antibody against the cytoplasmic domain of avian beta 1-integrin can produce potential artifacts in immunolocalization studies. However, these problems can be eliminated through use of starchless mutants or proper specimen preparation prior to electrophoresis. We also show that this antibody, when applied within the described parameters and with careful controls, identifies a large (100 kDa) integrin-like protein that is localized to plasma membrane fractions in Arabidopsis.

  8. Independent mobility of proteins and lipids in the plasma membrane of Escherichia coli.

    PubMed

    Nenninger, Anja; Mastroianni, Giulia; Robson, Alexander; Lenn, Tchern; Xue, Quan; Leake, Mark C; Mullineaux, Conrad W

    2014-06-01

    Fluidity is essential for many biological membrane functions. The basis for understanding membrane structure remains the classic Singer-Nicolson model, in which proteins are embedded within a fluid lipid bilayer and able to diffuse laterally within a sea of lipid. Here we report lipid and protein diffusion in the plasma membrane of live cells of the bacterium Escherichia coli, using Fluorescence Recovery after Photobleaching (FRAP) and Total Internal Reflection Fluorescence (TIRF) microscopy to measure lateral diffusion coefficients. Lipid and protein mobility within the membrane were probed by visualizing an artificial fluorescent lipid and a simple model membrane protein consisting of a single membrane-spanning alpha-helix with a Green Fluorescent Protein (GFP) tag on the cytoplasmic side. The effective viscosity of the lipid bilayer is strongly temperature-dependent, as indicated by changes in the lipid diffusion coefficient. Surprisingly, the mobility of the model protein was unaffected by changes in the effective viscosity of the bulk lipid, and TIRF microscopy indicates that it clusters in segregated, mobile domains. We suggest that this segregation profoundly influences the physical behaviour of the protein in the membrane, with strong implications for bacterial membrane function and bacterial physiology.

  9. Treatment of Second Order Structures of Protein on Medical Equipments Using Oxygen Plasma

    NASA Astrophysics Data System (ADS)

    Hayashi, Nobuya; Kitazaki, Satoshi; Goto, Masaaki; Yagyu, Yoshihito; Yonesu, Akira

    2009-10-01

    Removal of proteins from the surface of medical equipments are attempted using an RF plasma. Oxygen gas is introduced into a vacuum chamber with dimensions of 450 mm in length, 200 mm in diameter and 20L of capacity. When an RF power (13.56 MHz, 60W) is applied to an ICP type antenna, oxygen radicals (atomic oxygen and excited oxygen molecule) are produced below the antenna. The characteristics of removing protein from the medical equipments was investigated using casein and heat-resistive keratin proteins. Initial concentration of the proteins on a CaF2 substrate is several mg/cm2. The treatment effect of proteins is determined by the peak height of chemical bonds in amide and second order structures appeared on FTIR spectra. The second order structure of a protein such as alpha-helix and beta-sheet are decomposed with the treatment period. Complete treatment of proteins including the second order structure requires several hours avoiding the damage to medical equipments.

  10. EFR3s are palmitoylated plasma membrane proteins that control responsiveness to G-protein-coupled receptors.

    PubMed

    Bojjireddy, Naveen; Guzman-Hernandez, Maria Luisa; Reinhard, Nathalie Renée; Jovic, Marko; Balla, Tamas

    2015-01-01

    The yeast Efr3p protein is a main regulator of the Stt4p phosphatidylinositol 4-kinase at contact sites between the endoplasmic reticulum and the plasma membrane. A mutation in its fly homologue Rbo, leads to diminished light responses in the eye attributed to progressively impaired PLC signaling. Here, we find that Efr3s plays a role in maintaining responsiveness to the type-I angiotensin II (AngII) receptors. siRNA-mediated depletion of EFR3A and EFR3B impaired the sustained phase of cytosolic Ca(2+) response to high concentration of AngII in HEK293 cells that express wild type but not truncated AGTR1 (AT1a receptor), missing the phosphorylation sites. Efr3 depletion had minimal effect on the recovery of plasma membrane phosphoinositides during stimulation, and AT1 receptors still underwent ligand-induced internalization. A higher level of basal receptor phosphorylation and a larger response was observed after stimulation. Moreover, Gq activation more rapidly desensitized after AngII stimulation in Efr3 downregulated cells. A similar but less pronounced effect of EFR3 depletion was observed on the desensitization of the cAMP response after stimulation with isoproterenol. These data suggest that mammalian Efr3s contribute to the control of the phosphorylation state and, hence, desensitization of AT1a receptors, and could affect responsiveness of G-protein-coupled receptors in higher eukaryotes.

  11. EFR3s are palmitoylated plasma membrane proteins that control responsiveness to G-protein-coupled receptors

    PubMed Central

    Bojjireddy, Naveen; Guzman-Hernandez, Maria Luisa; Reinhard, Nathalie Renée; Jovic, Marko; Balla, Tamas

    2015-01-01

    ABSTRACT The yeast Efr3p protein is a main regulator of the Stt4p phosphatidylinositol 4-kinase at contact sites between the endoplasmic reticulum and the plasma membrane. A mutation in its fly homologue Rbo, leads to diminished light responses in the eye attributed to progressively impaired PLC signaling. Here, we find that Efr3s plays a role in maintaining responsiveness to the type-I angiotensin II (AngII) receptors. siRNA-mediated depletion of EFR3A and EFR3B impaired the sustained phase of cytosolic Ca2+ response to high concentration of AngII in HEK293 cells that express wild type but not truncated AGTR1 (AT1a receptor), missing the phosphorylation sites. Efr3 depletion had minimal effect on the recovery of plasma membrane phosphoinositides during stimulation, and AT1 receptors still underwent ligand-induced internalization. A higher level of basal receptor phosphorylation and a larger response was observed after stimulation. Moreover, Gq activation more rapidly desensitized after AngII stimulation in Efr3 downregulated cells. A similar but less pronounced effect of EFR3 depletion was observed on the desensitization of the cAMP response after stimulation with isoproterenol. These data suggest that mammalian Efr3s contribute to the control of the phosphorylation state and, hence, desensitization of AT1a receptors, and could affect responsiveness of G-protein-coupled receptors in higher eukaryotes. PMID:25380825

  12. Seminal plasma proteins interacting with sperm surface revert capacitation indicators in frozen-thawed ram sperm.

    PubMed

    Ledesma, Alba; Fernández-Alegre, Estela; Cano, Adriana; Hozbor, Federico; Martínez-Pastor, Felipe; Cesari, Andreína

    2016-10-01

    This study was conducted to evaluate the effects of interacting seminal plasma proteins (iSPP) obtained by AV or EE on frozen-thawed ram sperm in order to test the hypothesis whether this fraction could be sufficient to emulate the effect of complete seminal plasma (SP). Additionally, we evaluated whether these proteins have a differential effect between spermatozoa from high and low fertility rams and between breeding and non-breeding seasons. We assessed sperm motility, quality parameters (intracellular reactive oxygen species, membrane fluidity, plasma membrane permeability and mitochondrial activity) and capacitation status. The main findings from this work were: i) iSPP had no effect on sperm motility, whereas SP (AV or EE) addition produced the highest values of total motility (74.13±2.99 and 72.27±2.99 for AV and EE, respectively) and progressive motility (64.97±2.64 and 63.73±2.64 for AV and EE, respectively); ii) iSPP had no effect on sperm quality parameters (p>0.05), but whole SP improved all parameters evaluated. Moreover, SP collected by AV yielded significantly higher viability (44.60±2.87) and sperm with stable plasma membrane (44.56±2.49) comparing with the addition of SP collected by EE (35.80±2.47 and 36.67±1.71, respectively); iii) iSPP and SP collected by EE, but not by AV, reverted molecular signals of capacitation as protein tyrosine phosphorylation caused by freezing temperatures; iv) there were no effects of fertility or season in sperm quality parameters evaluated. This study demonstrated that, although the iSPP have a clear decapacitating effect, including the ability to revert cryo-capacitation indicators, they are not sufficient to emulate the effects of complete SP regarding sperm functional parameters. PMID:27570190

  13. Seminal plasma proteins interacting with sperm surface revert capacitation indicators in frozen-thawed ram sperm.

    PubMed

    Ledesma, Alba; Fernández-Alegre, Estela; Cano, Adriana; Hozbor, Federico; Martínez-Pastor, Felipe; Cesari, Andreína

    2016-10-01

    This study was conducted to evaluate the effects of interacting seminal plasma proteins (iSPP) obtained by AV or EE on frozen-thawed ram sperm in order to test the hypothesis whether this fraction could be sufficient to emulate the effect of complete seminal plasma (SP). Additionally, we evaluated whether these proteins have a differential effect between spermatozoa from high and low fertility rams and between breeding and non-breeding seasons. We assessed sperm motility, quality parameters (intracellular reactive oxygen species, membrane fluidity, plasma membrane permeability and mitochondrial activity) and capacitation status. The main findings from this work were: i) iSPP had no effect on sperm motility, whereas SP (AV or EE) addition produced the highest values of total motility (74.13±2.99 and 72.27±2.99 for AV and EE, respectively) and progressive motility (64.97±2.64 and 63.73±2.64 for AV and EE, respectively); ii) iSPP had no effect on sperm quality parameters (p>0.05), but whole SP improved all parameters evaluated. Moreover, SP collected by AV yielded significantly higher viability (44.60±2.87) and sperm with stable plasma membrane (44.56±2.49) comparing with the addition of SP collected by EE (35.80±2.47 and 36.67±1.71, respectively); iii) iSPP and SP collected by EE, but not by AV, reverted molecular signals of capacitation as protein tyrosine phosphorylation caused by freezing temperatures; iv) there were no effects of fertility or season in sperm quality parameters evaluated. This study demonstrated that, although the iSPP have a clear decapacitating effect, including the ability to revert cryo-capacitation indicators, they are not sufficient to emulate the effects of complete SP regarding sperm functional parameters.

  14. Investigating the effect of an arterial hypertension drug on the structural properties of plasma protein.

    PubMed

    Hassan, Natalia; Maldonado-Valderrama, Julia; Gunning, A Patrick; Morris, V J; Ruso, Juan M

    2011-10-15

    Propanolol is a betablocker drug used in the treatment of arterial hypertension related diseases. In order to achieve an optimal performance of this drug it is important to consider the possible interactions of propanolol with plasma proteins. In this work, we have used several experimental techniques to characterise the effect of addition of the betablocker propanolol on the properties of bovine plasma fibrinogen (FB). Differential scanning calorimeter (DSC), circular dichroism (CD), dynamic light scattering (DLS), surface tension techniques and atomic force microscopy (AFM) measurements have been combined to carry out a detailed physicochemical and surface characterization of the mixed system. As a result, DSC measurements show that propranolol can play two opposite roles, either acting as a structure stabilizer at low molar concentrations or as a structure destabilizer at higher concentrations, in different domains of fibrinogen. CD measurements have revealed that the effect of propanolol on the secondary structure of fibrinogen depends on the temperature and the drug concentration and the DLS analysis showed evidence for protein aggregation. Interestingly, surface tension measurements provided further evidence of the conformational change induced by propanolol on the secondary structure of FB by importantly increasing the surface tension of the system. Finally, AFM imaging of the fibrinogen system provided direct visualization of the protein structure in the presence of propanolol. Combination of these techniques has produced complementary information on the behavior of the mixed system, providing new insights into the structural properties of proteins with potential medical interest.

  15. Super-Resolution Imaging of Plasma Membrane Proteins with Click Chemistry

    PubMed Central

    Mateos-Gil, Pablo; Letschert, Sebastian; Doose, Sören; Sauer, Markus

    2016-01-01

    Besides its function as a passive cell wall, the plasma membrane (PM) serves as a platform for different physiological processes such as signal transduction and cell adhesion, determining the ability of cells to communicate with the exterior, and form tissues. Therefore, the spatial distribution of PM components, and the molecular mechanisms underlying it, have important implications in various biological fields including cell development, neurobiology, and immunology. The existence of confined compartments in the plasma membrane that vary on many length scales from protein multimers to micrometer-size domains with different protein and lipid composition is today beyond all questions. As much as the physiology of cells is controlled by the spatial organization of PM components, the study of distribution, size, and composition remains challenging. Visualization of the molecular distribution of PM components has been impeded mainly due to two problems: the specific labeling of lipids and proteins without perturbing their native distribution and the diffraction-limit of fluorescence microscopy restricting the resolution to about half the wavelength of light. Here, we present a bioorthogonal chemical reporter strategy based on click chemistry and metabolic labeling for efficient and specific visualization of PM proteins and glycans with organic fluorophores in combination with super-resolution fluorescence imaging by direct stochastic optical reconstruction microscopy (dSTORM) with single-molecule sensitivity. PMID:27668214

  16. Super-Resolution Imaging of Plasma Membrane Proteins with Click Chemistry

    PubMed Central

    Mateos-Gil, Pablo; Letschert, Sebastian; Doose, Sören; Sauer, Markus

    2016-01-01

    Besides its function as a passive cell wall, the plasma membrane (PM) serves as a platform for different physiological processes such as signal transduction and cell adhesion, determining the ability of cells to communicate with the exterior, and form tissues. Therefore, the spatial distribution of PM components, and the molecular mechanisms underlying it, have important implications in various biological fields including cell development, neurobiology, and immunology. The existence of confined compartments in the plasma membrane that vary on many length scales from protein multimers to micrometer-size domains with different protein and lipid composition is today beyond all questions. As much as the physiology of cells is controlled by the spatial organization of PM components, the study of distribution, size, and composition remains challenging. Visualization of the molecular distribution of PM components has been impeded mainly due to two problems: the specific labeling of lipids and proteins without perturbing their native distribution and the diffraction-limit of fluorescence microscopy restricting the resolution to about half the wavelength of light. Here, we present a bioorthogonal chemical reporter strategy based on click chemistry and metabolic labeling for efficient and specific visualization of PM proteins and glycans with organic fluorophores in combination with super-resolution fluorescence imaging by direct stochastic optical reconstruction microscopy (dSTORM) with single-molecule sensitivity.

  17. Super-Resolution Imaging of Plasma Membrane Proteins with Click Chemistry.

    PubMed

    Mateos-Gil, Pablo; Letschert, Sebastian; Doose, Sören; Sauer, Markus

    2016-01-01

    Besides its function as a passive cell wall, the plasma membrane (PM) serves as a platform for different physiological processes such as signal transduction and cell adhesion, determining the ability of cells to communicate with the exterior, and form tissues. Therefore, the spatial distribution of PM components, and the molecular mechanisms underlying it, have important implications in various biological fields including cell development, neurobiology, and immunology. The existence of confined compartments in the plasma membrane that vary on many length scales from protein multimers to micrometer-size domains with different protein and lipid composition is today beyond all questions. As much as the physiology of cells is controlled by the spatial organization of PM components, the study of distribution, size, and composition remains challenging. Visualization of the molecular distribution of PM components has been impeded mainly due to two problems: the specific labeling of lipids and proteins without perturbing their native distribution and the diffraction-limit of fluorescence microscopy restricting the resolution to about half the wavelength of light. Here, we present a bioorthogonal chemical reporter strategy based on click chemistry and metabolic labeling for efficient and specific visualization of PM proteins and glycans with organic fluorophores in combination with super-resolution fluorescence imaging by direct stochastic optical reconstruction microscopy (dSTORM) with single-molecule sensitivity. PMID:27668214

  18. Studies on rat liver plasma membrane. Altered protein and phospholipid metabolism after injection of D-galactosamine.

    PubMed Central

    Bachmann, W; Harms, E; Hassels, B; Henninger, H; Reuitter, W

    1977-01-01

    1. The metabolism of protein and phospholipid in rat liver plasma membranes isolated by the method of Neville [(1960) J. Biophys. Biochem. Cytol. 8, 413-422] was investigated 3 and 6 h after the injection of D-galactosamine in vivo. During this time, all the biochemical and morphological alterations associated with hepatitis developed. 2. After the injection of D-galactosamine the concentration of sphingomyelin in the plasma membrane decreased to below 60% of the control values. 3. The activity of 5'-nucleotidase (EC 3.1.3.5), which has been purified as a sphingomyelin-protein complex, decreased in the total homogenate as well as in the plasma-membrane fraction of livers of rats treated with galactosamine, to about 60% of the control values. 4. Protein synthesis, as measured by the incorporation of [14C]leucine into plasma membranes, was decreased to 45% of that of the controls. However, only small differences were observed in the amino acid composition of the plasma membrane after D-galactosamine treatment. 5. The protein composition of the plasma membranes was determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The results showed a change from low- to high-molecular-weight proteins after the injection of galactosamine. 6. These results demonstrate different metabolic processes of the plasma membrane altered during the induction of galactosamine hepatitis. Images Fig. 1. PMID:597240

  19. Expression and secretion of rabbit plasma cholesteryl ester transfer protein by Pichia pastoris.

    PubMed

    Kotake, H; Li, Q; Ohnishi, T; Ko, K W; Agellon, L B; Yokoyama, S

    1996-03-01

    The rabbit cholesteryl ester transfer protein (CETP) was expressed in the methylotrophic yeast Pichia pastoris by introducing the CETP cDNA under the control of the methanol-inducible alcohol oxidase promoter. The cDNA was cloned from in vitro amplified cDNA of rabbit liver mRNA. The nucleotide sequence of the cloned cDNA differed slightly from the previously published sequence that changed the amino acid sequence in six residues. Interestingly, five of these replacements are identical to the corresponding residues in human CEPT. In addition, the encoded mature N-terminal sequence was changed from Cys- to Arg-Glu-Phe- to link the CETP sequence to the yeast acid phosphatase signal peptide. The culture medium of the transformed cells induced with 1% methanol contained both cholesteryl ester and triglyceride transfer activity comparable to that of rabbit plasma. Like rabbit plasma, the lipid transfer activity in the medium could be inhibited by monoclonal antibodies that block CE/TG transfer or TG transfer alone. Immunoblot analysis of M(r) = 80 K and minor species of M(r) = 60-100 K. In spite of these differences, the specific transfer activity of the recombinant CETP was indistinguishable from that of rabbit plasma CETP of M(r) = 74 K. N-Glycosidase F treatment converted both the recombinant and plasma CETP to a single species of M(r) = 55 K. Both the plasma and recombinant CETP lost their activity after removal of N-linked carbohydrate and sialic acid. A single 55 K component was found in the cell-lysates. The intracellular form of the recombinant CETP was not modified by N-glycosidase F treatment. In conclusion, the recombinant CETP is synthesized as an inactive polypeptide that is processed and secreted as a functional glycoprotein. In addition, the N-terminal Cys residue of the plasma CETP is not required for its activity. PMID:8728322

  20. Maternal Plasma Retinol Binding Protein 4 in Acute Pyelonephritis during Pregnancy

    PubMed Central

    Vaisbuch, Edi; Romero, Roberto; Mazaki-Tovi, Shali; Kusanovic, Juan Pedro; Chaiworapongsa, Tinnakorn; Dong, Zhong; Kim, Sun Kwon; Ogge, Giovanna; Gervasi, Maria Teresa; Hassan, Sonia S.

    2010-01-01

    Objective Adipokines have been implicated in metabolic regulation and the immune response thus providing a molecular mechanism for the interaction between these two systems. Retinol binding protein 4 (RBP4) is a novel adipokine that plays a role in the pathophysiology of obesity-induced insulin resistance, as well as in the modulation of inflammation. The aim of this study was to determine whether there are changes in maternal plasma concentrations of RBP4 in pregnant women with acute pyelonephritis. Study design This cross-sectional study included pregnant women in the following groups: 1) normal pregnancy (n=80); 2) pyelonephritis (n=39). Maternal plasma RBP4 concentrations were determined by enzyme-linked immunoassays. Non-parametric statistics were used for analyses. Results 1) The median maternal plasma RBP4 concentration was lower in patients with acute pyelonephritis than in those with a normal pregnancy (3709.6 ng/mL, IQR 2917.7-5484.2 vs. 9167.6 ng/mL, IQR 7496.1-10384.1, p<0.001; 2) the median maternal plasma RBP4 concentration did not differ significantly between patients with acute pyelonephritis who had a positive blood culture and those with a negative culture (3285.3 ng/mL, IQR 2274.1-4741.1 vs. 3922.6 ng/mL, IQR 3126.8-5547.1, respectively, p=0.2); and 3) lower maternal plasma RBP4 concentrations were independently associated with pyelonephritis after adjustment for confounding factors. Conclusions In contrast to what has been reported in preeclampsia, acute pyelonephritis during pregnancy is associated with lower maternal plasma RBP4 concentrations than in normal pregnancy. This finding suggests that the acute maternal inflammatory process associated with pyelonephritis is fundamentally different from that of the chronic systemic inflammatory process suggested in preeclampsia, in which RBP4 concentrations were found to be elevated. PMID:20163326

  1. [Protein assay by the modified Dumas method applied to preparations of plasma proteins].

    PubMed

    Blondel, P; Vian, L

    1993-01-01

    Quantify protein according Pharmacopoeia method, based on Kjeldahl method, needs a long time to do. The development of an automaton which used the modified Dumas method divide the analysis time by 15 (6 minutes versus over 90 minutes). The results show no statistical differences between official method and this one. PMID:8154798

  2. Shotgun proteomics and network analysis between plasma membrane and extracellular matrix proteins from rat olfactory ensheathing cells.

    PubMed

    Liu, Yisong; Teng, Xiaohua; Yang, Xiaoxu; Song, Qing; Lu, Rong; Xiong, Jixian; Liu, Bo; Zeng, Nianju; Zeng, Yu; Long, Jia; Cao, Rui; Lin, Yong; He, Quanze; Chen, Ping; Lu, Ming; Liang, Songping

    2010-01-01

    Olfactory ensheathing cells (OECs) are a special type of glial cells that have characteristics of both astrocytes and Schwann cells. Evidence suggests that the regenerative capacity of OECs is induced by soluble, secreted factors that influence their microenvironment. These factors may regulate OECs self-renewal and/or induce their capacity to augment spinal cord regeneration. Profiling of plasma membrane and extracellular matrix through a high-throughput expression proteomics approach was undertaken to identify plasma membrane and extracellular matrix proteins of OECs under serum-free conditions. 1D-shotgun proteomics followed with gene ontology (GO) analysis was used to screen proteins from primary culture rat OECs. Four hundred and seventy nonredundant plasma membrane proteins and 168 extracellular matrix proteins were identified, the majority of which were never before reported to be produced by OECs. Furthermore, plasma membrane and extracellular proteins were classified based on their protein-protein interaction predicted by STRING quantitatively integrates interaction data. The proteomic profiling of the OECs plasma membrane proteins and their connection with the secretome in serum-free culture conditions provides new insights into the nature of their in vivo microenvironmental niche. Proteomic analysis for the discovery of clinical biomarkers of OECs mechanism warrants further study.

  3. Seminal plasma proteins inhibit in vitro- and cooling-induced capacitation in boar spermatozoa.

    PubMed

    Vadnais, Melissa L; Roberts, Kenneth P

    2010-01-01

    Dilute boar seminal plasma (SP) has been shown to inhibit in vitro capacitation and cooling-induced capacitation-like changes in boar spermatozoa, as assessed by the ability of the spermatozoa to undergo an ionophore-induced acrosome reaction. We hypothesised that the protein component of SP is responsible for this effect. To test this hypothesis, varying concentrations of total SP protein or SP proteins fractionated by heparin binding were assayed for their ability to inhibit in vitro capacitation, as well as cooling- and cryopreservation-induced capacitation-like changes. In vitro capacitation and cooling-induced capacitation-like changes were prevented by 10% whole SP, as well as by total proteins extracted from SP at concentrations greater than 500 microg mL(-1). No amount of SP protein was able to prevent cryopreservation-induced capacitation-like changes. Total SP proteins were fractionated based on their heparin-binding properties and the heparin-binding fraction was shown to possess capacitation inhibitory activity at concentrations as low as 250 microg mL(-1). The proteins in the heparin-binding fraction were subjected to mass spectrometry and identified. The predominant proteins were three members of the spermadhesin families, namely AQN-3, AQN-1 and AWN, and SP protein pB1. We conclude that one or more of these heparin-binding SP proteins is able to inhibit in vitro capacitation and cooling-induced capacitation-like changes, but not cryopreservation-induced capacitation-like changes, in boar spermatozoa. PMID:20591323

  4. Ubiquitin initiates sorting of Golgi and plasma membrane proteins into the vacuolar degradation pathway

    PubMed Central

    2012-01-01

    Background In yeast and mammals, many plasma membrane (PM) proteins destined for degradation are tagged with ubiquitin. These ubiquitinated proteins are internalized into clathrin-coated vesicles and are transported to early endosomal compartments. There, ubiquitinated proteins are sorted by the endosomal sorting complex required for transport (ESCRT) machinery into the intraluminal vesicles of multivesicular endosomes. Degradation of these proteins occurs after endosomes fuse with lysosomes/lytic vacuoles to release their content into the lumen. In plants, some PM proteins, which cycle between the PM and endosomal compartments, have been found to be ubiquitinated, but it is unclear whether ubiquitin is sufficient to mediate internalization and thus acts as a primary sorting signal for the endocytic pathway. To test whether plants use ubiquitin as a signal for the degradation of membrane proteins, we have translationally fused ubiquitin to different fluorescent reporters for the plasma membrane and analyzed their transport. Results Ubiquitin-tagged PM reporters localized to endosomes and to the lumen of the lytic vacuole in tobacco mesophyll protoplasts and in tobacco epidermal cells. The internalization of these reporters was significantly reduced if clathrin-mediated endocytosis was inhibited by the coexpression of a mutant of the clathrin heavy chain, the clathrin hub. Surprisingly, a ubiquitin-tagged reporter for the Golgi was also transported into the lumen of the vacuole. Vacuolar delivery of the reporters was abolished upon inhibition of the ESCRT machinery, indicating that the vacuolar delivery of these reporters occurs via the endocytic transport route. Conclusions Ubiquitin acts as a sorting signal at different compartments in the endomembrane system to target membrane proteins into the vacuolar degradation pathway: If displayed at the PM, ubiquitin triggers internalization of PM reporters into the endocytic transport route, but it also mediates

  5. Plasma protein biomarkers of Alzheimer's disease endophenotypes in asymptomatic older twins: early cognitive decline and regional brain volumes.

    PubMed

    Kiddle, S J; Steves, C J; Mehta, M; Simmons, A; Xu, X; Newhouse, S; Sattlecker, M; Ashton, N J; Bazenet, C; Killick, R; Adnan, J; Westman, E; Nelson, S; Soininen, H; Kloszewska, I; Mecocci, P; Tsolaki, M; Vellas, B; Curtis, C; Breen, G; Williams, S C R; Lovestone, S; Spector, T D; Dobson, R J B

    2015-06-16

    There is great interest in blood-based markers of Alzheimer's disease (AD), especially in its pre-symptomatic stages. Therefore, we aimed to identify plasma proteins whose levels associate with potential markers of pre-symptomatic AD. We also aimed to characterise confounding by genetics and the effect of genetics on blood proteins in general. Panel-based proteomics was performed using SOMAscan on plasma samples from TwinsUK subjects who are asymptomatic for AD, measuring the level of 1129 proteins. Protein levels were compared with 10-year change in CANTAB-paired associates learning (PAL; n = 195), and regional brain volumes (n = 34). Replication of proteins associated with regional brain volumes was performed in 254 individuals from the AddNeuroMed cohort. Across all the proteins measured, genetic factors were found to explain ~26% of the variability in blood protein levels on average. The plasma level of the mitogen-activated protein kinase (MAPK) MAPKAPK5 protein was found to positively associate with the 10-year change in CANTAB-PAL in both the individual and twin difference context. The plasma level of protein MAP2K4 was found to suggestively associate negatively (Q < 0.1) with the volume of the left entorhinal cortex. Future studies will be needed to assess the specificity of MAPKAPK5 and MAP2K4 to eventual conversion to AD.

  6. Plasma protein biomarkers of Alzheimer's disease endophenotypes in asymptomatic older twins: early cognitive decline and regional brain volumes

    PubMed Central

    Kiddle, S J; Steves, C J; Mehta, M; Simmons, A; Xu, X; Newhouse, S; Sattlecker, M; Ashton, N J; Bazenet, C; Killick, R; Adnan, J; Westman, E; Nelson, S; Soininen, H; Kloszewska, I; Mecocci, P; Tsolaki, M; Vellas, B; Curtis, C; Breen, G; Williams, S C R; Lovestone, S; Spector, T D; Dobson, R J B

    2015-01-01

    There is great interest in blood-based markers of Alzheimer's disease (AD), especially in its pre-symptomatic stages. Therefore, we aimed to identify plasma proteins whose levels associate with potential markers of pre-symptomatic AD. We also aimed to characterise confounding by genetics and the effect of genetics on blood proteins in general. Panel-based proteomics was performed using SOMAscan on plasma samples from TwinsUK subjects who are asymptomatic for AD, measuring the level of 1129 proteins. Protein levels were compared with 10-year change in CANTAB-paired associates learning (PAL; n=195), and regional brain volumes (n=34). Replication of proteins associated with regional brain volumes was performed in 254 individuals from the AddNeuroMed cohort. Across all the proteins measured, genetic factors were found to explain ~26% of the variability in blood protein levels on average. The plasma level of the mitogen-activated protein kinase (MAPK) MAPKAPK5 protein was found to positively associate with the 10-year change in CANTAB-PAL in both the individual and twin difference context. The plasma level of protein MAP2K4 was found to suggestively associate negatively (Q<0.1) with the volume of the left entorhinal cortex. Future studies will be needed to assess the specificity of MAPKAPK5 and MAP2K4 to eventual conversion to AD. PMID:26080319

  7. Plasma protein extravasation induced by mammalian tachykinins in rat skin: influence of anaesthetic agents and an acetylcholine antagonist.

    PubMed Central

    Couture, R.; Kérouac, R.

    1987-01-01

    The effect of mammalian tachykinins on plasma protein extravasation was assessed in the rat dorsal skin. Substance P (SP), neurokinin A (NKA) and neurokinin B (NKB) increased vascular permeability in a dose-related manner with a threshold dose of about 0.07 pmol in sodium pentobarbitone-anaesthetized animals. Plasma protein extravasation induced by the tachykinins was 100-500 times less in magnitude in animals anaesthetized with urethane. Plasma protein extravasation induced by SP (66 pmol) was significantly reduced (63%; P less than 0.001) by atropine (a muscarinic inhibitor) while that induced by NKA or NKB was unaffected by the inhibitor suggesting that a cholinergic component might only be involved in the vascular permeability elicited by SP. The rank order of potency for the tachykinins on plasma protein extravasation was: NKB greater than SP greater than NKA (in absence of atropine) and NKB greater than NKA greater than SP (in presence of atropine), suggesting that this vascular response is mediated by a SP-E receptor type. The amplitudes of the plasma protein extravasation induced by NKB and its hydrophilic analogue [Arg degrees]NKB were similar, indicating that the lipophilic features of the native peptide cannot account for its potent biological activity. Plasma protein extravasation was enhanced by the SP analogue [D-Pro4,Lys6,D-Trp7,9,10,Phe11]SP (4-11), thus showing the limitation of such SP analogues (antagonists) for characterizing the tachykinin receptors involved in vascular permeability. PMID:3475146

  8. Plasma protein extravasation induced by mammalian tachykinins in rat skin: influence of anaesthetic agents and an acetylcholine antagonist.

    PubMed

    Couture, R; Kérouac, R

    1987-06-01

    The effect of mammalian tachykinins on plasma protein extravasation was assessed in the rat dorsal skin. Substance P (SP), neurokinin A (NKA) and neurokinin B (NKB) increased vascular permeability in a dose-related manner with a threshold dose of about 0.07 pmol in sodium pentobarbitone-anaesthetized animals. Plasma protein extravasation induced by the tachykinins was 100-500 times less in magnitude in animals anaesthetized with urethane. Plasma protein extravasation induced by SP (66 pmol) was significantly reduced (63%; P less than 0.001) by atropine (a muscarinic inhibitor) while that induced by NKA or NKB was unaffected by the inhibitor suggesting that a cholinergic component might only be involved in the vascular permeability elicited by SP. The rank order of potency for the tachykinins on plasma protein extravasation was: NKB greater than SP greater than NKA (in absence of atropine) and NKB greater than NKA greater than SP (in presence of atropine), suggesting that this vascular response is mediated by a SP-E receptor type. The amplitudes of the plasma protein extravasation induced by NKB and its hydrophilic analogue [Arg degrees]NKB were similar, indicating that the lipophilic features of the native peptide cannot account for its potent biological activity. Plasma protein extravasation was enhanced by the SP analogue [D-Pro4,Lys6,D-Trp7,9,10,Phe11]SP (4-11), thus showing the limitation of such SP analogues (antagonists) for characterizing the tachykinin receptors involved in vascular permeability.

  9. Identification and properties of steroid-binding proteins in nesting Chelonia mydas plasma.

    PubMed

    Ikonomopoulou, M P; Bradley, A J; Whittier, J M; Ibrahim, K

    2006-11-01

    We report for the first time the presence of a sex steroid-binding protein in the plasma of green sea turtles Chelonia mydas, which provides an insight into reproductive status. A high affinity, low capacity sex hormone steroid-binding protein was identified in nesting C. mydas and its thermal profile was established. In nesting C. mydas testosterone and oestradiol bind at 4 degrees C with high affinity (K (a) = 1.49 +/- 0.09 x 10(9) M(-1); 0.17 +/- 0.02 x 10(7) M(-1)) and low binding capacity (B (max) = 3.24 +/- 0.84 x 10(-5) M; 0.33 +/- 0.06 x 10(-4) M). The binding affinity and capacity of testosterone at 23 and 36 degrees C, respectively were similar to those determined at 4 degrees C. However, oestradiol showed no binding activity at 36 degrees C. With competition studies we showed that oestradiol and oestrone do not compete for binding sites. Furthermore, in nesting C. mydas plasma no high-affinity binding was observed for adrenocortical steroids (cortisol and corticosterone) and progesterone. Our results indicate that in nesting C. mydas plasma temperature has a minimal effect on the high-affinity binding of testosterone to sex steroid-binding protein, however, the high affinity binding of oestradiol to sex steroid-binding protein is abolished at a hypothetically high (36 degrees C) sea/ambient/body temperature. This suggests that at high core body temperatures most of the oestradiol becomes biologically available to the tissues rather than remaining bound to a high-affinity carrier.

  10. Accuracy and Reproducibility in Quantification of Plasma Protein Concentrations by Mass Spectrometry without the Use of Isotopic Standards

    PubMed Central

    Kramer, Gertjan; Woolerton, Yvonne; van Straalen, Jan P.; Vissers, Johannes P. C.; Dekker, Nick; Langridge, James I.; Beynon, Robert J.; Speijer, Dave; Sturk, Auguste; Aerts, Johannes M. F. G.

    2015-01-01

    Background Quantitative proteomic analysis with mass spectrometry holds great promise for simultaneously quantifying proteins in various biosamples, such as human plasma. Thus far, studies addressing the reproducible measurement of endogenous protein concentrations in human plasma have focussed on targeted analyses employing isotopically labelled standards. Non-targeted proteomics, on the other hand, has been less employed to this end, even though it has been instrumental in discovery proteomics, generating large datasets in multiple fields of research. Results Using a non-targeted mass spectrometric assay (LCMSE), we quantified abundant plasma proteins (43 mg/mL—40 ug/mL range) in human blood plasma specimens from 30 healthy volunteers and one blood serum sample (ProteomeXchange: PXD000347). Quantitative results were obtained by label-free mass spectrometry using a single internal standard to estimate protein concentrations. This approach resulted in quantitative results for 59 proteins (cut off ≥11 samples quantified) of which 41 proteins were quantified in all 31 samples and 23 of these with an inter-assay variability of ≤ 20%. Results for 7 apolipoproteins were compared with those obtained using isotope-labelled standards, while 12 proteins were compared to routine immunoassays. Comparison of quantitative data obtained by LCMSE and immunoassays showed good to excellent correlations in relative protein abundance (r = 0.72–0.96) and comparable median concentrations for 8 out of 12 proteins tested. Plasma concentrations of 56 proteins determined by LCMSE were of similar accuracy as those reported by targeted studies and 7 apolipoproteins quantified by isotope-labelled standards, when compared to reference concentrations from literature. Conclusions This study shows that LCMSE offers good quantification of relative abundance as well as reasonable estimations of concentrations of abundant plasma proteins. PMID:26474480

  11. Site-specific O-Glycosylation Analysis of Human Blood Plasma Proteins*

    PubMed Central

    Hoffmann, Marcus; Marx, Kristina; Reichl, Udo; Wuhrer, Manfred; Rapp, Erdmann

    2016-01-01

    Site-specific glycosylation analysis is key to investigate structure-function relationships of glycoproteins, e.g. in the context of antigenicity and disease progression. The analysis, though, is quite challenging and time consuming, in particular for O-glycosylated proteins. In consequence, despite their clinical and biopharmaceutical importance, many human blood plasma glycoproteins have not been characterized comprehensively with respect to their O-glycosylation. Here, we report on the site-specific O-glycosylation analysis of human blood plasma glycoproteins. To this end pooled human blood plasma of healthy donors was proteolytically digested using a broad-specific enzyme (Proteinase K), followed by a precipitation step, as well as a glycopeptide enrichment and fractionation step via hydrophilic interaction liquid chromatography, the latter being optimized for intact O-glycopeptides carrying short mucin-type core-1 and -2 O-glycans, which represent the vast majority of O-glycans on human blood plasma proteins. Enriched O-glycopeptide fractions were subjected to mass spectrometric analysis using reversed-phase liquid chromatography coupled online to an ion trap mass spectrometer operated in positive-ion mode. Peptide identity and glycan composition were derived from low-energy collision-induced dissociation fragment spectra acquired in multistage mode. To pinpoint the O-glycosylation sites glycopeptides were fragmented using electron transfer dissociation. Spectra were annotated by database searches as well as manually. Overall, 31 O-glycosylation sites and regions belonging to 22 proteins were identified, the majority being acute-phase proteins. Strikingly, also 11 novel O-glycosylation sites and regions were identified. In total 23 O-glycosylation sites could be pinpointed. Interestingly, the use of Proteinase K proved to be particularly beneficial in this context. The identified O-glycan compositions most probably correspond to mono- and disialylated core-1

  12. Site-specific O-Glycosylation Analysis of Human Blood Plasma Proteins.

    PubMed

    Hoffmann, Marcus; Marx, Kristina; Reichl, Udo; Wuhrer, Manfred; Rapp, Erdmann

    2016-02-01

    Site-specific glycosylation analysis is key to investigate structure-function relationships of glycoproteins, e.g. in the context of antigenicity and disease progression. The analysis, though, is quite challenging and time consuming, in particular for O-glycosylated proteins. In consequence, despite their clinical and biopharmaceutical importance, many human blood plasma glycoproteins have not been characterized comprehensively with respect to their O-glycosylation. Here, we report on the site-specific O-glycosylation analysis of human blood plasma glycoproteins. To this end pooled human blood plasma of healthy donors was proteolytically digested using a broad-specific enzyme (Proteinase K), followed by a precipitation step, as well as a glycopeptide enrichment and fractionation step via hydrophilic interaction liquid chromatography, the latter being optimized for intact O-glycopeptides carrying short mucin-type core-1 and -2 O-glycans, which represent the vast majority of O-glycans on human blood plasma proteins. Enriched O-glycopeptide fractions were subjected to mass spectrometric analysis using reversed-phase liquid chromatography coupled online to an ion trap mass spectrometer operated in positive-ion mode. Peptide identity and glycan composition were derived from low-energy collision-induced dissociation fragment spectra acquired in multistage mode. To pinpoint the O-glycosylation sites glycopeptides were fragmented using electron transfer dissociation. Spectra were annotated by database searches as well as manually. Overall, 31 O-glycosylation sites and regions belonging to 22 proteins were identified, the majority being acute-phase proteins. Strikingly, also 11 novel O-glycosylation sites and regions were identified. In total 23 O-glycosylation sites could be pinpointed. Interestingly, the use of Proteinase K proved to be particularly beneficial in this context. The identified O-glycan compositions most probably correspond to mono- and disialylated core-1

  13. Plasma proteins as indices of response to nutritional therapy in cancer patients.

    PubMed

    Ota, D M; Frasier, P; Guevara, J; Foulkes, M

    1985-07-01

    The use of plasma albumin (ALB), transferrin (TFN), prealbumin (TBPA), retinol-binding protein (RBP), triceps skin fold (TSF), and midarm muscle circumference (MAMC) as determinants of response to nutritional therapy (TPN) was investigated in 40 cancer patients during preoperative TPN. Thirty-one patients received 90% or more of their anabolic caloric requirement (Harris-Benedict equation) by means of TPN. During this study period (average 11.1 +/- 4.7 days) nutritional assessments were completed before TPN and on the last day of TPN before surgery. Average weight loss based on usual body wt (UBW) and ideal body wt (IBW) was 19 +/- 11% and 9 +/- 15%, respectively (not significant, NS). Weight loss (UBW) correlated with ALB (P less than 0.001), TBPA (P less than 0.005) and RBP (P less than 0.02) but did not correlate with TFN (P less than 0.06), TSF, and MAMC. Weight loss (IBW) correlated with TSF (P less than 0.01) and MAMC (P less than 0.03) but did not correlate with plasma protein (PP). During TPN the average percent increases for PP were 0.1% (ALB, NS), 20% (TFN, NS), 60% (TBPA, P less than 0.02), and 116% (RBP, P less than 0.005). These results suggest that plasma TBPA and RBP are significant parameters of response to short-term nutritional therapy in cancer patients.

  14. Extracellular vesicles are rapidly purified from human plasma by PRotein Organic Solvent PRecipitation (PROSPR)

    PubMed Central

    Gallart-Palau, Xavier; Serra, Aida; Wong, Andrew See Weng; Sandin, Sara; Lai, Mitchell K. P.; Chen, Christopher P.; Kon, Oi Lian; Sze, Siu Kwan

    2015-01-01

    Extracellular vesicles (EVs) such as exosomes and microvesicles mediate intercellular communication and regulate a diverse range of crucial biological processes. Host cells that are damaged, infected or transformed release biomarker-containing EVs into the peripheral circulation, where they can be readily accessed for use in diagnostic or prognostic testing. However, current methods of EV isolation from blood plasma are complex and often require relatively large sample volumes, hence are inefficient for widespread use in clinical settings. Here, we report a novel and inexpensive method of rapidly isolating EVs from small volumes of human blood plasma by PRotein Organic Solvent PRecipitation (PROSPR). PROSPR encompasses a rapid three-step protocol to remove soluble proteins from plasma via precipitation in cold acetone, leaving the lipid-encapsulated EVs behind in suspension. This generates higher purity EVs that can then be obtained from filtration or classical ultracentrifugation methods. We foresee that PROSPR-based purification of EVs will significantly accelerate the discovery of new disease biomarkers and the characterization of EVs with potential for clinical applications. PMID:26419333

  15. Exchange of adsorbed serum proteins during adhesion of Staphylococcus aureus to an abiotic surface and Candida albicans hyphae--an AFM study.

    PubMed

    Ovchinnikova, Ekaterina S; van der Mei, Henny C; Krom, Bastiaan P; Busscher, Henk J

    2013-10-01

    Staphylococcus aureus and Candida albicans are the second and third most commonly isolated microorganisms in hospital-related-infections, that are often multi-species in nature causing high morbidity and mortality. Here, adhesion forces between a S. aureus strain and abiotic (tissue-culture-polystyrene, TCPS) or partly biotic (TCPS with adhering hyphae of C. albicans) surfaces were investigated in presence of fetal-bovine-serum or individual serum proteins and related with staphylococcal adhesion. Atomic-force-microscopy was used to measure adhesion forces between S. aureus and the abiotic and biotic surfaces. Adsorption of individual serum proteins like albumin and apo-transferrin to abiotic TCPS surfaces during 60min, impeded development of strong adhesion forces as compared to fibronectin, while 60min adsorption of proteins from fetal-bovine-serum yielded a decrease in adhesion force from -5.7nN in phosphate-buffered-saline to -0.6nN. Adsorption of albumin and apo-transferrin also decreased staphylococcal adhesion forces to hyphae as compared with fibronectin. During 60min exposure to fetal-bovine-serum however, initial (5min protein adsorption) staphylococcal adhesion forces were low (-1.6nN), but strong adhesion forces of around -5.5nN were restored within 60min. This suggests for the first time that in whole fetal-bovine-serum exchange of non-adhesive proteins by fibronectin occurs on biotic C. albicans hyphal surfaces. No evidence was found for such protein exchange on abiotic TCPS surfaces. Staphylococcal adhesion of abiotic and biotic surfaces varied in line with the adhesion forces and was low on TCPS in presence of fetal-bovine-serum. On partly biotic TCPS, staphylococci aggregated in presence of fetal-bovine-serum around adhering C. albicans hyphae.

  16. Plasma soluble prion protein, a potential biomarker for sport-related concussions: a pilot study.

    PubMed

    Pham, Nam; Akonasu, Hungbo; Shishkin, Rhonda; Taghibiglou, Changiz

    2015-01-01

    Sport-related mild traumatic brain injury (mTBI) or concussion is a significant health concern to athletes with potential long-term consequences. The diagnosis of sport concussion and return to sport decision making is one of the greatest challenges facing health care clinicians working in sports. Blood biomarkers have recently demonstrated their potential in assisting the detection of brain injury particularly, in those cases with no obvious physical injury. We have recently discovered plasma soluble cellular prion protein (PrP(C)) as a potential reliable biomarker for blast induced TBI (bTBI) in a rodent animal model. In order to explore the application of this novel TBI biomarker to sport-related concussion, we conducted a pilot study at the University of Saskatchewan (U of S) by recruiting athlete and non-athlete 18 to 30 year-old students. Using a modified quantitative ELISA method, we first established normal values for the plasma soluble PrP(C) in male and female students. The measured plasma soluble PrP(C) in confirmed concussion cases demonstrated a significant elevation of this analyte in post-concussion samples. Data collected from our pilot study indicates that the plasma soluble PrP(C) is a potential biomarker for sport-related concussion, which may be further developed into a clinical diagnostic tool to assist clinicians in the assessment of sport concussion and return-to-play decision making.

  17. Pharmacokinetics, urinary excretion and plasma protein binding of ofloxacin in water buffalo calves (Bubalus bubalis).

    PubMed

    Ola, Ajay K; Sandhu, Harpal S; Dumka, Vinod K; Ranjan, Bibhuti

    2013-04-18

    Pharmacokinetics and urinary excretion of an intravenous dose of 5 mg.kg-1 ofloxacin were investigated in water buffalo calves. Plasma concentrations of ofloxacin were determined by high-performance liquid chromatography. Ofloxacin was rapidly distributed from the central to the peripheral compartment as evidenced by a short distribution half-life (0.09 h ± 0.003 h) and high K12 (4.7 h(-1) ± 0.1 h(-1)), and was detected in plasma for 8 h. The large volume of distribution (2.48 L.kg(-1) ± 0.18 L.kg(-1)) obtained in this study indicated high distribution of ofloxacin in water buffalo calves. The elimination half-life, the area under the plasma drug concentration-time curve and total body clearance were 2.11 h ± 0.13 h, 6.20 µg.mL(-1) ± 0.23 µg.mL(-1).h and 0.81 mL.kg(-1).h(-1) ± 0.03 mL.kg(-1).h(-1), respectively. About 18.7% of administered drug was bound to plasma proteins and approximately 32.5% of the administered dose was recovered in urine within 48 h. The results of the study indicated a favourable pharmacokinetic profile of ofloxacin in water buffalo calves, which suggests that ofloxacin may be effective against urinary pathogens in this species.

  18. Irreversible binding of an anticancer compound (BI-94) to plasma proteins

    PubMed Central

    Gautam, Nagsen; Thakare, Rhishikesh; Rana, Sandeep; Natarajan, Amarnath; Alnouti, Yazen

    2015-01-01

    1. We investigated the mechanisms responsible for the in vivo instability of a benzofurazan compound BI-94 (NSC228148) with potent anti-cancer activity. 2. BI-94 was stable in MeOH, water, and in various buffers at pHs 2.5–5, regardless of the buffer composition. In contrast, BI-94 was unstable in NaOH and at pHs 7–9, regardless of the buffer composition. BI-94 disappeared immediately after spiking into mice, rat, monkey, and human plasma. BI-94 stability in plasma can be only partially restored by acidifying it, which indicated other mechanisms in addition to pH for BI-94 instability in plasma. 3. BI-94 formed adducts with the trapping agents, glutathione (GSH) and N-acetylcysteine (NAC), in vivo and in vitro via nucleophilic aromatic substitution reaction. The kinetics of adduct formation showed that neutral or physiological pHs enhanced and accelerated GSH and NAC adduct formation with BI-94, whereas acidic pHs prevented it. Therefore, physiological pHs not only altered BI-94 chemical stability but also enhanced adduct formation with endogenous nucleophiles. In addition, adduct formation with human serum albumin-peptide 3 (HSA-T3) at the Cys34 position was demonstrated. 4. In conclusion, BI-94 was unstable at physiological conditions due to chemical instability and irreversible binding to plasma proteins. PMID:25869245

  19. Plasma Surface Modification for Immobilization of Bone Morphogenic Protein-2 on Polycaprolactone Scaffolds

    NASA Astrophysics Data System (ADS)

    Kim, Byung Hoon; Myung, Sung Woon; Jung, Sang Chul; Ko, Yeong Mu

    2013-11-01

    The immobilization of recombinant human bone formation protein-2 (rhBMP-2) on polycaprolactone (PCL) scaffolds was performed by plasma polymerization. RhBMP-2, which induces osteoblast differentiation in various cell types, is a growth factor that plays an important role in bone formation and repair. The surface of the PCL scaffold was functionalized with the carboxyl groups of plasma-polymerized acrylic acid (PPAA) thin films. Plasma polymerization was carried out at a discharge power of 60 W at an acrylic acid flow rate of 7 sccm for 5 min. The PPAA thin film exhibited moderate hydrophilic properties and possessed a high density of carboxyl groups. Carboxyl groups and rhBMP-2 on the PCL scaffolds surface were identified by attenuated total reflection Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy, respectively. The alkaline phosphatase activity assay showed that the rhBMP-2 immobilized PCL scaffold increased the level of MG-63 cell differentiation. Plasma surface modification for the preparation of biomaterials, such as biofunctionalized polymer scaffolds, can be used for the binding of bioactive molecules in tissue engineering.

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

    PubMed

    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. PMID:26338058

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

    PubMed

    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.

  2. Determination of Dynamics of Plant Plasma Membrane Proteins with Fluorescence Recovery and Raster Image Correlation Spectroscopy.

    PubMed

    Laňková, Martina; Humpolíčková, Jana; Vosolsobě, Stanislav; Cit, Zdeněk; Lacek, Jozef; Čovan, Martin; Čovanová, Milada; Hof, Martin; Petrášek, Jan

    2016-04-01

    A number of fluorescence microscopy techniques are described to study dynamics of fluorescently labeled proteins, lipids, nucleic acids, and whole organelles. However, for studies of plant plasma membrane (PM) proteins, the number of these techniques is still limited because of the high complexity of processes that determine the dynamics of PM proteins and the existence of cell wall. Here, we report on the usage of raster image correlation spectroscopy (RICS) for studies of integral PM proteins in suspension-cultured tobacco cells and show its potential in comparison with the more widely used fluorescence recovery after photobleaching method. For RICS, a set of microscopy images is obtained by single-photon confocal laser scanning microscopy (CLSM). Fluorescence fluctuations are subsequently correlated between individual pixels and the information on protein mobility are extracted using a model that considers processes generating the fluctuations such as diffusion and chemical binding reactions. As we show here using an example of two integral PM transporters of the plant hormone auxin, RICS uncovered their distinct short-distance lateral mobility within the PM that is dependent on cytoskeleton and sterol composition of the PM. RICS, which is routinely accessible on modern CLSM instruments, thus represents a valuable approach for studies of dynamics of PM proteins in plants. PMID:27041337

  3. Outer Hair Cell Lateral Wall Structure Constrains the Mobility of Plasma Membrane Proteins.

    PubMed

    Yamashita, Tetsuji; Hakizimana, Pierre; Wu, Siva; Hassan, Ahmed; Jacob, Stefan; Temirov, Jamshid; Fang, Jie; Mellado-Lagarde, Marcia; Gursky, Richard; Horner, Linda; Leibiger, Barbara; Leijon, Sara; Centonze, Victoria E; Berggren, Per-Olof; Frase, Sharon; Auer, Manfred; Brownell, William E; Fridberger, Anders; Zuo, Jian

    2015-09-01

    Nature's fastest motors are the cochlear outer hair cells (OHCs). These sensory cells use a membrane protein, Slc26a5 (prestin), to generate mechanical force at high frequencies, which is essential for explaining the exquisite hearing sensitivity of mammalian ears. Previous studies suggest that Slc26a5 continuously diffuses within the membrane, but how can a freely moving motor protein effectively convey forces critical for hearing? To provide direct evidence in OHCs for freely moving Slc26a5 molecules, we created a knockin mouse where Slc26a5 is fused with YFP. These mice and four other strains expressing fluorescently labeled membrane proteins were used to examine their lateral diffusion in the OHC lateral wall. All five proteins showed minimal diffusion, but did move after pharmacological disruption of membrane-associated structures with a cholesterol-depleting agent and salicylate. Thus, our results demonstrate that OHC lateral wall structure constrains the mobility of plasma membrane proteins and that the integrity of such membrane-associated structures are critical for Slc26a5's active and structural roles. The structural constraint of membrane proteins may exemplify convergent evolution of cellular motors across species. Our findings also suggest a possible mechanism for disorders of cholesterol metabolism with hearing loss such as Niemann-Pick Type C diseases. PMID:26352669

  4. Outer Hair Cell Lateral Wall Structure Constrains the Mobility of Plasma Membrane Proteins

    PubMed Central

    Yamashita, Tetsuji; Hakizimana, Pierre; Wu, Siva; Hassan, Ahmed; Jacob, Stefan; Temirov, Jamshid; Fang, Jie; Mellado-Lagarde, Marcia; Gursky, Richard; Horner, Linda; Leibiger, Barbara; Leijon, Sara; Centonze, Victoria E.; Berggren, Per-Olof; Frase, Sharon; Auer, Manfred; Brownell, William E.; Fridberger, Anders; Zuo, Jian

    2015-01-01

    Nature’s fastest motors are the cochlear outer hair cells (OHCs). These sensory cells use a membrane protein, Slc26a5 (prestin), to generate mechanical force at high frequencies, which is essential for explaining the exquisite hearing sensitivity of mammalian ears. Previous studies suggest that Slc26a5 continuously diffuses within the membrane, but how can a freely moving motor protein effectively convey forces critical for hearing? To provide direct evidence in OHCs for freely moving Slc26a5 molecules, we created a knockin mouse where Slc26a5 is fused with YFP. These mice and four other strains expressing fluorescently labeled membrane proteins were used to examine their lateral diffusion in the OHC lateral wall. All five proteins showed minimal diffusion, but did move after pharmacological disruption of membrane-associated structures with a cholesterol-depleting agent and salicylate. Thus, our results demonstrate that OHC lateral wall structure constrains the mobility of plasma membrane proteins and that the integrity of such membrane-associated structures are critical for Slc26a5’s active and structural roles. The structural constraint of membrane proteins may exemplify convergent evolution of cellular motors across species. Our findings also suggest a possible mechanism for disorders of cholesterol metabolism with hearing loss such as Niemann-Pick Type C diseases. PMID:26352669

  5. Sulfur-based absolute quantification of proteins using isotope dilution inductively coupled plasma mass spectrometry

    NASA Astrophysics Data System (ADS)

    Lee, Hyun-Seok; Heun Kim, Sook; Jeong, Ji-Seon; Lee, Yong-Moon; Yim, Yong-Hyeon

    2015-10-01

    An element-based reductive approach provides an effective means of realizing International System of Units (SI) traceability for high-purity biological standards. Here, we develop an absolute protein quantification method using double isotope dilution (ID) inductively coupled plasma mass spectrometry (ICP-MS) combined with microwave-assisted acid digestion for the first time. We validated the method and applied it to certify the candidate protein certified reference material (CRM) of human growth hormone (hGH). The concentration of hGH was determined by analysing the total amount of sulfur in hGH. Next, the size-exclusion chromatography method was used with ICP-MS to characterize and quantify sulfur-containing impurities. By subtracting the contribution of sulfur-containing impurities from the total sulfur content in the hGH CRM, we obtained a SI-traceable certification value. The quantification result obtained with the present method based on sulfur analysis was in excellent agreement with the result determined via a well-established protein quantification method based on amino acid analysis using conventional acid hydrolysis combined with an ID liquid chromatography-tandem mass spectrometry. The element-based protein quantification method developed here can be generally used for SI-traceable absolute quantification of proteins, especially pure-protein standards.

  6. A Phospholipid-Protein Complex from Antarctic Krill Reduced Plasma Homocysteine Levels and Increased Plasma Trimethylamine-N-Oxide (TMAO) and Carnitine Levels in Male Wistar Rats

    PubMed Central

    Bjørndal, Bodil; Ramsvik, Marie S.; Lindquist, Carine; Nordrehaug, Jan E.; Bruheim, Inge; Svardal, Asbjørn; Nygård, Ottar; Berge, Rolf K.

    2015-01-01

    Seafood is assumed to be beneficial for cardiovascular health, mainly based on plasma lipid lowering and anti-inflammatory effects of n-3 polyunsaturated fatty acids. However, other plasma risk factors linked to cardiovascular disease are less studied. This study aimed to penetrate the effect of a phospholipid-protein complex (PPC) from Antarctic krill on one-carbon metabolism and production of trimethylamine-N-oxide (TMAO) in rats. Male Wistar rats were fed isoenergetic control, 6%, or 11% PPC diets for four weeks. Rats fed PPC had reduced total homocysteine plasma level and increased levels of choline, dimethylglycine and cysteine, whereas the plasma level of methionine was unchanged compared to control. PPC feeding increased the plasma level of TMAO, carnitine, its precursors trimethyllysine and γ-butyrobetaine. There was a close correlation between plasma TMAO and carnitine, trimethyllysine, and γ-butyrobetaine, but not between TMAO and choline. The present data suggest that PPC has a homocysteine lowering effect and is associated with altered plasma concentrations of metabolites related to one-carbon metabolism and B-vitamin status in rats. Moreover, the present study reveals a non-obligatory role of gut microbiota in the increased plasma TMAO level as it can be explained by the PPC’s content of TMAO. The increased level of carnitine and carnitine precursors is interpreted to reflect increased carnitine biosynthesis. PMID:26371012

  7. In vitro, in silico and integrated strategies for the estimation of plasma protein binding. A review.

    PubMed

    Lambrinidis, George; Vallianatou, Theodosia; Tsantili-Kakoulidou, Anna

    2015-06-23

    Plasma protein binding (PPB) strongly affects drug distribution and pharmacokinetic behavior with consequences in overall pharmacological action. Extended plasma protein binding may be associated with drug safety issues and several adverse effects, like low clearance, low brain penetration, drug-drug interactions, loss of efficacy, while influencing the fate of enantiomers and diastereoisomers by stereoselective binding within the body. Therefore in holistic drug design approaches, where ADME(T) properties are considered in parallel with target affinity, considerable efforts are focused in early estimation of PPB mainly in regard to human serum albumin (HSA), which is the most abundant and most important plasma protein. The second critical serum protein α1-acid glycoprotein (AGP), although often underscored, plays also an important and complicated role in clinical therapy and thus the last years it has been studied thoroughly too. In the present review, after an overview of the principles of HSA and AGP binding as well as the structure topology of the proteins, the current trends and perspectives in the field of PPB predictions are presented and discussed considering both HSA and AGP binding. Since however for the latter protein systematic studies have started only the last years, the review focuses mainly to HSA. One part of the review highlights the challenge to develop rapid techniques for HSA and AGP binding simulation and their performance in assessment of PPB. The second part focuses on in silico approaches to predict HSA and AGP binding, analyzing and evaluating structure-based and ligand-based methods, as well as combination of both methods in the aim to exploit the different information and overcome the limitations of each individual approach. Ligand-based methods use the Quantitative Structure-Activity Relationships (QSAR) methodology to establish quantitate models for the prediction of binding constants from molecular descriptors, while they provide

  8. One step physically adsorbed coating of silica capillary with excellent stability for the separation of basic proteins by capillary zone electrophoresis.

    PubMed

    Guo, Xiao-Feng; Guo, Xiao-Mei; Wang, Hong; Zhang, Hua-Shan

    2015-11-01

    The coating of capillary inner surface is considered to be an effective approach to suppress the adsorption of proteins on capillary inner surface in CE. However, most of coating materials reported are water-soluble, which may dissolve in BGE during the procedure of electrophoresis. In this study, a novel strategy for selection of physically coating materials has been illustrated to get coating layer with excellent stability using materials having poor solubility in commonly used solvents. Taking natural chitin as example (not hydrolyzed water soluble chitosan), a simple one step coating method using chitin solution in hexafluoroisopropanol was adopted within only 21 min with good coating reproducibility (RSDs of EOF for within-batch coated capillaries of 1.55% and between-batch coated capillaries of 2.31%), and a separation of four basic proteins on a chitin coated capillary was performed to evaluate the coating efficacy. Using chitin coating, the adsorption of proteins on capillary inner surface was successfully suppressed with reversed and stable EOF, and four basic proteins including lysozyme, cytochrome c, ribonuclease A and α-chymotrypsinogen A were baseline separated within 16 min with satisfied separation efficiency using 20 mM pH 2.0 H3PO4-Na2HPO4 as back ground electrolyte and 20 kV as separation voltage. What is more important, the chitin coating layer could be stable for more than two months during this study, which demonstrates that chitin is an ideal material for preparing semi-permanent coating on bare fused silica capillary inner wall and has hopeful potential in routine separation of proteins with CE.

  9. One step physically adsorbed coating of silica capillary with excellent stability for the separation of basic proteins by capillary zone electrophoresis.

    PubMed

    Guo, Xiao-Feng; Guo, Xiao-Mei; Wang, Hong; Zhang, Hua-Shan

    2015-11-01

    The coating of capillary inner surface is considered to be an effective approach to suppress the adsorption of proteins on capillary inner surface in CE. However, most of coating materials reported are water-soluble, which may dissolve in BGE during the procedure of electrophoresis. In this study, a novel strategy for selection of physically coating materials has been illustrated to get coating layer with excellent stability using materials having poor solubility in commonly used solvents. Taking natural chitin as example (not hydrolyzed water soluble chitosan), a simple one step coating method using chitin solution in hexafluoroisopropanol was adopted within only 21 min with good coating reproducibility (RSDs of EOF for within-batch coated capillaries of 1.55% and between-batch coated capillaries of 2.31%), and a separation of four basic proteins on a chitin coated capillary was performed to evaluate the coating efficacy. Using chitin coating, the adsorption of proteins on capillary inner surface was successfully suppressed with reversed and stable EOF, and four basic proteins including lysozyme, cytochrome c, ribonuclease A and α-chymotrypsinogen A were baseline separated within 16 min with satisfied separation efficiency using 20 mM pH 2.0 H3PO4-Na2HPO4 as back ground electrolyte and 20 kV as separation voltage. What is more important, the chitin coating layer could be stable for more than two months during this study, which demonstrates that chitin is an ideal material for preparing semi-permanent coating on bare fused silica capillary inner wall and has hopeful potential in routine separation of proteins with CE. PMID:26452799

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

    NASA Astrophysics Data System (ADS)

    Guo, Bihong; Li, Shaopeng; Song, Lusheng; Yang, Mo; Zhou, Wenfei; Tyagi, Deependra; Zhu, Jinsong

    2015-08-01

    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 improvement of the protein bioassay performance, rather than the chemical changes.

  11. Presence of membranous vesicles in cat seminal plasma: ultrastructural characteristics, protein profile and enzymatic activity.

    PubMed

    Polisca, A; Troisi, A; Minelli, A; Bellezza, I; Fontbonne, A; Zelli, R

    2015-02-01

    This study sought to verify the presence of membranous vesicles in cat seminal plasma by means of transmission electron microscopy and to identify protein profile and some of the enzymatic activities associated with these particles. The transmission electron microscopy observations showed the existence of different sized vesicular membranous structures of more or less spherical shape. These vesicles were surrounded by single-, double- or multiple-layered laminar membranes. The vesicle diameters ranged from 16.3 to 387.4 nm, with a mean of 116.5 ± 70.7 nm. Enzyme activity determinations showed the presence of dipeptilpeptidase IV, aminopeptidase, alkaline and acid phosphatase. To our knowledge, this is the first report that identifies and characterizes the membranous vesicles in cat seminal plasma. However, further studies are necessary to identify the exact site of production of these membranous vesicles in the cat male genital tract and to determine their specific roles in the reproductive events of this species.

  12. Measurement of canine gastric vascular permeability to plasma proteins in the normal and protein-losing states

    SciTech Connect

    Wood, J.G.; Davenport, H.W.

    1982-04-01

    An isolated segment of the greater curvature of a dog's stomach was perfused at constant flow through a single cannulated artery with donor blood containing 131I-albumin, 125I-fibrinogen, and papaverine. Perfusion pressure was 30-50 mmHg, and venous pressure was set at 15 mmHg. Venous blood was collected in 1-min samples for 60 min. Filtration of fluid and loss of labeled proteins were calculated as the difference between measured arterial inflow and venous outflow. Permeability-surface area products (PS) were calculated for the proteins, and reflection coefficients (sigma) were calculated from solute flux and filtration. Intraarterial infusion of histamine (1.6-1.9 microgram . ml-1) increased filtration and PS and decreased sigma for albumin but not fibrinogen. When protein-losing was established by topical irrigation with 10 mM dithiothreitol in neutral solution, filtration and PS increased, and sigma for albumin but not fibrinogen decreased. Irrigation of the mucosa with 10 mM salicylic acid in 100 mN HCl caused bleeding that was quantitated by addition of 51Cr-erythrocytes to perfusing blood. Filtration and PS increased, and sigma for albumin but not fibrinogen decreased. Hematocrit of blood lost remained low during extensive mucosal damage. Effects of histamine infusion were attenuated or abolished by cimetidine (4 mg . kg-1 loading, 1.4 mg . kg-1 . h-1 continuous infusion) or by pyrilamine maleate (5 mg . kg-1 bolus injection at beginning of irrigation, repeated at 40-50 min). Pyrilamine attenuated or abolished effects of topical dithiothreitol or salicylic acid. We conclude that during protein loss caused by dithiothreitol or salicylic acid, histamine released within the mucosa causes increased vascular permeability for plasma proteins.

  13. Type I antifreeze proteins expressed in snailfish skin are identical to their plasma counterparts.

    PubMed

    Evans, Robert P; Fletcher, Garth L

    2005-10-01

    Type I antifreeze proteins (AFPs) are usually small, Ala-rich alpha-helical polypeptides found in right-eyed flounders and certain species of sculpin. These proteins are divided into two distinct subclasses, liver type and skin type, which are encoded by separate gene families. Blood plasma from Atlantic (Liparis atlanticus) and dusky (Liparis gibbus) snailfish contain type I AFPs that are significantly larger than all previously described type I AFPs. In this study, full-length cDNA clones that encode snailfish type I AFPs expressed in skin tissues were generated using a combination of library screening and PCR-based methods. The skin clones, which lack both signal and pro-sequences, produce proteins that are identical to circulating plasma AFPs. Although all fish examined consistently express antifreeze mRNA in skin tissue, there is extreme individual variation in liver expression - an unusual phenomenon that has never been reported previously. Furthermore, genomic Southern blot analysis revealed that snailfish AFPs are products of multigene families that consist of up to 10 gene copies per genome. The 113-residue snailfish AFPs do not contain any obvious amino acid repeats or continuous hydrophobic face which typify the structure of most other type I AFPs. These structural differences might have implications for their ice-crystal binding properties. These results are the first to demonstrate a dual liver/skin role of identical type I AFP expression which may represent an evolutionary intermediate prior to divergence into distinct gene families.

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

    PubMed

    Poulsen, Nicklas N; Andersen, Nina Z; Østergaard, Jesper; Zhuang, Guisheng; Petersen, Nickolaj J; Jensen, Henrik

    2015-07-01

    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 the presence of the analyte the apparent diffusivity of the indicator changes due to complexation. This change in diffusivity is used to quantify the analyte. This approach, termed Flow Induced Dispersion Analysis (FIDA), is characterized by being fast (minutes), selective (quantification is possible 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-analyte dissociation constant which in favourable cases is in the sub-nanomolar to picomolar range for antibody-antigen interactions. PMID:26031223

  15. Site-Specific Zwitterionic Polymer Conjugates of a Protein Have Long Plasma Circulation.

    PubMed

    Bhattacharjee, Somnath; Liu, Wenge; Wang, Wei-Han; Weitzhandler, Isaac; Li, Xinghai; Qi, Yizhi; Liu, Jinyao; Pang, Yan; Hunt, Donald F; Chilkoti, Ashutosh

    2015-11-01

    Many proteins suffer from suboptimal pharmacokinetics (PK) that limit their utility as drugs. The efficient synthesis of polymer conjugates of protein drugs with tunable PK to optimize their in vivo efficacy is hence critical. We report here the first study of the in vivo behavior of a site-specific conjugate of a zwitterionic polymer and a protein. To synthesize the conjugate, we first installed an initiator for atom-transfer radical polymerization (ATRP) at the N terminus of myoglobin (Mb-N-Br). Subsequently, in situ ATRP was carried out in aqueous buffer to grow an amine-functionalized polymer from Mb-N-Br. The cationic polymer was further derivatized to two zwitterionic polymers by treating the amine groups of the cationic polymer with iodoacetic acid to obtain poly(carboxybetaine methacrylate) with a one-carbon spacer (PCBMA; C1 ), and sequentially with 3-iodopropionic acid and iodoacetic acid to obtain PCBMA(mix) with a mixture of C1 and C2 spacers. The Mb-N-PCBMA polymer conjugates had a longer in vivo plasma half-life than a PEG-like comb polymer conjugate of similar molecular weights (MW). The structure of the zwitterion plays a role in controlling the in vivo behavior of the conjugate, as the PCBMA conjugate with a C1 spacer had significantly longer plasma circulation than the conjugate with a mixture of C1 and C2 spacers.

  16. Change in N-Glycosylation of Plasma Proteins in Japanese Semisupercentenarians.

    PubMed

    Miura, Yuri; Hashii, Noritaka; Tsumoto, Hiroki; Takakura, Daisuke; Ohta, Yuki; Abe, Yukiko; Arai, Yasumichi; Kawasaki, Nana; Hirose, Nobuyoshi; Endo, Tamao

    2015-01-01

    An N-glycomic analysis of plasma proteins was performed in Japanese semisupercentenarians (SSCs) (mean 106.7 years), aged controls (mean 71.6 years), and young controls (mean 30.2 years) by liquid chromatography/mass spectrometry (LC/MS) using a graphitized carbon column. Characteristic N-glycans in SSCs were discriminated using a multivariate analysis; orthogonal projections to latent structures (O-PLS). The results obtained showed that multi-branched and highly sialylated N-glycans as well as agalacto- and/or bisecting N-glycans were increased in SSCs, while biantennary N-glycans were decreased. Since multi-branched and highly sialylated N-glycans have been implicated in anti-inflammatory activities, these changes may play a role in the enhanced chronic inflammation observed in SSCs. The levels of inflammatory proteins, such as CRP, adiponectin, IL-6, and TNF-α, were elevated in SSCs. These results suggested that responses to inflammation may play an important role in extreme longevity and healthy aging in humans. This is the first study to show that the N-glycans of plasma proteins were associated with extreme longevity and healthy aging in humans. PMID:26559536

  17. Change in N-Glycosylation of Plasma Proteins in Japanese Semisupercentenarians

    PubMed Central

    Tsumoto, Hiroki; Takakura, Daisuke; Ohta, Yuki; Abe, Yukiko; Arai, Yasumichi; Kawasaki, Nana; Hirose, Nobuyoshi; Endo, Tamao

    2015-01-01

    An N-glycomic analysis of plasma proteins was performed in Japanese semisupercentenarians (SSCs) (mean 106.7 years), aged controls (mean 71.6 years), and young controls (mean 30.2 years) by liquid chromatography/mass spectrometry (LC/MS) using a graphitized carbon column. Characteristic N-glycans in SSCs were discriminated using a multivariate analysis; orthogonal projections to latent structures (O-PLS). The results obtained showed that multi-branched and highly sialylated N-glycans as well as agalacto- and/or bisecting N-glycans were increased in SSCs, while biantennary N-glycans were decreased. Since multi-branched and highly sialylated N-glycans have been implicated in anti-inflammatory activities, these changes may play a role in the enhanced chronic inflammation observed in SSCs. The levels of inflammatory proteins, such as CRP, adiponectin, IL-6, and TNF-α, were elevated in SSCs. These results suggested that responses to inflammation may play an important role in extreme longevity and healthy aging in humans. This is the first study to show that the N-glycans of plasma proteins were associated with extreme longevity and healthy aging in humans. PMID:26559536

  18. C-reactive protein collaborates with plasma lectins to boost immune response against bacteria.

    PubMed

    Ng, Patricia M L; Le Saux, Agnès; Lee, Chia M; Tan, Nguan S; Lu, Jinhua; Thiel, Steffen; Ho, Bow; Ding, Jeak L

    2007-07-25

    Although human C-reactive protein (CRP) becomes upregulated during septicemia, its role remains unclear, since purified CRP showed no binding to many common pathogens. Contrary to previous findings, we show that purified human CRP (hCRP) binds to Salmonella enterica, and that binding is enhanced in the presence of plasma factors. In the horseshoe crab, Carcinoscorpius rotundicauda, CRP is a major hemolymph protein. Incubation of hemolymph with a range of bacteria resulted in CRP binding to all the bacteria tested. Lipopolysaccharide-affinity chromatography of the hemolymph co-purified CRP, galactose-binding protein (GBP) and carcinolectin-5 (CL5). Yeast two-hybrid and pull-down assays suggested that these pattern recognition receptors (PRRs) form pathogen recognition complexes. We show the conservation of PRR crosstalk in humans, whereby hCRP interacts with ficolin (CL5 homologue). This interaction stabilizes CRP binding to bacteria and activates the lectin-mediated complement pathway. We propose that CRP does not act alone but collaborates with other plasma PRRs to form stable pathogen recognition complexes when targeting a wide range of bacteria for destruction. PMID:17581635

  19. Evaluation of Serum Pregnancy Associated Plasma Protein-A & Plasma D-Dimer in Acute Coronary Syndrome

    PubMed Central

    Thomas, Vivian Samuel

    2016-01-01

    Introduction Acute coronary syndrome (ACS), a spectrum comprising unstable angina pectoris, ST Elevated Myocardial Infarction (STEMI) & Non ST Elevated Myocardial Infarction (NSTEMI) is the major cause of presentation in Emergency Department today. Though ECG and cardiac enzymes are used for diagnosis, they mislead the diagnosis sometimes and delay in treatment initiation. This leads us to search certain new parameters which reflect the pathophysiology of ACS. Markers of plaque stability like Pregnancy Associated Plasma Protein-A and D-Dimer, a marker of ongoing thrombosis are found to be better markers in early diagnosis. Aim To evaluate the diagnostic competence of PAPP-A and D-Dimer in acute coronary syndrome over CK-MB and to compare with the inflammatory marker High Sensitive C-Reactive Protein (hs-CRP) which is associated with atherosclerosis. Materials and Methods Fifty patients presenting with acute onset of chest pain to Emergency Department with or without ECG changes served as cases and 50 healthy people served as controls. Serum PAPP-A is measured by Enzyme Linked Immunosorbent Assay (ELISA), D-Dimer and hs-CRP by using Latex Turbidimetry method. Results A statistical significant difference of PAPP-A and D-Dimer was noted between the ACS and controls (p < 0.001) whereas CK-MB shows no much difference (p 0.09). Statistically significant positive correlation is noted between parameters. Conclusion PAPP-A marker of plaque instability and D-Dimer marker of ongoing thrombosis are raised in acute coronary syndrome and thus can be considered as one of the marker in ACS for diagnosis. PMID:26894054

  20. Intrinsic stability of Brassicaceae plasma membrane in relation to changes in proteins and lipids as a response to salinity.

    PubMed

    Chalbi, Najla; Martínez-Ballesta, Ma Carmen; Youssef, Nabil Ben; Carvajal, Micaela

    2015-03-01

    Changes in plasma membrane lipids, such as sterols and fatty acids, have been observed as a result of salt stress. These alterations, together with modification of the plasma membrane protein profile, confer changes in the physical properties of the membrane to be taken into account for biotechnological uses. In our experiments, the relationship between lipids and proteins in three different Brassicaceae species differing in salinity tolerance (Brassica oleracea, B. napus and Cakile maritima) and the final plasma membrane stability were studied. The observed changes in the sterol (mainly an increase in sitosterol) and fatty acid composition (increase in RUFA) in each species led to physical adaptation of the plasma membrane to salt stress. The in vitro vesicles stability was higher in the less tolerant (B. oleracea) plants together with low lipoxygenase activity. These results indicate that the proteins/lipids ratio and lipid composition is an important aspect to take into account for the use of natural vesicles in plant biotechnology. PMID:25544590

  1. Intrinsic stability of Brassicaceae plasma membrane in relation to changes in proteins and lipids as a response to salinity.

    PubMed

    Chalbi, Najla; Martínez-Ballesta, Ma Carmen; Youssef, Nabil Ben; Carvajal, Micaela

    2015-03-01

    Changes in plasma membrane lipids, such as sterols and fatty acids, have been observed as a result of salt stress. These alterations, together with modification of the plasma membrane protein profile, confer changes in the physical properties of the membrane to be taken into account for biotechnological uses. In our experiments, the relationship between lipids and proteins in three different Brassicaceae species differing in salinity tolerance (Brassica oleracea, B. napus and Cakile maritima) and the final plasma membrane stability were studied. The observed changes in the sterol (mainly an increase in sitosterol) and fatty acid composition (increase in RUFA) in each species led to physical adaptation of the plasma membrane to salt stress. The in vitro vesicles stability was higher in the less tolerant (B. oleracea) plants together with low lipoxygenase activity. These results indicate that the proteins/lipids ratio and lipid composition is an important aspect to take into account for the use of natural vesicles in plant biotechnology.

  2. Plasma IgG autoantibody against actin-related protein 3 in liver fluke Opisthorchis viverrini infection.

    PubMed

    Rucksaken, R; Haonon, O; Pinlaor, P; Pairojkul, C; Roytrakul, S; Yongvanit, P; Selmi, C; Pinlaor, S

    2015-07-01

    Opisthorchiasis secondary to Opisthorchis viverrini infection leads to cholangiocellular carcinoma through chronic inflammation of the bile ducts and possibly inducing autoimmunity. It was hypothesized that plasma autoantibodies directed against self-proteins are biomarkers for opisthorchiasis. Plasma from patients with opisthorchiasis was tested using proteins derived from immortalized cholangiocyte cell lines, and spots reacting with plasma were excised and subjected to LC-MS/MS. Seven protein spots were recognized by IgG autoantibodies, and the highest matching scored protein was actin-related protein 3 (ARP3). The antibody against ARP3 was tested in plasma from 55 O. viverrini-infected patients, 24 patients with others endemic parasitic infections and 17 healthy controls using Western blot and ELISA. Immunoreactivity against recombinant ARP3 was significantly more prevalent in opisthorchiasis compared to healthy controls at Western blotting and ELISA (P < 0.05). Plasma ARP3 autoantibody titres were also higher in opisthorchiasis compared to healthy individuals (P < 0.01) and other parasitic infections including Strongyloides stercoralis (P < 0.001), echinostome (P < 0.05), hookworms (P < 0.001) and Taenia spp. (P < 0.05). It was further characterized in that the ARP3 autoantibody titre had a sensitivity of 78.18% and specificity of 100% for opisthorchiasis. In conclusion, it may be suggested that plasma anti-ARP3 might represent a new diagnostic antibody for opisthorchiasis. PMID:25809205

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

    PubMed Central

    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

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

    PubMed

    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.

  5. Plasma protein binding of (99m)Tc-labeled hydrazino nicotinamide derivatized polypeptides and peptides.

    PubMed

    Ono, M; Arano, Y; Mukai, T; Uehara, T; Fujioka, Y; Ogawa, K; Namba, S; Nakayama, M; Saga, T; Konishi, J; Horiuchi, K; Yokoyama, A; Saji, H

    2001-02-01

    6-Hydrazinopyridine-3-carboxylic acid (HYNIC) constitutes one of the most attractive reagents to prepare (99m)Tc-labeled polypeptides and peptides of various molecular weights in combination with two tricine molecules as coligands. Indeed, (99m)Tc-HYNIC-conjugated IgG showed biodistribution of radioactivity similar to that of (111)In-DTPA-conjugated IgG. However, recent studies indicated significant plasma protein binding when the (99m)Tc labeling procedure was expanded to low molecular weight peptides. In this study, pharmacokinetics of (99m)Tc-HYNIC-conjugated IgG, Fab and RC160 using tricine were compared with their radioiodinated counterparts to evaluate this (99m)Tc-labeling method. In mice, [(99m)Tc](HYNIC-IgG)(tricine)(2) and [(99m)Tc](HYNIC-Fab)(tricine)(2) showed persistent localization of radioactivity in tissues when compared with their (125)I-labeled counterparts. [(99m)Tc](HYNIC-IgG)(tricine)(2) eliminated from the blood at a rate similar to that of (125)I-labeled IgG, while [(99m)Tc](HYNIC-Fab)(tricine)(2) showed significantly slower clearance of the radioactivity than (125)I-labeled Fab. On size-exclusion HPLC analyses, little changes were observed in radiochromatograms after incubation of [(99m)Tc](HYNIC-IgG)(tricine)(2) in murine plasma. However, [(99m)Tc](HYNIC-Fab)(tricine)(2) and [(99m)Tc](HYNIC-RC160)(tricine)(2) demonstrated significant increases in the radioactivity in higher molecular weight fractions in plasma. Formation of higher molecular weight species was reduced when [(99m)Tc](HYNIC-RC160)(tricine)(2) was stabilized with nicotinic acid (NIC) to generate [(99m)Tc](HYNIC-RC160)(tricine)(NIC). [(99m)Tc](HYNIC-RC160)(tricine)(NIC) also demonstrated significantly faster clearance of the radioactivity from the blood than [(99m)Tc](HYNIC-RC160)(tricine)(2). These findings suggested that one of the tricine coligands in (99m)Tc-HYNIC-labeled (poly)peptides would be replaced with plasma proteins to generate higher molecular weight species that

  6. KDEL-Containing Auxin-Binding Protein Is Secreted to the Plasma Membrane and Cell Wall.

    PubMed Central

    Jones, A. M.; Herman, E. M.

    1993-01-01

    The auxin-binding protein ABP1 has been postulated to mediate auxin-induced cellular changes associated with cell expansion. This protein contains the endoplasmic reticulum (ER) retention signal, the tetrapeptide lysine-aspartic acid-glutamic acid-leucine (KDEL), at its carboxy terminus, consistent with previous subcellular fractionation data that indicated an ER location for ABP1. We used electron microscopic immunocytochemistry to identify the subcellular localization of ABP1. Using maize (Zea mays) coleoptile tissue and a black Mexican sweet (BMS) maize cell line, we found that ABP1 is located in the ER as expected, but is also on or closely associated with the plasma membrane and within the cell wall. Labeling of the Golgi apparatus suggests that the transport of ABP1 to the cell wall occurs via the secretory system. Inhibition of secretion of an ABP homolog into the medium of BMS cell cultures by brefeldin A, a drug that specifically blocks secretion, is consistent with this secretion pathway. The secreted protein was recognized by an anti-KDEL peptide antibody, strongly supporting the interpretation that movement of this protein out of the ER does not involve loss of the carboxy-terminal signal. Cells starved for 2,4-dichlorophenoxyacetic acid for 72 h retained less ABP in the cell and secreted more of it into the medium. The significance of our observations is 2-fold. We have identified a KDEL-containing protein that specifically escapes the ER retention system, and we provide an explanation for the apparent discrepancy that most of the ABP is located in the ER, whereas ABP and auxin act at the plasma membrane. PMID:12231715

  7. Overexpression of BAX INHIBITOR-1 Links Plasma Membrane Microdomain Proteins to Stress1[OPEN

    PubMed Central

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

    2015-01-01

    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. PMID:26297139

  8. Determination of plasma protein binding of positron emission tomography radioligands by high-performance frontal analysis.

    PubMed

    Amini, Nahid; Nakao, Ryuji; Schou, Magnus; Halldin, Christer

    2014-09-01

    Positron emission tomography (PET) is an imaging technique based on the use of radioligands labeled with short lived radionuclides, such as (11)C (t½=20.4min) and (18)F (t½=109.8min), which as a consequence often requires rapid plasma protein binding analysis methods. In addition, PET radioligands can suffer from non-specific binding to the membrane when ultrafiltraion, which is the most commonly used method for measuring protein binding in PET, is employed. In this study a high-performance frontal analysis (HPFA) method based on incorporation of a gel filtration column (discovery(®) BIO GFC 100, 50mm×4.6mm, 5μm, 100Å) into a radio-LC system with phosphate buffered saline (PBS, pH 7.4) at a flow rate of 3ml/min as mobile phase was developed and investigated for four PET radioligands. The minimum injection volume (MIV) of plasma, which is a crucial factor in HPFA, was determined to be 200μl (human), 500μl (monkey), 700μl (human) and 1000μl (monkey) for these four radioligands. The MIV values increased as a higher fraction of the radioligand was present in the protein-free form. The protein binding results obtained were in good agreement with ultrafiltration and the method did not suffer from non-specific binding. The short analysis time (<12min) allowed multiple protein binding measurements during time course of a human [(11)C]PBR28 PET study. PMID:24922085

  9. Lipid-protein interactions in plasma membranes of fiber cells isolated from the human eye lens.

    PubMed

    Raguz, Marija; Mainali, Laxman; O'Brien, William J; Subczynski, Witold K

    2014-03-01

    The protein content in human lens membranes is extremely high, increases with age, and is higher in the nucleus as compared with the cortex, which should strongly affect the organization and properties of the lipid bilayer portion of intact membranes. To assess these effects, the intact cortical and nuclear fiber cell plasma membranes isolated from human lenses from 41- to 60-year-old donors were studied using electron paramagnetic resonance spin-labeling methods. Results were compared with those obtained for lens lipid membranes prepared from total lipid extracts from human eyes of the same age group [Mainali, L., Raguz, M., O'Brien, W. J., and Subczynski, W. K. (2013) Biochim. Biophys. Acta]. Differences were considered to be mainly due to the effect of membrane proteins. The lipid-bilayer portions of intact membranes were significantly less fluid than lipid bilayers of lens lipid membranes, prepared without proteins. The intact membranes were found to contain three distinct lipid environments termed the bulk lipid domain, boundary lipid domain, and trapped lipid domain. However, the cholesterol bilayer domain, which was detected in cortical and nuclear lens lipid membranes, was not detected in intact membranes. The relative amounts of bulk and trapped lipids were evaluated. The amount of lipids in domains uniquely formed due to the presence of membrane proteins was greater in nuclear membranes than in cortical membranes. Thus, it is evident that the rigidity of nuclear membranes is greater than that of cortical membranes. Also the permeability coefficients for oxygen measured in domains of nuclear membranes were significantly lower than appropriate coefficients measured in cortical membranes. Relationships between the organization of lipids into lipid domains in fiber cells plasma membranes and the organization of membrane proteins are discussed.

  10. Lipid-Protein Interactions in Plasma Membranes of Fiber Cells Isolated from the Human Eye Lens

    PubMed Central

    Raguz, Marija; Mainali, Laxman; O’Brien, William J.; Subczynski, Witold K.

    2014-01-01

    The protein content in human lens membranes is extremely high, increases with age, and is higher in the nucleus as compared with the cortex, which should strongly affect the organization and properties of the lipid bilayer portion of intact membranes. To assess these effects, the intact cortical and nuclear fiber cell plasma membranes isolated from human lenses from 41- to 60-year-old donors were studied using electron paramagnetic resonance spin-labeling methods. Results were compared with those obtained for lens lipid membranes prepared from total lipid extracts from human eyes of the same age group [Mainali,L., Raguz, M., O’Brien, W. J., and Subczynski, W. K. (2013) Biochim. Biophys. Acta]. Differences were considered to be mainly due to the effect of membrane proteins. The lipid-bilayer portions of intact membranes were significantly less fluid than lipid bilayers of lens lipid membranes, prepared without proteins. The intact membranes were found to contain three distinct lipid environments termed the bulk lipid domain, boundary lipid domain, and trapped lipid domain. However, the cholesterol bilayer domain, which was detected in cortical and nuclear lens lipid membranes, was not detected in intact membranes. The relative amounts of bulk and trapped lipids were evaluated. The amount of lipids in domains uniquely formed due to the presence of membrane proteins was greater in nuclear membranes than in cortical membranes. Thus, it is evident that the rigidity of nuclear membranes is greater than that of cortical membranes. Also the permeability coefficients for oxygen measured in domains of nuclear membranes were significantly lower than appropriate coefficients measured in cortical membranes. Relationships between the organization of lipids into lipid domains in fiber cells plasma membranes and the organization of membrane proteins are discussed. PMID:24486794

  11. Protein receptor-independent plasma membrane remodeling by HAMLET: A tumoricidal protein-lipid complex

    SciTech Connect

    Nadeem, Aftab; Sanborn, Jeremy; Gettel, Douglas L.; James, Ho C. S.; Rydström, Anna; Ngassam, Viviane N.; Klausen, Thomas Kjaer; Pedersen, Stine Falsig; Lam, Matti; Parikh, Atul N.; Svanborg, Catharina

    2015-11-12

    A central tenet of signal transduction in eukaryotic cells is that extra-cellular ligands activate specific cell surface receptors, which orchestrate downstream responses. This ‘’protein-centric” view is increasingly challenged by evidence for the involvement of specialized membrane domains in signal transduction. Here, we propose that membrane perturbation may serve as an alternative mechanism to activate a conserved cell-death program in cancer cells. This view emerges from the extraordinary manner in which HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells) kills a wide range of tumor cells in vitro and demonstrates therapeutic efficacy and selectivity in cancer models and clinical studies. We identify a ‘’receptor independent” transformation of vesicular motifs in model membranes, which is paralleled by gross remodeling of tumor cell membranes. Furthermore, we find that HAMLET accumulates within these de novo membrane conformations and define membrane blebs as cellular compartments for direct interactions of HAMLET with essential target proteins such as the Ras family of GTPases. In conclusion, we demonstrate lower sensitivity of healthy cell membranes to HAMLET challenge. These features suggest that HAMLET-induced curvature-dependent membrane conformations serve as surrogate receptors for initiating signal transduction cascades, ultimately leading to cell death.

  12. Protein receptor-independent plasma membrane remodeling by HAMLET: a tumoricidal protein-lipid complex

    PubMed Central

    Nadeem, Aftab; Sanborn, Jeremy; Gettel, Douglas L.; James, Ho C. S.; Rydström, Anna; Ngassam, Viviane N.; Klausen, Thomas Kjær; Pedersen, Stine Falsig; Lam, Matti; Parikh, Atul N.; Svanborg, Catharina

    2015-01-01

    A central tenet of signal transduction in eukaryotic cells is that extra-cellular ligands activate specific cell surface receptors, which orchestrate downstream responses. This ‘’protein-centric” view is increasingly challenged by evidence for the involvement of specialized membrane domains in signal transduction. Here, we propose that membrane perturbation may serve as an alternative mechanism to activate a conserved cell-death program in cancer cells. This view emerges from the extraordinary manner in which HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells) kills a wide range of tumor cells in vitro and demonstrates therapeutic efficacy and selectivity in cancer models and clinical studies. We identify a ‘’receptor independent” transformation of vesicular motifs in model membranes, which is paralleled by gross remodeling of tumor cell membranes. Furthermore, we find that HAMLET accumulates within these de novo membrane conformations and define membrane blebs as cellular compartments for direct interactions of HAMLET with essential target proteins such as the Ras family of GTPases. Finally, we demonstrate lower sensitivity of healthy cell membranes to HAMLET challenge. These features suggest that HAMLET-induced curvature-dependent membrane conformations serve as surrogate receptors for initiating signal transduction cascades, ultimately leading to cell death. PMID:26561036

  13. Protein receptor-independent plasma membrane remodeling by HAMLET: a tumoricidal protein-lipid complex.

    PubMed

    Nadeem, Aftab; Sanborn, Jeremy; Gettel, Douglas L; James, Ho C S; Rydström, Anna; Ngassam, Viviane N; Klausen, Thomas Kjær; Pedersen, Stine Falsig; Lam, Matti; Parikh, Atul N; Svanborg, Catharina

    2015-11-12

    A central tenet of signal transduction in eukaryotic cells is that extra-cellular ligands activate specific cell surface receptors, which orchestrate downstream responses. This ''protein-centric" view is increasingly challenged by evidence for the involvement of specialized membrane domains in signal transduction. Here, we propose that membrane perturbation may serve as an alternative mechanism to activate a conserved cell-death program in cancer cells. This view emerges from the extraordinary manner in which HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells) kills a wide range of tumor cells in vitro and demonstrates therapeutic efficacy and selectivity in cancer models and clinical studies. We identify a ''receptor independent" transformation of vesicular motifs in model membranes, which is paralleled by gross remodeling of tumor cell membranes. Furthermore, we find that HAMLET accumulates within these de novo membrane conformations and define membrane blebs as cellular compartments for direct interactions of HAMLET with essential target proteins such as the Ras family of GTPases. Finally, we demonstrate lower sensitivity of healthy cell membranes to HAMLET challenge. These features suggest that HAMLET-induced curvature-dependent membrane conformations serve as surrogate receptors for initiating signal transduction cascades, ultimately leading to cell death.

  14. Protein receptor-independent plasma membrane remodeling by HAMLET: A tumoricidal protein-lipid complex

    DOE PAGES

    Nadeem, Aftab; Sanborn, Jeremy; Gettel, Douglas L.; James, Ho C. S.; Rydström, Anna; Ngassam, Viviane N.; Klausen, Thomas Kjaer; Pedersen, Stine Falsig; Lam, Matti; Parikh, Atul N.; et al

    2015-11-12

    A central tenet of signal transduction in eukaryotic cells is that extra-cellular ligands activate specific cell surface receptors, which orchestrate downstream responses. This ‘’protein-centric” view is increasingly challenged by evidence for the involvement of specialized membrane domains in signal transduction. Here, we propose that membrane perturbation may serve as an alternative mechanism to activate a conserved cell-death program in cancer cells. This view emerges from the extraordinary manner in which HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells) kills a wide range of tumor cells in vitro and demonstrates therapeutic efficacy and selectivity in cancer models and clinical studies. Wemore » identify a ‘’receptor independent” transformation of vesicular motifs in model membranes, which is paralleled by gross remodeling of tumor cell membranes. Furthermore, we find that HAMLET accumulates within these de novo membrane conformations and define membrane blebs as cellular compartments for direct interactions of HAMLET with essential target proteins such as the Ras family of GTPases. In conclusion, we demonstrate lower sensitivity of healthy cell membranes to HAMLET challenge. These features suggest that HAMLET-induced curvature-dependent membrane conformations serve as surrogate receptors for initiating signal transduction cascades, ultimately leading to cell death.« less

  15. Protein, cell and bacterial response to atmospheric pressure plasma grafted hyaluronic acid on poly(methylmethacrylate).

    PubMed

    D'Sa, Raechelle A; Raj, Jog; Dickinson, Peter J; McMahon, M Ann S; McDowell, David A; Meenan, Brian J

    2015-11-01

    Hyaluronic acid (HA) has been immobilised on poly(methyl methacrylate) (PMMA) surfaces using a novel dielectric barrier discharge (DBD) plasma process for the purposes of repelling protein, cellular and bacterial adhesion in the context of improving the performance of ophthalmic devices. Grafting was achieved by the following steps: (1) treatment of the PMMA with a DBD plasma operating at atmospheric pressure, (2) amine functionalisation of the activated polymer surface by exposure to a 3-aminopropyltrimethoxysilane (APTMS) linker molecule and (3) reaction of HA with the surface bound amine. The mechanism and effectiveness of the grafting process was verified by surface analysis. XPS data indicates that the APTMS linker molecule binds to PMMA via the Si-O chemistry and has the required pendant amine moiety. The carboxylic acid moiety on HA then binds with this -NH2 group via standard carbodiimide chemistry. ToF-SIMS confirms the presence of a coherent HA layer the microstructure of which is verified by AFM. The plasma grafted HA coating surfaces showed a pronounced decrease in protein and cellular adhesion when tested with bovine serum albumin and human corneal epithelial cells, respectively. The ability of these coatings to resist bacterial adhesion was established using Staphylococcus aureus NTC8325. Interestingly, the coatings did not repel bacterial adhesion, indicating that the mechanism of adhesion of bacterial cells is different to that for the surface interactions of mammalian cells. It is proposed that this difference is a consequence of the specific HA conformation that occurs under the conditions employed here. Hence, it is apparent that the microstructure/architecture of the HA coatings is an important factor in fabricating surfaces intended to repel proteins, mammalian and bacterial cells.

  16. Protein, cell and bacterial response to atmospheric pressure plasma grafted hyaluronic acid on poly(methylmethacrylate).

    PubMed

    D'Sa, Raechelle A; Raj, Jog; Dickinson, Peter J; McMahon, M Ann S; McDowell, David A; Meenan, Brian J

    2015-11-01

    Hyaluronic acid (HA) has been immobilised on poly(methyl methacrylate) (PMMA) surfaces using a novel dielectric barrier discharge (DBD) plasma process for the purposes of repelling protein, cellular and bacterial adhesion in the context of improving the performance of ophthalmic devices. Grafting was achieved by the following steps: (1) treatment of the PMMA with a DBD plasma operating at atmospheric pressure, (2) amine functionalisation of the activated polymer surface by exposure to a 3-aminopropyltrimethoxysilane (APTMS) linker molecule and (3) reaction of HA with the surface bound amine. The mechanism and effectiveness of the grafting process was verified by surface analysis. XPS data indicates that the APTMS linker molecule binds to PMMA via the Si-O chemistry and has the required pendant amine moiety. The carboxylic acid moiety on HA then binds with this -NH2 group via standard carbodiimide chemistry. ToF-SIMS confirms the presence of a coherent HA layer the microstructure of which is verified by AFM. The plasma grafted HA coating surfaces showed a pronounced decrease in protein and cellular adhesion when tested with bovine serum albumin and human corneal epithelial cells, respectively. The ability of these coatings to resist bacterial adhesion was established using Staphylococcus aureus NTC8325. Interestingly, the coatings did not repel bacterial adhesion, indicating that the mechanism of adhesion of bacterial cells is different to that for the surface interactions of mammalian cells. It is proposed that this difference is a consequence of the specific HA conformation that occurs under the conditions employed here. Hence, it is apparent that the microstructure/architecture of the HA coatings is an important factor in fabricating surfaces intended to repel proteins, mammalian and bacterial cells. PMID:26449450

  17. Purification of a sarcoplasmic reticulum protein that binds Ca2+ and plasma lipoproteins

    SciTech Connect

    Hofmann, S.L.; Brown, M.S.; Lee, E.; Pathak, R.K.; Anderson, R.G.; Goldstein, J.L. )

    1989-05-15

    A protein in the sarcoplasmic reticulum of rabbit skeletal and cardiac muscle was identified because of its ability to bind 125I-labeled low density lipoprotein (LDL) with high affinity after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This protein, referred to as the 165-kDa protein, is restricted to striated muscle. It was not detected in 14 other tissues, including several that contain smooth muscle, but it appears in rat L6 myoblasts when they differentiate into myocytes. Immunofluorescence and immunoelectron microscopic studies revealed that the protein is present throughout the sarcoplasmic reticulum and the terminal cisternae. It binds 45Ca2+ on nitrocellulose blots and stains metachromatically with Stains-all, a cationic dye that stains Ca2+-binding proteins. It does not appear to be a glycoprotein, and it appears slightly larger than the 160-kDa glycoprotein previously described in sarcoplasmic reticulum. The 165-kDa protein binds LDL, beta-migrating very low density lipoprotein, and a cholesterol-induced high density lipoprotein particle that contains apoprotein E as its sole apoprotein with much higher affinity than it binds high density lipoprotein. The protein is stable to boiling and to treatment with sodium dodecyl sulfate, but it becomes sensitive to these treatments when its cystine residues are reduced and alkylated. The protein was purified 1300-fold to apparent homogeneity from rabbit skeletal muscle membranes. It differs from the cell surface LDL receptor in that (1) its apparent molecular weight is not changed by reduction and alkylation; (2) it is present in Watanabe-heritable hyperlipidemic rabbits, which lack functional LDL receptors; (3) binding of lipoproteins is not inhibited by EDTA; and (4) it is located within the lumen of the sarcoplasmic reticulum where it has no access to plasma lipoproteins.

  18. Effect of degree of hydrolysis of whey protein on in vivo plasma amino acid appearance in humans.

    PubMed

    Farup, Jean; Rahbek, Stine Klejs; Storm, Adam C; Klitgaard, Søren; Jørgensen, Henry; Bibby, Bo M; Serena, Anja; Vissing, Kristian

    2016-01-01

    Whey protein is generally found to be faster digested and to promote faster and higher increases in plasma amino acid concentrations during the immediate ~60 min following protein ingestion compared to casein. The aim of the present study was to compare three different whey protein hydrolysates with varying degrees of hydrolysis (DH, % cleaved peptide bonds) to evaluate if the degree of whey protein hydrolysis influences the rate of amino acid plasma appearance in humans. A casein protein was included as reference. The three differentially hydrolysed whey proteins investigated were: High degree of hydrolysis (DH, DH = 48 %), Medium DH (DH = 27 %), and Low DH (DH = 23 %). The casein protein was intact. Additionally, since manufacturing of protein products may render some amino acids unavailable for utilisation in the body the digestibility and the biological value of all four protein fractions were evaluated in a rat study. A two-compartment model for the description of the postprandial plasma amino acid kinetics was applied to investigate the rate of postprandial total amino acid plasma appearance of the four protein products. The plasma amino acid appearance rates of the three whey protein hydrolysates (WPH) were all significantly higher than for the casein protein, however, the degree of hydrolysis of the WPH products did not influence plasma total amino acid appearance rate (estimates of DH and 95 % confidence intervals [CI] (mol L(-1) min(-1)): High DH 0.0585 [0.0454, 0.0754], Medium DH 0.0594 [0.0495, 0.0768], Low DH 0.0560 [0.0429, 0.0732], Casein 0.0194 [0.0129, 0.0291]). The four protein products were all highly digestible, while the biological value decreased with increasing degree of hydrolysis. In conclusion, the current study does not provide evidence that the degree of whey protein hydrolysis is a strong determinant for plasma amino acid appearance rate within the studied range of hydrolysis and protein dose. PMID:27065230

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

    PubMed Central

    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

  20. Plasma protein thiolation index (PTI) as a biomarker of thiol-specific oxidative stress in haemodialyzed patients.

    PubMed

    Colombo, Graziano; Reggiani, Francesco; Podestà, Manuel A; Garavaglia, Maria Lisa; Portinaro, Nicola M; Milzani, Aldo; Badalamenti, Salvatore; Dalle-Donne, Isabella

    2015-12-01

    The role of oxidative stress in patients with end stage renal disease (ESRD), which occurs at significantly higher levels than in the general population, is often underestimated in clinical practice. Emerging evidence highlights the strong correlation of oxidative stress with chronic inflammation and cardiovascular disease, which are highly prevalent in most patients on maintenance haemodialysis (HD) and are a major risk factor for mortality in this population. In this study, total plasma thiols and plasma S-thiolated proteins were measured in patients with ESRD, before and after a regular HD session, and compared to age-matched healthy subjects. We found a significant decrease in the level of total plasma thiols and, conversely, a significant increase in the level of S-thiolated proteins in these patients. In most patients, post-HD plasma level of total thiols did not differ from the one in healthy subjects, whereas plasma level of S-thiolated proteins was lower in HD patients than in age-matched healthy controls. This suggests that a single HD session restores plasma thiol redox status and re-establishes the antioxidant capacity of plasma thiols. Additionally, we determined protein thiolation index (PTI), i.e., the molar ratio between the sum of all low molecular mass thiols bound to S-thiolated plasma proteins and protein free cysteinyl residues. Patients with ESRD had a significantly higher PTI compared to age-matched healthy subjects and HD was associated with a decrease in PTI to normal, or lower than normal, levels. Although this study is limited in size, our results suggest that PTI is a useful indicator of thiol-specific oxidative stress in patients with ESRD on maintenance HD. This study also emphasizes that PTI determination is a cheap and simple tool suitable for large-scale clinical studies that could be used for routine screening of thiol-specific oxidative stress.

  1. The intracellular carboxyl terminal domain of Vangl proteins contains plasma membrane targeting signals

    PubMed Central

    Iliescu, Alexandra; Gros, Philippe

    2014-01-01

    Vangl1 and Vangl2 are integral membrane proteins that play a critical role in establishing planar cell polarity (PCP) in epithelial cells and are required for convergent extension (CE) movements during embryogenesis. Their proper targeting to the plasma membrane (PM) is required for function. We created discrete deletions at the amino and carboxy termini of Vangl1 and monitored the effect of the mutations on PM targeting in Madin–Darby canine kidney cells. Our results show that the Vangl1 amino terminus lacks PM targeting determinants, and these are restricted to the carboxy terminus, including the predicted PDZBM motif at the C-terminus. PMID:24452931

  2. [Mechanism of changes in amine binding to plasma proteins during allergy].

    PubMed

    Shleĭkin, A G; Gor'kova, L B; Pozhilenkova, K S; Zvezdochkin, A G

    1989-01-01

    Blood plasma of healthy persons bound 13.13 +/- 1.07 mmol/l of p-phenylene diamine (PDA), histamine pectic index constituted 33.3 +/- 2.18%. In patients with neurodermitis and eczema both these patterns were markedly reduced. Unitiol (10(-3) mol/l) increased PDA binding and histamine level in vitro. The same concentration of cystamine decreased histamine pectic index in healthy persons. Importance of protein SH-groups in binding of amines is discussed. PMID:2741420

  3. On the mechanism of the cold ethanol precipitation method of plasma protein fractionation.

    PubMed

    van Oss, C J

    1989-10-01

    Given the negligible difference in the value of the dielectric constant of water at 20 degrees C and that of ethanol solutions at low temperatures, the often advanced explanation for the precipitation of plasma proteins by the cold ethanol process, as being due to a reduction of the dielectric constant and the resulting increase in interprotein charge interactions, is not tenable. It is shown by a surface-thermodynamic approach that, upon dehydration by ethanol, isoelectric serum albumin molecules as well as isoelectric serum gamma globulin molecules will attract each other to a sufficient degree by van der Waals forces to become insoluble in the ethanol-water mixtures used.

  4. A plasma protein corona enhances the biocompatibility of Au@Fe3O4 Janus particles.

    PubMed

    Landgraf, Lisa; Christner, Carolin; Storck, Wiebke; Schick, Isabel; Krumbein, Ines; Dähring, Heidi; Haedicke, Katja; Heinz-Herrmann, Karl; Teichgräber, Ulf; Reichenbach, Jürgen R; Tremel, Wolfgang; Tenzer, Stefan; Hilger, Ingrid

    2015-11-01

    Au@Fe3O4 Janus particles (JPs) are heteroparticles with discrete domains defined by different materials. Their tunable composition and morphology confer multimodal and versatile capabilities for use as contrast agents and drug carriers in future medicine. Au@Fe3O4 JPs have colloidal properties and surface characteristics leading to interactions with proteins in biological fluids. The resulting protein adsorption layer ("protein corona") critically affects their interaction with living matter. Although Au@Fe3O4 JPs displayed good biocompatibility in a standardized in vitro situation, an in-depth characterization of the protein corona is of prime importance to unravel underlying mechanisms affecting their pathophysiology and biodistribution in vitro and in vivo. Here, we comparatively analyzed the human plasma corona of Au-thiol@Fe3O4-SiO2-PEG JPs (NH2-functionalized and non-functionalized) and spherical magnetite (Fe3O4-SiO2-PEG) particles and investigated its effects on colloidal stability, biocompatibility and cellular uptake. Label-free quantitative proteomic analyses revealed that complex coronas including almost 180 different proteins were formed within only one minute. Remarkably, in contrast to spherical magnetite particles with surface NH2 groups, the Janus structure prevented aggregation and the adhesion of opsonins. This resulted in an enhanced biocompatibility of corona sheathed JPs compared to spherical magnetite particles and corona-free JPs.

  5. Influence of plasma protein on the potencies of inhibitors of cyclooxygenase-1 and -2.

    PubMed

    Warner, Timothy D; Vojnovic, Ivana; Bishop-Bailey, David; Mitchell, Jane A

    2006-03-01

    It is widely believed that the potencies of nonsteroid anti-inflammatory drugs (NSAIDs) as inhibitors of cyclooxygenase (COX) are influenced by protein binding in the extracellular fluid, since NSAIDs are bound to circulating albumin by well over 95%. This is an important point because the protein concentrations in synovial fluid and the central nervous system, which are sites of NSAID action, are markedly different from those in plasma. Here we have used a modified whole-blood assay to compare the potencies of aspirin, celecoxib, diclofenac, indomethacin, lumiracoxib, meloxicam, naproxen, rofecoxib, sodium salicylate, and SC560 as inhibitors of COX-1 and COX-2 in the presence of differing concentrations of protein. The potencies of diclofenac, naproxen, rofecoxib, and salicylate, but not aspirin, celecoxib, indomethacin, lumiracoxib, meloxicam, or SC560, against COX-1 (human platelets) increased as protein concentrations were reduced. Varying protein concentrations did not affect the potencies of any of the drugs against COX-2, with the exception of sodium salicylate (A549 cells). Clearly, our findings show that the selectivity of inhibitors for COX-1 and COX-2, which are taken to be linked to their efficacy and side effects, may change in different extracellular fluid conditions. In particular, selectivity in one body compartment does not demonstrate selectivity in another. Thus, whole-body safety or toxicity cannot be linked to one definitive measure of COX selectivity.

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

    PubMed

    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. PMID:3934350

  7. Quantification of horse plasma proteins altered by xylazine using the fluorogenic derivatization-liquid chromatography-tandem mass spectrometry

    PubMed Central

    MORI, Miwako; ICHIBANGASE, Tomoko; YAMASHITA, Shozo; KIJIMA-SUDA, Isao; KAWAHARA, Masahiro; IMAI, Kazuhiro

    2016-01-01

    ABSTRACT In the doping tests currently used in horse racing, prohibited substances or their metabolites are usually directly detected in urine or blood samples. However, despite their lasting pharmaceutical effects, some prohibited substances are rapidly eliminated from horse urine and blood, making them difficult to detect. Therefore, new indirect biomarkers for doping, such as plasma proteins that are increased by the prohibited substances, have recently attracted much attention. Here, a fluorogenic derivatization-liquid chromatography-tandem mass spectrometry (FD-LC-MS/MS) method was adopted for horse plasma proteomics analysis, in order to identify plasma proteins whose concentrations were altered in response to xylazine in Thoroughbred horses. Xylazine, which is rapidly absorbed and eliminated and has possibility of the change in the levels of plasma proteins, was selected as a model drug. Of the ten plasma proteins identified, four proteins, including three acute phase proteins (haptoglobin, ceruloplasmin, and α-2-macroglobulin-like), were significantly increased after xylazine administration. Therefore, our present approach might be useful in identifying indirect biomarkers of drug administration. PMID:26858580

  8. [Development of online conventional array-based two-dimensional liquid chromatographic system for proteins separation in human plasma].

    PubMed

    Huang, Zhi; Hong, Guangfeng; Gao, Mingxia; Zhang, Xiangmin

    2014-04-01

    Human plasma is one of the proteins-containing samples most difficult to characterize on account of the wide dynamic concentration range of its intact proteins. Herein, we developed a high-throughput conventional array-based two-dimensional liquid chromatographic system for proteins separation in human plasma in online mode. In the system, a conventional strong-anion exchange chromatographic column was used as the first separation dimension and eight parallel conventional reversed-phase liquid chromatographic columns were integrated as the second separation dimension. The fractions from the first dimension were sequentially transferred into the corresponding reversed-phase liquid chromatographic precolumns for retention and enrichment using a 10-port electrically actuated multi-position valve. The second dimensional solvent flow was directly and identically split into 8 channels. The fractions were concurrently back-flushed from the precolumns into the 8 conventional RP columns and were separated simultaneously. An 8-channel fraction collector was refitted to collect the reversed-phase liquid chromatographic fractions for further investigation. Bicinchoninic acid (BCA) dyein solution was conveniently used for high-abundance protein location. Two separation dimensions were relatively independent parts, as well as each channel of the second dimensional array separation. Therefore, the new system could improve the separation throughput and total peak capacity. The system was successfully applied for the separation of human plasma intact proteins. The results indicated the established system is an effective method for removing high abundance proteins in plasma and in-depth research in plasma proteomics.

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

    SciTech Connect

    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

    2013-06-15

    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

  10. Characterization of Membrane Protein Interactions in Plasma Membrane Derived Vesicles with Quantitative Imaging FRET

    PubMed Central

    Sarabipour, Sarvenaz; Del Piccolo, Nuala; Hristova, Kalina

    2016-01-01

    CONSPECTUS Here we describe an experimental tool, termed Quantitative Imaging Förster Resonance Energy Transfer (QI-FRET), which enables the quantitative characterization of membrane protein interactions. The QI-FRET methodology allows us to acquire binding curves and calculate association constants for complex membrane proteins in the native plasma membrane environment. The method utilizes FRET detection, and thus requires that the proteins of interest are labeled with florescent proteins, either FRET donors or FRET acceptors. Since plasma membranes of cells have complex topologies precluding the acquisition of two-dimensional binding curves, the FRET measurements are performed in plasma membrane derived vesicles which bud off cells as a result of chemical or osmotic stress. The results overviewed here are acquired in vesicles produced with an osmotic vesiculation buffer developed in our laboratory, which does not utilize harsh chemicals. The concentrations of the donor-labeled and the acceptor-labeled proteins are determined, along with the FRET efficiencies, in each vesicle. The experiments utilize transient transfection, such that a wide variety of concentrations is sampled. Then, data from hundreds of vesicles are combined to yield dimerization curves. Here we discuss recent findings about the dimerization of receptor tyrosine kinases (RTKs), membrane proteins that control cell growth and differentiation via lateral dimerization in the plasma membrane. We focus on the dimerization of fibroblast growth factor receptor 3 (FGFR3), an RTK that plays a critically important role in skeletal development. We study the role of different FGFR3 domains in FGFR3 dimerization in the absence of ligand, and we show that FGFR3 extracellular domains inhibit unliganded dimerization, while contacts between the juxtamembrane domains, which connect the transmembrane domains to the kinase domains, stabilize the unliganded FGFR3 dimers. Since FGFR3 has been documented to harbor

  11. Receptor communication within the lymphocyte plasma membrane: a role for the thrombospondin family of matricellular proteins.

    PubMed

    Forslöw, A; Liu, Z; Sundqvist, K-G

    2007-01-01

    Lymphocytes, the principal cells of the immune system, carry out immune surveillance throughout the body by their unique capacity to constantly reposition themselves between a free-floating vascular state and a tissue state characterized by migration and frequent adhesive interactions with endothelial cells and components of the extracellular matrix. Therefore, mechanisms co-ordinating adhesion and migration with signals delivered through antigen recognition probably play a pivotal role for the regulation of lymphocyte behaviour and function. Endogenous thrombospondin-1 (TSP-1) seems to be the hub in such a mechanism for autocrine regulation of T cell adhesion and migration. TSP-1 functions as a mediator of cis interaction of vital receptors within the T lymphocyte plasma membrane, including integrins, low density lipoprotein receptor-related protein, calreticulin and integrin-associated protein.

  12. Relationships between plasma lipids, proteins, surface tension and post-dive bubbles.

    PubMed

    Schellart, Nico A M; Rozložník, Miroslav; Balestra, Costantino

    2015-01-01

    Decompression sickness (DCS) in divers is caused by bubbles of inert gas. When DCS occurs, most bubbles can be found in the venous circulation: venous gas emboli (VGE). Bubbles are thought to be stabilized by low molecular weight surfactant reducing the plasma-air surface tension (γ). Proteins may play a role as well. We studied the interrelations between these substances, γ and VGE, measured before and after a dry dive simulation. VGE of 63 dive simulations (21-msw/40-minute profile) of 52 divers was examined 40, 80, 120 and 160 minutes after surfacing (precordial Doppler method) and albumin, total protein, triglycerides, total cholesterol and free fatty acids were determined pre- and post-exposure. To manipulate blood plasma composition, half of the subjects obtained a fat-rich breakfast, while the other half got a fat-poor breakfast pre-dive. Eleven subjects obtained both. VGE scores measured with the precordial Doppler method were transformed to the logarithm of Kisman Integrated Severity Scores. With statistical analysis, including (partial) correlations, it could not be established whether γ as well as VGE scores are related to albumin, total protein or total cholesterol. With triglycerides and fatty acids correlations were also lacking, despite the fact that these compounds varied substantially. The same holds true for the paired differences between the two exposures of the 11 subjects. Moreover, no correlation between surface tension and VGE could be shown. From these findings and some theoretical considerations it seems likely that proteins lower surface tension rather than lipids. Since the findings are not in concordance with the classical surfactant hypothesis, reconsideration seems necessary.

  13. Plasma Fatty Acid Binding Protein 4 and Risk of Sudden Cardiac Death in Older Adults

    PubMed Central

    Djoussé, Luc; Maziarz, Marlena; Biggs, Mary L.; Ix, Joachim H.; Zieman, Susan J.; Kizer, Jorge R.; Lemaitre, Rozenn N.; Mozaffarian, Dariush; Tracy, Russell P.; Mukamal, Kenneth J.; Siscovick, David S.; Sotoodehnia, Nona

    2013-01-01

    Although fatty acid binding protein 4 (FABP4) may increase risk of diabetes and exert negative cardiac inotropy, it is unknown whether plasma concentrations of FABP4 are associated with incidence of sudden cardiac death (SCD). We prospectively analyzed data on 4,560 participants of the Cardiovascular Health Study. FABP4 was measured at baseline using ELISA, and SCD events were adjudicated through review of medical records. We used Cox proportional hazards to estimate effect measures. During a median followup of 11.8 years, 146 SCD cases occurred. In a multivariable model adjusting for demographic, lifestyle, and metabolic factors, relative risk of SCD associated with each higher standard deviation (SD) of plasma FABP4 was 1.15 (95% CI: 0.95–1.38), P = 0.15. In a secondary analysis stratified by prevalent diabetes status, FABP4 was associated with higher risk of SCD in nondiabetic participants, (RR per SD higher FABP4: 1.33 (95% CI: 1.07–1.65), P = 0.009) but not in diabetic participants (RR per SD higher FABP4: 0.88 (95% CI: 0.62–1.27), P = 0.50), P for diabetes-FABP4 interaction 0.049. In summary, a single measure of plasma FABP4 obtained later in life was not associated with the risk of SCD in older adults overall. Confirmation of our post-hoc results in nondiabetic people in other studies is warranted. PMID:24455402

  14. Effects of storage time on total protein and globulin concentrations in bovine fresh frozen plasma obtained for transfusion.

    PubMed

    Proverbio, D; Spada, E; Baggiani, L; Bagnagatti De Giorgi, G; Roggero, N; Belloli, A; Pravettoni, D; Perego, R

    2015-01-01

    To evaluate the effects of storage conditions on total protein (TP) and globulin fractions in fresh frozen bovine plasma units prepared and stored for transfusion, TP and globulin fractions were evaluated in fresh plasma and at 1 month and 6 and 12 months after blood collection in plasma stored at -20°C. Significant differences in concentrations were found in the median concentration of total protein (P=0.0336), between 0 months and 1 month (P=0.0108), 0 and 6 months (P=0.0023), and 0 and 12 months (P=0.0027), in mean concentration (g/dL) of albumin (P=0.0394), between 0 months and 1 month (P=0.0131), 0 and 6 months (P=0.0035), and 0 and 12 months (P=0.0038), and beta-2 fraction (P=0.0401), between 0 and 6 months (P=0.0401) and 0 and 12 months (P=0.0230). This study suggests that total gamma globulin concentration in bovine frozen plasma is stable for 12 months at -20°C. Total protein, ALB, and beta-2 fraction have significantly different concentrations (g/dL) when compared to prestorage. This study has shown IgG protein fraction stability in bovine fresh frozen plasma collected for transfusion; therefore, bovine fresh frozen plasma seems to be suitable for the treatment of hypogammaglobulinemia (failure of passive transfer) in calves when stored for 12 months at -20°C.

  15. Involvement of various organs in the initial plasma clearance of differently glycosylated rat liver secretory proteins.

    PubMed

    Gross, V; Heinrich, P C; vom Berg, D; Steube, K; Andus, T; Tran-Thi, T A; Decker, K; Gerok, W

    1988-05-01

    The initial plasma clearance and organ distribution of alpha 1-acid glycoprotein and alpha 2-macroglobulin carrying different types of oligosaccharide, side chains was studied in rats. The differently glycosylated proteins were synthesized by rat hepatocytes in culture in the presence of tunicamycin (unglycosylated form), swainsonine (hybrid type), or 1-deoxymannojirimycin (high-mannose type). Deglycosylated glycoproteins (Asn-GlcNAc) were obtained by endoglucosaminidase H treatment of high-mannose-type glycoproteins. Ten minutes after intravenous injection 3% of complex type, 26% of hybrid type, 84% of high-mannose type. 64% of unglycosylated and 80% of deglycosylated alpha 1-acid glycoprotein disappeared from the plasma. The respective values for alpha 2-macroglobulin were 26%, 42%, 59% and 67%. When the clearance of total hepatic secretory proteins was examined, major differences between glycosylated and unglycosylated (glyco)proteins were found, particularly in the case of low-molecular-mass polypeptides. Whereas complex-type alpha 1-acid glycoprotein and alpha 2-macroglobulin showed no accumulation in various organs, hybrid-type alpha 1-acid glycoprotein and alpha 2-macroglobulin were present in spleen and liver. High-mannose-type alpha 1-acid glycoprotein and alpha 2-macroglobulin also accumulated mainly in spleen and liver. Spleen had the highest specific activity; liver, due to its larger organ mass, represented the major organ for the uptake of high-mannose-type glycoproteins. Competition experiments with mannan and GlcNAc-bovine-serum-albumin showed a mannose/GlcNAc receptor-mediated removal. Whereas unglycosylated alpha 1-acid glycoprotein was taken up by the kidney, unglycosylated alpha 2-macroglobulin was found in the spleen. Deglycosylated glycoproteins (Asn-GlcNAc) were removed from the plasma via two different mechanisms: firstly, clearance by the kidney similar to the unglycosylated glycoproteins; secondly, clearance by a mannose/GlcNAc receptor

  16. Short-Lived Cages Restrict Protein Diffusion in the Plasma Membrane

    PubMed Central

    Goiko, Maria; de Bruyn, John R.; Heit, Bryan

    2016-01-01

    The plasma membrane is a heterogeneous environment characterized by anomalous diffusion and the presence of microdomains that are molecularly distinct from the bulk membrane. Using single particle tracking of the C-type lectin CD93, we have identified for the first time the transient trapping of transmembrane proteins in cage-like microdomains which restrict protein diffusion. These cages are stabilized by actin-dependent confinement regions, but are separate structures with sizes and lifespans uncorrelated to those of the underlying actin corral. These membrane cages require cholesterol for their strength and stability, with cholesterol depletion decreasing both. Despite this, cages are much larger in size and are longer lived than lipid rafts, suggesting instead that cholesterol-dependent effects on membrane fluidity or molecular packing play a role in cage formation. This diffusional compartment in the plasma membrane has characteristics of both a diffusional barrier and a membrane microdomain, with a size and lifespan intermediate between short-lived microdomains such as lipid rafts and long-lasting diffusional barriers created by the actin cytoskeleton. PMID:27725698

  17. A physiologically based pharmacokinetic model linking plasma protein binding interactions with drug disposition.

    PubMed

    Buur, J L; Baynes, R E; Smith, G W; Riviere, J E

    2009-04-01

    Combination drug therapy increases the chance for an adverse drug reactions due to drug-drug interactions. Altered disposition for sulfamethazine (SMZ) when concurrently administered with flunixin meglumine (FLU) in swine could lead to increased tissue residues. There is a need for a pharmacokinetic modeling technique that can predict the consequences of possible drug interactions. A physiologically based pharmacokinetic model was developed that links plasma protein binding interactions to drug disposition for SMZ and FLU in swine. The model predicted a sustained decrease in total drug and a temporary increase in free drug concentration. An in vivo study confirmed the presence of a drug interaction. Neither the model nor the in vivo study revealed clinically significant changes that alter tissue disposition. This novel linkage approach has use in the prediction of the clinical impact of plasma protein binding interactions. Ultimately it could be used in the design of dosing regimens and in the protection of the food supply through prediction and minimization of tissue residues. PMID:18721993

  18. Species-dependent enantioselective plasma protein binding of MK-571, a potent leukotriene D4 antagonist.

    PubMed

    Lin, J H; deLuna, F A; Ulm, E H; Tocco, D J

    1990-01-01

    The plasma protein binding of the enantiomers of MK-571 was stereoselective and the stereoselectivity was species dependent. The 12 mammalian species studied could be classified into three groups: those that bind the S-(+)-enantiomer to a greater extent than the R-(-)-enantiomer (human, baboon, monkey, cow, dog, and cat); those that bind the R-(-)-enantiomer more extensively (rat, guinea pig, and sheep); and those that show no stereoselectivity (rabbit, hamster, and mouse). The stereoselective binding appears to have no phylogenetic relationship. Using serum albumin instead of plasma, a similar degree of stereoselective binding was observed for human, dog, sheep, and rat, suggesting that albumin is the major binding component for MK-571 enantiomers, and that species differences in stereoselective binding are likely due to structural differences in the albumin molecule. Displacement studies with [14C] diazepam, [14C]warfarin, and [3H]digitoxin indicated that the enantioselective differences in protein binding are most likely due to the differences in binding affinity rather than to different binding sites. PMID:1976072

  19. Electrochemical aptasensor for lung cancer-related protein detection in crude blood plasma samples

    PubMed Central

    Zamay, Galina S.; Zamay, Tatiana N.; Kolovskii, Vasilii A.; Shabanov, Alexandr V.; Glazyrin, Yury E.; Veprintsev, Dmitry V.; Krat, Alexey V.; Zamay, Sergey S.; Kolovskaya, Olga S.; Gargaun, Ana; Sokolov, Alexey E.; Modestov, Andrey A.; Artyukhov, Ivan P.; Chesnokov, Nikolay V.; Petrova, Marina M.; Berezovski, Maxim V.; Zamay, Anna S.

    2016-01-01

    The development of an aptamer-based electrochemical sensor for lung cancer detection is presented in this work. A highly specific DNA-aptamer, LC-18, selected to postoperative lung cancer tissues was immobilized onto a gold microelectrode and electrochemical measurements were performed in a solution containing the redox marker ferrocyanide/ferricyanide. The aptamer protein targets were harvested from blood plasma of lung cancer patients by using streptavidin paramagnetic beads and square wave voltammetry of the samples was performed at various concentrations. In order to enhance the sensitivity of the aptasensor, silica-coated iron oxide magnetic beads grafted with hydrophobic C8 and C4 alkyl groups were used in a sandwich detection approach. Addition of hydrophobic beads increased the detection limit by 100 times. The detection limit of the LC-18 aptasensor was enhanced by the beads to 0.023 ng/mL. The formation of the aptamer – protein – bead sandwich on the electrode surface was visualized by electron microcopy. As a result, the electrochemical aptasensor was able to detect cancer-related targets in crude blood plasma of lung cancer patients. PMID:27694916

  20. High precision quantification of human plasma proteins using the automated SISCAPA Immuno-MS workflow.

    PubMed

    Razavi, Morteza; Leigh Anderson, N; Pope, Matthew E; Yip, Richard; Pearson, Terry W

    2016-09-25

    Efficient robotic workflows for trypsin digestion of human plasma and subsequent antibody-mediated peptide enrichment (the SISCAPA method) were developed with the goal of improving assay precision and throughput for multiplexed protein biomarker quantification. First, an 'addition only' tryptic digestion protocol was simplified from classical methods, eliminating the need for sample cleanup, while improving reproducibility, scalability and cost. Second, methods were developed to allow multiplexed enrichment and quantification of peptide surrogates of protein biomarkers representing a very broad range of concentrations and widely different molecular masses in human plasma. The total workflow coefficients of variation (including the 3 sequential steps of digestion, SISCAPA peptide enrichment and mass spectrometric analysis) for 5 proteotypic peptides measured in 6 replicates of each of 6 different samples repeated over 6 days averaged 3.4% within-run and 4.3% across all runs. An experiment to identify sources of variation in the workflow demonstrated that MRM measurement and tryptic digestion steps each had average CVs of ∼2.7%. Because of the high purity of the peptide analytes enriched by antibody capture, the liquid chromatography step is minimized and in some cases eliminated altogether, enabling throughput levels consistent with requirements of large biomarker and clinical studies.

  1. Cholesterol and F-actin are required for clustering of recycling synaptic vesicle proteins in the presynaptic plasma membrane.

    PubMed

    Dason, Jeffrey S; Smith, Alex J; Marin, Leo; Charlton, Milton P

    2014-02-15

    Synaptic vesicles (SVs) and their proteins must be recycled for sustained synaptic transmission. We tested the hypothesis that SV cholesterol is required for proper sorting of SV proteins during recycling in live presynaptic terminals. We used the reversible block of endocytosis in the Drosophila temperature-sensitive dynamin mutant shibire-ts1 to trap exocytosed SV proteins, and then examined the effect of experimental treatments on the distribution of these proteins within the presynaptic plasma membrane by confocal microscopy. SV proteins synaptotagmin, vglut and csp were clustered following SV trapping in control experiments but dispersed in samples treated with the cholesterol chelator methyl-β-cyclodextrin to extract SV cholesterol. There was accumulation of phosphatidylinositol (4,5)-bisphosphate (PIP2) in presynaptic terminals following SV trapping and this was reduced following SV cholesterol extraction. Reduced PIP2 accumulation was associated with disrupted accumulation of actin in presynaptic terminals. Similar to vesicular cholesterol extraction, disruption of actin by latrunculin A after SV proteins had been trapped on the plasma membrane resulted in the dispersal of SV proteins and prevented recovery of synaptic transmission due to impaired endocytosis following relief of the endocytic block. Our results demonstrate that vesicular cholesterol is required for aggregation of exocytosed SV proteins in the presynaptic plasma membrane and are consistent with a mechanism involving regulation of PIP2 accumulation and local actin polymerization by cholesterol. Thus, alteration of membrane or SV lipids may affect the ability of synapses to undergo sustained synaptic transmission by compromising the recycling of SV proteins.

  2. Characterization of a calcium- and lipid-dependent protein kinase associated with the plasma membrane of oat

    SciTech Connect

    Schaller, G.E.; Sussman, M.R. ); Harmon, A.C. )

    1992-02-18

    A protein kinase that is activated by calcium and lipid has been partially purified from the plasma membrane of oat roots. This protein kinase cross-reacts with four monoclonal antibodies directed against a soluble calcium-dependent protein kinase from soybean described previously indicating that the oat enzyme is a member of this calcium-dependent protein kinase family. Immunoblots demonstrate that the membrane-derived protein kinase is slightly larger than that observed in the cytosolic fraction of oat. Limited digestion of the membrane-derived kinase with trypsin generates a smaller water-soluble kinase that is still activated by calcium but is no longer activated by lipid. When posthomogenization proteolysis is minimized, the bulk of the immunoreactive kinase material is localized in the membrane. These results suggest that a calcium-dependent protein kinase observed in the supernatant fraction of oat extracts may originate in situ from a calcium- and lipid-dependent protein kinase which is associated with the oat plasma membrane. They further indicate that, in contrast to animal cells, the predominant calcium- and lipid-dependent protein kinase associated with the plasma membrane of plant cells has biochemical properties and amino acid sequence unlike protein kinase C.

  3. Plasma levels of the chemokines monocyte chemotactic proteins-1 and -2 are elevated in human sepsis.

    PubMed

    Bossink, A W; Paemen, L; Jansen, P M; Hack, C E; Thijs, L G; Van Damme, J

    1995-11-15

    Because of their effects on monocytes, monocyte chemotactic proteins-1 and -2 (MCP-1 and MCP-2) may participate in the pathophysiology of sepsis. We measured circulating MCP-1 and MCP-2 levels in 42 septic patients having positive local or blood cultures. MCP-1 and MCP-2 levels were elevated in 24 (57%) and 25 (59%) of 42 septic patients, respectively, compared with healthy volunteers. Both patients with gram-positive and gram-negative infections had elevated MCP-1 plasma levels (P = .0001) and P < .0001), respectively; Mann-Whitney-U test), whereas patients with gram-positive infection, but not those with gram-negative infection, had increased MCP-2 plasma levels (P= .0182). No relative differences in MCP-1 and MCP-2 plasma levels were observed between several subgroups of patients (sepsis v septic shock; survivors v nonsurvivors), although levels of MCP-1 were the highest in patients with the more severe forms of sepsis, ie, those with shock or a lethal outcome. Serial observations showed that MCP-1 and MCP-2 plasma levels remained elevated for at least 48 hours. MCP-1 correlated weakly with interleukin-8 and MCP-2, the correlations for which were most pronounced in patients with septic shock. MCP-2 correlated with interleukin-8, and surprisingly, with the complement activation product C3a; these correlations further improved when analyzing patients with septic shock or when applying gram-positive infections. Thus, our results not only show increased MCP-1 and MCP-2 levels in patients with sepsis, but also suggest that the synthesis and release of MCP-1 and MCP-2 in sepsis are differently regulated in part.

  4. Plasma C-Reactive Protein and Clinical Outcomes after Acute Ischemic Stroke: A Prospective Observational Study

    PubMed Central

    Matsuo, Ryu; Ago, Tetsuro; Hata, Jun; Wakisaka, Yoshinobu; Kuroda, Junya; Kuwashiro, Takahiro; Kitazono, Takanari; Kamouchi, Masahiro

    2016-01-01

    Background and Purpose Although plasma C-reactive protein (CRP) is elevated in response to inflammation caused by brain infarction, the association of CRP with clinical outcomes after acute ischemic stroke remains uncertain. This study examined whether plasma high-sensitivity CRP (hsCRP) levels at onset were associated with clinical outcomes after acute ischemic stroke independent of conventional risk factors and acute infections after stroke. Methods We prospectively included 3653 patients with first-ever ischemic stroke who had been functionally independent and were hospitalized within 24 h of onset. Plasma hsCRP levels were measured on admission and categorized into quartiles. The association between hsCRP levels and clinical outcomes, including neurological improvement, neurological deterioration, and poor functional outcome (modified Rankin scale ≥3 at 3 months), were investigated using a logistic regression analysis. Results Higher hsCRP levels were significantly associated with unfavorable outcomes after adjusting for age, sex, baseline National Institutes of Health Stroke Scale score, stroke subtype, conventional risk factors, intravenous thrombolysis and endovascular therapy, and acute infections during hospitalization (multivariate-adjusted odds ratios [95% confidence interval] in the highest quartile versus the lowest quartile as a reference: 0.80 [0.65–0.97] for neurological improvement, 1.72 [1.26–2.34] for neurological deterioration, and 2.03 [1.55–2.67] for a poor functional outcome). These associations were unchanged after excluding patients with infectious diseases occurring during hospitalization, or those with stroke recurrence or death. These trends were similar irrespective of stroke subtypes or baseline stroke severity, but more marked in patients aged <70 years (Pheterogeneity = 0.001). Conclusions High plasma hsCRP is independently associated with unfavorable clinical outcomes after acute ischemic stroke. PMID:27258004

  5. Hemocompatible mixed-charge copolymer brushes of pseudozwitterionic surfaces resistant to nonspecific plasma protein fouling.

    PubMed

    Chang, Yung; Shu, Shih-Hung; Shih, Yu-Ju; Chu, Chih-Wei; Ruaan, Ruoh-Chyu; Chen, Wen-Yih

    2010-03-01

    In this work, the hemocompatibility of a sulfobetaine-like copolymer brush resulting from a mixed-charge copolymerization of the positively charged 11-mercapto-N,N,N-trimethylammonium chloride (TMA) and negatively charged 11-mercaptoundecylsulfonic acid (SA) was studied. Mixed charge distribution in the prepared poly(TMA-co-SA) copolymer brushes was controlled by the regulation of the reaction rate of the surface-initiated atom transfer radical polymerization (ATRP). The adsorption behavior of plasma proteins on a surface grafted with poly(TMA-co-SA) was measured by a surface plasmon resonance (SPR) sensor. The effects of varying temperature, solution pH, and ionic strength on the antifouling characteristics of the mixed-charge copolymer brushes were systematically evaluated, and the protein-fouling resistance was discussed in detail, especially with respect to the effect of ionic strength on the intra- and intermolecular interactions of the poly(TMA-co-SA) with proteins. The adhesion and activation of blood cells on the poly(TMA-co-SA)-grafted surface in contact with human whole blood was also demonstrated. The results suggest that mixed-charge copolymer brushes of poly(TMA-co-SA), which, like zwitterionic homopolymer brushes, have overall charge neutrality, can be used in similar applications for protein-fouling resistance and have excellent hemocompatibility with human whole blood at physiologic temperatures. PMID:19947616

  6. Vacuolar sorting receptor (VSR) proteins reach the plasma membrane in germinating pollen tubes.

    PubMed

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

    2011-09-01

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

  7. A major integral protein of the plant plasma membrane binds flavin.

    PubMed

    Lorenz, Astrid; Kaldenhoff, Ralf; Hertel, Rainer

    2003-05-01

    Abundant flavin binding sites have been found in membranes of plants and fungi. With flavin mononucleotide-agarose affinity columns, riboflavin-binding activity from microsomes of Cucurbita pepoL. hypocotyls was purified and identified as a specific PIP1-homologous protein of the aquaporin family. Sequences such as gi|2149955 in Phaseolus vulgaris, PIP1b of Arabidopsis thaliana, and NtAQP1 of tobacco are closely related. The identification as a riboflavin-binding protein was confirmed by binding tests with an extract of Escherichia coli cells expressing the tobacco NtAQP1 as well as leaves of transgenic tobacco plants that overexpress NtAQP1 or were inhibited in PIP1 expression by antisense constructs. When binding was assayed in the presence of dithionite, the reduced flavin formed a relatively stable association with the protein. Upon dilution under oxidizing conditions, the adduct was resolved, and free flavin reappeared with a half time of about 30 min. Such an association can also be induced photochemically, with oxidized flavin by blue light at 450 nm, in the presence of an electron donor. Several criteria, localization in the plasma membrane, high abundance, affinity to roseoflavin, and photochemistry, argue for a role of the riboflavin-binding protein PIP1 as a photoreceptor. PMID:12768338

  8. Binding of plasma proteins to titanium dioxide nanotubes with different diameters

    PubMed Central

    Kulkarni, Mukta; Flašker, Ajda; Lokar, Maruša; Mrak-Poljšak, Katjuša; Mazare, Anca; Artenjak, Andrej; Čučnik, Saša; Kralj, Slavko; Velikonja, Aljaž; Schmuki, Patrik; Kralj-Iglič, Veronika; Sodin-Semrl, Snezna; Iglič, Aleš

    2015-01-01

    Titanium and titanium alloys are considered to be one of the most applicable materials in medical devices because of their suitable properties, most importantly high corrosion resistance and the specific combination of strength with biocompatibility. In order to improve the biocompatibility of titanium surfaces, the current report initially focuses on specifying the topography of titanium dioxide (TiO2) nanotubes (NTs) by electrochemical anodization. The zeta potential (ζ-potential) of NTs showed a negative value and confirmed the agreement between the measured and theoretically predicted dependence of ζ-potential on salt concentration, whereby the absolute value of ζ-potential diminished with increasing salt concentrations. We investigated binding of various plasma proteins with different sizes and charges using the bicinchoninic acid assay and immunofluorescence microscopy. Results showed effective and comparatively higher protein binding to NTs with 100 nm diameters (compared to 50 or 15 nm). We also showed a dose-dependent effect of serum amyloid A protein binding to NTs. These results and theoretical calculations of total available surface area for binding of proteins indicate that the largest surface area (also considering the NT lengths) is available for 100 nm NTs, with decreasing surface area for 50 and 15 nm NTs. These current investigations will have an impact on increasing the binding ability of biomedical devices in the body leading to increased durability of biomedical devices. PMID:25733829

  9. Binding of plasma proteins to titanium dioxide nanotubes with different diameters.

    PubMed

    Kulkarni, Mukta; Flašker, Ajda; Lokar, Maruša; Mrak-Poljšak, Katjuša; Mazare, Anca; Artenjak, Andrej; Čučnik, Saša; Kralj, Slavko; Velikonja, Aljaž; Schmuki, Patrik; Kralj-Iglič, Veronika; Sodin-Semrl, Snezna; Iglič, Aleš

    2015-01-01

    Titanium and titanium alloys are considered to be one of the most applicable materials in medical devices because of their suitable properties, most importantly high corrosion resistance and the specific combination of strength with biocompatibility. In order to improve the biocompatibility of titanium surfaces, the current report initially focuses on specifying the topography of titanium dioxide (TiO2) nanotubes (NTs) by electrochemical anodization. The zeta potential (ζ-potential) of NTs showed a negative value and confirmed the agreement between the measured and theoretically predicted dependence of ζ-potential on salt concentration, whereby the absolute value of ζ-potential diminished with increasing salt concentrations. We investigated binding of various plasma proteins with different sizes and charges using the bicinchoninic acid assay and immunofluorescence microscopy. Results showed effective and comparatively higher protein binding to NTs with 100 nm diameters (compared to 50 or 15 nm). We also showed a dose-dependent effect of serum amyloid A protein binding to NTs. These results and theoretical calculations of total available surface area for binding of proteins indicate that the largest surface area (also considering the NT lengths) is available for 100 nm NTs, with decreasing surface area for 50 and 15 nm NTs. These current investigations will have an impact on increasing the binding ability of biomedical devices in the body leading to increased durability of biomedical devices.

  10. Dietary aspartame with protein on plasma and brain amino acids, brain monoamines and behavior in rats.

    PubMed

    Torii, K; Mimura, T; Takasaki, Y; Ichimura, M

    1986-01-01

    Aspartame (APM; L-aspartyl-L-phenylalanine methyl ester), was investigated for its ability to alter levels of the large neutral amino acids and monoamines in overnight fasted rats allowed to consume meals with or without protein for two hours. Additionally, the possible long term behavioral consequences of APM in 25% casein diets with or without 10% sucrose were determined. Acute APM ingestion increased both plasma and brain phenylalanine and tyrosine levels, but brain tryptophan levels were not altered regardless of dietary protein. Brain norepinephrine and dopamine levels were unaltered by any of the diet while serotonin levels were slightly increased when a protein-free diet was consumed. But APM and/or protein ingestion minimized this increase of brain serotonin levels as much as controls. Chronic APM ingestion failed to influence diurnal feeding patterns, meal size distributions, or diurnal patterns of spontaneous motor activity. The chronic ingestion of abuse doses of APM produced no significant chemical changes in brain capable of altering behavioral parameters believed to be controlled by monoamines in rats.

  11. Scavenger receptor-mediated recognition of maleyl bovine plasma albumin and the demaleylated protein in human monocyte macrophages.

    PubMed Central

    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 125I-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. The demaleylated protein suppressed 75% of the hydrolysis of 125I-labeled malondialdehyde LDL and greater than 80% of 125I-labeled maleyl bovine plasma albumin. The ability of the demaleylated protein to compete was abolished after treatment with guanidine hydrochloride. Although ligands recognized by the scavenger receptor typically are anionic, we 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). The altered conformation of the modified protein apparently persists after removal of the maleyl groups. We 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. PMID:3857610

  12. Effect of caffeine on the plasma protein binding and the disposition of ceftriaxone.

    PubMed

    Kwon, K I; Bourne, D W; Ho, P C

    1985-11-01

    The effects of caffeine on the in-vitro protein binding and the pharmacokinetics of ceftriaxone (a highly protein bound cephalosporin) were investigated. Caffeine failed to decrease in-vitro protein binding of ceftriaxone. Rabbit plasma concentrations of ceftriaxone (30 mg kg-1 i.v.) were elevated significantly (P less than 0.05 at 0.3, 0.6 and 1 h after injection) when caffeine 5 or 10 mg kg-1 i.v. was co-administered compared with ceftriaxone given alone. Caffeine increased the volume of distribution of the central compartment (V1) for ceftriaxone significantly from 49 +/- 38 ml kg-1 (mean +/- s.d., n = 6) to 97 +/- 33 ml kg-1 (caffeine 5 mg kg-1, P less than 0.05), and 94 +/- 8 ml kg-1 (caffeine 10 mg kg-1, P less than 0.05) and decreased the volume of distribution of the peripheral compartment (V2) from 145 +/- 106 ml kg-1 (mean +/- s.d., n = 6) to 31 +/- 18 ml kg-1 (caffeine 5 mg kg-1, P less than 0.5) and 36 +/- 31 ml kg-1 (caffeine 10 mg kg-1, P less than 0.1). The rate of transfer of ceftriaxone to the peripheral compartment (k12) was also decreased significantly (P less than 0.05) after caffeine. The elevated plasma concentration of ceftriaxone, increased V1 value and the decreased V2 and k12 values are probably the result of caffeine altering the distribution of ceftriaxone to the central and the peripheral compartments. PMID:2867172

  13. Uptake of copper from plasma proteins in cells where expression of CTR1 has been modulated.

    PubMed

    Kidane, Theodros Z; Farhad, Ramin; Lee, Kyoung Jin; Santos, Abraham; Russo, Eric; Linder, Maria C

    2012-08-01

    Plasma proteins rather than amino acid chelates are the direct sources of copper for mammalian cells. In continuing studies on the mechanisms by which albumin and transcuprein deliver copper and the potential involvement of CTR1, rates of uptake from these proteins and Cu-histidine were compared in cells with/without CTR1 knockdown or knockout. siRNA knocked down expression of CTR1 mRNA 60-85% in human mammary epithelial and hepatic cell models, but this had little or no effect on uptake of 1 μM Cu(II) attached to pure human albumin or alpha-2-macroglobulin. Mouse embryonic fibroblasts that did/did not express Ctr1 took up Cu(II) bound to albumin about as readily as from the histidine complex at physiological concentrations and by a single saturable process. Uptake from mouse albumin achieved a 2-4-fold higher Vmax (with a lower Km) than from heterologous human albumin. Maximum uptake rates from Cu(I)-histidine were >12-fold higher (with higher Km) than for Cu(II), suggesting mediation by a reductase. The presence of cell surface Cu(II) and Fe(III) reductase activity responding only slightly to dehydroascorbate was verified. Excess Fe(III) inhibited uptake from albumin-Cu(II). Ag(I) also inhibited, but kinetics were not or un-competitive. In general there was little difference in rates/kinetics of uptake in the Ctr1+/+ and -/- cells. Endocytosis was not involved. We conclude that plasma proteins deliver Cu(II) to homologous cells with greater efficiency than ionic copper at physiological concentrations, probably through the mediation of a Steap Cu(II)-reductase, and confirm the existence of an additional copper uptake system in mammalian cells.

  14. Multiple sequence signals determine the distribution of glycosylphosphatidylinositol proteins between the plasma membrane and cell wall in Saccharomyces cerevisiae.

    PubMed

    Frieman, Matthew B; Cormack, Brendan P

    2004-10-01

    Glycosylphosphatidylinositol (GPI)-anchored cell wall proteins (GPI-CWPs) play an important role in the structure and function of the cell wall in Saccharomyces cerevisiae and other fungi. While the majority of characterized fungal GPI-anchored proteins localize to the cell wall, a subset of GPI proteins are thought to reside at the plasma membrane and not to traffic significantly to the cell wall. The amino acids immediately upstream of the site of GPI anchor addition (the omega site) are the primary signal determining whether a GPI protein localizes to the cell wall or to the plasma membrane. Here, evidence was found that in addition to this omega-proximal signal, other sequences in the protein can impact the distribution of GPI proteins between cell wall and membrane. In particular, it was found that long regions rich in serine and threonine residues (a feature of many cell wall proteins) can override the omega-proximal signal and redirect a model GPI plasma membrane protein to the cell wall.

  15. Evaluation of Multi-Protein Immunoaffinity Subtraction for Plasma Proteomics and Candidate Biomarker Discovery Using Mass Spectrometry

    PubMed Central

    Liu, Tao; Qian, Wei-Jun; Mottaz, Heather M.; Gritsenko, Marina A.; Norbeck, Angela D.; Moore, Ronald J.; Purvine, Samuel O.; Camp, David G.; Smith, Richard D.

    2007-01-01

    SUMMARY Strategies for removal of high-abundance proteins have been increasingly utilized in proteomic studies of serum/plasma and other body fluids to enhance the detection of low-abundance proteins and achieve broader proteome coverage; however, both the reproducibility and specificity of the high-abundance protein depletion process still represent common concerns. Here, we report a detailed evaluation of immunoaffinity subtraction performed applying the ProteomeLab IgY-12 system which is commonly used in human serum/plasma proteome characterization in combination with high resolution LC-MS/MS. Plasma samples were repeatedly processed implementing this system, and the resulting flow-through fractions and bound fractions were individually analyzed for comparison. The removal of target proteins by the immunoaffinity subtraction system and the overall process was highly reproducible. Non-target proteins, including one spiked protein standard (rabbit glyceraldehyde-3-phosphate dehydrogenase), were also observed to bind to the column at different levels, but in a reproducible manner. The results suggest that multi-protein immunoaffinity subtraction systems can be readily integrated into quantitative strategies to enhance detection of low-abundance proteins in biomarker discovery studies. PMID:16854842

  16. Ram seminal plasma proteins contribute to sperm capacitation and modulate sperm-zona pellucida interaction.

    PubMed

    Luna, C; Colás, C; Casao, A; Serrano, E; Domingo, J; Pérez-Pé, R; Cebrián-Pérez, J A; Muiño-Blanco, T

    2015-03-01

    Incubation of ram spermatozoa in capacitating conditions with cAMP-elevating agents promotes a progressive time-dependent increase in the capacitated sperm subpopulation. In this study, the fertilizing capacity of ram spermatozoa (ability to bind to the zona pellucida, ZBA rate) capacitated in these conditions was determined. The results showed an increase (P < 0.001) in ZBA rate related to control samples in basal medium that contained BSA, calcium, and bicarbonate (1.97 ± 0.19 vs. 1.31 ± 0.09 sperm bound/oocyte, respectively). A significant correlation between protein tyrosine phosphorylation and ZBA rate (P < 0.05, r = 0.501) corroborated that incubation in a "high-cAMP" environment improves the fertilizing ability of ram spermatozoa. Likewise, the presence of two seminal plasma (SP) proteins able to protect sperm against cold shock (RSVP14 and RSVP20) was evidenced in both SP and the ram sperm surface, and their influence in the fertilizing ability of spermatozoa capacitated in basal medium or with cAMP-elevating agents was determined. The results verified that RSVP14 and RSVP20 act as decapacitating factors given that their addition to SP-free sperm samples previously to capacitation maintained high proportions of the noncapacitated sperm pattern with no increase in protein tyrosine phosphorylation. However, the obtained ZBA rate in the high-cAMP-containing samples was increased in the presence of RSVP20 (P < 0.05). These findings would indicate that the stimulating effect exerted by this protein on the sperm-oocyte binding occurs downstream from the cAMP generation and that the mechanisms by which RSVP20 promotes the zona pellucida binding might be independent of protein tyrosine phosphorylation. PMID:25515364

  17. Ram seminal plasma proteins contribute to sperm capacitation and modulate sperm-zona pellucida interaction.

    PubMed

    Luna, C; Colás, C; Casao, A; Serrano, E; Domingo, J; Pérez-Pé, R; Cebrián-Pérez, J A; Muiño-Blanco, T

    2015-03-01

    Incubation of ram spermatozoa in capacitating conditions with cAMP-elevating agents promotes a progressive time-dependent increase in the capacitated sperm subpopulation. In this study, the fertilizing capacity of ram spermatozoa (ability to bind to the zona pellucida, ZBA rate) capacitated in these conditions was determined. The results showed an increase (P < 0.001) in ZBA rate related to control samples in basal medium that contained BSA, calcium, and bicarbonate (1.97 ± 0.19 vs. 1.31 ± 0.09 sperm bound/oocyte, respectively). A significant correlation between protein tyrosine phosphorylation and ZBA rate (P < 0.05, r = 0.501) corroborated that incubation in a "high-cAMP" environment improves the fertilizing ability of ram spermatozoa. Likewise, the presence of two seminal plasma (SP) proteins able to protect sperm against cold shock (RSVP14 and RSVP20) was evidenced in both SP and the ram sperm surface, and their influence in the fertilizing ability of spermatozoa capacitated in basal medium or with cAMP-elevating agents was determined. The results verified that RSVP14 and RSVP20 act as decapacitating factors given that their addition to SP-free sperm samples previously to capacitation maintained high proportions of the noncapacitated sperm pattern with no increase in protein tyrosine phosphorylation. However, the obtained ZBA rate in the high-cAMP-containing samples was increased in the presence of RSVP20 (P < 0.05). These findings would indicate that the stimulating effect exerted by this protein on the sperm-oocyte binding occurs downstream from the cAMP generation and that the mechanisms by which RSVP20 promotes the zona pellucida binding might be independent of protein tyrosine phosphorylation.

  18. A general method for fractionation of plasma proteins. Dye-ligand affinity chromatography on immobilized Cibacron blue F3-GA.

    PubMed Central

    Gianazza, E; Arnaud, P

    1982-01-01

    The chromatographic behaviour of 27 different plasma proteins on fractionation of human plasma on immobilized Cibacron Blue F3-GA was studied. The column was eluted by using a three-step procedure. First, a low-molarity buffer (30 mM-H3PO4/Na3PO4, pH 7.0, I0.053) was used, then a linear salt gradient (0-1 M-NaCl in the buffer above) was applied, followed by a wash with two bed volumes of 1.0 M-NaCl. Finally, bound proteins were 'stripped' with 0.5 M-NaSCN. Up to 1 ml of whole plasma could be loaded per 5 ml bed volume. No denaturation of proteinase inhibitors or complement fractions was observed. The recovery of individual proteins ranged between 52 and greater than 95%. Enrichment of four individual plasma components (alpha 1-antitrypsin, caeruloplasmin, antithrombin III and haemopexin) was between 10-fold and 75-fold. These results indicate that chromatography on immobilized Cibacron Blue F3-GA can be a useful initial step in the purification of plasma proteins. Images Fig. 2. Fig. 3. Fig. 4. PMID:7082279

  19. Binding of lithium and boron to human plasma proteins II: results for a bipolar patient not on lithium therapy.

    PubMed

    Clarke, W Brian; Guscott, Richard; Lindstrom, Richard M

    2004-02-01

    We report further measurements of lithium and boron bound to human plasma proteins using the techniques of gel chromatography, thermal-neutron activation, and high-sensitivity helium isotope mass spectrometry. The plasma sample was donated by a bipolar patient who had never been on lithium therapy. The plasma lithium-binding pattern for the bipolar patient is distinctly different from that previously observed in this laboratory for plasma donated by a normal individual. In the bipolar case, virtually all of the lithium is bound to low-molecular-weight proteins (approx 1000 amu), whereas in the normal case, most of the lithium eluted from the gel column was bound to five high-molecular-weight proteins (approx 50,000 amu to approx 1,000,000 amu). The gel elution profiles for boron were roughly similar for the normal and bipolar cases. The lithium results are in agreement with our previous speculation that lithium-binding plasma proteins are missing or exist in very low concentrations in some individuals suffering from affective disorders.

  20. Synthesis and evaluation of radioactive and fluorescent residualizing labels for identifying sites of plasma protein catabolism

    SciTech Connect

    Maxwell, J.L.; Baynes, J.W.; Thorpe, S.R.

    1986-05-01

    Inulin and lactose were each coupled to tyramine by reductive amination with NaBH/sub 3/CN and the tyramine then labeled with /sup 125/I. Dilactitol-/sup 125/I-tyramine (DLT) and inulin-/sup 125/I-tyramine (InTn) were coupled by reductive amination and cyanuric chloride, respectively, to asialofetuin (ASF), fetuin and rat serum albumin (RSA). Attachment of either label had no effect on the circulating half-lives of the proteins. Radioactivity from labeled ASF was recovered in rat liver (> 90%) by 1 h post-injection and remained in liver with half-lives of 2 and 6 days, respectively, for the DLT and InTn labels. Whole body recoveries of radioactivity from DLT- and InTn labels. Whole body recoveries of radioactivity from DLT- and InTn-labeled RSA were 5 and 6.5 days, respectively, again indicating that the larger glycoconjugate label residualized more efficiently in cells following protein degradation. (Lactitol)/sub 2/-N-CH/sub 2/-CH/sub 2/-NH-fluroescein (DLF) was also coupled to ASF by reductive amination and recovered quantitatively in liver at 1 h post-injection. Native ASF was an effective competitor for clearance of DLF-ASF from the circulation. Fluorescent degradation products were retained in liver with a half-life of 1.2 days. Residualizing fluorescent labels should be useful for identification and sorting of cells active in the degradation of plasma proteins.

  1. Rapid changes in plasma membrane protein phosphorylation during initiation of cell wall digestion

    SciTech Connect

    Blowers, D.P.; Boss, W.F.; Trewavas, A.J. )

    1988-02-01

    Plasma membrane vesicles from wild carrot cells grown in suspension culture were isolated by aqueous two-phase partitioning, and ATP-dependent phosphorylation was measured with ({gamma}-{sup 32}P)ATP in the presence and absence of calcium. Treatment of the carrot cells with the cell wall digestion enzymes, driselase, in a sorbitol osmoticum for 1.5 min altered the protein phosphorylation pattern compared to that of cells treated with sorbitol alone. Driselase treatment resulted in decreased phosphorylation of a band of M{sub r} 80,000 which showed almost complete calcium dependence in the osmoticum treated cells; decreased phosphorylation of a band of M{sub r} 15,000 which showed little calcium activation, and appearance of a new band of calcium-dependent phosphorylation at M{sub r} 22,000. However, protein phosphorylation was decreased. Adding driselase to the in vitro reaction mixture caused a general decrease in the membrane protein phosphorylation either in the presence or absence of calcium which did not mimic the in vivo response. Cells labeled in vivo with inorganic {sup 32}P also showed a response to the Driselase treatment. An enzymically active driselas preparation was required for the observed responses.

  2. Purification and identification of the fusicoccin binding protein from oat root plasma membrane

    NASA Technical Reports Server (NTRS)

    de Boer, A. H.; Watson, B. A.; Cleland, R. E.

    1989-01-01

    Fusicoccin (FC), a fungal phytotoxin, stimulates the H(+) -ATPase located in the plasma membrane (PM) of higher plants. The first event in the reaction chain leading to enhanced H(+) -efflux seems to be the binding of FC to a FC-binding protein (FCBP) in the PM. We solubilized 90% of the FCBP from oat (Avena sativa L. cv Victory) root PM in an active form with 1% octyl-glucoside. The FCBP was stabilized by the presence of protease inhibitors. The FCBP was purified by affinity chromatography using FC-linked adipic acid dihydrazide agarose (FC-AADA). Upon elution with 8 molar urea, two major protein bands on sodium dodecyl sulfate-polyaerylamide gel electrophoresis with molecular weights of 29,700 and 31,000 were obtained. Successive chromatography on BBAB Bio-Gel A, hexyl agarose, and FC-AADA resulted in the same two bands when the FC-AADA was eluted with sodium dodecyl sulfate. A direct correlation was made between 3H-FC-binding activity and the presence of the two protein bands. The stoichiometry of the 29,700 and 31,000 molecular weight bands was 1:2. This suggests that the FCBP occurs in the native form as a heterotrimer with an apparent molecular weight of approximately 92,000.

  3. Myelin Basic Protein Induces Neuron-Specific Toxicity by Directly Damaging the Neuronal Plasma Membrane

    PubMed Central

    Zheng, Sixin; Liu, Xiao; Jin, Jinghua; Ren, Yi; Luo, Jianhong

    2014-01-01

    The central nervous system (CNS) insults may cause massive demyelination and lead to the release of myelin-associated proteins including its major component myelin basic protein (MBP). MBP is reported to induce glial activation but its effect on neurons is still little known. Here we found that MBP specifically bound to the extracellular surface of the neuronal plasma membrane and induced neurotoxicity in vitro. This effect of MBP on neurons was basicity-dependent because the binding was blocked by acidic lipids and competed by other basic proteins. Further studies revealed that MBP induced damage to neuronal membrane integrity and function by depolarizing the resting membrane potential, increasing the permeability to cations and other molecules, and decreasing the membrane fluidity. At last, artificial liposome vesicle assay showed that MBP directly disturbed acidic lipid bilayer and resulted in increased membrane permeability. These results revealed that MBP induces neurotoxicity through its direct interaction with acidic components on the extracellular surface of neuronal membrane, which may suggest a possible contribution of MBP to the pathogenesis in the CNS disorders with myelin damage. PMID:25255088

  4. Purification and identification of the fusicoccin binding protein from oat root plasma membrane.

    PubMed

    de Boer, A H; Watson, B A; Cleland, R E

    1989-01-01

    Fusicoccin (FC), a fungal phytotoxin, stimulates the H(+) -ATPase located in the plasma membrane (PM) of higher plants. The first event in the reaction chain leading to enhanced H(+) -efflux seems to be the binding of FC to a FC-binding protein (FCBP) in the PM. We solubilized 90% of the FCBP from oat (Avena sativa L. cv Victory) root PM in an active form with 1% octyl-glucoside. The FCBP was stabilized by the presence of protease inhibitors. The FCBP was purified by affinity chromatography using FC-linked adipic acid dihydrazide agarose (FC-AADA). Upon elution with 8 molar urea, two major protein bands on sodium dodecyl sulfate-polyaerylamide gel electrophoresis with molecular weights of 29,700 and 31,000 were obtained. Successive chromatography on BBAB Bio-Gel A, hexyl agarose, and FC-AADA resulted in the same two bands when the FC-AADA was eluted with sodium dodecyl sulfate. A direct correlation was made between 3H-FC-binding activity and the presence of the two protein bands. The stoichiometry of the 29,700 and 31,000 molecular weight bands was 1:2. This suggests that the FCBP occurs in the native form as a heterotrimer with an apparent molecular weight of approximately 92,000. PMID:11537448

  5. Aggregation analysis of Con A binding proteins of human seminal plasma: a dynamic light scattering study.

    PubMed

    Tomar, Anil Kumar; Sooch, Balwinder Singh; Singh, Sarman; Yadav, Savita

    2013-02-01

    Concanavalin A (Con A) binding fraction of human seminal plasma is vital as it shows decapacitating activity and contains proteins which have critical roles in fertility related processes. Con A binding proteins were isolated by lectin affinity chromatography. These proteins form high molecular weight aggregates at near physiological pH (7.0) as inferred by gel filtration. Aggregation analysis was performed by dynamic light scattering (DLS). DLS analysis was also performed at different pH values and in presence of various additives including NaCl, EDTA, cholesterol and sugars, such as d-glucose, d-fructose and d-mannose to identify their effect on aggregation size. The results indicate that degree of aggregation was highly reduced in presence of d-fructose, EDTA and at lower and higher pH values as depicted by lowering of hydrodynamic radii. This aggregation behaviour might be decisive for fertility related events with a suggestive role towards inhibition of premature capacitation.

  6. Mutational scanning reveals the determinants of protein insertion and association energetics in the plasma membrane

    PubMed Central

    Elazar, Assaf; Weinstein, Jonathan; Biran, Ido; Fridman, Yearit; Bibi, Eitan; Fleishman, Sarel Jacob

    2016-01-01

    Insertion of helix-forming segments into the membrane and their association determines the structure, function, and expression levels of all plasma membrane proteins. However, systematic and reliable quantification of membrane-protein energetics has been challenging. We developed a deep mutational scanning method to monitor the effects of hundreds of point mutations on helix insertion and self-association within the bacterial inner membrane. The assay quantifies insertion energetics for all natural amino acids at 27 positions across the membrane, revealing that the hydrophobicity of biological membranes is significantly higher than appreciated. We further quantitate the contributions to membrane-protein insertion from positively charged residues at the cytoplasm-membrane interface and reveal large and unanticipated differences among these residues. Finally, we derive comprehensive mutational landscapes in the membrane domains of Glycophorin A and the ErbB2 oncogene, and find that insertion and self-association are strongly coupled in receptor homodimers. DOI: http://dx.doi.org/10.7554/eLife.12125.001 PMID:26824389

  7. Plasma membrane protein polarity and trafficking in RPE cells: Past, present and future

    PubMed Central

    Lehmann, Guillermo L.; Benedicto, Ignacio; Philp, Nancy J.; Rodriguez-Boulan, Enrique

    2015-01-01

    The retinal pigment epithelium (RPE) comprises a monolayer of polarized pigmented epithelial cells that is strategically interposed between the neural retina and the fenestrated choroid capillaries. The RPE performs a variety of vectorial transport functions (water, ions, metabolites, nutrients and waste products) that regulate the composition of the subretinal space and support the functions of photoreceptors (PRs) and other cells in the neural retina. To this end, RPE cells display a polarized distribution of channels, transporters and receptors in their plasma membrane (PM) that is remarkably different from that found in conventional extra-ocular epithelia, e.g. intestine, kidney, and gall bladder. This characteristic PM protein polarity of RPE cells depends on the interplay of sorting signals in the RPE PM proteins and sorting mechanisms and biosynthetic/recycling trafficking routes in the RPE cell. Although considerable progress has been made in our understanding of the RPE trafficking machinery, most available data have been obtained from immortalized RPE cell lines that only partially maintain the RPE phenotype and by extrapolation of data obtained in the prototype Madin–Darby Canine Kidney (MDCK) cell line. The increasing availability of RPE cell cultures that more closely resemble the RPE in vivo together with the advent of advanced live imaging microscopy techniques provides a platform and an opportunity to rapidly expand our understanding of how polarized protein trafficking contributes to RPE PM polarity. PMID:25152359

  8. Identification of cDNA clones encoding valosin-containing protein and other plant plasma membrane-associated proteins by a general immunoscreening strategy.

    PubMed Central

    Shi, J; Dixon, R A; Gonzales, R A; Kjellbom, P; Bhattacharyya, M K

    1995-01-01

    An approach was developed for the isolation and characterization of soybean plasma membrane-associated proteins by immunoscreening of a cDNA expression library. An antiserum was raised against purified plasma membrane vesicles. In a differential screening of approximately 500,000 plaque-forming units with the anti-(plasma membrane) serum and DNA probes derived from highly abundant clones isolated in a preliminary screening, 261 clones were selected from approximately 1,200 antiserum-positive plaques. These clones were classified into 40 groups by hybridization analysis and 5'- and 3'-terminal sequencing. By searching nucleic acid and protein sequence data bases, 11 groups of cDNAs were identified, among which valosin-containing protein (VCP), clathrin heavy chain, phospholipase C, and S-adenosylmethionine:delta 24-sterol-C-methyltransferase have not to date been cloned from plants. The remaining 29 groups did not match any current data base entries and may, therefore, represent additional or yet uncharacterized genes. A full-length cDNA encoding the soybean VCP was sequenced. The high level of amino acid identity with vertebrate VCP and yeast CDC48 protein indicates that the soybean protein is a plant homolog of vertebrate VCP and yeast CDC48 protein. Images Fig. 1 Fig. 2 PMID:7753826

  9. A Common Missense Variant in the Glucokinase Regulatory Protein Gene (GCKR) Is Associated with Increased Plasma Triglyceride and C-Reactive Protein but Lower Fasting Glucose Concentrations

    Technology Transfer Automated Retrieval System (TEKTRAN)

    OBJECTIVE-Using the genome-wide-association approach, we recently identified the glucokinase regulatory protein gene (GCKR, rs780094) region as a novel quantitative trait locus for plasma triglyceride concentration in Europeans. Here, we sought to study the association of GCKR variants with metaboli...

  10. Supplementation of Pork Patties with Bovine Plasma Protein Hydrolysates Augments Antioxidant Properties and Improves Quality

    PubMed Central

    Seo, Hyun-Woo

    2016-01-01

    This study investigated the effects of bovine plasma protein (PP) hydrolysates on the antioxidant and quality properties of pork patties during storage. Pork patties were divided into 4 groups: without butylated hydroxytoluene (BHT) and PP hydrolysates (control), 0.02% BHT (T1), 1% PP hydrolysates (T2), and 2% PP hydrolysates (T3). Pork patty supplemented with PP hydrolysates had higher pH values and lower weight loss during cooking than the control patties. Results showed that lightness and hardness both decreased upon the addition of PP hydrolysates. All samples containing BHT and PP hydrolysates had reduced TBARS and peroxide values during storage. In particular, 2% PP hydrolysates were more effective in delaying lipid oxidation than were the other treatments. It was concluded that treatment with 2% PP hydrolysates can enhance the acceptance of pork patty. PMID:27194928

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

    PubMed

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

    1988-05-01

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

  12. Intrathecal administration of substance P enhances cutaneous plasma protein extravasation in pentobarbital ana