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Sample records for human plasma protein

  1. Radioimmunoassay of human plasma protein C

    International Nuclear Information System (INIS)

    Wang Bocheng; Li Jinquan; Jing Jian; Zhang Manda

    1995-01-01

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

  2. Informing the Human Plasma Protein Binding of ...

    Science.gov (United States)

    The free fraction of a xenobiotic in plasma (Fub) is an important determinant of chemical adsorption, distribution, metabolism, elimination, and toxicity, yet experimental plasma protein binding data is scarce for environmentally relevant chemicals. The presented work explores the merit of utilizing available pharmaceutical data to predict Fub for environmentally relevant chemicals via machine learning techniques. Quantitative structure-activity relationship (QSAR) models were constructed with k nearest neighbors (kNN), support vector machines (SVM), and random forest (RF) machine learning algorithms from a training set of 1045 pharmaceuticals. The models were then evaluated with independent test sets of pharmaceuticals (200 compounds) and environmentally relevant ToxCast chemicals (406 total, in two groups of 238 and 168 compounds). The selection of a minimal feature set of 10-15 2D molecular descriptors allowed for both informative feature interpretation and practical applicability domain assessment via a bounded box of descriptor ranges and principal component analysis. The diverse pharmaceutical and environmental chemical sets exhibit similarities in terms of chemical space (99-82% overlap), as well as comparable bias and variance in constructed learning curves. All the models exhibit significant predictability with mean absolute errors (MAE) in the range of 0.10-0.18 Fub. The models performed best for highly bound chemicals (MAE 0.07-0.12), neutrals (MAE 0

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

    International Nuclear Information System (INIS)

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

    1998-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1981-08-27

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

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

    International Nuclear Information System (INIS)

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

    1981-01-01

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

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

    International Nuclear Information System (INIS)

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

    1988-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Shen Changyu

    2008-04-01

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

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

    International Nuclear Information System (INIS)

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

    2009-01-01

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

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

    NARCIS (Netherlands)

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

    1953-01-01

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

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

    DEFF Research Database (Denmark)

    Havelund, Jesper F; Wojdyla, Katarzyna; Davies, Michael J

    2017-01-01

    Protein carbonylation is an irreversible protein oxidation correlated with oxidative stress, various diseases and ageing. Here we describe a peptide-centric approach for identification and characterisation of up to 14 different types of carbonylated amino acids in proteins. The modified residues...... in vitro metal ion-catalysed oxidation. Furthermore, we assigned 133 carbonylated sites in 36 proteins in native human plasma protein samples. The optimised workflow enabled detection of 10 hitherto undetected types of carbonylated amino acids in proteins: aldehyde and ketone modifications of leucine...

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

    DEFF Research Database (Denmark)

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

    2008-01-01

    BACKGROUND: The immense diagnostic potential of human plasma has prompted great interest and effort in cataloging its contents, exemplified by the Human Proteome Organization (HUPO) Plasma Proteome Project (PPP) pilot project. Due to challenges in obtaining a reliable blood plasma protein list......-trap-Fourier transform (LTQ-FT) and a linear ion trap-Orbitrap (LTQ-Orbitrap) for mass spectrometry (MS) analysis. Both instruments allow the measurement of peptide masses in the low ppm range. Furthermore, we employed a statistical score that allows database peptide identification searching using the products of two...... consecutive stages of tandem mass spectrometry (MS3). The combination of MS3 with very high mass accuracy in the parent peptide allows peptide identification with orders of magnitude more confidence than that typically achieved. RESULTS: Herein we established a high confidence set of 697 blood plasma proteins...

  12. Enhanced Detection of Human Plasma Proteins on Nanostructured Silver Surfaces

    Directory of Open Access Journals (Sweden)

    Zuzana Orságová Králová

    2013-08-01

    enhancement factor of 3.6×102 was achieved for a band with a Raman shift of 2104cm‐1 for globulin deposited onto silver nanostructured film on unpolished stainless steel substrate. The detection limit was 400g/mL. Plasma or serum could present a preferable material for non‐ invasive cancer disease diagnosis using the SERS method.

  13. Differential proteomics of human seminal plasma: A potential target for searching male infertility marker proteins.

    Science.gov (United States)

    Tomar, Anil Kumar; Sooch, Balwinder Singh; Singh, Sarman; Yadav, Savita

    2012-04-01

    The clinical fertility tests, available in the market, fail to define the exact cause of male infertility in almost half of the cases and point toward a crucial need of developing better ways of infertility investigations. The protein biomarkers may help us toward better understanding of unknown cases of male infertility that, in turn, can guide us to find better therapeutic solutions. Many clinical attempts have been made to identify biomarkers of male infertility in sperm proteome but only few studies have targeted seminal plasma. Human seminal plasma is a rich source of proteins that are essentially required for development of sperm and successful fertilization. This viewpoint article highlights the importance of human seminal plasma proteome in reproductive physiology and suggests that differential proteomics integrated with functional analysis may help us in searching potential biomarkers of male infertility. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

    International Nuclear Information System (INIS)

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

    2010-01-01

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

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

    DEFF Research Database (Denmark)

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

    2015-01-01

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

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

    International Nuclear Information System (INIS)

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

    1985-01-01

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

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

    Science.gov (United States)

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

    2012-05-01

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

  18. Lipid-protein interactions in plasma membranes of fiber cells isolated from the human eye lens.

    Science.gov (United States)

    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. Copyright © 2014 Elsevier Ltd. All rights reserved.

  19. An acid phosphatase in the plasma membranes of human astrocytoma showing marked specificity toward phosphotyrosine protein.

    Science.gov (United States)

    Leis, J F; Kaplan, N O

    1982-11-01

    The plasma membrane from the human tumor astrocytoma contains an active acid phosphatase activity based on hydrolysis of p-nitrophenyl phosphate. Other acid phosphatase substrates--beta-glycerophosphate, O-phosphorylcholine, and 5'-AMP--are not hydrolyzed significantly. The phosphatase activity is tartrate insensitive and is stimulated by Triton X-100 and EDTA. Of the three known phosphoamino acids, only free O-phosphotyrosine is hydrolyzed by the membrane phosphatase activity. Other acid phosphatases tested from potato, wheat germ, milk, and bovine prostate did not show this degree of specificity. The plasma membrane activity also dephosphorylated phosphotyrosine histone at a much greater rate than did the other acid phosphatases. pH profiles for free O-phosphotyrosine and phosphotyrosine histone showed a shift toward physiological pH, indicating possible physiological significance. Phosphotyrosine histone dephosphorylation activity was nearly 10 times greater than that seen for phosphoserine histone dephosphorylation, and Km values were much lower for phosphotyrosine histone dephosphorylation (0.5 microM vs. 10 microM). Fluoride and zinc significantly inhibited phosphoserine histone dephosphorylation. Vanadate, on the other hand, was a potent inhibitor of phosphotyrosine histone dephosphorylation (50% inhibition at 0.5 microM) but not of phosphoserine histone. ATP stimulated phosphotyrosine histone dephosphorylation (160-250%) but inhibited phosphoserine histone dephosphorylation (95%). These results suggest the existence of a highly specific phosphotyrosine protein phosphatase activity associated with the plasma membrane of human astrocytoma.

  20. Fractionation separation of human plasma proteins using HPLC with a homemade iron porphyrin based monolithic column.

    Science.gov (United States)

    Zhang, Doudou; Zhao, Yu; Lan, Dandan; Pang, Xiaomin; Bai, Ligai; Liu, Haiyan; Yan, Hongyuan

    2017-11-15

    In this work a polymer monolithic column was fabricated within the confines of a stainless steel column (50×4.6mm i.d.) via radical polymerization by using iron porphyrin and butyl methacrylate as co-monomers, ethylene glycol dimethacrylate as crosslinking agent, ethylene glycol, isopropyl alcohol and N, N-dimethylformamide as tri-porogens, benzoyl peroxide and N,N-dimethylaniline as initiators. The resulting monolithic column was characterized by elemental analysis, scanning electron microscopy, nitrogen adsorption BET surface area, and mercury intrusion porosimetry, respectively. Results showed that the homemade monolith occupied relatively uniform pore structure, low back pressure, and enhanced selectivity for proteins in complex bio-samples. The present work described a simple and efficient method for "fractionation separation" of human plasma proteins, and it is a promising separation method for complex bio-samples in proteomic research. Copyright © 2017 Elsevier B.V. All rights reserved.

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

    International Nuclear Information System (INIS)

    Brassinne, A.

    1979-01-01

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

  2. A two-site immunoradiometric assay for human pregnancy-associated plasma protein A (PAPP-A) using monoclonal antibodies

    International Nuclear Information System (INIS)

    Mowles, E.A.; Pinto-Furtado, L.G.; Bolton, A.E.

    1986-01-01

    A rapid, sensitive immunoradiometric assay has been developed for human pregnancy-associated plasma protein A (PAPP-A) using a purified mouse monoclonal antibody as the tracer and a rabbit polyclonal antibody to this protein in the solid-phase antibody preparation. The assay showed no measurable cross-reaction (< 0.1%) against a range of purified human placental proteins, and a good correlation with a previously described radioimmunoassay procedure when tested on samples taken throughout normal human pregnancies. No PAPP-A-like immunological activity could be detected in sera from non-pregnant women, confirming the absence of this protein from the circulation outside pregnancy. (Auth.)

  3. Mesoporous silica particles modified with graphitic carbon: interaction with human red blood cells and plasma proteins

    Energy Technology Data Exchange (ETDEWEB)

    Martinez, Diego Stefani Teodoro; Franqui, Lidiane Silva; Bettini, Jefferson; Strauss, Mathias, E-mail: diego.martinez@lnnano.cnpem.br [Centro Nacional de Pesquisa em Energia e Materiais (CNPEM), Campinas, SP (Brazil); Damasceno, Joao Paulo Vita; Mazali, Italo Odone [Universidade Estadual de Campinas (UNICAMP), SP (Brazil)

    2016-07-01

    Full text: In this work the interaction of the mesoporous silica particles (SBA-15, ∼700 nm) modified with graphitic carbon (SBA-15/C) on human red blood cells (hemolysis) and plasma proteins (protein corona formation) is studied. XPS and CHN analysis showed that the carbon content on the SBA-15/C samples varied from 2 to 10% and was tuned by the functionalization step. The formed carbon structures where associated to graphitic nanodomains coating the pores surface as verified by Raman spectroscopy and {sup 13}C NMR. Advanced TEM/EELS analysis showed that the carbon structures are distributed along the SBA-15 mesopores. SAXS and textural analyses were used to confirm that the porous structure of the silica support is kept after the modification procedure and to calculate the number of graphitic carbon stacked layers coating the mesopores. After incubation of SBA-15 with human red blood cells (RBCs), it was observed a dose-dependent hemolytic effect, probably, due to binding of the material silanol-rich surface to the phosphatidylcholine molecules from the RBC membrane. The graphitic carbon modifications have mitigated this effect, indicating that the graphitic carbon coating protected the silanol groups of the particle surface hindering the hemolysis. Considering the protein corona formation, selective biomolecular interaction of proteins was observed for the different materials using gel electrophoresis (SDS-PAGE) analysis. Besides, graphitic carbon modification decreased the amount of proteins on the corona. Together, the in vitro hemolysis and protein corona assays are promising biological models to understand the influence of silica surface functionalization on their bionano-interactions. Finally, our work contributes to the development of fundamental research on such nanomaterials chemistry in the emerging field of nanobioscience and nanotoxicology. (author)

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

    Science.gov (United States)

    Luo, Shen; Zhang, Baolin

    2015-01-01

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

  5. Human plasma phospholipid transfer protein increases the antiatherogenic potential of high density lipoproteins in transgenic mice

    NARCIS (Netherlands)

    M.J. van Haperen (Rien); A. van Tol (Arie); P. Vermeulen; M. Jauhiainen; T. van Gent (Teus); P.M. van den Berg (Paul); S. Ehnholm (Sonja); A.W.M. van der Kamp (Arthur); M.P.G. de Crom (Rini); F.G. Grosveld (Frank)

    2000-01-01

    textabstractPlasma phospholipid transfer protein (PLTP) transfers phospholipids between lipoprotein particles and alters high density lipoprotein (HDL) subfraction patterns in vitro, but its physiological function is poorly understood. Transgenic mice that overexpress

  6. Insulin sensitivity is independent of lipid binding protein trafficking at the plasma membrane in human skeletal muscle

    DEFF Research Database (Denmark)

    Jordy, Andreas Børsting; Serup, Annette Karen; Karstoft, Kristian

    2014-01-01

    The aim of the present study was to investigate lipid-induced regulation of lipid binding proteins in human skeletal muscle and the impact hereof on insulin sensitivity. Eleven healthy male subjects underwent a 3-day hyper-caloric and high-fat diet regime. Muscle biopsies were taken before......-regulated by increased fatty acid availability. This suggests a time dependency in the up-regulation of FAT/CD36 and FABPpm protein during high availability of plasma fatty acids. Furthermore, we did not detect FATP1 and FATP4 protein in giant sarcolemmal vesicles obtained from human skeletal muscle. In conclusion......, this study shows that a short-term lipid-load increases mRNA content of key lipid handling proteins in human muscle. However, decreased insulin sensitivity after high-fat diet is not accompanied with relocation of FAT/CD36 or FABPpm protein to the sarcolemma. Finally, FATP1 and FATP4 protein could...

  7. Phospholipid transfer from vesicles to high density lipoproteins, catalyzed by human plasma phospholipid transfer protein

    International Nuclear Information System (INIS)

    Sweeny, S.A.

    1985-01-01

    Human plasma phospholipid transfer protein (PLTP) catalyzes the mass transfer of phosphatidylcholine (PC). Partial purification of PLTP yielded proteins with apparent M/sub r/ = 59,000 and 40,000 by SDS-PAGE. PLTP activity was measured by transfer of [ 14 C]L-α-dipalmitoyl PC from egg-PC vesicles to HDL. Activity was enhanced at low pH (4.5) upon addition of β-mercaptoethanol while Ca +2 and Na + had no effect. E/sub act/ for facilitated PC transfer was 18.2 +/- 2 kcal/mol. The donor specificity of PLTP was examined using vesicles containing egg-PC plus cholesterol or sphingomyelin. The fluidity of the donor membrane (measured by fluorescence polarization of diphenylhexatriene) correlated strongly with a decrease in PLTP activity. Phosphatidic acid did not affect activity. Increase in vesicle size reduced activity. The acceptor specificity of PLTP was examined using chemically modified HDL. PLTP activity increased up to 1.7-fold with an initial increase in negative charge and then decreased upon extensive modification. A mechanism is proposed where PLTP binds to vesicls and enhances the diffusion of PC into the medium where it is adsorbed by HDL

  8. Pregnancy-associated plasma protein A in human ovarian follicles and its association with intrafollicular hormone levels

    DEFF Research Database (Denmark)

    Bøtkjær, Jane Alrø; Jeppesen, Janni Vikkelsø; Wissing, Marie Louise

    2015-01-01

    To evaluate follicular fluid (FF) levels of pregnancy-associated plasma protein A (PAPP-A) in relation to levels of intrafollicular hormones. Furthermore, immunostaining of human follicles of varying diameters was studied for PAPP-A, antimüllerian hormone (AMH), and aromatase, and the biological...... activity of PAPP-A in FF was evaluated....

  9. The human multidrug resistance-associated protein MRP is a plasma membrane drug-efflux pump

    NARCIS (Netherlands)

    Zaman, G. J.; Flens, M. J.; van Leusden, M. R.; de Haas, M.; Mülder, H. S.; Lankelma, J.; Pinedo, H. M.; Scheper, R. J.; Baas, F.; Broxterman, H. J.

    1994-01-01

    The multidrug-resistance associated protein MRP is a 180- to 195-kDa membrane protein associated with resistance of human tumor cells to cytotoxic drugs. We have investigated how MRP confers drug resistance in SW-1573 human lung carcinoma cells by generating a subline stably transfected with an

  10. Delineation of concentration ranges and longitudinal changes of human plasma protein variants.

    Directory of Open Access Journals (Sweden)

    Olgica Trenchevska

    Full Text Available Human protein diversity arises as a result of alternative splicing, single nucleotide polymorphisms (SNPs and posttranslational modifications. Because of these processes, each protein can exists as multiple variants in vivo. Tailored strategies are needed to study these protein variants and understand their role in health and disease. In this work we utilized quantitative mass spectrometric immunoassays to determine the protein variants concentration of beta-2-microglobulin, cystatin C, retinol binding protein, and transthyretin, in a population of 500 healthy individuals. Additionally, we determined the longitudinal concentration changes for the protein variants from four individuals over a 6 month period. Along with the native forms of the four proteins, 13 posttranslationally modified variants and 7 SNP-derived variants were detected and their concentration determined. Correlations of the variants concentration with geographical origin, gender, and age of the individuals were also examined. This work represents an important step toward building a catalog of protein variants concentrations and examining their longitudinal changes.

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

    Science.gov (United States)

    Staempfli, Henry R; Constable, Peter D

    2003-08-01

    The mechanism for an acid-base disturbance can be determined by using the strong ion approach, which requires species-specific values for the total concentration of plasma nonvolatile buffers (Atot) and the effective dissociation constant for plasma weak acids (Ka). The aim of this study was to experimentally determine Atot and Ka values for human plasma by using in vitro CO2 tonometry. Plasma Pco2 was systematically varied from 25 to 145 Torr at 37 degrees C, thereby altering plasma pH over the physiological range of 6.90-7.55, and plasma pH, Pco2, and concentrations of quantitatively important strong ions (Na+, K+, Ca2+, Mg2+, Cl-, lactate) and buffer ions (total protein, albumin, phosphate) were measured. Strong ion difference was estimated, and nonlinear regression was used to calculate Atot and Ka from the measured pH and Pco2 and estimated strong ion difference; the Atot and Ka values were then validated by using a published data set (Figge J, Rossing TH, and Fencl V, J Lab Clin Med 117: 453-467, 1991). The values (mean +/- SD) were as follows: Atot = 17.2 +/- 3.5 mmol/l (equivalent to 0.224 mmol/g of protein or 0.378 mmol/g of albumin); Ka = 0.80 +/- 0.60 x 10-7; negative log of Ka = 7.10. Mean estimates were obtained for strong ion difference (37 meq/l) and net protein charge (13+.0 meq/l). The experimentally determined values for Atot, Ka, and net protein charge should facilitate the diagnosis and treatment of acid-base disturbances in critically ill humans.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2017-08-11

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

  13. Metastasis-related plasma membrane proteins of human breast cancer cells identified by comparative quantitative mass spectrometry

    DEFF Research Database (Denmark)

    Leth-Larsen, Rikke; Lund, Rikke; Hansen, Helle V

    2009-01-01

    The spread of cancer cells from a primary tumor to form metastasis at distant sites is a complex multi-step process. The cancer cell proteins, and plasma membrane proteins in particular, involved in this process are poorly defined and a study of the very early events of the metastatic process using...... clinical samples or in vitro assays is not feasible. We have used a unique model system consisting of two isogenic human breast cancer cell lines that are equally tumorigenic in mice, but while one gives rise to metastasis, the other disseminates single cells that remain dormant at distant organs. Membrane...... purification and comparative quantitative LC-MS/MS proteomic analysis identified 13 membrane proteins that were expressed at higher levels and 3 that were under-expressed in the metastatic compared to the non-metastatic cell line from a total of 1919 identified protein entries. Among the proteins were ecto-5...

  14. A practical guide for the identification of membrane and plasma membrane proteins in human embryonic stem cells and human embryonal carcinoma cells.

    NARCIS (Netherlands)

    Dormeyer, W.; van Hoof, D.; Mummery, C.L.; Krijgsveld, J.; Heck, A.

    2008-01-01

    The identification of (plasma) membrane proteins in cells can provide valuable insights into the regulation of their biological processes. Pluripotent cells such as human embryonic stem cells and embryonal carcinoma cells are capable of unlimited self-renewal and share many of the biological

  15. An acid phosphatase in the plasma membranes of human astrocytoma showing marked specificity toward phosphotyrosine protein.

    OpenAIRE

    Leis, J F; Kaplan, N O

    1982-01-01

    The plasma membrane from the human tumor astrocytoma contains an active acid phosphatase activity based on hydrolysis of p-nitrophenyl phosphate. Other acid phosphatase substrates--beta-glycerophosphate, O-phosphorylcholine, and 5'-AMP--are not hydrolyzed significantly. The phosphatase activity is tartrate insensitive and is stimulated by Triton X-100 and EDTA. Of the three known phosphoamino acids, only free O-phosphotyrosine is hydrolyzed by the membrane phosphatase activity. Other acid pho...

  16. A practical guide for the identification of membrane and plasma membrane proteins in human embryonic stem cells and human embryonal carcinoma cells.

    Science.gov (United States)

    Dormeyer, Wilma; van Hoof, Dennis; Mummery, Christine L; Krijgsveld, Jeroen; Heck, Albert J R

    2008-10-01

    The identification of (plasma) membrane proteins in cells can provide valuable insights into the regulation of their biological processes. Pluripotent cells such as human embryonic stem cells and embryonal carcinoma cells are capable of unlimited self-renewal and share many of the biological mechanisms that regulate proliferation and differentiation. The comparison of their membrane proteomes will help unravel the biological principles of pluripotency, and the identification of biomarker proteins in their plasma membranes is considered a crucial step to fully exploit pluripotent cells for therapeutic purposes. For these tasks, membrane proteomics is the method of choice, but as indicated by the scarce identification of membrane and plasma membrane proteins in global proteomic surveys it is not an easy task. In this minireview, we first describe the general challenges of membrane proteomics. We then review current sample preparation steps and discuss protocols that we found particularly beneficial for the identification of large numbers of (plasma) membrane proteins in human tumour- and embryo-derived stem cells. Our optimized assembled protocol led to the identification of a large number of membrane proteins. However, as the composition of cells and membranes is highly variable we still recommend adapting the sample preparation protocol for each individual system.

  17. Scavenger receptor-mediated recognition of maleyl bovine plasma albumin and the demaleylated protein in human monocyte macrophages

    International Nuclear Information System (INIS)

    Haberland, M.E.; Fogelman, A.M.

    1985-01-01

    Maleyl bovine plasma albumin competed on an equimolar basis with malondialdehyde low density lipoprotein (LDL) in suppressing the lysosomal hydrolysis of 125 I-labeled malondialdehyde LDL mediated by the scavenger receptor of human monocyte macrophages. Maleyl bovine plasma albumin, in which 94% of the amino groups were modified, exhibited an anodic mobility in agarose electrophoresis 1.7 times that of the native protein. Incubation of maleyl bovine plasma albumin at pH 3.5 regenerated the free amino groups and restored the protein to the same electrophoretic mobility as native albumin. Although ligands recognized by the scavenger receptor typically are anionic, the authors propose that addition of new negative charge achieved by maleylation, rather than directly forming the receptor binding site(s), induces conformational changes in albumin as a prerequisite to expression of the recognition domain(s). They conclude that the primary sequence of albumin, rather than addition of new negative charge, provides the recognition determinant(s) essential for interaction of maleyl bovine plasma albumin with the scavenger receptor

  18. Study on the interaction of plasma protein binding rate between edaravone and taurine in human plasma based on HPLC analysis coupled with ultrafiltration technique.

    Science.gov (United States)

    Tang, Dao-quan; Li, Yin-jie; Li, Zheng; Bian, Ting-ting; Chen, Kai; Zheng, Xiao-xiao; Yu, Yan-yan; Jiang, Shui-shi

    2015-08-01

    In this work, two high-performance liquid chromatography (HPLC) assays were developed and validated for the independent determination of edaravone and taurine using 3-methyl-1-p-tolyl-5-pyrazolone and L-glutamine as internal standards. In in vitro experiments, human plasma was separately spiked with a mixture of edaravone and taurine, edaravone or taurine alone. Plasma was precipitated with acetonitrile containing 0.1% formic acid. Ultrafiltration was employed to obtain the unbound ingredients of the two drugs. The factors that might influence the ultrafiltration effiency were elaborately optimized. Plasma supernatant and ultrafiltrate containing taurine were derivated with o-phthalaldehyde and ethanethiol in the presence of 40 mmol/L sodium borate buffer (pH 10.2) at room temperature within 1 min. Chromatographic separations were achieved on an InertSustain C18 column (250 × 4.6 mm, 5 µm). Isocratic 50 mmol/L ammonium acetate-acetonitrile and gradient 50 mmol/L sodium acetate (pH 5.3)-methanol were respectively selected as the mobile phase for the determination of edaravone and taurine. All of the validation data including linearity, extraction recovery, precision, accuracy and stability conformed to the requirements. Results showed that there were no significant alterations in the plasma protein binding rate of taurine and edaravone, implying that the proposed combination therapy was pharmacologically feasible. Copyright © 2014 John Wiley & Sons, Ltd.

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

    Science.gov (United States)

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

    2014-11-01

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

  20. Proteins at interfaces : the adsorption of human plasma albumin and bovine pancreas ribonuclease on polystyrene latices

    NARCIS (Netherlands)

    Norde, W.

    1976-01-01

    The adsorption from (aqueous) solution of proteins is very complex. The interfacial behaviour of proteins is determined by the properties of, and the mutual interactions between, the adsorbing interface, the protein molecules, the solvent (water) molecules and other solutes (e.g. ions).

  1. Protein C inhibitor acts as a procoagulant by inhibiting the thrombomodulin-induced activation of protein C in human plasma

    NARCIS (Netherlands)

    Elisen, M. G.; von dem Borne, P. A.; Bouma, B. N.; Meijers, J. C.

    1998-01-01

    Protein C inhibitor (PCI), which was originally identified as an inhibitor of activated protein C, also efficiently inhibits coagulation factors such as factor Xa and thrombin. Recently it was found, using purified proteins, that the anticoagulant thrombin-thrombomodulin complex was also inhibited

  2. First trimester pregnancy-associated plasma protein A and human chorionic gonadotropin-beta in early and late pre-eclampsia

    DEFF Research Database (Denmark)

    Karahasanovic, Azra; Sørensen, Steen; Nilas, Lisbeth

    2014-01-01

    Abstract Background: The aim of this study was to compare pregnancy-associated plasma protein A (PAPP-A) and the β-subunit of human chorionic gonadotropin (hCGβ) measured in maternal plasma at the first trimester screening, in women who later developed early or late pre-eclampsia (PE) to women...... with normal pregnancies. Methods: In the study were included 161 cases with PE and 88 controls with uncomplicated pregnancies. Plasma PAPP-A and hCGβ were measured between gestational age (GA) 8+2 and 14+0, and cases with early and late onset PE were compared with controls. Results: Median of hCGβ (Mo...

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

    African Journals Online (AJOL)

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

  4. Evaluation of metabolism, plasma protein binding and other biological parameters after administration of (−)-[18 F]Flubatine in humans

    International Nuclear Information System (INIS)

    Patt, Marianne; Becker, Georg A.; Grossmann, Udo; Habermann, Bernd; Schildan, Andreas; Wilke, Stephan; Deuther-Conrad, Winnie; Graef, Susanne; Fischer, Steffen; Smits, René; Hoepping, Alexander; Wagenknecht, Gudrun; Steinbach, Jörg; Gertz, Hermann-Josef; Hesse, Swen; Schönknecht, Peter

    2014-01-01

    Introduction: (−)-[ 18 F]Flubatine is a PET tracer with high affinity and selectivity for the nicotinic acetylcholine α 4 β 2 receptor subtype. A clinical trial assessing the availability of this subtype of nAChRs was performed. From a total participant number of 21 Alzheimer’s disease (AD) patients and 20 healthy controls (HCs), the following parameters were determined: plasma protein binding, metabolism and activity distribution between plasma and whole blood. Methods: Plasma protein binding and fraction of unchanged parent compound were assessed by ultracentrifugation and HPLC, respectively. The distribution of radioactivity (parent compound + metabolites) between plasma and whole blood was determined ex vivo at different time-points after injection by gamma counting after separation of whole blood by centrifugation into the cellular and non-cellular components. In additional experiments in vitro, tracer distribution between these blood components was assessed for up to 90 min. Results: A fraction of 15% ± 2% of (−)-[ 18 F]Flubatine was found to be bound to plasma proteins. Metabolic degradation of (−)-[ 18 F]Flubatine was very low, resulting in almost 90% unchanged parent compound at 90 min p.i. with no significant difference between AD and HC. The radioactivity distribution between plasma and whole blood changed in vivo only slightly over time from 0.82 ± 0.03 at 3 min p.i. to 0.87 ± 0.03 at 270 min p.i. indicating the contribution of only a small amount of metabolites. In vitro studies revealed that (−)-[ 18 F]Flubatine was instantaneously distributed between cellular and non-cellular blood parts. Discussion: (−)-[ 18 F]Flubatine exhibits very favourable characteristics for a PET radiotracer such as slow metabolic degradation and moderate plasma protein binding. Equilibrium of radioactivity distribution between plasma and whole blood is reached instantaneously and remains almost constant over time allowing both convenient sample handling and

  5. Application of pressure ultrafiltration in determining the binding capacity of drugs to human albumin and to plasma proteins of intact and irradiated rat females

    International Nuclear Information System (INIS)

    Zima, M.

    1976-01-01

    The significance of the binding of drugs to plasma proteins has repeatedly been demonstrated and draws the interest of many pharmacologists. The described experiments served to study the binding of isoniazid (INH) to human albumin of various dilution and to whole plasma proteins of irradiated (on the Oth, 3rd and 6th day after exposure to 154.8 mC/kg=600 R) and non-irradiated rats using the technique of modified accelerated ultrafiltration through cellophane. The total characteristics of the binding and its changes were demonstrated by the equilibrium constant, the numbers of binding sites and the changes of free binding energy. The results show that the dilution of human albumin affects the strength of the INH binding on this albumin and further that the normally weak INH binding is diminished even more in irradiated rats. This cannot be explained by the change in the percentage composition of the rat plasma. (author)

  6. New low-flux mixed matrix membranes that offer superior removal of protein-bound toxins from human plasma

    Science.gov (United States)

    Pavlenko, Denys; van Geffen, Esmée; van Steenbergen, Mies J.; Glorieux, Griet; Vanholder, Raymond; Gerritsen, Karin G. F.; Stamatialis, Dimitrios

    2016-10-01

    Hemodialysis is a widely available and well-established treatment for patients with End Stage Renal Disease (ESRD). However, although life-sustaining, patient mortality rates are very high. Several recent studies corroborated the link between dialysis patients’ outcomes and elevated levels of protein-bound uremic toxins (PBUT) that are poorly removed by conventional hemodialysis. Therefore, new treatments are needed to improve their removal. Recently, our group showed that the combination of dialysis and adsorption on one membrane, the mixed matrix membrane (MMM), can effectively remove those toxins from human plasma. However, these first MMMs were rather large in diameter and their mass transport characteristics needed improvement before application in the clinical setting. Therefore, in this study we developed a new generation of MMMs that have a smaller diameter and optimized characteristics offering superior ability in removing the PBUT indoxyl sulfate (IS) and p-cresyl sulfate (pCS) in comparison to first generation MMMs (30 and 125% respectively), as well as, a commercial dialysis membrane (more than 100% better removal).

  7. The proteolytic activity of pregnancy-associated plasma protein-A is potentially regulated by stanniocalcin-1 and -2 during human ovarian follicle development

    DEFF Research Database (Denmark)

    Jepsen, Malene R.; Kløverpris, Søren; Bøtkjær, Jane A.

    2016-01-01

    STUDY QUESTION: Is the proteolytic activity of pregnancy-associated plasma protein-A (PAPP-A) regulated by the stanniocalcins (STC1 and STC2) during human follicle maturation? SUMMARY ANSWER: The STCs and PAPP-A show similar expression by immunohistochemistry in developing follicles, and regulation...... of PAPP-A proteolytic activity is suggested by the identification of inhibited protein complexes between PAPP-A and STC1 or STC2 in human follicular fluid (FF). WHAT IS KNOWN ALREADY: The insulin-like growth factor (IGF)-regulating proteinase PAPP-A is secreted by the granulosa cells of estrogen...

  8. A rapid chemical method of labelling human plasma proteins with sup(99m)Tc-pertechnetate at pH 7.4

    International Nuclear Information System (INIS)

    Wong, D.W.; Mishkin, F.; Lee, T.

    1978-01-01

    A successful method for labelling human plasma proteins with sup(99m)Tc-pertechnetate by chemical means is described. The labelling methodology involves the production of Sup(99m)Tc-(Sn)citrate complex species with high protein binding capacity at pH 7.4 condition following initial chemical reduction of sodium sup(99m)Tc-pertechnetate by stannous chloride. A combined labelling efficiency range of 95-99% for sup(99m)Tc-labelled fibrinogen, immune gamma globulin and serum albumin is achieved. The actual amount of labelled protein content in the product is found to be 85-95% when assayed by ITLC and 74-85% by TCAA protein precipitation. In vitro experimental data indicate that sup(99m)Tc-fibrinogen contains an average of 85% clottable protein with an average clottability of 95%. This strongly suggests that the radioactive proteins retain much of their biological and physiological activities after the labelling process. (author)

  9. Cysteine-rich secretory protein 3 is a ligand of alpha1B-glycoprotein in human plasma

    DEFF Research Database (Denmark)

    Udby, Lene; Sørensen, Ole E; Pass, Jesper

    2004-01-01

    Human cysteine-rich secretory protein 3 (CRISP-3; also known as SGP28) belongs to a family of closely related proteins found in mammals and reptiles. Some mammalian CRISPs are known to be involved in the process of reproduction, whereas some of the CRISPs from reptiles are neurotoxin...

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

    Directory of Open Access Journals (Sweden)

    Vera Ignjatovic

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

  11. Insulin does not stimulate muscle protein synthesis during increased plasma branched-chain amino acids alone but still decreases whole body proteolysis in humans.

    Science.gov (United States)

    Everman, Sarah; Meyer, Christian; Tran, Lee; Hoffman, Nyssa; Carroll, Chad C; Dedmon, William L; Katsanos, Christos S

    2016-10-01

    Insulin stimulates muscle protein synthesis when the levels of total amino acids, or at least the essential amino acids, are at or above their postabsorptive concentrations. Among the essential amino acids, branched-chain amino acids (BCAA) have the primary role in stimulating muscle protein synthesis and are commonly sought alone to stimulate muscle protein synthesis in humans. Fourteen healthy young subjects were studied before and after insulin infusion to examine whether insulin stimulates muscle protein synthesis in relation to the availability of BCAA alone. One half of the subjects were studied in the presence of postabsorptive BCAA concentrations (control) and the other half in the presence of increased plasma BCAA (BCAA). Compared with that prior to the initiation of the insulin infusion, fractional synthesis rate of muscle protein (%/h) did not change (P > 0.05) during insulin in either the control (0.04 ± 0.01 vs 0.05 ± 0.01) or the BCAA (0.05 ± 0.02 vs. 0.05 ± 0.01) experiments. Insulin decreased (P BCAA (0.89 ± 0.07 vs 0.61 ± 0.03) experiments, but the change was not different between the two experiments (P > 0.05). In conclusion, insulin does not stimulate muscle protein synthesis in the presence of increased circulating levels of plasma BCAA alone. Insulin's suppressive effect on proteolysis is observed independently of the levels of circulating plasma BCAA. Copyright © 2016 the American Physiological Society.

  12. Nanocomposites based on graphene oxide and mesoporous silica nanoparticles: Preparation, characterization and nanobiointeractions with red blood cells and human plasma proteins

    Science.gov (United States)

    Fonseca, Leandro C.; de Araújo, Maciel M.; de Moraes, Ana Carolina M.; da Silva, Douglas S.; Ferreira, Ariane G.; Franqui, Lidiane S.; Martinez, Diego Stéfani T.; Alves, Oswaldo L.

    2018-04-01

    The current work refers to the development of a novel nanocomposite based on graphene oxide (GO) and mesoporous amino silica nanoparticles (H2N-MSNs) and its biological interaction with red blood cells (RBCs) and human blood plasma toward the investigation of nanobiointeractions. Silica nanoparticles and several graphene oxide-based materials are, separately, known for their high hemolytic potential and strong interaction with human plasma proteins. In this context, the GO-MSN interaction and its influence in minimizing the reported effects were investigated. The materials were synthesized by covalently attaching H2N-MSNs onto the surface of GO through an amidation reaction. GO-MSN nanocomposites were obtained by varying the mass of H2N-MSNs and were characterized by FTIR, NMR, XRD, TGA, zeta potential and TEM. The characterization results confirm that nanocomposites were obtained, suggest covalent bond attachment mostly by amine-epoxy reactions and evidence an unexpected reduction reaction of GO by H2N-MSNs, whose mechanism is proposed. Biological assays showed a decrease of hemolysis (RBC lysis) and a minimization of the interaction with human plasma proteins (protein corona formation). These are important findings toward achieving in vivo biocompatibility and understanding the nanobiointeractions. Finally, this work opens possibilities for new nanomedicine applications of GO-MSN nanocomposites, such as drug delivery system.

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

    Directory of Open Access Journals (Sweden)

    Seong-Beom Ahn

    2014-06-01

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

  14. Human conglutinin-like protein

    DEFF Research Database (Denmark)

    Jensenius, J C; Thiel, S; Baatrup, G

    1985-01-01

    The presence in human plasma of a molecule homologous to bovine conglutinin is indicated by the results of biological and immunochemical analysis. The human conglutinin-like protein shows calcium-dependent binding to complement-treated solid phase IgG and immunological cross-reaction with chicken...... anti-bovine conglutinin. The binding of the human protein to complement-treated IgG was inhibited by N-acetyl-D-glucosamine but not by other sugars. Analysis by SDS-PAGE and Western blotting showed reaction of anti-conglutinin with molecules of similar mobility to the monomer and hexamer of bovine...

  15. Zirconium silicate assisted removal of residual proteins after organic solvent deproteinization of human plasma, enhancing the stability of the LC–ESI-MS response for the bioanalysis of small molecules

    Energy Technology Data Exchange (ETDEWEB)

    Hussain, Shah; Pezzei, Cornelia [Institute of Analytical Chemistry and Radiochemistry, CCB-Center for Chemistry and Biomedicine, Leopold-Franzens University, Innrain 80/82, 6020 Innsbruck (Austria); Güzel, Yüksel [Institute of Analytical Chemistry and Radiochemistry, CCB-Center for Chemistry and Biomedicine, Leopold-Franzens University, Innrain 80/82, 6020 Innsbruck (Austria); ADSI-Austrian Drug Screening Institute, Innrain 66a, 6020 Innsbruck (Austria); Rainer, Matthias [Institute of Analytical Chemistry and Radiochemistry, CCB-Center for Chemistry and Biomedicine, Leopold-Franzens University, Innrain 80/82, 6020 Innsbruck (Austria); Huck, Christian W., E-mail: Christian.W.Huck@uibk.ac.at [Institute of Analytical Chemistry and Radiochemistry, CCB-Center for Chemistry and Biomedicine, Leopold-Franzens University, Innrain 80/82, 6020 Innsbruck (Austria); Bonn, Günther K. [Institute of Analytical Chemistry and Radiochemistry, CCB-Center for Chemistry and Biomedicine, Leopold-Franzens University, Innrain 80/82, 6020 Innsbruck (Austria); ADSI-Austrian Drug Screening Institute, Innrain 66a, 6020 Innsbruck (Austria)

    2014-12-10

    Highlights: • A novel sample preparation technique for isolation of small molecules from human plasma. • Effectiveness of zirconium silicate for the removal of residual proteins after protein precipitation. • Abolishing the consumption of salts for the depletion of residual proteins after protein precipitation. • More than 99.6% removal of plasma proteins. - Abstract: An efficient blood plasma clean-up method was developed, where methanol protein precipitation was applied, followed by zirconium silicate assisted exclusion of residual proteins. A strong binding of zirconium (IV) silicate to the proteins enabled the elimination of remaining proteins after solvent deproteinization through a rapid solid-phase extraction (SPE) procedure. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI/TOF MS) was used for monitoring the proteins during clean-up practice applied to human plasma samples. The proteins were quantified by colorimetric detection using the bicinchoninic acid (BCA) assay. The presented analytical strategy resulted in the depletion of >99.6% proteins from human plasma samples. Furthermore, high-performance liquid chromatography hyphenated to diode-array and electrospray ionization mass spectrometric detection (HPLC–DAD/ESI MS) was applied for qualitative and quantitative analysis of the caffeoylquinic acids (CQAs) and their metabolites in human plasma. The procedure demonstrated high recoveries for the standard compounds spiked at different concentrations. Cynarin and chlorogenic acid were recovered in the range of 81–86% and 78–83%, respectively. Caffeic acid was extracted in the excess of 89–92%, while ferulic acid and dihydroxyhydrocinnamic acid showed a recovery of 87–91% and 92–95%, respectively. The method was partially validated in accordance with FDA-Industry Guidelines for Bioanalytical Method Validation (2001). The presented scheme improves the clean-up efficacy of the methanol deproteinization

  16. Zirconium silicate assisted removal of residual proteins after organic solvent deproteinization of human plasma, enhancing the stability of the LC–ESI-MS response for the bioanalysis of small molecules

    International Nuclear Information System (INIS)

    Hussain, Shah; Pezzei, Cornelia; Güzel, Yüksel; Rainer, Matthias; Huck, Christian W.; Bonn, Günther K.

    2014-01-01

    Highlights: • A novel sample preparation technique for isolation of small molecules from human plasma. • Effectiveness of zirconium silicate for the removal of residual proteins after protein precipitation. • Abolishing the consumption of salts for the depletion of residual proteins after protein precipitation. • More than 99.6% removal of plasma proteins. - Abstract: An efficient blood plasma clean-up method was developed, where methanol protein precipitation was applied, followed by zirconium silicate assisted exclusion of residual proteins. A strong binding of zirconium (IV) silicate to the proteins enabled the elimination of remaining proteins after solvent deproteinization through a rapid solid-phase extraction (SPE) procedure. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI/TOF MS) was used for monitoring the proteins during clean-up practice applied to human plasma samples. The proteins were quantified by colorimetric detection using the bicinchoninic acid (BCA) assay. The presented analytical strategy resulted in the depletion of >99.6% proteins from human plasma samples. Furthermore, high-performance liquid chromatography hyphenated to diode-array and electrospray ionization mass spectrometric detection (HPLC–DAD/ESI MS) was applied for qualitative and quantitative analysis of the caffeoylquinic acids (CQAs) and their metabolites in human plasma. The procedure demonstrated high recoveries for the standard compounds spiked at different concentrations. Cynarin and chlorogenic acid were recovered in the range of 81–86% and 78–83%, respectively. Caffeic acid was extracted in the excess of 89–92%, while ferulic acid and dihydroxyhydrocinnamic acid showed a recovery of 87–91% and 92–95%, respectively. The method was partially validated in accordance with FDA-Industry Guidelines for Bioanalytical Method Validation (2001). The presented scheme improves the clean-up efficacy of the methanol deproteinization

  17. Niemann-Pick C2 protein regulates sterol transport between plasma membrane and late endosomes in human fibroblasts

    DEFF Research Database (Denmark)

    Berzina, Zane; Solanko, Lukasz M; Mehadi, Ahmed S

    2018-01-01

    /LYSs is currently unknown. We show that the close cholesterol analog dehydroergosterol (DHE), when delivered to the plasma membrane (PM) accumulates in LE/LYSs of human fibroblasts lacking functional NPC2. We measured two different time scales of sterol diffusion; while DHE rich LE/LYSs moved by slow anomalous...... but not of DHE is reduced 10-fold in disease fibroblasts compared to control cells. Internalized NPC2 rescued the sterol storage phenotype and strongly expanded the dynamic sterol pool seen in FRAP experiments. Together, our study shows that cholesterol esterification and trafficking of sterols between the PM...

  18. Informing the Human Plasma Protein Binding of Environmental Chemicals by Machine Learning in the Pharmaceutical Space: Applicability Domain and Limits of Predictability

    Science.gov (United States)

    The free fraction of a xenobiotic in plasma (Fub) is an important determinant of chemical adsorption, distribution, metabolism, elimination, and toxicity, yet experimental plasma protein binding data is scarce for environmentally relevant chemicals. The presented work explores th...

  19. Simultaneous determination of glucose, triglycerides, urea, cholesterol, albumin and total protein in human plasma by Fourier transform infrared spectroscopy: direct clinical biochemistry without reagents.

    Science.gov (United States)

    Jessen, Torben E; Höskuldsson, Agnar T; Bjerrum, Poul J; Verder, Henrik; Sørensen, Lars; Bratholm, Palle S; Christensen, Bo; Jensen, Lene S; Jensen, Maria A B

    2014-09-01

    Direct measurement of chemical constituents in complex biologic matrices without the use of analyte specific reagents could be a step forward toward the simplification of clinical biochemistry. Problems related to reagents such as production errors, improper handling, and lot-to-lot variations would be eliminated as well as errors occurring during assay execution. We describe and validate a reagent free method for direct measurement of six analytes in human plasma based on Fourier-transform infrared spectroscopy (FTIR). Blood plasma is analyzed without any sample preparation. FTIR spectrum of the raw plasma is recorded in a sampling cuvette specially designed for measurement of aqueous solutions. For each analyte, a mathematical calibration process is performed by a stepwise selection of wavelengths giving the optimal least-squares correlation between the measured FTIR signal and the analyte concentration measured by conventional clinical reference methods. The developed calibration algorithms are subsequently evaluated for their capability to predict the concentration of the six analytes in blinded patient samples. The correlation between the six FTIR methods and corresponding reference methods were 0.87albumin and total protein in human plasma. Copyright © 2014 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

  20. Multijunction Capillary Isoelectric Focusing Device Combined with Online Membrane-Assisted Buffer Exchanger Enables Isoelectric Point Fractionation of Intact Human Plasma Proteins for Biomarker Discovery.

    Science.gov (United States)

    Pirmoradian, Mohammad; Astorga-Wells, Juan; Zubarev, Roman A

    2015-12-01

    Prefractionation of proteins is often employed to improve analysis specificity in proteomics. Prefractionation based on the isoelectric point (pI) is particularly attractive because pI is a well-defined parameter and it is orthogonal to hydrophobicity on which reversed-phase chromatography is based. However, direct capillary electrophoresis of blood proteins is challenging due to its high content of salts and charged small molecules. Here, we couple an online desalinator device to our multijunction capillary isoelectric focusing (MJ-CIEF) instrument and perform direct isoelectric separation of human blood plasma. In a proof-of-principle experiment, pooled samples of patients with progressive mild cognitive impairment and corresponding healthy controls were investigated. Injection of 3 μL of plasma containing over 100 μg of proteins into the desalinator was followed by pI fractionation with MJ-CIEF in less than 1 h. Shotgun proteomics of 12 collected fractions from each of the 5 replicates of pooled samples resulted in the identification and accurate quantification (median CV between the replicates is <4%) of nearly 365 protein groups from 4030 unique peptides (with <1% FDR for both peptides and proteins). The obtained results include several proteins previously reported as AD markers. The isoelectric point of each quantified protein was calculated using a set of 7 synthetic peptides spiked into the samples. Several proteins with a significant pI shift between their isoforms in the patient and control samples were identified. The presented method is straightforward, robust, and scalable; therefore, it can be used in both biological and clinical applications.

  1. PLASMA PROTEIN AND HEMOGLOBIN PRODUCTION

    Science.gov (United States)

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

    1947-01-01

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

  2. Monoclonal anti-human factor VII antibodies. Detection in plasma of a second protein antigenically and genetically related to factor VII.

    OpenAIRE

    Broze, G J; Hickman, S; Miletich, J P

    1985-01-01

    Several murine monoclonal anti-human Factor VII antibodies were produced using hybridoma technology. Two noncompetitive monoclonal antibodies were used to examine by Western blotting the Factor VII cross-reactive material (CRM) in normal human plasma and three commercially available congenitally Factor VII-deficient plasmas, and to construct a facile "sandwich" immunoassay for plasma Factor VII. A second, previously undescribed, form of Factor VII CRM was detected in human plasma, which on We...

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

    Science.gov (United States)

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

    2013-02-01

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

  4. A direct thrombin inhibitor suppresses protein C activation and factor Va degradation in human plasma: Possible mechanisms of paradoxical enhancement of thrombin generation.

    Science.gov (United States)

    Kamisato, Chikako; Furugohri, Taketoshi; Morishima, Yoshiyuki

    2016-05-01

    We have demonstrated that antithrombin (AT)-independent thrombin inhibitors paradoxically increase thrombin generation (TG) in human plasma in a thrombomodulin (TM)- and protein C (PC)-dependent manner. We determined the effects of AT-independent thrombin inhibitors on the negative-feedback system, activation of PC and production and degradation of factor Va (FVa), as possible mechanisms underlying the paradoxical enhancement of TG. TG in human plasma containing 10nM TM was assayed by means of the calibrated automated thrombography. As an index of PC activation, plasma concentration of activated PC-PC inhibitor complex (aPC-PCI) was measured. The amounts of FVa heavy chain and its degradation product (FVa(307-506)) were examined by western blotting. AT-independent thrombin inhibitors, melagatran and dabigatran (both at 25-600nM) and 3-30μg/ml active site-blocked thrombin (IIai), increased peak levels of TG. Melagatran, dabigatran and IIai significantly decreased plasma concentration of aPC-PCI complex at 25nM or more, 75nM or more, and 10 and 30μg/ml, respectively. Melagatran (300nM) significantly increased FVa and decreased FVa(307-506). In contrast, a direct factor Xa inhibitor edoxaban preferentially inhibited thrombin generation (≥25nM), and higher concentrations were required to inhibit PC activation (≥150nM) and FVa degradation (300nM). The present study suggests that the inhibitions of protein C activation and subsequent degradation of FVa and increase in FVa by antithrombin-independent thrombin inhibitors may contribute to the paradoxical TG enhancement, and edoxaban may inhibit PC activation and FVa degradation as a result of TG suppression. Copyright © 2016 Elsevier Ltd. All rights reserved.

  5. Prediction of inflammatory responses induced by biomaterials in contact with human blood using protein fingerprint from plasma.

    Science.gov (United States)

    Engberg, Anna E; Nilsson, Per H; Huang, Shan; Fromell, Karin; Hamad, Osama A; Mollnes, Tom Eirik; Rosengren-Holmberg, Jenny P; Sandholm, Kerstin; Teramura, Yuji; Nicholls, Ian A; Nilsson, Bo; Ekdahl, Kristina N

    2015-01-01

    Inappropriate complement activation is often responsible for incompatibility reactions that occur when biomaterials are used. Complement activation is therefore a criterion included in legislation regarding biomaterials testing. However, no consensus is yet available regarding appropriate complement-activation-related test parameters. We examined protein adsorption in plasma and complement activation/cytokine release in whole blood incubated with well-characterized polymers. Strong correlations were found between the ratio of C4 to its inhibitor C4BP and generation of 10 (mainly pro-inflammatory) cytokines, including IL-17, IFN-γ, and IL-6. The levels of complement activation products correlated weakly (C3a) or not at all (C5a, sC5b-9), confirming their poor predictive values. We have demonstrated a direct correlation between downstream biological effects and the proteins initially adhering to an artificial surface after contact with blood. Consequently, we propose the C4/C4BP ratio as a robust, predictor of biocompatibility with superior specificity and sensitivity over the current gold standard. Copyright © 2014 Elsevier Ltd. All rights reserved.

  6. Application of a UPLC–MS/MS method to the protein binding study of TM-2 in rat, human and beagle dog plasma

    Directory of Open Access Journals (Sweden)

    Hui Liu

    2016-02-01

    Full Text Available TM-2 known as a potential antitumor drug is a novel semi-synthetic taxane derivative. As drug–protein interactions contribute to insights into pharmacokinetic and pharmacodynamic properties, we elucidated the binding of TM-2 to plasma protein. In this study, a simple, rapid and reliable method was developed and validated employing equilibrium dialysis for the separation of bound and unbound drugs and ultra-performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS for the quantitation. Protein binding reached equilibrium within 24 h of incubation at 37 °C. After liquid–liquid extraction with methyl tert-butyl ether, the samples were separated on Thermo Syncronis UPLC® C18 (2.1 mm×50 mm, 1.7 µm, and acquisition of mass spectrometric data was performed in multiple reaction monitoring (MRM mode via positive electrospray ionization. The assay was linear over the concentration rang of 5–2000 ng/mL. The intra- and inter-day precisions were 0.1%–14.8%, and the accuracy was from −6.4% to 7.0%. This assay has been successfully applied to a protein binding study of TM-2 in rat, human and beagle dog plasma. TM-2 showed high protein binding of 81.4%±6.5% (rat, 87.9%±3.6% (human and 79.4%±4.0% (beagle dog. The results revealed that there was an insignificant difference among the three species.

  7. The effect of casein, hydrolyzed casein and whey proteins on urinary and postprandial plasma metabolites in overweight and moderately obese human subjects

    DEFF Research Database (Denmark)

    Schmedes, Mette S; Bendtsen, Line Quist; Gomes, Sisse

    2018-01-01

    , hydrolyzed casein and whey proteins in overweight and moderately obese men and women by investigating select urinary and blood plasma metabolites. RESULTS: A total of 21 urinary and 23 plasma metabolites were identified by NMR spectroscopy. The postprandial plasma metabolites revealed a significant diet...

  8. Simple protein precipitation extraction technique followed by validated chromatographic method for linezolid analysis in real human plasma samples to study its pharmacokinetics.

    Science.gov (United States)

    Mohammed, Samah A; Eissa, Maya S; Ahmed, Hytham M

    2017-02-01

    Fast and sensitive HPLC method was developed, optimized and validated for quantification of linezolid (LNZ) in human plasma using guaifenesin as an internal standard (IS). Analyte and IS were extracted from plasma by simple protein precipitation extraction technique using methanol as the precipitating solvent. The pretreated samples were injected in a mobile phase formed of acetonitrile:water:methanol (20:70:10v/v/v) in an isocratic mode at a flow rate of 1.5mL/min with UV detection at 251nm. Separation was done using Aglient ODS C 18 . The method showed linearity in the range of 0.75-50μg/mL with correlation coefficients equals to 0.9991. Precision and accuracy were in conformity with the criteria normally accepted in bio-analytical method validation. The RSDs for intra- and inter-day assays were <3.56 and 4.63%, respectively. The intra- and inter-day accuracies were 94.67-98.28% and 91.25-96.18%, respectively. The mean absolute recoveries ranged from 92.56±1.78 to 95.24±2.84. According to stability results, LNZ was stable in human plasma during the storage and analysis. LNZ a pharmacokinetic behavior was studied by applying the proposed analytical method. Copyright © 2016 Elsevier B.V. All rights reserved.

  9. Liquid-liquid extraction of strongly protein bound BMS-299897 from human plasma and cerebrospinal fluid, followed by high-performance liquid chromatography/tandem mass spectrometry.

    Science.gov (United States)

    Xue, Y J; Pursley, Janice; Arnold, Mark

    2007-04-11

    BMS-299897 is a gamma-secretase inhibitor that is being developed for the treatment of Alzheimer's disease. Liquid-liquid extraction (LLE), chromatographic/tandem mass spectrometry (LC/MS/MS) methods have been developed and validated for the quantitation of BMS-299897 in human plasma and cerebrospinal fluid (CSF). Both methods utilized (13)C6-BMS-299897, the stable label isotope analog, as the internal standard. For the human plasma extraction method, two incubation steps were required after the addition of 5 mM ammonium acetate and the internal standard in acetonitrile to release the analyte bound to proteins prior to LLE with toluene. For the human CSF extraction method, after the addition of 0.5 N HCl and the internal standard, CSF samples were extracted with toluene and no incubation was required. The organic layers obtained from both extraction methods were removed and evaporated to dryness. The residues were reconstituted and injected into the LC/MS/MS system. Chromatographic separation was achieved isocratically on a MetaChem C18 Hypersil BDS column (2.0 mm x 50 mm, 3 microm). The mobile phase contained 10 mM ammonium acetate pH 5 and acetonitrile. Detection was by negative ion electrospray tandem mass spectrometry. The standard curves ranged from 1 to 1000 ng/ml for human plasma and 0.25-100 ng/ml for human CSF. Both standard curves were fitted to a 1/x weighted quadratic regression model. For both methods, the intra-assay precision was within 8.2% CV, the inter-assay precision was within 5.4% CV, and assay accuracy was within +/-7.4% of the nominal values. The validation and sample analysis results demonstrated that both methods had acceptable precision and accuracy across the calibration ranges.

  10. Plasma proteins and proteinuria in gestational malaria

    OpenAIRE

    Fisayo, Asaolu Modupe

    2007-01-01

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

  11. Genomics meets glycomics-the first GWAS study of human N-Glycome identifies HNF1α as a master regulator of plasma protein fucosylation.

    Directory of Open Access Journals (Sweden)

    Gordan Lauc

    2010-12-01

    Full Text Available Over half of all proteins are glycosylated, and alterations in glycosylation have been observed in numerous physiological and pathological processes. Attached glycans significantly affect protein function; but, contrary to polypeptides, they are not directly encoded by genes, and the complex processes that regulate their assembly are poorly understood. A novel approach combining genome-wide association and high-throughput glycomics analysis of 2,705 individuals in three population cohorts showed that common variants in the Hepatocyte Nuclear Factor 1α (HNF1α and fucosyltransferase genes FUT6 and FUT8 influence N-glycan levels in human plasma. We show that HNF1α and its downstream target HNF4α regulate the expression of key fucosyltransferase and fucose biosynthesis genes. Moreover, we show that HNF1α is both necessary and sufficient to drive the expression of these genes in hepatic cells. These results reveal a new role for HNF1α as a master transcriptional regulator of multiple stages in the fucosylation process. This mechanism has implications for the regulation of immunity, embryonic development, and protein folding, as well as for our understanding of the molecular mechanisms underlying cancer, coronary heart disease, and metabolic and inflammatory disorders.

  12. Development of a displacement immunoassay for human heart-type fatty acid-binding protein in plasma : the basic conditions

    NARCIS (Netherlands)

    van der Voort, D; Pelsers, MMAL; Korf, J; Hermens, WT; Glatz, JFC

    2003-01-01

    To risk-stratify patients with chest pain who are admitted to emergency rooms and for whom initial evaluation is not conclusive, the use of cardiac markers has become a standard procedure. A recently introduced early plasma marker for acute myocardial infarction (AMI) is the 14.5-kDa cytoplasmic

  13. Measurement of HNE-protein adducts in human plasma and serum by ELISA—Comparison of two primary antibodies

    Directory of Open Access Journals (Sweden)

    Daniela Weber

    2013-01-01

    After modification and validation of the protocol for both antibodies, samples of two groups were analyzed: apparently healthy obese (n=62 and non-obese controls (n=15. Although the detected absolute values of HNE–protein adducts were different, depending on the antibody used, both ELISA methods showed significantly higher values of HNE–protein adducts in the obese group.

  14. Differential plasma protein binding to metal oxide nanoparticles

    International Nuclear Information System (INIS)

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

    2009-01-01

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

  15. Body Position Modulates Gastric Emptying and Affects the Post-Prandial Rise in Plasma Amino Acid Concentrations Following Protein Ingestion in Humans

    Directory of Open Access Journals (Sweden)

    Andrew M. Holwerda

    2016-04-01

    Full Text Available Dietary protein digestion and amino acid absorption kinetics determine the post-prandial muscle protein synthetic response. Body position may affect gastrointestinal function and modulate the post-prandial rise in plasma amino acid availability. We aimed to assess the impact of body position on gastric emptying rate and the post-prandial rise in plasma amino acid concentrations following ingestion of a single, meal-like amount of protein. In a randomized, cross-over design, eight healthy males (25 ± 2 years, 23.9 ± 0.8 kg·m−2 ingested 22 g protein and 1.5 g paracetamol (acetaminophen in an upright seated position (control and in a −20° head-down tilted position (inversion. Blood samples were collected during a 240-min post-prandial period and analyzed for paracetamol and plasma amino acid concentrations to assess gastric emptying rate and post-prandial amino acid availability, respectively. Peak plasma leucine concentrations were lower in the inversion compared with the control treatment (177 ± 15 vs. 236 ± 15 mmol·L−1, p < 0.05, which was accompanied by a lower plasma essential amino acid (EAA response over 240 min (31,956 ± 6441 vs. 50,351 ± 4015 AU; p < 0.05. Peak plasma paracetamol concentrations were lower in the inversion vs. control treatment (5.8 ± 1.1 vs. 10.0 ± 0.6 mg·L−1, p < 0.05. Gastric emptying rate and post-prandial plasma amino acid availability are significantly decreased after protein ingestion in a head-down tilted position. Therefore, upright body positioning should be considered when aiming to augment post-prandial muscle protein accretion in both health and disease.

  16. Temporal effects of maternal and pregnancy characteristics on serum pregnancy-associated plasma protein-A and free β-human chorionic gonadotropin at 7-14 weeks' gestation

    DEFF Research Database (Denmark)

    Ball, S; Ekelund, C; Wright, D

    2013-01-01

    OBJECTIVE: The aim of this study was to investigate gestational age-dependent effects of racial origin, smoking status and mode of conception on maternal serum levels of free β-human chorionic gonadotropin (β-hCG) and pregnancy-associated plasma protein-A (PAPP-A) at 7-14 weeks' gestation. METHODS...

  17. Zeolite Nanoparticles for Selective Sorption of Plasma Proteins.

    Science.gov (United States)

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

    2015-11-30

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

  18. Hypochlorite-induced oxidation of proteins in plasma

    DEFF Research Database (Denmark)

    Hawkins, C L; Davies, Michael Jonathan

    1999-01-01

    Activated phagocyte cells generate hypochlorite (HOCl) via the release of H2O2 and the enzyme myeloperoxidase. Plasma proteins are major targets for HOCl, although little information is available about the mechanism(s) of oxidation. In this study the reaction of HOCl (at least 50 microM) with dil......Activated phagocyte cells generate hypochlorite (HOCl) via the release of H2O2 and the enzyme myeloperoxidase. Plasma proteins are major targets for HOCl, although little information is available about the mechanism(s) of oxidation. In this study the reaction of HOCl (at least 50 micro......M) with diluted fresh human plasma has been shown to generate material that oxidizes 5-thio-2-nitrobenzoic acid; these oxidants are believed to be chloramines formed from the reaction of HOCl with protein amine groups. Chloramines have also been detected with isolated plasma proteins treated with HOCl. In both...... more efficient. The reaction of fresh diluted plasma with HOCl also gives rise to protein-derived nitrogen-centred radicals in a time- and HOCl-concentration-dependent manner; these have been detected by EPR spin trapping. Identical radicals have been detected with isolated HOCl-treated plasma proteins...

  19. Analysis of native human plasma proteins and haemoglobin for the presence of bityrosine by high-performance liquid chromatography

    DEFF Research Database (Denmark)

    Daneshvar, B; Frandsen, H; Dragsted, L O

    1997-01-01

    fluorescent substance, bityrosine. High-performance liquid chromatography (HPLC) analysis of acid hydrolyzed serum albumin after oxidation with peroxidase/H2O2 or with Cu++/H2O2 showed that bityrosine had been formed whereas oxidation of this protein with Fe(III)/ascorbate did not result in the formation...

  20. Plasma proteins of Barbus holubi and Clarias gariepinus

    African Journals Online (AJOL)

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

  1. Interaction of spin-labeled HPMA-based nanoparticles with human blood plasma proteins - the introduction of protein-corona-free polymer nanomedicine

    Czech Academy of Sciences Publication Activity Database

    Klepac, Damir; Kostková, Hana; Petrova, Svetlana; Chytil, Petr; Etrych, Tomáš; Kereiche, S.; Raška, I.; Weitz, D. A.; Filippov, Sergey K.

    2018-01-01

    Roč. 10, č. 13 (2018), s. 6194-6204 ISSN 2040-3364 R&D Projects: GA MŠk(CZ) LH15213; GA ČR(CZ) GA17-07164S Institutional support: RVO:61389013 Keywords : EPR * nanoparticles * protein s Subject RIV: CD - Macromolecular Chemistry OBOR OECD: Polymer science Impact factor: 7.367, year: 2016

  2. Plasma protein haptoglobin modulates renal iron loading

    DEFF Research Database (Denmark)

    Fagoonee, Sharmila; Gburek, Jakub; Hirsch, Emilio

    2005-01-01

    Haptoglobin is the plasma protein with the highest binding affinity for hemoglobin. The strength of hemoglobin binding and the existence of a specific receptor for the haptoglobin-hemoglobin complex in the monocyte/macrophage system clearly suggest that haptoglobin may have a crucial role in heme...... distribution of hemoglobin in haptoglobin-deficient mice resulted in abnormal iron deposits in proximal tubules during aging. Moreover, iron also accumulated in proximal tubules after renal ischemia-reperfusion injury or after an acute plasma heme-protein overload caused by muscle injury, without affecting...... morphological and functional parameters of renal damage. These data demonstrate that haptoglobin crucially prevents glomerular filtration of hemoglobin and, consequently, renal iron loading during aging and following acute plasma heme-protein overload....

  3. Human ovolution and plasma oxytocin

    International Nuclear Information System (INIS)

    Kumaresan, Perianna; Kumaresan, Malathi; Hossini, Mahmood; Arellano, Carolina; Vasicka, Alois

    1983-01-01

    Plasma oxytocin (OT) concentrations were measured by radioimmunoassay (RIA) method without extracting plasma in 11 normal menstruating women. Mean plasma OT level began to increase steadily from the 7th day of the menstrual cycle and this level rose up to 20+-5 μU/ml (Mean+-S.E.) on the 10th day of the cycle. OT level declined to 13+-6 μU/ml on the day of the LH peak and continuously declined for another 2 days - then rose. The OT level was higher during the follicular phase than during the luteal phase. In 1 individual OT measured in 2 cycles a year apart showed the highest level of OT coincided with LH and FSH peak and abruptly declined. When there was the highest level of progesterone, the OT level was measurable 1 out of 11 cycles. From this study, we conclude that OT may have a role in human ovulation either synergistically or alone with other ovulatory mechanisms and ovarian estradiol and progesterone control the secretion of OT and also suggests that OT may play some role in the regulation of the luteolysis and the menstrual cycle in women. (author)

  4. Functionalization of alkyne-terminated thermally hydrocarbonized porous silicon nanoparticles with targeting peptides and antifouling polymers: effect on the human plasma protein adsorption.

    Science.gov (United States)

    Wang, Chang-Fang; Mäkilä, Ermei M; Bonduelle, Colin; Rytkönen, Jussi; Raula, Janne; Almeida, Sérgio; Närvänen, Ale; Salonen, Jarno J; Lecommandoux, Sebastien; Hirvonen, Jouni T; Santos, Hélder A

    2015-01-28

    Porous silicon (PSi) nanomaterials combine a high drug loading capacity and tunable surface chemistry with various surface modifications to meet the requirements for biomedical applications. In this work, alkyne-terminated thermally hydrocarbonized porous silicon (THCPSi) nanoparticles were fabricated and postmodified using five bioactive molecules (targeting peptides and antifouling polymers) via a single-step click chemistry to modulate the bioactivity of the THCPSi nanoparticles, such as enhancing the cellular uptake and reducing the plasma protein association. The size of the nanoparticles after modification was increased from 176 to 180-220 nm. Dextran 40 kDa modified THCPSi nanoparticles showed the highest stability in aqueous buffer. Both peptide- and polymer-functionalized THCPSi nanoparticles showed an extensive cellular uptake which was dependent on the functionalized moieties presented on the surface of the nanoparticles. The plasma protein adsorption study showed that the surface modification with different peptides or polymers induced different protein association profiles. Dextran 40 kDa functionalized THCPSi nanoparticles presented the least protein association. Overall, these results demonstrate that the "click" conjugation of the biomolecules onto the alkyne-terminated THCPSi nanoparticles is a versatile and simple approach to modulate the surface chemistry, which has high potential for biomedical applications.

  5. Modulating Protein Adsorption on Oxygen Plasma Modified Polysiloxane Surfaces

    International Nuclear Information System (INIS)

    Marletta, G.

    2006-01-01

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

  6. Radioimmunoassay for pregnancy-associated plasma protein A

    International Nuclear Information System (INIS)

    Sinosich, M.J.; Teisner, B.; Folkerson, J.; Saunders, D.M.; Grudzinskas, J.G.

    1982-01-01

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

  7. A radioimmunoassay for neurotensin in human plasma

    International Nuclear Information System (INIS)

    Blackburn, A.M.; Bloom, S.R.

    1979-01-01

    A radioimmunoassay was developed for detecting the neurotensin peptide in human plasma. The plasma was specific for neurotensin as no cross-reaction was found with any of the other gut hormones tested. Changes of 5 pmol/l could be detected with 95% confidence. Neurotensin was unstable in both blood and plasma but considerable protection was afforded by addition of aprotinin, rapid separation of plasma and immediate deep freezing. Neurotensin-like immunoreactivity was detected in human plasma in both a small and large molecular form. The mean fasting level of plasma neurotensin-like immunoreactivity in 36 healthy volunteers was 29 +- 3 pmol/l. A significant increment of 27 +- 8 pmol/l plasma neurotensin immunoreactivity was detected after a large meal in nine healthy men. In view of the present results in man and also of neurotensin's potent pharmacological actions in experimental animals, neurotensin appears to fulfil some of the criteria needed for a hormone. (UK)

  8. Radioimmunoassay of sodium cromoglycate in human plasma

    Energy Technology Data Exchange (ETDEWEB)

    Brown, K; Gardner, J J; Lockley, W J.S.; Preston, J R; Wilkinson, D J [Fisons plc, Loughborough (UK). Pharmaceutical Div.

    1983-01-01

    A sensitive radioimmunoassay method for sodium cromoglycate in human plasma is described. The lowest quantifiable concentration of sodium cromoglycate is 0.93 nmol/l when 0.1 ml plasma samples are analysed. The range of the method is limited; both 0.01 and 0.1 ml volumes of plasma must be analysed to encompass the concentration range 0.93-139 nmol/l which may be encountered in plasma samples from patients and human volunteers. The method is specific for sodium cromoglycate as indicated by a low cross-reactivity of the anti-cromoglycate antiserum with a number of drugs.

  9. Phosphorylation of human link proteins

    International Nuclear Information System (INIS)

    Oester, D.A.; Caterson, B.; Schwartz, E.R.

    1986-01-01

    Three link proteins of 48, 44 and 40 kDa were purified from human articular cartilage and identified with monoclonal anti-link protein antibody 8-A-4. Two sets of lower molecular weight proteins of 30-31 kDa and 24-26 kDa also contained link protein epitopes recognized by the monoclonal antibody and were most likely degradative products of the intact link proteins. The link proteins of 48 and 40 kDa were identified as phosphoproteins while the 44 kDa link protein did not contain 32 P. The phosphorylated 48 and 40 kDa link proteins contained approximately 2 moles PO 4 /mole link protein

  10. [Production of human proteins in the blood of transgenic animals

    NARCIS (Netherlands)

    Massoud, M.; Bischoff, Rainer; Dalemans, W.; Pointu, H.; Attal, J.; Schultz, H.; Clesse, D.; Stinnakre, M.G.; Pavirani, A.; Houdebine, L.M.

    1990-01-01

    The human alpha 1-antitrypsin gene has been microinjected into rabbit embryos. A line of transgenic rabbits has thus been established. Human alpha 1-antitrypsin was found in the blood of transgenic animals at the concentration of 1 mg/ml plasma. The human protein was active and separable from its

  11. Palmitoylation of POTE family proteins for plasma membrane targeting

    International Nuclear Information System (INIS)

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

    2007-01-01

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

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

    International Nuclear Information System (INIS)

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

    1966-01-01

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

  13. Viral Organization of Human Proteins

    Science.gov (United States)

    Wuchty, Stefan; Siwo, Geoffrey; Ferdig, Michael T.

    2010-01-01

    Although maps of intracellular interactions are increasingly well characterized, little is known about large-scale maps of host-pathogen protein interactions. The investigation of host-pathogen interactions can reveal features of pathogenesis and provide a foundation for the development of drugs and disease prevention strategies. A compilation of experimentally verified interactions between HIV-1 and human proteins and a set of HIV-dependency factors (HDF) allowed insights into the topology and intricate interplay between viral and host proteins on a large scale. We found that targeted and HDF proteins appear predominantly in rich-clubs, groups of human proteins that are strongly intertwined among each other. These assemblies of proteins may serve as an infection gateway, allowing the virus to take control of the human host by reaching protein pathways and diversified cellular functions in a pronounced and focused way. Particular transcription factors and protein kinases facilitate indirect interactions between HDFs and viral proteins. Discerning the entanglement of directly targeted and indirectly interacting proteins may uncover molecular and functional sites that can provide novel perspectives on the progression of HIV infection and highlight new avenues to fight this virus. PMID:20827298

  14. Irradiation of porcine plasma protein powder, 1

    International Nuclear Information System (INIS)

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

    1987-01-01

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

  15. Type 2 diabetes mellitus is associated with differential effects on plasma cholesteryl ester transfer protein and phospholipid transfer protein activities and concentrations

    NARCIS (Netherlands)

    Dullaart, RPF; De Vries, R; Scheek, L; Borggreve, SE; Van Gent, T; Dallinga-Thie, GM; Ito, M; Nagano, M; Sluiter, WJ; Hattori, H; Van Tol, A

    Background: Human plasma contains two lipid transfer proteins, cholesteryl ester transfer protein (CETP) and phospholipid transfer protein (PLTP), which are crucial in reverse cholesterol transport. Methods: Plasma CETP and PLTP activity levels and concentrations in 16 type 2 diabetic patients and

  16. Endogenous pyrogen activity in human plasma after exercise.

    Science.gov (United States)

    Cannon, J G; Kluger, M J

    1983-05-06

    Plasma obtained from human subjects after exercise and injected intraperitoneally into rats elevated rat rectal temperature and depressed plasma iron and zinc concentrations. The pyrogenic component was heat-denaturable and had an apparent molecular weight of 14,000 daltons. Human mononuclear leukocytes obtained after exercise and incubated in vitro released a factor into the medium that also elevated body temperature in rats and reduced trace metal concentrations. These results suggest that endogenous pyrogen, a protein mediator of fever and trace metal metabolism during infection, is released during exercise.

  17. An enzymatic deglycosylation scheme enabling identification of core fucosylated N-glycans and O-glycosylation site mapping of human plasma proteins

    DEFF Research Database (Denmark)

    Hägglund, Per; Matthiesen, R.; Elortza, F.

    2007-01-01

    and N-acetyl-β-glucosaminidase) are also included. The two strategies were here applied to identify 103 N-glycosylation sites in the Cohn IV fraction of human plasma. In addition, Endo D/H digestion uniquely enabled identification of 23 fucosylated N-glycosylation sites. Several O-glycosylated peptides......, thereby reducing the complexity and facilitating glycosylation site determinations. Here, we have used two different enzymatic deglycosylation strategies for N-glycosylation site analysis. (1) Removal of entire N-glycan chains by peptide- N-glycosidase (PNGase) digestion, with concomitant deamidation...... of the released asparagine residue. The reaction is carried out in H218O to facilitate identification of the formerly glycosylated peptide by incorporatation of 18O into the formed aspartic acid residue. (2) Digestion with two endo-β- N-acetylglucosaminidases (Endo D and Endo H) that cleave the glycosidic bond...

  18. Regulation of pregnancy-associated plasma protein A2 (PAPPA2 in a human placental trophoblast cell line (BeWo

    Directory of Open Access Journals (Sweden)

    Christians Julian K

    2011-04-01

    Full Text Available Abstract Background Pregnancy-associated plasma protein A2 (PAPPA2 is an insulin-like growth factor-binding protein (IGFBP protease expressed at high levels in the placenta and upregulated in pregnancies complicated by preeclampsia and HELLP (Hemolytic anemia, Elevated Liver enzymes, and Low Platelet count syndrome. However, it is unclear whether elevated PAPPA2 expression causes abnormal placental development, or whether upregulation compensates for placental pathology. In the present study, we investigate whether PAPPA2 expression is affected by hypoxia, oxidative stress, syncytialization factors or substances known to affect the expression of PAPPA2's paralogue, PAPPA. Methods BeWo cells, a model of placental trophoblasts, were treated with one of the following: hypoxia (2% O2, oxidative stress (20 microM hydrogen peroxide, forskolin (10 microM and 100 microM, TGF-beta (10 and 50 ng/mL, TNF-alpha (100 ng/mL, IL-1beta (100 ng/mL or PGE2 (1 microM. We used quantitative RT-PCR (qRT-PCR to quantify the mRNA levels of PAPPA2, as well as those of PAPPA and ADAM12 since these proteases have similar substrates and are also highly expressed in the placenta. Where we observed significant effects on PAPPA2 mRNA levels, we tested for effects at the protein level using an in-cell Western assay. Results Hypoxia, but not oxidative stress, caused a 47-fold increase in PAPPA2 mRNA expression, while TNF-alpha resulted in a 6-fold increase, and both of these effects were confirmed at the protein level. PGE2 resulted in a 14-fold upregulation of PAPPA2 mRNA but this was not reflected at the protein level. Forskolin, TGF-beta and IL-1beta had no significant effect on PAPPA2 mRNA expression. We observed no effects of any treatment on PAPPA or ADAM12 expression. Conclusion Our study demonstrates that factors previously known to be highly expressed in preeclamptic placentae (PGE2 and TNF-alpha, contribute to the upregulation of PAPPA2. Hypoxia, known to occur in

  19. Proteins aggregation and human diseases

    Science.gov (United States)

    Hu, Chin-Kun

    2015-04-01

    Many human diseases and the death of most supercentenarians are related to protein aggregation. Neurodegenerative diseases include Alzheimer's disease (AD), Huntington's disease (HD), Parkinson's disease (PD), frontotemporallobar degeneration, etc. Such diseases are due to progressive loss of structure or function of neurons caused by protein aggregation. For example, AD is considered to be related to aggregation of Aβ40 (peptide with 40 amino acids) and Aβ42 (peptide with 42 amino acids) and HD is considered to be related to aggregation of polyQ (polyglutamine) peptides. In this paper, we briefly review our recent discovery of key factors for protein aggregation. We used a lattice model to study the aggregation rates of proteins and found that the probability for a protein sequence to appear in the conformation of the aggregated state can be used to determine the temperature at which proteins can aggregate most quickly. We used molecular dynamics and simple models of polymer chains to study relaxation and aggregation of proteins under various conditions and found that when the bending-angle dependent and torsion-angle dependent interactions are zero or very small, then protein chains tend to aggregate at lower temperatures. All atom models were used to identify a key peptide chain for the aggregation of insulin chains and to find that two polyQ chains prefer anti-parallel conformation. It is pointed out that in many cases, protein aggregation does not result from protein mis-folding. A potential drug from Chinese medicine was found for Alzheimer's disease.

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

    DEFF Research Database (Denmark)

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

    2015-01-01

    The objective of the study was to define the specific plasma protein signature that predicts the increase of the inflammation marker C-reactive protein from index day to next-day using proteome analysis and novel bioinformatics tools. We performed a prospective study of 91 incident kidney....... The prediction model selected and validated 82 plasma proteins which determined increased next-day C-reactive protein (area under receiver-operator-characteristics curve, 0.772; 95% confidence interval, 0.669 to 0.876; P signature (P ....001) was associated with observed increased next-day C-reactive protein. The 82-plex protein signature outperformed routine clinical procedures. The category-free net reclassification index improved with 82-plex plasma protein signature (total net reclassification index, 88.3%). Using the 82-plex plasma protein...

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

    African Journals Online (AJOL)

    Dr Olaleye Samuel

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

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

    OpenAIRE

    Motta, Guacyara; Tersariol, Ivarne L. S.

    2017-01-01

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

  3. Protein Crystal Recombinant Human Insulin

    Science.gov (United States)

    1994-01-01

    The comparison of protein crystal, Recombiant Human Insulin; space-grown (left) and earth-grown (right). On STS-60, Spacehab II indicated that space-grown crystals are larger and of greater optical clarity than their earth-grown counterparts. Recombiant Human Insulin facilitates the incorporation of glucose into cells. In diabetics, there is either a decrease in or complete lack of insulin, thereby leading to several harmful complications. Principal Investigator is Larry DeLucas.

  4. Site specific modification of the human plasma proteome by methylglyoxal

    International Nuclear Information System (INIS)

    Kimzey, Michael J.; Kinsky, Owen R.; Yassine, Hussein N.; Tsaprailis, George; Stump, Craig S.; Monks, Terrence J.; Lau, Serrine S.

    2015-01-01

    Increasing evidence identifies dicarbonyl stress from reactive glucose metabolites, such as methylglyoxal (MG), as a major pathogenic link between hyperglycemia and complications of diabetes. MG covalently modifies arginine residues, yet the site specificity of this modification has not been thoroughly investigated. Sites of MG adduction in the plasma proteome were identified using LC–MS/MS analysis in vitro following incubation of plasma proteins with MG. Treatment of plasma proteins with MG yielded 14 putative MG hotspots from five plasma proteins (albumin [nine hotspots], serotransferrin, haptoglobin [2 hotspots], hemopexin, and Ig lambda-2 chain C regions). The search results revealed two versions of MG-arginine modification, dihydroxyimidazolidine (R + 72) and hydroimidazolone (R + 54) adducts. One of the sites identified was R257 in human serum albumin, which is a critical residue located in drug binding site I. This site was validated as a target for MG modification by a fluorescent probe displacement assay, which revealed significant drug dissociation at 300 μM MG from a prodan–HSA complex (75 μM). Moreover, twelve human plasma samples (six male, six female, with two type 2 diabetic subjects from both genders) were analyzed using multiple reaction monitoring (MRM) tandem mass spectrometry and revealed the presence of the MG-modified albumin R257 peptide. These data provide insights into the nature of the site-specificity of MG modification of arginine, which may be useful for therapeutic treatments that aim to prevent MG-mediated adverse responses in patients. - Highlights: • Methylglyoxal (MG) selectively modifies arginine sites in human plasma proteome. • Dihydroxyimidazolidine and hydroimidazolone adducts on serum albumin identified • MG modification on albumin R257 associated with loss of drug site I binding capacity • MRM-tandem mass spectrometry enables sensitive detection of albumin MG-R257. • Site-specific MG modification may

  5. Site specific modification of the human plasma proteome by methylglyoxal

    Energy Technology Data Exchange (ETDEWEB)

    Kimzey, Michael J.; Kinsky, Owen R. [Southwest Environmental Health Sciences Center, Department of Pharmacology & Toxicology, College of Pharmacy, The University of Arizona, Tucson, AZ 85721 (United States); Yassine, Hussein N. [Department of Medicine, The University of Arizona, Tucson, AZ 85721 (United States); Tsaprailis, George [Southwest Environmental Health Sciences Center, Department of Pharmacology & Toxicology, College of Pharmacy, The University of Arizona, Tucson, AZ 85721 (United States); Stump, Craig S. [Department of Medicine, The University of Arizona, Tucson, AZ 85721 (United States); Southern Arizona VA Health Care System, Tucson, AZ 85723 (United States); Monks, Terrence J. [Southwest Environmental Health Sciences Center, Department of Pharmacology & Toxicology, College of Pharmacy, The University of Arizona, Tucson, AZ 85721 (United States); Lau, Serrine S., E-mail: lau@pharmacy.arizona.edu [Southwest Environmental Health Sciences Center, Department of Pharmacology & Toxicology, College of Pharmacy, The University of Arizona, Tucson, AZ 85721 (United States)

    2015-12-01

    Increasing evidence identifies dicarbonyl stress from reactive glucose metabolites, such as methylglyoxal (MG), as a major pathogenic link between hyperglycemia and complications of diabetes. MG covalently modifies arginine residues, yet the site specificity of this modification has not been thoroughly investigated. Sites of MG adduction in the plasma proteome were identified using LC–MS/MS analysis in vitro following incubation of plasma proteins with MG. Treatment of plasma proteins with MG yielded 14 putative MG hotspots from five plasma proteins (albumin [nine hotspots], serotransferrin, haptoglobin [2 hotspots], hemopexin, and Ig lambda-2 chain C regions). The search results revealed two versions of MG-arginine modification, dihydroxyimidazolidine (R + 72) and hydroimidazolone (R + 54) adducts. One of the sites identified was R257 in human serum albumin, which is a critical residue located in drug binding site I. This site was validated as a target for MG modification by a fluorescent probe displacement assay, which revealed significant drug dissociation at 300 μM MG from a prodan–HSA complex (75 μM). Moreover, twelve human plasma samples (six male, six female, with two type 2 diabetic subjects from both genders) were analyzed using multiple reaction monitoring (MRM) tandem mass spectrometry and revealed the presence of the MG-modified albumin R257 peptide. These data provide insights into the nature of the site-specificity of MG modification of arginine, which may be useful for therapeutic treatments that aim to prevent MG-mediated adverse responses in patients. - Highlights: • Methylglyoxal (MG) selectively modifies arginine sites in human plasma proteome. • Dihydroxyimidazolidine and hydroimidazolone adducts on serum albumin identified • MG modification on albumin R257 associated with loss of drug site I binding capacity • MRM-tandem mass spectrometry enables sensitive detection of albumin MG-R257. • Site-specific MG modification may

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

    Science.gov (United States)

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

    2017-12-01

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

  7. LC-MS/MS Based Quantitation of ABC and SLC Transporter Proteins in Plasma Membranes of Cultured Primary Human Retinal Pigment Epithelium Cells and Immortalized ARPE19 Cell Line.

    Science.gov (United States)

    Pelkonen, Laura; Sato, Kazuki; Reinisalo, Mika; Kidron, Heidi; Tachikawa, Masanori; Watanabe, Michitoshi; Uchida, Yasuo; Urtti, Arto; Terasaki, Tetsuya

    2017-03-06

    The retinal pigment epithelium (RPE) forms the outer blood-retinal barrier between neural retina and choroid. The RPE has several important vision supporting functions, such as transport mechanisms that may also modify pharmacokinetics in the posterior eye segment. Expression of plasma membrane transporters in the RPE cells has not been quantitated. The aim of this study was to characterize and compare transporter protein expression in the ARPE19 cell line and hfRPE (human fetal RPE) cells by using quantitative targeted absolute proteomics (QTAP). Among 41 studied transporters, 16 proteins were expressed in hfRPE and 13 in ARPE19 cells. MRP1, MRP5, GLUT1, 4F2hc, TAUT, CAT1, LAT1, and MATE1 proteins were detected in both cell lines within 4-fold differences. MPR7, OAT2 and RFC1 were detected in the hfRPE cells, but their expression levels were below the limit of quantification in ARPE19 cells. PCFT was detected in both studied cell lines, but the expression was over 4-fold higher in hfRPE cells. MCT1, MCT4, MRP4, and Na + /K + ATPase were upregulated in the ARPE19 cell line showing over 4-fold differences in the quantitative expression values. Expression levels of 25 transporters were below the limit of quantification in both cell models. In conclusion, we present the first systematic and quantitative study on transporter protein expression in the plasma membranes of ARPE19 and hfRPE cells. Overall, transporter expression in the ARPE19 and hfRPE cells correlated well and the absolute expression levels were similar, but not identical. The presented quantitative expression levels could be a useful basis for further studies on drug permeation in the outer blood-retinal barrier.

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

    DEFF Research Database (Denmark)

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

    2017-01-01

    Recent advances in highly multiplexed immunoassays have allowed systematic large-scale measurement of hundreds of plasma proteins in large cohort studies. In combination with genotyping, such studies offer the prospect to 1) identify mechanisms involved with regulation of protein expression...... in plasma, and 2) determine whether the plasma proteins are likely to be causally implicated in disease. We report here the results of genome-wide association (GWA) studies of 83 proteins considered relevant to cardiovascular disease (CVD), measured in 3,394 individuals with multiple CVD risk factors. We...... on coronary artery disease, and highlight several potentially causal associations. Overall, a majority of the plasma proteins studied showed evidence of regulation at the genetic level. Our results enable future studies of the causal architecture of human disease, which in turn should aid discovery of new...

  9. Influence of ionizing radiation on the plasma membrane proteins

    International Nuclear Information System (INIS)

    Dreval', V.I.

    1992-01-01

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

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

    NARCIS (Netherlands)

    van der Scheer, Albert

    1978-01-01

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

  11. Plasma myelin basic protein assay using Gilford enzyme immunoassay cuvettes.

    Science.gov (United States)

    Groome, N P

    1981-10-01

    The assay of myelin basic protein in body fluids has potential clinical importance as a routine indicator of demyelination. Preliminary details of a competitive enzyme immunoassay for this protein have previously been published by the author (Groome, N. P. (1980) J. Neurochem. 35, 1409-1417). The present paper now describes the adaptation of this assay for use on human plasma and various aspects of routine data processing. A commercially available cuvette system was found to have advantages over microtitre plates but required a permuted arrangement of sample replicates for consistent results. For dose interpolation, the standard curve could be fitted to a three parameter non-linear equation by regression analysis or linearised by the logit/log transformation.

  12. Proteins aggregation and human diseases

    International Nuclear Information System (INIS)

    Hu, Chin-Kun

    2015-01-01

    Many human diseases and the death of most supercentenarians are related to protein aggregation. Neurodegenerative diseases include Alzheimer's disease (AD), Huntington's disease (HD), Parkinson's disease (PD), frontotemporallobar degeneration, etc. Such diseases are due to progressive loss of structure or function of neurons caused by protein aggregation. For example, AD is considered to be related to aggregation of Aβ40 (peptide with 40 amino acids) and Aβ42 (peptide with 42 amino acids) and HD is considered to be related to aggregation of polyQ (polyglutamine) peptides. In this paper, we briefly review our recent discovery of key factors for protein aggregation. We used a lattice model to study the aggregation rates of proteins and found that the probability for a protein sequence to appear in the conformation of the aggregated state can be used to determine the temperature at which proteins can aggregate most quickly. We used molecular dynamics and simple models of polymer chains to study relaxation and aggregation of proteins under various conditions and found that when the bending-angle dependent and torsion-angle dependent interactions are zero or very small, then protein chains tend to aggregate at lower temperatures. All atom models were used to identify a key peptide chain for the aggregation of insulin chains and to find that two polyQ chains prefer anti-parallel conformation. It is pointed out that in many cases, protein aggregation does not result from protein mis-folding. A potential drug from Chinese medicine was found for Alzheimer's disease. (paper)

  13. Clinical relevance of drug binding to plasma proteins

    Science.gov (United States)

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

    2014-12-01

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

  14. Standardization for cortisol determination in human blood by competitive protein-binding

    International Nuclear Information System (INIS)

    Okada, H.

    1978-01-01

    Standardization for determination of cortisol from human plasma (17-hydroxycorticosteroids) using competitive protein-binding method is presented. Activated carbon coated with dextrans is used for separation of the hormone-protein complexe and hormone labelled free [pt

  15. Dietary modulation of plasma angiopoietin-like protein 4 concentrations in healthy volunteers and in patients with type 2 diabetes

    NARCIS (Netherlands)

    Jonker, Jacqueline T.; Smit, Johannes W. A.; Hammer, Sebastiaan; Snel, Marieke; van der Meer, Rutger W.; Lamb, Hildo J.; Mattijssen, Frits; Mudde, Karin; Jazet, Ingrid M.; Dekkers, Olaf M.; de Roos, Albert; Romijn, Johannes A.; Kersten, Sander; Rensen, Patrick C. N.

    2013-01-01

    Angiopoietin-like protein 4 (ANGPTL4) has been identified as an inhibitor of lipoprotein lipase. Preliminary data suggest that plasma nonesterified fatty acids (NEFAs) raise plasma ANGPTL4 concentrations in humans. The objective was to assess plasma ANGPTL4 concentrations after various nutritional

  16. Dietary modulation of plasma angiopoietin-like protein 4 concentrations in healthy volunteers and in patients with type 2 diabetes

    NARCIS (Netherlands)

    Jonker, J.T.; Smit, J.W.A.; Hammer, S.; Snel, M.; Meer, R.W. van der; Lamb, H.J.; Mattijssen, F.; Mudde, K.; Jazet, I.M.; Dekkers, O.M.; Roos, A. de; Romijn, J.A.; Kersten, S.; Rensen, P.C.

    2013-01-01

    BACKGROUND: Angiopoietin-like protein 4 (ANGPTL4) has been identified as an inhibitor of lipoprotein lipase. Preliminary data suggest that plasma nonesterified fatty acids (NEFAs) raise plasma ANGPTL4 concentrations in humans. OBJECTIVE: The objective was to assess plasma ANGPTL4 concentrations

  17. Dietary modulation of plasma angiopoietin-like protein 4 concentrations in healthy volunteers and in patients with type 2 diabetes

    NARCIS (Netherlands)

    Jonker, J.T.; Smit, J.W.A.; Hammer, S.; Snel, M.; Meer, van der R.; Lamb, H.J.; Mattijssen, F.B.J.; Mudde, C.M.; Jazet, I.M.; Dekkers, O.M.; Roos, de A.; Romijn, J.A.; Kersten, A.H.; Rensen, P.C.N.

    2013-01-01

    Background: Angiopoietin-like protein 4 (ANGPTL4) has been identified as an inhibitor of lipoprotein lipase. Preliminary data suggest that plasma nonesterified fatty acids (NEFAs) raise plasma ANGPTL4 concentrations in humans. Objective: The objective was to assess plasma ANGPTL4 concentrations

  18. Radioimmunoassay study of neurophysins in human plasma

    International Nuclear Information System (INIS)

    Reinharz, A.C.; Tissot-Berthet, M.-C.; Vallotton, M.B.

    1978-01-01

    Using a homologous system we have developed a specific and sensitive radioimmunoassay for the measurement of one of the human neurophysins in unextracted human plasma. This neurophysin is specifically secreted in response to oestrogen and has therefore been referred to as human oestrogen-stimulated neurophysin (h-OeSN). The plasma concentration was 0.57 plus minus 0.17 ng/ml (SD) in females and 0.88 plus minus 0.76 ng/ml (SD) in males. This difference is not significant. In women on oral contraceptives, plasma h-OeSN was 2.0 plus minus 1.1 ng/ml. During pregnancy h-OeSN increased progressively to 3.7 plus minus 2.9 ng/ml at the end of the first trimester, and 5.2 plus minus 2.8 ng/ml at term. Plasma h-OeSN concentrations increased rapidly and markedly in men treated with ethinyl-oestradiol. We have also demonstrated the presence of h-OeSN in amniotic and cerebrospinal fluids. A second human neurophysin, which is stimulated by nicotine but not by oestrogen, was also measured. This neurophysin was monitored by a heterologous system using antiserum raised against bovine neurophysin II (b-NII), and b-NII as the standard and tracer. (author)

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

    Directory of Open Access Journals (Sweden)

    Lulu Jia

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

  20. Human plasminogen binding protein tetranectin

    DEFF Research Database (Denmark)

    Kastrup, J S; Rasmussen, H; Nielsen, B B

    1997-01-01

    The recombinant human plasminogen binding protein tetranectin (TN) and the C-type lectin CRD of this protein (TN3) have been crystallized. TN3 crystallizes in the tetragonal space group P4(2)2(1)2 with cell dimensions a = b = 64.0, c = 75.7 A and with one molecule per asymmetric unit. The crystals...... to at least 2.5 A. A full data set has been collected to 3.0 A. The asymmetric unit contains one monomer of TN. Molecular replacement solutions for TN3 and TN have been obtained using the structure of the C-type lectin CRD of rat mannose-binding protein as search model. The rhombohedral space group indicates...

  1. Cold Atmospheric Plasma Manipulation of Proteins in Food Systems

    DEFF Research Database (Denmark)

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

    2017-01-01

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

  2. The Complex Exogenous RNA Spectra in Human Plasma: An Interface with Human Gut Biota?

    Science.gov (United States)

    Wang, Kai; Li, Hong; Yuan, Yue; Etheridge, Alton; Zhou, Yong; Huang, David; Wilmes, Paul; Galas, David

    2012-01-01

    Human plasma has long been a rich source for biomarker discovery. It has recently become clear that plasma RNA molecules, such as microRNA, in addition to proteins are common and can serve as biomarkers. Surveying human plasma for microRNA biomarkers using next generation sequencing technology, we observed that a significant fraction of the circulating RNA appear to originate from exogenous species. With careful analysis of sequence error statistics and other controls, we demonstrated that there is a wide range of RNA from many different organisms, including bacteria and fungi as well as from other species. These RNAs may be associated with protein, lipid or other molecules protecting them from RNase activity in plasma. Some of these RNAs are detected in intracellular complexes and may be able to influence cellular activities under in vitro conditions. These findings raise the possibility that plasma RNAs of exogenous origin may serve as signaling molecules mediating for example the human-microbiome interaction and may affect and/or indicate the state of human health. PMID:23251414

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

    Science.gov (United States)

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

    1983-01-01

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

  4. A radioimmunoassay of gastric inhibitory polypeptide in human plasma

    International Nuclear Information System (INIS)

    Sarson, D.L.; Bryant, M.G.; Bloom, S.R.

    1980-01-01

    A sensitive radioimmunoassay for the measurement of human gastric inhibitory polypeptide (GIP), using pure porcine GIP, has been developed. Cross-reactivity of the antiserum with all available mammalian gut peptide preparations was negligible with the exception of glucagon when it was approximately 1%. Two major molecular forms of GIP were detectable in plasma and tissue extracts, one of large molecular size and the other corresponding to the elution coefficient of pure porcine standard. Concentrations of GIP in plasma from 50 normal subjects after overnight fasting were 9+-1.0(S.E.M.) pmol/1 rising to a peak of 34+-2.8 pmol/1 following the ingestion of a small mixed test meal. Ingestion of glucose or fat resulted in a similar rise of plasma GIP, whereas no change was observed after the ingestion of protein. (author)

  5. A Modified Method for Determination of Lumefantrine in Human Plasma by HPLC-UV and Combination of Protein Precipitation and Solid-Phase Extraction: Application to a Pharmacokinetic Study

    Directory of Open Access Journals (Sweden)

    Liusheng Huang

    2010-03-01

    Full Text Available An HPLC-UV method was developed and validated for the determination of lumefantrine in human plasma. Lumefantrine and its internal standard halofantrine were extracted from plasma samples using protein precipitation with acetonitrile (0.2% perchloric acid followed by solid-phase extraction with Hypersep C8 cartridges. Chromatographic separation was performed on a Zorbax SB-CN HPLC column (3.0 × 150 mm, 3.5 μm with water/methanol (0.1% TFA as the mobile phases in a gradient elution mode. Detection was performed using UV/vis detector at λ = 335 nm. The method showed to be linear over a range of 50–10,000 ng/mL with acceptable intra- and inter-day precision and accuracy. The mean recoveries were 88.2% for lumefatrine and 84.5% for the I.S. The internal standard halofantrine is readily available from commercial sources. This method was successfully applied to a pharmacokinetic interaction study between a first-line antimalarial combination (artemether—lumefantrine and antiretroviral therapy.

  6. A high-performance liquid chromatography-tandem mass spectrometry method coupled with protein precipitation for determination of granisetron in human plasma and its application to a comparative pharmacokinetic study.

    Science.gov (United States)

    Zhou, Ying; Jiang, Ji; Hu, Pei; Wang, Hongyun

    2014-12-01

    A rapid, simple and validated method based on liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) has been developed for the determination of granisetron in human plasma. Plasma samples were pre-purified by protein precipitation procedure. The chromatographic separation was achieved with Synergi Polar-RP (75 × 2 mm, 4 µm) column using a mixture of 5 mm pH4.0 ammonium formate and methanol (300:316, v/v) under isocratic conditions at a flow rate of 0.3 mL/min. The detection was performed on a triple quadrupole tandem mass spectrometer in multiple reaction monitoring mode using positive electrospray ionization. The analysis time was about 2.5 min. The method was fully validated over the concentration range 0.1-10 ng/mL. The lower limit of quantification was 0.1 ng/mL. Inter- and intra-batch precision was granisetron in Chinese healthy subjects. Copyright © 2014 John Wiley & Sons, Ltd.

  7. Protein tyrosine adduct in humans self-poisoned by chlorpyrifos

    Science.gov (United States)

    Li, Bin; Eyer, Peter; Eddleston, Michael; Jiang, Wei; Schopfer, Lawrence M.; Lockridge, Oksana

    2013-01-01

    Studies of human cases of self-inflicted poisoning suggest that chlorpyrifos oxon reacts not only with acetylcholinesterase and butyrylcholinesterase but also with other blood proteins. A favored candidate is albumin because in vitro and animal studies have identified tyrosine 411 of albumin as a site covalently modified by organophosphorus poisons. Our goal was to test this proposal in humans by determining whether plasma from humans poisoned by chlorpyrifos has adducts on tyrosine. Plasma samples from 5 self-poisoned humans were drawn at various time intervals after ingestion of chlorpyrifos for a total of 34 samples. All 34 samples were analyzed for plasma levels of chlorpyrifos and chlorpyrifos oxon (CPO) as a function of time post-ingestion. Eleven samples were analyzed for the presence of diethoxyphosphorylated tyrosine by mass spectrometry. Six samples yielded diethoxyphosphorylated tyrosine in pronase digests. Blood collected as late as 5 days after chlorpyrifos ingestion was positive for CPO-tyrosine, consistent with the 20-day half-life of albumin. High plasma CPO levels did not predict detectable levels of CPO-tyrosine. CPO-tyrosine was identified in pralidoxime treated patients as well as in patients not treated with pralidoxime, indicating that pralidoxime does not reverse CPO binding to tyrosine in humans. Plasma butyrylcholinesterase was a more sensitive biomarker of exposure than adducts on tyrosine. In conclusion, chlorpyrifos oxon makes a stable covalent adduct on the tyrosine residue of blood proteins in humans who ingested chlorpyrifos. PMID:23566956

  8. Characterization of the human plasma phosphoproteome using linear ion trap mass spectrometry and multiple search engines.

    Science.gov (United States)

    Carrascal, Montserrat; Gay, Marina; Ovelleiro, David; Casas, Vanessa; Gelpí, Emilio; Abian, Joaquin

    2010-02-05

    Major plasma protein families play different roles in blood physiology and hemostasis and in immunodefense. Other proteins in plasma can be involved in signaling as chemical messengers or constitute biological markers of the status of distant tissues. In this respect, the plasma phosphoproteome holds potentially relevant information on the mechanisms modulating these processes through the regulation of protein activity. In this work we describe for the first time a collection of phosphopeptides identified in human plasma using immunoaffinity separation of the seven major serum protein families from other plasma proteins, SCX fractionation, and TiO(2) purification prior to LC-MS/MS analysis. One-hundred and twenty-seven phosphosites in 138 phosphopeptides mapping 70 phosphoproteins were identified with FDR search engines.

  9. Proteomic analysis of heparin-binding proteins from human seminal ...

    Indian Academy of Sciences (India)

    Prakash

    (MALDI TOF/MS) for protein analysis of human HBPs. We resolved 70 ... Thus, the combined effects of seminal plasma components support the survival of ...... The BBXB motif of RANTES is the principal site for heparin binding and controls ...

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

    Data.gov (United States)

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

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

    African Journals Online (AJOL)

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

  12. Radioimmunoassay of arginine vasopressin in human plasma

    International Nuclear Information System (INIS)

    Uhlich, E.; Weber, P.; Groeschel-Stewart, U.; Roeschlau, T.; Wuerzburg Univ.

    1975-01-01

    Antibodies for the radioimmunoassay of arginine vasopressin (AVP) described here were produced in rabbits using synthetic AVP coupled to rabbit γ-globulin with carbodiimide. In three out of six rabbits, significant antibody titres were obtained. Using the best antisera produced, 40% of labelled AVP was bound at a final dilution of 1 : 50,000. After iodination of synthetic AVP with 125 I using the chloramin-T method, a gel filtration on Sephadex G-25 was performed to purify the iodinated AVP. For separation of antibody bound and free hormone, a second antibody precipitation was used. There was no crossreactivity with oxytocin. AVP was extracted from plasma after ammoniumsulfate precipitation of the proteins by adsorption to Florisil. The recovery of AVP added to plasma in amounts of 5-25 pg/ml was 60 +- 15% (n = 6). The minimum amount of AVP detectable was 1 pg per ml plasma. The plasma level in normal adults under standard conditions was 3.4 +- 2.2 pg/ml. This is in agreement with data recently published by other researchers. The applicability and reproducibility was further tested in measurements of samples taken hourly during the entire day under water diuresis and after hormonal stimulation of AVP. (orig.) [de

  13. Double Stranded RNA in Human Seminal Plasma

    Directory of Open Access Journals (Sweden)

    Maxim V. Zagoskin

    2017-10-01

    Full Text Available Recently, human semen was shown to contain cell-free nucleic acids, such as DNA, long single stranded RNA, and small RNAs–miRNA and piRNA. The RNAs have been suggested to have potential biological roles as communication molecules between cells and in the temporal and spatial regulation of gene expression in the male reproductive system. Here we demonstrate that human seminal plasma contains a variety of cell-free dsRNAs, describe a robust method to isolate this type of nucleic acid in preparative amounts, and discuss the potential biological roles of these molecules in inheritance. dsRNA plays a role in a variety of biological processes, including gene regulation, is extremely stable and can gain access to cells from the extracellular medium. We suggest that one of the possible functions of dsRNA in human seminal plasma may be to influence human oocytes and therefore, influence the offspring. It also remains possible that these dsRNAs might have potential use as biomarkers for the study of human physiopathological conditions and genetic variation.

  14. Protein tyrosine adduct in humans self-poisoned by chlorpyrifos

    International Nuclear Information System (INIS)

    Li, Bin; Eyer, Peter; Eddleston, Michael; Jiang, Wei; Schopfer, Lawrence M.; Lockridge, Oksana

    2013-01-01

    Studies of human cases of self-inflicted poisoning suggest that chlorpyrifos oxon reacts not only with acetylcholinesterase and butyrylcholinesterase but also with other blood proteins. A favored candidate is albumin because in vitro and animal studies have identified tyrosine 411 of albumin as a site covalently modified by organophosphorus poisons. Our goal was to test this proposal in humans by determining whether plasma from humans poisoned by chlorpyrifos has adducts on tyrosine. Plasma samples from 5 self-poisoned humans were drawn at various time intervals after ingestion of chlorpyrifos for a total of 34 samples. All 34 samples were analyzed for plasma levels of chlorpyrifos and chlorpyrifos oxon (CPO) as a function of time post-ingestion. Eleven samples were analyzed for the presence of diethoxyphosphorylated tyrosine by mass spectrometry. Six samples yielded diethoxyphosphorylated tyrosine in pronase digests. Blood collected as late as 5 days after chlorpyrifos ingestion was positive for CPO-tyrosine, consistent with the 20-day half-life of albumin. High plasma CPO levels did not predict detectable levels of CPO-tyrosine. CPO-tyrosine was identified in pralidoxime treated patients as well as in patients not treated with pralidoxime, indicating that pralidoxime does not reverse CPO binding to tyrosine in humans. Plasma butyrylcholinesterase was a more sensitive biomarker of exposure than adducts on tyrosine. In conclusion, chlorpyrifos oxon makes a stable covalent adduct on the tyrosine residue of blood proteins in humans who ingested chlorpyrifos. - Highlights: • Chlorpyrifos-poisoned patients have adducts on protein tyrosine. • Diethoxyphosphate-tyrosine does not lose an alkyl group. • Proteins in addition to AChE and BChE are modified by organophosphates

  15. Protein tyrosine adduct in humans self-poisoned by chlorpyrifos

    Energy Technology Data Exchange (ETDEWEB)

    Li, Bin, E-mail: binli@unmc.edu [Eppley Institute, University of Nebraska Medical Center, Omaha, NE 68198-5950 (United States); Eyer, Peter, E-mail: peter.eyer@lrz.uni-muenchen.de [Walther-Straub-Institut Für Pharmakologie und Toxikologie, Ludwig-Maximilians-Universität München, 80336 München (Germany); Eddleston, Michael, E-mail: M.Eddleston@ed.ac.uk [Clinical Pharmacology Unit, University of Edinburgh, Edinburgh (United Kingdom); Jiang, Wei, E-mail: wjiang@unmc.edu [Eppley Institute, University of Nebraska Medical Center, Omaha, NE 68198-5950 (United States); Schopfer, Lawrence M., E-mail: lmschopf@unmc.edu [Eppley Institute, University of Nebraska Medical Center, Omaha, NE 68198-5950 (United States); Lockridge, Oksana, E-mail: olockrid@unmc.edu [Eppley Institute, University of Nebraska Medical Center, Omaha, NE 68198-5950 (United States)

    2013-06-15

    Studies of human cases of self-inflicted poisoning suggest that chlorpyrifos oxon reacts not only with acetylcholinesterase and butyrylcholinesterase but also with other blood proteins. A favored candidate is albumin because in vitro and animal studies have identified tyrosine 411 of albumin as a site covalently modified by organophosphorus poisons. Our goal was to test this proposal in humans by determining whether plasma from humans poisoned by chlorpyrifos has adducts on tyrosine. Plasma samples from 5 self-poisoned humans were drawn at various time intervals after ingestion of chlorpyrifos for a total of 34 samples. All 34 samples were analyzed for plasma levels of chlorpyrifos and chlorpyrifos oxon (CPO) as a function of time post-ingestion. Eleven samples were analyzed for the presence of diethoxyphosphorylated tyrosine by mass spectrometry. Six samples yielded diethoxyphosphorylated tyrosine in pronase digests. Blood collected as late as 5 days after chlorpyrifos ingestion was positive for CPO-tyrosine, consistent with the 20-day half-life of albumin. High plasma CPO levels did not predict detectable levels of CPO-tyrosine. CPO-tyrosine was identified in pralidoxime treated patients as well as in patients not treated with pralidoxime, indicating that pralidoxime does not reverse CPO binding to tyrosine in humans. Plasma butyrylcholinesterase was a more sensitive biomarker of exposure than adducts on tyrosine. In conclusion, chlorpyrifos oxon makes a stable covalent adduct on the tyrosine residue of blood proteins in humans who ingested chlorpyrifos. - Highlights: • Chlorpyrifos-poisoned patients have adducts on protein tyrosine. • Diethoxyphosphate-tyrosine does not lose an alkyl group. • Proteins in addition to AChE and BChE are modified by organophosphates.

  16. Bovine plasma protein fractionation by ion exchange chromatography.

    Science.gov (United States)

    Moure, F; Rendueles, M; Díaz, M

    2004-12-01

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

  17. Transport proteins of the plant plasma membrane

    Science.gov (United States)

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

    1996-01-01

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

  18. Posttranslational modifications in human plasma MBL and human recombinant MBL

    DEFF Research Database (Denmark)

    Jensen, Pia Hønnerup; Laursen, Inga; Matthiesen, Finn

    2007-01-01

    the intact protein in its active conformation. For the first time, positions and occupation frequency of partial hydroxylations and partial glycosylations are reported in MBL. Hydroxylation and glycosylation patterns of both recombinant and plasma derived MBL were determined, using a combination of mass......Mannan-binding lectin (MBL) is a complex serum protein that plays an important role in innate immunity. In addition to assuming several different oligomeric forms, the polypeptide itself is highly heterogeneous. This heterogeneity is due to post-translational modifications, which help to stabilize......(202)) was modified in trace amounts to dehydroalanine, as detected by a 34 Da mass loss. This impairs the formation of a disulphide bond in the carbohydrate recognition domain. The dehydroalanine was identified in similar small amounts in both recombinant and plasma-derived MBL....

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

    DEFF Research Database (Denmark)

    Gaidamauskas, Ervinas

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

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

    Science.gov (United States)

    Luschnig, Christian; Vert, Grégory

    2014-08-01

    Plants are permanently situated in a fixed location and thus are well adapted to sense and respond to environmental stimuli and developmental cues. At the cellular level, several of these responses require delicate adjustments that affect the activity and steady-state levels of plasma membrane proteins. These adjustments involve both vesicular transport to the plasma membrane and protein internalization via endocytic sorting. A substantial part of our current knowledge of plant plasma membrane protein sorting is based on studies of PIN-FORMED (PIN) auxin transport proteins, which are found at distinct plasma membrane domains and have been implicated in directional efflux of the plant hormone auxin. Here, we discuss the mechanisms involved in establishing such polar protein distributions, focusing on PINs and other key plant plasma membrane proteins, and we highlight the pathways that allow for dynamic adjustments in protein distribution and turnover, which together constitute a versatile framework that underlies the remarkable capabilities of plants to adjust growth and development in their ever-changing environment. © 2014. Published by The Company of Biologists Ltd.

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

    Science.gov (United States)

    Koldsø, Heidi; Sansom, Mark S P

    2015-11-25

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

  2. Human Antimicrobial Peptides and Proteins

    Directory of Open Access Journals (Sweden)

    Guangshun Wang

    2014-05-01

    Full Text Available As the key components of innate immunity, human host defense antimicrobial peptides and proteins (AMPs play a critical role in warding off invading microbial pathogens. In addition, AMPs can possess other biological functions such as apoptosis, wound healing, and immune modulation. This article provides an overview on the identification, activity, 3D structure, and mechanism of action of human AMPs selected from the antimicrobial peptide database. Over 100 such peptides have been identified from a variety of tissues and epithelial surfaces, including skin, eyes, ears, mouths, gut, immune, nervous and urinary systems. These peptides vary from 10 to 150 amino acids with a net charge between −3 and +20 and a hydrophobic content below 60%. The sequence diversity enables human AMPs to adopt various 3D structures and to attack pathogens by different mechanisms. While α-defensin HD-6 can self-assemble on the bacterial surface into nanonets to entangle bacteria, both HNP-1 and β-defensin hBD-3 are able to block cell wall biosynthesis by binding to lipid II. Lysozyme is well-characterized to cleave bacterial cell wall polysaccharides but can also kill bacteria by a non-catalytic mechanism. The two hydrophobic domains in the long amphipathic α-helix of human cathelicidin LL-37 lays the basis for binding and disrupting the curved anionic bacterial membrane surfaces by forming pores or via the carpet model. Furthermore, dermcidin may serve as ion channel by forming a long helix-bundle structure. In addition, the C-type lectin RegIIIα can initially recognize bacterial peptidoglycans followed by pore formation in the membrane. Finally, histatin 5 and GAPDH(2-32 can enter microbial cells to exert their effects. It appears that granulysin enters cells and kills intracellular pathogens with the aid of pore-forming perforin. This arsenal of human defense proteins not only keeps us healthy but also inspires the development of a new generation of personalized

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

    International Nuclear Information System (INIS)

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

    1979-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2017-04-01

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

  5. A catalogue of human secreted proteins and its implications

    Directory of Open Access Journals (Sweden)

    Shivakumar Keerthikumar

    2016-11-01

    Full Text Available Under both normal and pathological conditions, cells secrete variety of proteins through classical and non-classical secretory pathways into the extracellular space. Majority of these proteins represent pathophysiology of the cell from which it is secreted. Recently, though more than 92% of the protein coding genes has been mapped by human proteome map project, but number of those proteins that constitutes secretome of the cell still remains elusive. Secreted proteins or the secretome can be accessible in bodily fluids and hence are considered as potential biomarkers to discriminate between healthy and diseased individuals. In order to facilitate the biomarker discovery and to further aid clinicians and scientists working in these arenas, we have compiled and catalogued secreted proteins from the human proteome using integrated bioinformatics approach. In this study, nearly 14% of the human proteome is likely to be secreted through classical and non-classical secretory pathways. Out of which, ~38% of these secreted proteins were found in extracellular vesicles including exosomes and shedding microvesicles. Among these secreted proteins, 94% were detected in human bodily fluids including blood, plasma, serum, saliva, semen, tear and urine. We anticipate that this high confidence list of secreted proteins could serve as a compendium of candidate biomarkers. In addition, the catalogue may provide functional insights in understanding the molecular mechanisms involved in various physiological and pathophysiological conditions of the cell.

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

    DEFF Research Database (Denmark)

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

    1996-01-01

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

  7. Studies on the interaction of lidocaine with plasma proteins

    International Nuclear Information System (INIS)

    Adotey, J.

    1985-01-01

    This study sought to quantitate lidocaine's interaction with alpha-1-acid glycoprotein (AAG), human serum albumin (HSA), and AAG in the presence of HSA, and to determine the extent of displacement of lidocaine from its binding site(s) by selected cardiovascular drugs (dipyridamole, disopyramide and quinidine). Since the limited experimental work reported in this area has involved the use of a single lidocaine concentration, this study involved the evaluation of a range of lidocaine concentrations. Lidocaine interaction with plasma proteins (AAG and HSA) was studied at 37 0 C using an isothermal equilibrium dialysis system and 14 C-lidocaine HCl. A dialysis membrane (M.W. cutoff 12,000 to 14,000) separated the two chambers of each dialysis cell. The extent of 14 C-lidocaine dialysis was studied with respect to both drug and protein concentrations. Aliquots of each chamber of each of the cells were subjected to liquid scintillation counting (LSC) analyses for 14 C-lidocaine. The ratio of bound to free (R/F) lidocaine was evaluated as a function of AAG concentration from the LSC data. Scatchard and/or Rosenthal analyses were employed to evaluate n and k values where appropriate. Linear and multiple linear regression analyses of the data were appropriately performed

  8. Human plasma lecithin-cholesterol acyltransferase

    International Nuclear Information System (INIS)

    Jauhiainen, M.; Stevenson, K.J.; Dolphin, P.J.

    1988-01-01

    Lecithin-cholesterol acyltransferase (LCAT) is a plasma enzyme which catalyzes the transacylation of the fatty acid at the sn-2 position of lecithin to cholesterol forming lysolecithin and cholesteryl ester. The substrates for and products of this reaction are present within the plasma lipoproteins upon which the enzyme acts to form the majority of cholesteryl ester in human plasma. The authors proposed a covalent catalytic mechanism of action for LCAT in which serine and histidine residues mediate lecithin cleavage and two cysteine residues cholesterol esterification. With the aid of sulfhydryl reactive trivalent organoarsenical compounds which are specific for vicinal thiols they have probed the geometry of the catalytic site. They conclude that the two catalytic cysteine residues of LCAT (Cys 31 and Cys 184 ) are vicinal with a calculated distance between their sulfur atoms of 3.50-3.62 A. The additional residue alkylated by teh bifunctional reagent is within the catalytic site and may represent a previously identified catalytic serine or histidine residue

  9. Bioassay of procoagulant albumin in human plasma.

    Science.gov (United States)

    Grosset, A; Liu, L; Parker, C J; Rodgers, G M

    1994-09-01

    Procoagulant albumin (P-Al) is present in normal human plasma and increases monocyte and endothelial cell expression of tissue factor activity. To develop a bioassay for P-Al, we partially purified plasma from healthy volunteers and several patient groups using BaCl2 and (NH4)2SO4 precipitation. The samples were assayed for tissue factor (TF) inducing activity, expressed as a percentage increase compared to a serum-free media control. Over six months, the assay was reproducible in stored samples and in serial samples from normal volunteers. The plasma P-Al activities of 35 volunteers averaged 141 +/- 8.2% (SEM). There was no diurnal variation. There was no difference in the P-Al activity after a 12 hour fast and 2 hours after a large meal in 4 healthy volunteers. There was no increase in activity (r = 0.16) with the subject's age. The average activity from 16 poorly-controlled diabetics was 131 +/- 11% (SEM). No alteration in activity was seen with samples from patients with uremia, liver dysfunction, hemophilia, thrombotic events, or adenocarcinoma. These results indicate that P-Al activity can be bioassayed in individual patient samples; however, pathologic states associated with abnormal P-Al-induced tissue factor activity presently remain unidentified.

  10. New validated method for piracetam HPLC determination in human plasma.

    Science.gov (United States)

    Curticapean, Augustin; Imre, Silvia

    2007-01-10

    The new method for HPLC determination of piracetam in human plasma was developed and validated by a new approach. The simple determination by UV detection was performed on supernatant, obtained from plasma, after proteins precipitation with perchloric acid. The chromatographic separation of piracetam under a gradient elution was achieved at room temperature with a RP-18 LiChroSpher 100 column and aqueous mobile phase containing acetonitrile and methanol. The quantitative determination of piracetam was performed at 200 nm with a lower limit of quantification LLQ=2 microg/ml. For this limit, the calculated values of the coefficient of variation and difference between mean and the nominal concentration are CV%=9.7 and bias%=0.9 for the intra-day assay, and CV%=19.1 and bias%=-7.45 for the between-days assay. For precision, the range was CV%=1.8/11.6 in the intra-day and between-days assay, and for accuracy, the range was bias%=2.3/14.9 in the intra-day and between-days assay. In addition, the stability of piracetam in different conditions was verified. Piracetam proved to be stable in plasma during 4 weeks at -20 degrees C and for 36 h at 20 degrees C in the supernatant after protein precipitation. The new proposed method was used for a bioequivalence study of two medicines containing 800 mg piracetam.

  11. Plasma metabolism of apolipoprotein A-IV in humans

    International Nuclear Information System (INIS)

    Ghiselli, G.; Krishnan, S.; Beigel, Y.; Gotto, A.M. Jr.

    1986-01-01

    As assessed by molecular sieve chromatography and quantitation by a specific radioimmunoassay, apoA-IV is associated in plasma with the triglyceride-rich lipoproteins, to a high density lipoprotein (HDL) subfraction of smaller size than HDL3, and to the plasma lipoprotein-free fraction (LFF). In this study, the turnover of apoA-IV associated to the triglyceride-rich lipoproteins, HDL and LFF was investigated in vivo in normal volunteers. Human apoA-IV isolated from the thoracic duct lymph chylomicrons was radioiodinated and incubated with plasma withdrawn from normal volunteers after a fatty meal. Radioiodinated apoA-IV-labeled triglyceride-rich lipoproteins, HDL, and LFF were then isolated by chromatography on an AcA 34 column. Shortly after the injection of the radioiodinated apoA-IV-labeled triglyceride-rich lipoproteins, most of the radioactivity could be recovered in the HDL and LFF column fractions. On the other hand, when radioiodinated apoA-IV-labeled HDL or LFF were injected, the radioactivity remained with the originally injected fractions at all times. The residence time in plasma of 125 I-labeled apoA-IV, when injected in association with HDL or LFF, was 1.61 and 0.55 days, respectively. When 125 I-labeled apoA-IV was injected as a free protein, the radioactivity distributed rapidly among the three plasma pools in proportion to their mass. The overall fractional catabolic rate of apoA-IV in plasma was measured in the three normal subjects and averaged 1.56 pools per day. The mean degradation rate of apoA-IV was 8.69 mg/kg X day

  12. A rapid method for selecting suitable animal species for studying pathogen interactions with plasma protein ligands in vivo.

    Science.gov (United States)

    Naudin, Clément; Schumski, Ariane; Salo-Ahen, Outi M H; Herwald, Heiko; Smeds, Emanuel

    2017-05-01

    Species tropism constitutes a serious problem for developing relevant animal models of infection. Human pathogens can express virulence factors that show specific selectivity to human proteins, while their affinity for orthologs from other species can vary significantly. Suitable animal species must be used to analyse whether virulence factors are potential targets for drug development. We developed an assay that rapidly predicts applicable animal species for studying virulence factors binding plasma proteins. We used two well-characterized Staphylococcus aureus proteins, SSL7 and Efb, to develop an ELISA-based inhibition assay using plasma from different animal species. The interaction between SSL7 and human C5 and the binding of Efb to human fibrinogen and human C3 was studied. Affinity experiments and Western blot analyses were used to validate the assay. Human, monkey and cat plasma interfered with binding of SSL7 to human C5. Binding of Efb to human fibrinogen was blocked in human, monkey, gerbil and pig plasma, while human, monkey, gerbil, rabbit, cat and guinea pig plasma inhibited the binding of Efb to human C3. These results emphasize the importance of choosing correct animal models, and thus, our approach is a rapid and cost-effective method that can be used to prevent unnecessary animal experiments. © 2017 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

  13. 2 D gel based analysis of biological variability of the human plasma proteome

    DEFF Research Database (Denmark)

    Rentsch, Maria Louise; Jessen, Flemming

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

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

    Science.gov (United States)

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

    2009-04-13

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

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

    Directory of Open Access Journals (Sweden)

    Rodica Căpriță

    2013-10-01

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

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

    Science.gov (United States)

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

    2014-11-01

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

  17. LC determination of praziquantel in human plasma.

    Science.gov (United States)

    Ridtitid, Wibool; Wongnawa, Malinee; Mahatthanatrakul, Werawath; Punyo, Jarurat; Sunbhanich, Methi

    2002-04-01

    A simple high-performance liquid chromatographic (HPLC) method for the determination of praziquantel in human plasma was developed and validated. The present method was described by adding drop-wise 0.2 M Zinc sulfate and acetonitrile to plasma sample for deproteinization. This method used a reversed-phase Spherisorb ODS 2 column (5 microm), 250 x 4.6 mm i.d. as a stationary phase with a mobile phase consisting of acetonitrile- methanol-water (36:10:54, v/v/v), a flow rate of 1.5 ml/min and UV detection wavelength of 217 nm. Diazepam was used as internal standard. The standard calibration curve was linear over the concentration range of 100-2000 ng/ml (r=0.999). The equation of a linear regression line was y=8.05E-04+7.25E-04x with slope and intercept values of 0.0007 and 0.0008, respectively. The limit of detection was 12.25 ng/ml and the limit of quantification was set at 100 ng/ml. The intra- and inter-day assay coefficients of variation (CV) were 3.0+/-1.7 and 6.3+/-1.9%, respectively. The percentage of recovery was 102.1+/-5.6. Therefore, the HPLC method described here was simple, rapid and reproducible since it did not require extraction and evaporation processes in sample preparation, which will reduce time-consuming or expensive sample preparation.

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

    NARCIS (Netherlands)

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

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

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

    Science.gov (United States)

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

    2008-12-01

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

  20. Novel Parvovirus and Related Variant in Human Plasma

    Science.gov (United States)

    Fryer, Jacqueline F.; Kapoor, Amit; Minor, Philip D.; Delwart, Eric

    2006-01-01

    We report a novel parvovirus (PARV4) and related variants in pooled human plasma used in the manufacture of plasma-derived medical products. Viral DNA was detected by using highly selective polymerase chain reaction assays; 5% of pools tested positive, and amounts of DNA ranged from 106 copies/mL plasma. PMID:16494735

  1. Protein phosphorylation systems in postmortem human brain

    International Nuclear Information System (INIS)

    Walaas, S.I.; Perdahl-Wallace, E.; Winblad, B.; Greengard, P.

    1989-01-01

    Protein phosphorylation systems regulated by cyclic adenosine 3',5'-monophosphate (cyclic AMP), or calcium in conjunction with calmodulin or phospholipid/diacylglycerol, have been studied by phosphorylation in vitro of particulate and soluble fractions from human postmortem brain samples. One-dimensional or two-dimensional gel electrophoretic protein separations were used for analysis. Protein phosphorylation catalyzed by cyclic AMP-dependent protein kinase was found to be highly active in both particulate and soluble preparations throughout the human CNS, with groups of both widely distributed and region-specific substrates being observed in different brain nuclei. Dopamine-innervated parts of the basal ganglia and cerebral cortex contained the phosphoproteins previously observed in rodent basal ganglia. In contrast, calcium/phospholipid-dependent and calcium/calmodulin-dependent protein phosphorylation systems were less prominent in human postmortem brain than in rodent brain, and only a few widely distributed substrates for these protein kinases were found. Protein staining indicated that postmortem proteolysis, particularly of high-molecular-mass proteins, was prominent in deeply located, subcortical regions in the human brain. Our results indicate that it is feasible to use human postmortem brain samples, when obtained under carefully controlled conditions, for qualitative studies on brain protein phosphorylation. Such studies should be of value in studies on human neurological and/or psychiatric disorders

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

    Science.gov (United States)

    Vosolsobě, Stanislav; Petrášek, Jan; Schwarzerová, Kateřina

    2017-05-01

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

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

    African Journals Online (AJOL)

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

  4. Human leukocyte antigen-G in the male reproductive system and in seminal plasma

    DEFF Research Database (Denmark)

    Horup Larsen, Margit; Bzorek, Michael; Pass, Malene B.

    2011-01-01

    -eclampsia. We have investigated whether HLA-G protein is present in human seminal plasma and in different tissue samples of the male reproductive system.Western blot technique and a soluble HLA-G (sHLA-G) assay were used to detect sHLA-G in human seminal plasma samples. Immunohistochemical staining...... was performed on paraffin-embedded tissue samples. We detected sHLA-G protein in seminal plasma, and HLA-G expression in normal testis and in epididymal tissue of the male reproductive system but not in the seminal vesicle. Furthermore, the results indicated a weak expression of HLA–G in hyperplastic prostatic...... tissue. In summary, several of the findings reported in this study suggest an immunoregulatory role of HLA-G in the male reproductive system and in seminal plasma....

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

    DEFF Research Database (Denmark)

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

    2006-01-01

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

  6. Characterization of a cocaine binding protein in human placenta

    International Nuclear Information System (INIS)

    Ahmed, M.S.; Zhou, D.H.; Maulik, D.; Eldefrawi, M.E.

    1990-01-01

    [ 3 H]-Cocaine binding sites are identified in human placental villus tissue plasma membranes. These binding sites are associated with a protein and show saturable and specific binding of [ 3 H]-cocaine with a high affinity site of 170 fmole/mg protein. The binding is lost with pretreatment with trypsin or heat. The membrane bound protein is solubilized with the detergent 3-(3-cholamidopropyl)dimethyl-ammonio-1-propane sulphonate (CHAPS) with retention of its saturable and specific binding of [ 3 H]-cocaine. The detergent-protein complex migrates on a sepharose CL-6B gel chromatography column as a protein with an apparent molecular weight of 75,900. The protein has an S 20,w value of 5.1. The binding of this protein to norcocaine, pseudococaine, nomifensine, imipramine, desipramine, amphetamine and dopamine indicates that it shares some, but not all, the properties of the brain cocaine receptor. The physiologic significance of this protein in human placenta is currently unclear

  7. Robustness of solvent/detergent treatment of plasma derivatives: a data collection from Plasma Protein Therapeutics Association member companies.

    Science.gov (United States)

    Dichtelmüller, Herbert O; Biesert, Lothar; Fabbrizzi, Fabrizio; Gajardo, Rodrigo; Gröner, Albrecht; von Hoegen, Ilka; Jorquera, Juan I; Kempf, Christoph; Kreil, Thomas R; Pifat, Dominique; Osheroff, Wendy; Poelsler, Gerhard

    2009-09-01

    Solvent/detergent (S/D) treatment is an established virus inactivation technology that has been applied in the manufacture of medicinal products derived from human plasma for more than 20 years. Data on the inactivation of enveloped viruses by S/D treatment collected from seven Plasma Protein Therapeutics Association member companies demonstrate the robustness, reliability, and efficacy of this virus inactivation method. The results from 308 studies reflecting production conditions as well as technical variables significantly beyond the product release specification were evaluated for virus inactivation, comprising different combinations of solvent and detergent (tri(n-butyl) phosphate [TNBP]/Tween 80, TNBP/Triton X-100, TNBP/Na-cholate) and different products (Factor [F]VIII, F IX, and intravenous and intramuscular immunoglobulins). Neither product class, process temperature, protein concentration, nor pH value has a significant impact on virus inactivation. A variable that did appear to be critical was the concentration of solvent and detergent. The data presented here demonstrate the robustness of virus inactivation by S/D treatment for a broad spectrum of enveloped test viruses and process variables. Our data substantiate the fact that no transmission of viruses such as human immunodeficiency virus, hepatitis B virus, hepatitis C virus, or of other enveloped viruses was reported for licensed plasma derivatives since the introduction of S/D treatment.

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

    International Nuclear Information System (INIS)

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

    1979-01-01

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

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

    Science.gov (United States)

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

    2013-07-01

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

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

    KAUST Repository

    Capriotti, Anna Laura

    2012-03-01

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

  11. Heat shock proteins on the human sperm surface.

    Science.gov (United States)

    Naaby-Hansen, Soren; Herr, John C

    2010-01-01

    The sperm plasma membrane is known to be critical to fertilization and to be highly regionalized into domains of head, mid- and principal pieces. However, the molecular composition of the sperm plasma membrane and its alterations during genital tract passage, capacitation and the acrosome reaction remains to be fully dissected. A two-dimensional gel-based proteomic study previously identified 98 human sperm proteins which were accessible for surface labelling with both biotin and radioiodine. In this report twelve dually labelled protein spots were excised from stained gels or PDVF membranes and analysed by mass spectrometry (MS) and Edman degradation. Seven members from four different heat shock protein (HSP) families were identified including HYOU1 (ORP150), HSPC1 (HSP86), HSPA5 (Bip), HSPD1 (HSP60), and several isoforms of the two testis-specific HSP70 chaperones HSPA2 and HSPA1L. An antiserum raised against the testis-specific HSPA2 chaperone reacted with three 65kDa HSPA2 isoforms and three high molecular weight surface proteins (78-79kDa, 84kDa and 90-93kDa). These proteins, together with seven 65kDa HSP70 forms, reacted with human anti-sperm IgG antibodies that blocked in vitro fertilization in humans. Three of these surface biotinylated human sperm antigens were immunoprecipitated with a rabbit antiserum raised against a linear peptide epitope in Chlamydia trachomatis HSP70. The results indicate diverse HSP chaperones are accessible for surface labelling on human sperm. Some of these share epitopes with C. trachomatis HSP70, suggesting an association between genital tract infection, immunity to HSP70 and reproductive failure. 2009 Elsevier Ireland Ltd. All rights reserved.

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

    Science.gov (United States)

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

    2014-03-21

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

  13. Human cancer protein-protein interaction network: a structural perspective.

    Directory of Open Access Journals (Sweden)

    Gozde Kar

    2009-12-01

    Full Text Available Protein-protein interaction networks provide a global picture of cellular function and biological processes. Some proteins act as hub proteins, highly connected to others, whereas some others have few interactions. The dysfunction of some interactions causes many diseases, including cancer. Proteins interact through their interfaces. Therefore, studying the interface properties of cancer-related proteins will help explain their role in the interaction networks. Similar or overlapping binding sites should be used repeatedly in single interface hub proteins, making them promiscuous. Alternatively, multi-interface hub proteins make use of several distinct binding sites to bind to different partners. We propose a methodology to integrate protein interfaces into cancer interaction networks (ciSPIN, cancer structural protein interface network. The interactions in the human protein interaction network are replaced by interfaces, coming from either known or predicted complexes. We provide a detailed analysis of cancer related human protein-protein interfaces and the topological properties of the cancer network. The results reveal that cancer-related proteins have smaller, more planar, more charged and less hydrophobic binding sites than non-cancer proteins, which may indicate low affinity and high specificity of the cancer-related interactions. We also classified the genes in ciSPIN according to phenotypes. Within phenotypes, for breast cancer, colorectal cancer and leukemia, interface properties were found to be discriminating from non-cancer interfaces with an accuracy of 71%, 67%, 61%, respectively. In addition, cancer-related proteins tend to interact with their partners through distinct interfaces, corresponding mostly to multi-interface hubs, which comprise 56% of cancer-related proteins, and constituting the nodes with higher essentiality in the network (76%. We illustrate the interface related affinity properties of two cancer-related hub

  14. A Phospholipidomic Analysis of All Defined Human Plasma Lipoproteins

    NARCIS (Netherlands)

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

    2011-01-01

    Since plasma lipoproteins contain both protein and phospholipid components, either may be involved in processes such as atherosclerosis. In this study the identification of plasma lipoprotein-associated phospholipids, which is essential for understanding these processes at the molecular level, are

  15. A phospholipidomic analysis of all defined human plasma lipoproteins

    NARCIS (Netherlands)

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

    2011-01-01

    Since plasma lipoproteins contain both protein and phospholipid components, either may be involved in processes such as atherosclerosis. In this study the identification of plasma lipoprotein-associated phospholipids, which is essential for understanding these processes at the molecular level, are

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

    Directory of Open Access Journals (Sweden)

    Poonam Gautam

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

  17. Purification and preliminary X-ray crystallographic studies of β-microseminoprotein from human seminal plasma

    International Nuclear Information System (INIS)

    Kumar, Vijay; Roske, Yvette; Singh, Nagendra; Heinemann, Udo; Singh, Tej P.; Yadav, Savita

    2009-01-01

    The preliminary X-ray data of β-microseminoprotein isolated from human seminal plasma at 2.4 Å resolution are reported. β-Microseminoprotein (β-MSP) is a small cysteine-rich protein with a molecular mass of 10 kDa. It was first isolated from human seminal plasma and has subsequently been identified from several species. Comparison of the amino-acid sequences of β-MSP proteins suggests that the protein is a rapidly evolving protein. The function of β-MSP is poorly understood. Furthermore, no crystal structure has been reported of any β-MSP; therefore, determination of the crystal structure of β-MSP is the foremost task in order to understand the function of this protein completely. Here, the purification, crystallization and preliminary X-ray diffraction analysis of β-MSP from human seminal plasma are described. The protein was purified using anion-exchange and size-exclusion chromatography and the purified protein was crystallized using 0.1 M ammonium sulfate, 0.1 M HEPES buffer pH 7.0 and 20%(w/v) PEG 3350. The crystals belonged to the tetragonal space group P4 3 22 and contained three β-MSP molecules in the asymmetric unit. X-ray intensity data were collected to 2.4 Å resolution

  18. Antioxidant capacity of plasma after red wine intake in Human

    Directory of Open Access Journals (Sweden)

    M. Gianmmanco

    2009-01-01

    Full Text Available Aim: Antioxidant effects after consumption of red wine have been investigated in several studies but results are contradictory and the difference in the plasma antioxidant capacity (AC after intake of red wine between women and men has never been studies. This work purpose is manifold: to ascertain whether red wine intake modifies the human plasma AC; to study the behaviour of plasma AC of women in comparison with men and finally to investigate on the plasma uric acid concentration and its relationships with the plasma AC after red wine intake.

  19. Protein Misfolding and Human Disease

    DEFF Research Database (Denmark)

    Gregersen, Niels; Bross, Peter Gerd; Vang, Søren

    2006-01-01

    phenylketonuria, Parkinson's disease, α-1-antitrypsin deficiency, familial neurohypophyseal diabetes insipidus, and short-chain acyl-CoA dehydrogenase deficiency. Despite the differences, an emerging paradigm suggests that the cellular effects of protein misfolding provide a common framework that may contribute...

  20. Assignment of the human gene for pregnancy-associated plasma protein A (PAPPA) to 9q33.1 by fluorescence in situ hybridization to mitotic and meiotic chromosomes

    DEFF Research Database (Denmark)

    Silahtaroglu, A N; Tümer, Z; Kristensen, Torsten

    1993-01-01

    Low levels of pregnancy-associated plasma protein A (PAPPA) during the first trimester has been suggested as a biochemical indicator of pregnancies with aneuploid fetuses. Furthermore, the complete absence of PAPPA in pregnancies associated with Cornelia de Lange syndrome (CL) has suggested...... a causal connection between PAPPA and the development of CL. We have assigned the locus for PAPPA to chromosome region 9q33.1 on mitotic and meiotic chromosomes by fluorescence in situ hybridization, using a 3.7-kb partial PAPPA cDNA probe...

  1. Imparting albumin-binding affinity to a human protein by mimicking the contact surface of a bacterial binding protein.

    Science.gov (United States)

    Oshiro, Satoshi; Honda, Shinya

    2014-04-18

    Attachment of a bacterial albumin-binding protein module is an attractive strategy for extending the plasma residence time of protein therapeutics. However, a protein fused with such a bacterial module could induce unfavorable immune reactions. To address this, we designed an alternative binding protein by imparting albumin-binding affinity to a human protein using molecular surface grafting. The result was a series of human-derived 6 helix-bundle proteins, one of which specifically binds to human serum albumin (HSA) with adequate affinity (KD = 100 nM). The proteins were designed by transferring key binding residues of a bacterial albumin-binding module, Finegoldia magna protein G-related albumin-binding domain (GA) module, onto the human protein scaffold. Despite 13-15 mutations, the designed proteins maintain the original secondary structure by virtue of careful grafting based on structural informatics. Competitive binding assays and thermodynamic analyses of the best binders show that the binding mode resembles that of the GA module, suggesting that the contacting surface of the GA module is mimicked well on the designed protein. These results indicate that the designed protein may act as an alternative low-risk binding module to HSA. Furthermore, molecular surface grafting in combination with structural informatics is an effective approach for avoiding deleterious mutations on a target protein and for imparting the binding function of one protein onto another.

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

    Directory of Open Access Journals (Sweden)

    Vegard Lysne

    2015-06-01

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

  3. Mannan-binding proteins from boar seminal plasma

    Czech Academy of Sciences Publication Activity Database

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

    2004-01-01

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

  4. Knowns and unknowns of plasma membrane protein degradation in plants.

    Science.gov (United States)

    Liu, Chuanliang; Shen, Wenjin; Yang, Chao; Zeng, Lizhang; Gao, Caiji

    2018-07-01

    Plasma membrane (PM) not only creates a physical barrier to enclose the intracellular compartments but also mediates the direct communication between plants and the ever-changing environment. A tight control of PM protein homeostasis by selective degradation is thus crucial for proper plant development and plant-environment interactions. Accumulated evidences have shown that a number of plant PM proteins undergo clathrin-dependent or membrane microdomain-associated endocytic routes to vacuole for degradation in a cargo-ubiquitination dependent or independent manner. Besides, several trans-acting determinants involved in the regulation of endocytosis, recycling and multivesicular body-mediated vacuolar sorting have been identified in plants. More interestingly, recent findings have uncovered the participation of selective autophagy in PM protein turnover in plants. Although great progresses have been made to identify the PM proteins that undergo dynamic changes in subcellular localizations and to explore the factors that control the membrane protein trafficking, several questions remain to be answered regarding the molecular mechanisms of PM protein degradation in plants. In this short review article, we briefly summarize recent progress in our understanding of the internalization, sorting and degradation of plant PM proteins. More specifically, we focus on discussing the elusive aspects underlying the pathways of PM protein degradation in plants. Copyright © 2018 Elsevier B.V. All rights reserved.

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

    Science.gov (United States)

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

    2010-06-01

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

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

    Science.gov (United States)

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

    2018-02-28

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

  7. [3H]cholesteryl ester labeling and transfer among human and honhuman primate plasma lipoproteins

    International Nuclear Information System (INIS)

    Thomas, M.S.; Rudel, L.L.

    1983-01-01

    Aliquots of human and nonhuman primate plasma containing 5,5'-dithiobis (2-nitrobenzoic acid) were incubated at 37 0 C in tubes previously coated with trace amounts of tritium-labeled cholesteryl oleate ([ 3 H]CO). Initially, cholesteryl esters were transferred at a rapid rate into plasma after which the rate slowed. During 24 h of incubation, an average of 55% of the [ 3 H]CO transferred from the side of the tube into African green monkey plasma, 44% into human plasma and 21% into rat plasma. Greater than 98% of the radioactive ester transferred into plasma was found to be associated with plasma lipoproteins that were then rapidly separated using vertical rotor density gradient ultracentrifugation. In very low density lipoprotein (VLDL)-poor plasma after 30 min incubations, high density lipoproteins (HDL) contained most of the [ 3 H]CO while 5- to 24-h incubations resulted in increased labeling of low density proteins (LDL). In VLDL-rich plasma, it was found that in addition to the labeling of HDL, VLDL contained about 25% of the labeled cholesteryl esters after 30-min incubations and, as above, the proportion in LDL subsequently increased. Compositional analyses showed that intermediate-sized LDL (ILDL) were accumulating cholesteryl ester mass while transfer occurred. LDL labeled using this method were injected intravenously into monkeys and their removal from plasma was found to be similar to that found for LDL labeled in vivo. It was concluded that this method of plasma lipoprotein cholesteryl ester labeling, presumably a result of cholesteryl ester transfer protein activity, was efficient, resulted in lipoproteins labeled only in the cholesteryl ester moiety, and induced minimal modification of lipoprotein particles that did not alter their biological activity

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

    Directory of Open Access Journals (Sweden)

    Daniel J Sobczynski

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

  9. Comparative studies of human and chicken retinol-binding proteins and prealbumins.

    Science.gov (United States)

    Kopelman, M; Mokady, S; Cogan, U

    1976-08-09

    Microheterogeneity of retinol-binding proteins of human plasma and urine, and of chicken plasma was studied by polyacrylamide gel electrophoresis. All three protein systems were found microheterogenous. Incorporation of retinol into the protein preparations on the one hand, and depletion of these proteins from retinol on the other hand, enabled us to clarify the extent to which the presence or absence of the ligand affects the apparent heterogeneity. Upon electrophoresis, each of the native proteins displayed two pairs of protein zones. It appeared that within each pair the fast moving band corresponded to aporetinol-binding protein which upon binding of retinol was converted to a holoprotein with a slightly lower mobility. However, it did not seem that proteins of one pair were converted to proteins of the second pair upon binding of retinol, substantiating ghe microheterogenous character of this protein system. A rapid, two step procedure for isolation of prealbumins from plasma is described. The method which consists of DEAE-cellulose chromatography follwed by preparative electrophoresis was utilized to separate human and chicken prealbumins. Routine dodecyl sulphate electrophoresis resulted in partial dissociation of human prealbumin but in no dissociation of the chicken protein. More drastic treatments prior to electrophoresis were needed to effect complete disruption of both proteins into subunits.

  10. Inferring high-confidence human protein-protein interactions

    Directory of Open Access Journals (Sweden)

    Yu Xueping

    2012-05-01

    Full Text Available Abstract Background As numerous experimental factors drive the acquisition, identification, and interpretation of protein-protein interactions (PPIs, aggregated assemblies of human PPI data invariably contain experiment-dependent noise. Ascertaining the reliability of PPIs collected from these diverse studies and scoring them to infer high-confidence networks is a non-trivial task. Moreover, a large number of PPIs share the same number of reported occurrences, making it impossible to distinguish the reliability of these PPIs and rank-order them. For example, for the data analyzed here, we found that the majority (>83% of currently available human PPIs have been reported only once. Results In this work, we proposed an unsupervised statistical approach to score a set of diverse, experimentally identified PPIs from nine primary databases to create subsets of high-confidence human PPI networks. We evaluated this ranking method by comparing it with other methods and assessing their ability to retrieve protein associations from a number of diverse and independent reference sets. These reference sets contain known biological data that are either directly or indirectly linked to interactions between proteins. We quantified the average effect of using ranked protein interaction data to retrieve this information and showed that, when compared to randomly ranked interaction data sets, the proposed method created a larger enrichment (~134% than either ranking based on the hypergeometric test (~109% or occurrence ranking (~46%. Conclusions From our evaluations, it was clear that ranked interactions were always of value because higher-ranked PPIs had a higher likelihood of retrieving high-confidence experimental data. Reducing the noise inherent in aggregated experimental PPIs via our ranking scheme further increased the accuracy and enrichment of PPIs derived from a number of biologically relevant data sets. These results suggest that using our high

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

    Directory of Open Access Journals (Sweden)

    Guacyara Motta

    2017-07-01

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

  12. Structural analysis of recombinant human protein QM

    International Nuclear Information System (INIS)

    Gualberto, D.C.H.; Fernandes, J.L.; Silva, F.S.; Saraiva, K.W.; Affonso, R.; Pereira, L.M.; Silva, I.D.C.G.

    2012-01-01

    Full text: The ribosomal protein QM belongs to a family of ribosomal proteins, which is highly conserved from yeast to humans. The presence of the QM protein is necessary for joining the 60S and 40S subunits in a late step of the initiation of mRNA translation. Although the exact extra-ribosomal functions of QM are not yet fully understood, it has been identified as a putative tumor suppressor. This protein was reported to interact with the transcription factor c-Jun and thereby prevent c-Jun actives genes of the cellular growth. In this study, the human QM protein was expressed in bacterial system, in the soluble form and this structure was analyzed by Circular Dichroism and Fluorescence. The results of Circular Dichroism showed that this protein has less alpha helix than beta sheet, as described in the literature. QM protein does not contain a leucine zipper region; however the ion zinc is necessary for binding of QM to c-Jun. Then we analyzed the relationship between the removal of zinc ions and folding of protein. Preliminary results obtained by the technique Fluorescence showed a gradual increase in fluorescence with the addition of increasing concentration of EDTA. This suggests that the zinc is important in the tertiary structure of the protein. More studies are being made for better understand these results. (author)

  13. Calibrated kallikrein generation in human plasma

    DEFF Research Database (Denmark)

    Biltoft, D; Sidelmann, J J; Olsen, L F

    2016-01-01

    generation method as a template. RESULTS: A suitable kallikrein specific fluorogenic substrate was identified (KM=0.91mM, kcat=19s(-1)), and kallikrein generation could be measured in undiluted plasma when silica was added as activator. Disturbing effects, including substrate depletion and the inner......-filter effect, however, affected the signal. These problems were corrected for by external calibration with α2-macroglobulin-kallikrein complexes. Selectivity studies of the substrate, experiments with FXII and PK depleted plasmas, and plasma with high or low complement C1-esterase inhibitor activity indicated...

  14. A Phospholipidomic Analysis of All Defined Human Plasma Lipoproteins

    Science.gov (United States)

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

    2011-01-01

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

  15. Circulating growth hormone (GH)-binding protein complex: a major constituent of plasma GH in man

    International Nuclear Information System (INIS)

    Baumann, G.; Amburn, K.; Shaw, M.A.

    1988-01-01

    The recent discovery of a specific binding protein for human GH (hGH) in human plasma suggests that hGH circulates in part as a complex in association with the binding protein(s). However, the magnitude of the complexed fraction prevailing under physiological conditions is unknown because of 1) dissociation of the complex during analysis and 2) potential differences in the binding characteristics of radiolabeled and native hGH. We conducted experiments designed to minimize dissociation during analysis (gel filtration in prelabeled columns, frontal analysis, and batch molecular sieving) with both native and radioiodinated hGH. All three methods yielded similar estimates for the complexed fraction. In normal plasma the bound fraction for 22 K hGH averaged 50.1% (range, 39-59%), that for 20 K hGH averaged 28.5% (range, 26-31%). Above a hGH level of about 20 ng/ml the bound fraction declines in concentration-dependent manner due to saturation of the binding protein. We conclude that a substantial part of circulating hGH is complexed with carrier proteins. This concept has important implications for the metabolism, distribution, and biological activity of hGH

  16. Plasma Protein Profiles Differ Between Women Diagnosed with Cervical Intraepithelial Neoplasia (CIN 1 and 3

    Directory of Open Access Journals (Sweden)

    Edward E. Partridge

    2006-01-01

    Full Text Available Early detection of precancerous cells in the cervix and their clinical management is the main purpose of cervical cancer prevention and treatment programs. Cytological findings or testing for high risk (HR-human papillomavirus (HPV are inadequately sensitive for use in triage of women at high risk for cervical cancer. The current study is an exploratory study to identify candidate surface-enhanced laser desorption/ionization (SELDI time of flight (TOF mass spectrometry (MS protein profiles in plasma that may distinguish cervical intraepithelial neoplasia (CIN 3 from CIN 1 among women infected with HR-HPV. We evaluated the SELDI-TOF-MS plasma protein profiles of HR-HPV positive 32 women with CIN 3 (cases and 28 women with CIN1 (controls. Case-control status was kept blinded and triplicates of each sample and quality control plasma samples were randomized and after robotic sample preparations were run on WCX2 chips. After alignment of mass/charge (m-z values, an iterative method was used to develop a classifier on a training data set that had 28 cases and 22 controls. The classifier developed was used to classify the subjects in a test data set that has six cases and six controls. The classifier separated the cases from controls in the test set with 100% sensitivity and 100% specificity suggesting the possibility of using plasma SELDI protein profiles to identify women who are likely to have CIN 3 lesions.

  17. Human leukocyte antigen-G in the male reproductive system and in seminal plasma.

    Science.gov (United States)

    Larsen, Margit Hørup; Bzorek, Michael; Pass, Malene B; Larsen, Lise Grupe; Nielsen, Mette Weidinger; Svendsen, Signe Goul; Lindhard, Anette; Hviid, Thomas Vauvert F

    2011-12-01

    One of the non-classical human leukocyte antigen (HLA) class Ib proteins, HLA-G, is believed to exert important immunoregulatory functions, especially during pregnancy. The presence of HLA protein in paternal seminal fluid has been suggested to have an influence on the risk of developing pre-eclampsia. We have investigated whether HLA-G protein is present in human seminal plasma and in different tissue samples of the male reproductive system. Western blot technique and a soluble HLA-G (sHLA-G) assay were used to detect sHLA-G in human seminal plasma samples. Immunohistochemical staining was performed on paraffin-embedded tissue samples. We detected sHLA-G protein in seminal plasma, and HLA-G expression in normal testis and in epididymal tissue of the male reproductive system but not in the seminal vesicle. Furthermore, the results indicated a weak expression of HLA-G in hyperplastic prostatic tissue. In summary, several of the findings reported in this study suggest an immunoregulatory role of HLA-G in the male reproductive system and in seminal plasma.

  18. Quantitative, multiplexed workflow for deep analysis of human blood plasma and biomarker discovery by mass spectrometry.

    Science.gov (United States)

    Keshishian, Hasmik; Burgess, Michael W; Specht, Harrison; Wallace, Luke; Clauser, Karl R; Gillette, Michael A; Carr, Steven A

    2017-08-01

    Proteomic characterization of blood plasma is of central importance to clinical proteomics and particularly to biomarker discovery studies. The vast dynamic range and high complexity of the plasma proteome have, however, proven to be serious challenges and have often led to unacceptable tradeoffs between depth of coverage and sample throughput. We present an optimized sample-processing pipeline for analysis of the human plasma proteome that provides greatly increased depth of detection, improved quantitative precision and much higher sample analysis throughput as compared with prior methods. The process includes abundant protein depletion, isobaric labeling at the peptide level for multiplexed relative quantification and ultra-high-performance liquid chromatography coupled to accurate-mass, high-resolution tandem mass spectrometry analysis of peptides fractionated off-line by basic pH reversed-phase (bRP) chromatography. The overall reproducibility of the process, including immunoaffinity depletion, is high, with a process replicate coefficient of variation (CV) of 4,500 proteins are detected and quantified per patient sample on average, with two or more peptides per protein and starting from as little as 200 μl of plasma. The approach can be multiplexed up to 10-plex using tandem mass tags (TMT) reagents, further increasing throughput, albeit with some decrease in the number of proteins quantified. In addition, we provide a rapid protocol for analysis of nonfractionated depleted plasma samples analyzed in 10-plex. This provides ∼600 quantified proteins for each of the ten samples in ∼5 h of instrument time.

  19. Radioimmunoassay of cholecystokinin in human plasma

    International Nuclear Information System (INIS)

    Byrnes, D.J.; Henderson, L.; Borody, T.; Rehfeld, J.F.

    1981-01-01

    A sensitive radioimmunoassay for cholecystokinin (CCK) has been developed. Porcine CCK-33 was labelled by conjugation with 125 I-hydroxyphenyl-propionic acid succinimide ester. Antibodies were raised against porcine CCK-33 covalently coupled to egg albumin. Plasma samples were extracted with 96% ethanol prior to assay. Free and bound hormone were separated by dextran-coated charcoal. The antibodies bound CCK-8 and CCK-33 with equimolar potency. The assay detection limit was 1 pmol/l plasma. Within and between assay coefficients of variation were +-12.7 and 13.0% at mean plasma CCK concentrations of 13.2 and 13.6 pmol/l. The concentration of CCK in 47 normal fasting subjects ranged from undetectable to 22 pmol/l. Ingestion of a mixed meal in 9 normal subjects increased the plasma concentration from 8.3 +- 2.5 S.E. to 24.4 +- 6.5 pmol/l. (Auth.)

  20. A simple and highly sensitive on-line column extraction liquid chromatography-tandem mass spectrometry method for the determination of protein-unbound tacrolimus in human plasma samples.

    Science.gov (United States)

    Bittersohl, Heike; Schniedewind, Björn; Christians, Uwe; Luppa, Peter B

    2018-04-27

    Therapeutic drug monitoring (TDM) of the immunosuppressive drug tacrolimus is essential to avoid side effects and rejection of the allograft after transplantation. In the blood circulation, tacrolimus is largely located inside erythrocytes or bound to plasma proteins and less than 0.1% is protein-unbound (free). One basic principle of clinical pharmacology is that only free drug is pharmacologically active and monitoring this portion has the potential to better reflect the drug effect than conventional measurements of total tacrolimus in whole blood. To address this, a highly sensitive and straightforward on-line liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed, validated and applied to patient plasma samples. The sample preparation included ultracentrifugation and addition of the stable isotope labeled drug analogue D2,13C-tacrolimus, followed by on-line sample extraction and measurement using a Sciex QTRAP ® 6500 in the multiple reaction monitoring mode. Due to very low concentrations of protein-unbound tacrolimus, it was important to develop a highly sensitive, precise and accurate assay. Here, we first report the efficient formation of tacrolimus lithium adduct ions, which greatly increased assay sensitivity. A lower limit of quantification (LLOQ) of 1 pg/mL (10 fg on column) was achieved and the assay was linear between 1 and 200 pg/mL. There was no carry-over detected. The inaccuracy ranged from -9.8 to 7.4% and the greatest imprecision was 7.5%. The matrix factor was found to be smaller than 1.1%. In summary, this method represents a suitable tool to investigate the potential clinical value of free tacrolimus monitoring in organ transplant recipients. Copyright © 2018 Elsevier B.V. All rights reserved.

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

    Science.gov (United States)

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

    2016-09-01

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

  2. Bystander apoptosis in human cells mediated by irradiated blood plasma

    Energy Technology Data Exchange (ETDEWEB)

    Vinnikov, Volodymyr, E-mail: vlad.vinnikov@mail.ru [Grigoriev Institute for Medical Radiology of the National Academy of Medical Science of Ukraine (Ukraine); Lloyd, David; Finnon, Paul [Centre for Radiation, Chemical and Environmental Hazards of the Health Protection Agency of the United Kingdom (United Kingdom)

    2012-03-01

    Following exposure to high doses of ionizing radiation, due to an accident or during radiotherapy, bystander signalling poses a potential hazard to unirradiated cells and tissues. This process can be mediated by factors circulating in blood plasma. Thus, we assessed the ability of plasma taken from in vitro irradiated human blood to produce a direct cytotoxic effect, by inducing apoptosis in primary human peripheral blood mononuclear cells (PBM), which mainly comprised G{sub 0}-stage lymphocytes. Plasma was collected from healthy donors' blood irradiated in vitro to 0-40 Gy acute {gamma}-rays. Reporter PBM were separated from unirradiated blood with Histopaque and held in medium with the test plasma for 24 h at 37 Degree-Sign C. Additionally, plasma from in vitro irradiated and unirradiated blood was tested against PBM collected from blood given 4 Gy. Apoptosis in reporter PBM was measured by the Annexin V test using flow cytometry. Plasma collected from unirradiated and irradiated blood did not produce any apoptotic response above the control level in unirradiated reporter PBM. Surprisingly, plasma from irradiated blood caused a dose-dependent reduction of apoptosis in irradiated reporter PBM. The yields of radiation-induced cell death in irradiated reporter PBM (after subtracting the respective values in unirradiated reporter PBM) were 22.2 {+-} 1.8% in plasma-free cultures, 21.6 {+-} 1.1% in cultures treated with plasma from unirradiated blood, 20.2 {+-} 1.4% in cultures with plasma from blood given 2-4 Gy and 16.7 {+-} 3.2% in cultures with plasma from blood given 6-10 Gy. These results suggested that irradiated blood plasma did not cause a radiation-induced bystander cell-killing effect. Instead, a reduction of apoptosis in irradiated reporter cells cultured with irradiated blood plasma has implications concerning oncogenic risk from mutated cells surviving after high dose in vivo irradiation (e.g. radiotherapy) and requires further study.

  3. Bystander apoptosis in human cells mediated by irradiated blood plasma

    International Nuclear Information System (INIS)

    Vinnikov, Volodymyr; Lloyd, David; Finnon, Paul

    2012-01-01

    Following exposure to high doses of ionizing radiation, due to an accident or during radiotherapy, bystander signalling poses a potential hazard to unirradiated cells and tissues. This process can be mediated by factors circulating in blood plasma. Thus, we assessed the ability of plasma taken from in vitro irradiated human blood to produce a direct cytotoxic effect, by inducing apoptosis in primary human peripheral blood mononuclear cells (PBM), which mainly comprised G 0 -stage lymphocytes. Plasma was collected from healthy donors’ blood irradiated in vitro to 0–40 Gy acute γ-rays. Reporter PBM were separated from unirradiated blood with Histopaque and held in medium with the test plasma for 24 h at 37 °C. Additionally, plasma from in vitro irradiated and unirradiated blood was tested against PBM collected from blood given 4 Gy. Apoptosis in reporter PBM was measured by the Annexin V test using flow cytometry. Plasma collected from unirradiated and irradiated blood did not produce any apoptotic response above the control level in unirradiated reporter PBM. Surprisingly, plasma from irradiated blood caused a dose-dependent reduction of apoptosis in irradiated reporter PBM. The yields of radiation-induced cell death in irradiated reporter PBM (after subtracting the respective values in unirradiated reporter PBM) were 22.2 ± 1.8% in plasma-free cultures, 21.6 ± 1.1% in cultures treated with plasma from unirradiated blood, 20.2 ± 1.4% in cultures with plasma from blood given 2–4 Gy and 16.7 ± 3.2% in cultures with plasma from blood given 6–10 Gy. These results suggested that irradiated blood plasma did not cause a radiation-induced bystander cell-killing effect. Instead, a reduction of apoptosis in irradiated reporter cells cultured with irradiated blood plasma has implications concerning oncogenic risk from mutated cells surviving after high dose in vivo irradiation (e.g. radiotherapy) and requires further study.

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

    Science.gov (United States)

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

    2014-01-01

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

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

    International Nuclear Information System (INIS)

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

    1987-01-01

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

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

    Science.gov (United States)

    Eisenberg, Sharon; Henis, Yoav I

    2008-01-01

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

  7. Identification of actin binding protein, ABP-280, as a binding partner of human Lnk adaptor protein.

    Science.gov (United States)

    He, X; Li, Y; Schembri-King, J; Jakes, S; Hayashi, J

    2000-08-01

    Human Lnk (hLnk) is an adaptor protein with multiple functional domains that regulates T cell activation signaling. In order to identify cellular Lnk binding partners, a yeast two-hybrid screening of human spleen cDNA library was carried out using human hLnk as bait. A polypeptide sequence identical to the C-terminal segment of the actin binding protein (ABP-280) was identified as a hLnk binding protein. The expressed hLnk and the FLAG tagged C-terminal 673 amino acid residues of ABP-280 or the endogenous ABP-280 in COS-7 cells could be co-immunoprecipitated using antibodies either to hLnk, FLAG or ABP-280, respectively. Furthermore, immunofluorescence confocal microscope showed that hLnk and ABP-280 co-localized at the plasma membrane and at juxtanuclear region of COS-7 cells. In Jurkat cells, the endogenous hLnk also associates with the endogenous ABP-280 indicating that the association of these two proteins is physiological. The interacting domains of both proteins were mapped using yeast two-hybrid assays. Our results indicate that hLnk binds to the residues 2006-2454 (repeats 19-23C) of ABP-280. The domain in hLnk that associates with ABP-280 was mapped to an interdomain region of 56 amino acids between pleckstrin homology and Src homology 2 domains. These results suggest that hLnk may exert its regulatory role through its association with ABP-280.

  8. Protein Detection Using the Multiplexed Proximity Extension Assay (PEA) from Plasma and Vaginal Fluid Applied to the Indicating FTA Elute Micro CardTM

    OpenAIRE

    Berggrund, Malin; Ekman, Daniel; Gustavsson, Inger; Sundfeldt, Karin; Olovsson, Matts; Enroth, Stefan; Gyllensten, Ulf

    2016-01-01

    The indicating FTA elute micro cardTM has been developed to collect and stabilize the nucleic acid in biological samples and is widely used in human and veterinary medicine and other disciplines. This card is not recommended for protein analyses, since surface treatment may denature proteins. We studied the ability to analyse proteins in human plasma and vaginal fluid as applied to the indicating FTA elute micro cardTM using the sensitive proximity extension assay (PEA). Among 92 proteins in ...

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

    Science.gov (United States)

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

    2016-06-01

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

  10. Identification of low abundance cyclophilins in human plasma.

    Science.gov (United States)

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

    2016-11-01

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

  11. [Cow's milk protein allergy through human milk].

    Science.gov (United States)

    Denis, M; Loras-Duclaux, I; Lachaux, A

    2012-03-01

    Cow's milk protein allergy (CMPA) is the first allergy that affects infants. In this population, the incidence rate reaches 7.5%. The multiplicity and aspecificity of the symptoms makes its diagnosis sometimes complicated, especially in the delayed type (gastrointestinal, dermatological, and cutaneous). CMPA symptoms can develop in exclusively breastfed infants with an incidence rate of 0.5%. It, therefore, raises questions about sensitization to cow's milk proteins through breast milk. Transfer of native bovine proteins such as β-lactoglobulin into the breast milk is controversial: some authors have found bovine proteins in human milk but others point to cross-reactivity between human milk proteins and cow's milk proteins. However, it seems that a small percentage of dietary proteins can resist digestion and become potentially allergenic. Moreover, some authors suspect the transfer of some of these dietary proteins from the maternal bloodstream to breast milk, but the mechanisms governing sensitization are still being studied. Theoretically, CMPA diagnosis is based on clinical observations, prick-test or patch-test results, and cow's milk-specific IgE antibody concentration. A positive food challenge test usually confirms the diagnosis. No laboratory test is available to make a certain diagnosis, but the detection of eosinophil cationic protein (ECP) in the mother's milk, for example, seems to be advantageous since it is linked to CMA. Excluding cow's milk from the mother's diet is the only cure when she still wants to breastfeed. Usually, cow's milk proteins are reintroduced after 6 months of exclusion. Indeed, the prognosis for infants is very good: 80% acquire a tolerance before the age of 3 or 4 years. Mothers should not avoid dairy products during pregnancy and breastfeeding as preventive measures against allergy. Copyright © 2011 Elsevier Masson SAS. All rights reserved.

  12. Comparison of two methods forecasting binding rate of plasma protein.

    Science.gov (United States)

    Hongjiu, Liu; Yanrong, Hu

    2014-01-01

    By introducing the descriptors calculated from the molecular structure, the binding rates of plasma protein (BRPP) with seventy diverse drugs are modeled by a quantitative structure-activity relationship (QSAR) technique. Two algorithms, heuristic algorithm (HA) and support vector machine (SVM), are used to establish linear and nonlinear models to forecast BRPP. Empirical analysis shows that there are good performances for HA and SVM with cross-validation correlation coefficients Rcv(2) of 0.80 and 0.83. Comparing HA with SVM, it was found that SVM has more stability and more robustness to forecast BRPP.

  13. Sterilization of Normal Human Plasma and Some of its Fractions by Means of Gamma Rays

    International Nuclear Information System (INIS)

    López Martínez de Alva, L.; Crespo, Y M.

    1967-01-01

    Owing to the frequency with which normal human plasma transmits hepatitis, various methods of sterilization have been tried. The method most used, but which has been shown to be ineffective, is sterilization of the liquid plasma with ultraviolet rays. The other method is the use of ß-propiolactone, but this has also been discontinued because of the changes it produces in the structure of plasma proteins. The object of the present study, which should be regarded as a preliminary report, is to present the results obtained by sterilizing, by means of gamma rays ( 60 Co, 1.3316 MeV) at doses of 2, 2. 5 and 3 Mrad, a substance which, like plasma, contains highly labile proteins easily able to undergo structural changes under irradiation. Pure fibrinogen, pure gamma globulin, and albumin of human origin were subjected to the same doses; the results were very satisfactory, in that no appreciable change could be demonstrated as regards the structure, solubility or chemical characteristics of the substances concerned. It was shown by means of simple coagulation tests that some of the proteins involved in this mechanism, such as prothrombin, die Power-Stuart factor and the Hageman factor, were practically unchanged. All the plasma samples and the various proteins were previously lyophilized to give a maximum moisture content of 0.034%, in order to avoid ionizing the water content into oxygenated water, which would modify and oxidize the proteins. It was shown that lyophilized plasmas initially contaminated with different strains and viruses remained sterile with doses as low as 2 Mrad. Finally, it was shown that this method is simple and practical, since sterilization can be checked in the final packaging. (author) [es

  14. Dataset of protein species from human liver

    Directory of Open Access Journals (Sweden)

    Stanislav Naryzhny

    2017-06-01

    Full Text Available This article contains data related to the research article entitled “Zipf׳s law in proteomics” (Naryzhny et al., 2017 [1]. The protein composition in the human liver or hepatocarcinoma (HepG2 cells extracts was estimated using a filter-aided sample preparation (FASP protocol. The protein species/proteoform composition in the human liver was determined by two-dimensional electrophoresis (2-DE followed by Electrospray Ionization Liquid Chromatography-Tandem Mass Spectrometry (ESI LC-MS/MS. In the case of two-dimensional electrophoresis (2-DE, the gel was stained with Coomassie Brilliant Blue R350, and image analysis was performed with ImageMaster 2D Platinum software (GE Healthcare. The 96 sections in the 2D gel were selected and cut for subsequent ESI LC-MS/MS and protein identification. If the same protein was detected in different sections, it was considered to exist as different protein species/proteoforms. A list of human liver proteoforms detected in this way is presented.

  15. Serum Copper and Plasma Protein Status in Normal Pregnancy

    Directory of Open Access Journals (Sweden)

    Nushrat Noor, Nasim Jahan, Nayma Sultana

    2012-12-01

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

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

    Science.gov (United States)

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

    2016-05-15

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1966-07-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1966-07-01

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

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

    African Journals Online (AJOL)

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

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

    Science.gov (United States)

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

    2014-03-01

    Phospholipids are the major components of cell membranes, but they have functional roles beyond forming lipid bilayers. In particular, acidic phospholipids form microdomains in the plasma membrane and can ionically interact with proteins via polybasic sequences, which can have functional consequences for the protein. The list of proteins regulated by ionic protein-lipid interaction has been quickly expanding, and now includes membrane proteins, cytoplasmic soluble proteins, and viral proteins. Here we review how acidic phospholipids in the plasma membrane regulate protein structure and function via ionic interactions, and how Ca(2+) regulates ionic protein-lipid interactions via direct and indirect mechanisms. Copyright © 2014 Elsevier Ltd. All rights reserved.

  1. Purification of human alpha uterine protein.

    Science.gov (United States)

    Sutcliffe, R G; Bolton, A E; Sharp, F; Nicholson, L V; MacKinnon, R

    1980-03-01

    Human alpha uterine protein (AUP) has been prepared from extracts of decudua by antibody affinity chromatography, DEAE Sepharose chromatography and by filtration through Sephadex G-150. This procedure yielded a protein fraction containing AUP, which was labelled with 125I by chloramine T. When analysed by SDS gel electrophoresis this radioiodinated protein fraction was found to contain predominantly a single species of protein which was precipitated by antibodies against AUP in antibody-antigen crossed electrophoresis. Rabbit anti-AUP precipitated 55-65% of the tracer in a double-antibody system. Sephadex G150 gel filtration of AUP obtained before and after affinity chromatography provided a molecular weight estimate of 50000. Since SDS gel electrophoresis revealed a polypeptide molecular weight of 23000-25000, it is suggested that AUP is a dimer.

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

    Science.gov (United States)

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

    2013-09-01

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

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

    Directory of Open Access Journals (Sweden)

    Fagundes, B.

    2011-07-01

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

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

    Science.gov (United States)

    Ye, Min; Nagar, Swati; Korzekwa, Ken

    2015-01-01

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

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

    Science.gov (United States)

    Ye, Min; Nagar, Swati; Korzekwa, Ken

    2016-04-01

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

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

    Directory of Open Access Journals (Sweden)

    Julie Bachmann

    2014-04-01

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

  7. Radioimmunoassay and characterization of atrial natriuretic peptide in human plasma

    International Nuclear Information System (INIS)

    Yandle, T.G.; Espiner, E.A.; Nicholls, M.G.; Duff, H.

    1986-01-01

    A RIA for alpha-human atrial natriuretic peptide (alpha hANP) in plasma was developed and used to study the immunoreactive components secreted by the heart and circulating in peripheral venous plasma. The assay used [125I]diiodotyrosyl-alpha hANP, purified by high pressure liquid chromatography (HPLC), and a C-terminal-specific antiserum purchased from Peninsula Laboratories. Serial dilution curves of coronary sinus plasma samples were parallel with the standard curve, but significant nonparallelism was found in peripheral plasma samples of low immunoreactivity. When plasma was extracted using C-18 Sep-Pak cartridges, serial dilution curves from both coronary sinus and peripheral plasma samples were parallel to the standard curve. Although values for plasma samples assayed before and after extraction agreed closely (r = 0.99; n = 76), immunoreactive ANP in unextracted plasma was consistently greater (70-79 pmol/liter) than in extracts of plasma, suggesting non-specific interference by a component in plasma when assayed without extraction. Mean plasma immunoreactive ANP in 19 normal subjects consuming a normal salt intake was 14 +/- 1 (+/- SE) pmol/liter. In 5 normal men, increasing dietary sodium intake from 10 to 200 mmol sodium/day was associated with a 2-fold increment in ANP levels, and similar changes accompanied acute sodium loading using iv saline. Elevated values were found in patients with congestive heart failure (mean, 58 pmol/liter; range, 0-200; n = 9), chronic renal failure (mean, 118 pmol/liter; range, 30-290; n = 8), and primary aldosteronism (range, 32-90 pmol/liter; n = 3). HPLC and gel chromatographic analysis of the immunoreactive material found in coronary sinus plasma extracts showed that a large amount of the material eluted in the position of alpha hANP

  8. Regulation of human protein S gene (PROS1) transcription

    NARCIS (Netherlands)

    Wolf, Cornelia de

    2006-01-01

    This thesis describes the investigation of the transcriptional regulation of the gene for anticoagulant plasma Protein S, PROS1. Protein S is a cofactor for Protein C in the Protein C anticoagulant pathway. The coagulation cascade is negatively regulated by this pathway through inactivation of

  9. Competitive protein adsorption to polymer surface from human serum

    DEFF Research Database (Denmark)

    Holmberg, Maria; Jensen, Karin Bagger Stibius; Larsen, Niels Bent

    2008-01-01

    Surface modification by "soft" plasma polymerisation to obtain a hydrophilic and non-fouling polymer surface has been validated using radioactive labelling. Adsorption to unmodified and modified polymer surfaces, from both single protein and human serum solutions, has been investigated. By using...... different radioisotopes, albumin and Immunoglobulin G (IgG) adsorption has been monitored simultaneously during competitive adsorption processes, which to our knowledge has not been reported in the literature before. Results show that albumin and IgG adsorption is dependent on adsorption time...... and on the presence and concentration of other proteins in bulk solutions during adsorption. Generally, lower albumin and IgG adsorption was observed on the modified and more hydrophilic polymer surfaces, but otherwise the modified and unmodified polymer surfaces showed the same adsorption characteristics....

  10. Plasma membrane organization promotes virulence of the human fungal pathogen Candida albicans.

    Science.gov (United States)

    Douglas, Lois M; Konopka, James B

    2016-03-01

    Candida albicans is a human fungal pathogen capable of causing lethal systemic infections. The plasma membrane plays key roles in virulence because it not only functions as a protective barrier, it also mediates dynamic functions including secretion of virulence factors, cell wall synthesis, invasive hyphal morphogenesis, endocytosis, and nutrient uptake. Consistent with this functional complexity, the plasma membrane is composed of a wide array of lipids and proteins. These components are organized into distinct domains that will be the topic of this review. Some of the plasma membrane domains that will be described are known to act as scaffolds or barriers to diffusion, such as MCC/eisosomes, septins, and sites of contact with the endoplasmic reticulum. Other zones mediate dynamic processes, including secretion, endocytosis, and a special region at hyphal tips that facilitates rapid growth. The highly organized architecture of the plasma membrane facilitates the coordination of diverse functions and promotes the pathogenesis of C. albicans.

  11. Plasma membrane organization promotes virulence of the human fungal pathogen Candida albicans

    Science.gov (United States)

    Douglas, Lois M.; Konopka, James. B.

    2017-01-01

    Candida albicans is a human fungal pathogen capable of causing lethal systemic infections. The plasma membrane plays key roles in virulence because it not only functions as a protective barrier, it also mediates dynamic functions including secretion of virulence factors, cell wall synthesis, invasive hyphal morphogenesis, endocytosis, and nutrient uptake. Consistent with this functional complexity, the plasma membrane is composed of a wide array of lipids and proteins. These components are organized into distinct domains that will be the topic of this review. Some of the plasma membrane domains that will be described are known to act as scaffolds or barriers to diffusion, such as MCC/eisosomes, septins, and sites of contact with the endoplasmic reticulum. Other zones mediate dynamic processes, including secretion, endocytosis, and a special region at hyphal tips that facilitates rapid growth. The highly organized architecture of the plasma membrane facilitates the coordination of diverse functions and promotes the pathogenesis of C. albicans. PMID:26920878

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

    Science.gov (United States)

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

    2015-11-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2017-12-06

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

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

    Science.gov (United States)

    Sun, Bingyun; Hood, Leroy

    2014-06-06

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

  15. Determination of albendazole sulfoxide in human plasma by using liquid chromatography-tandem mass spectrometry.

    Science.gov (United States)

    Saraner, Nihal; Özkan, Güler Yağmur; Güney, Berrak; Alkan, Erkin; Burul-Bozkurt, Nihan; Sağlam, Onursal; Fikirdeşici, Ezgi; Yıldırım, Mevlüt

    2016-06-01

    A rapid, simple and sensitive method was developed and validated using liquid chromatography-tandem mass spectrometry (LC-MS/MS) for determination of albendazole sulfoxide (ABZOX) in human plasma. The plasma samples were extracted by protein precipitation using albendazole sulfoxide-d3 as internal standard (IS). The chromatographic separation was performed on Waters Xbridge C18Column (100×4.6mm, 3.5μm) with a mobile phase consisting of ammonia solution, water and methanol at a flow rate of 0.70mL/min. ABZOX was detected and identified by mass spectrometry with electrospray ionization (ESI) in positive ion and multiple-reaction monitoring (MRM) mode. The method was linear in the range of 3-1500ng/mL for ABZOX. This method was successfully applied to the bioequivalence study in human plasma samples. Copyright © 2016 Elsevier B.V. All rights reserved.

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

    Science.gov (United States)

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

    2012-02-13

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

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

    Science.gov (United States)

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

    2016-01-04

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

  18. [Blood plasma protein adsorption capacity of perfluorocarbon emulsion stabilized by proxanol 268 (in vitro and in vivo studies)].

    Science.gov (United States)

    Sklifas, A N; Zhalimov, V K; Temnov, A A; Kukushkin, N I

    2012-01-01

    The adsorption abilities of the perfluorocarbon emulsion stabilized by Proxanol 268 were investigated in vitro and in vivo. In vitro, the saturation point for the blood plasma proteins was nearly reached after five minutes of incubation of the emulsion with human/rabbit blood plasma and was stable for all incubation periods studied. The decrease in volume ratio (emulsion/plasma) was accompanied by the increase in the adsorptive capacity of the emulsion with maximal values at 1/10 (3.2 and 1.5 mg of proteins per 1 ml of the emulsion, for human and rabbit blood plasma, respectively) that was unchanged at lower ratios. In vivo, in rabbits, intravenously injected with the emulsion, the proteins with molecular masses of 12, 25, 32, 44, 55, 70, and 200 kDa were adsorbed by the emulsion (as in vitro) if it was used 6 hours or less before testing. More delayed testing (6 h) revealed elimination of proteins with molecular masses of 25 and 44 kDa and an additional pool of adsorpted new ones of 27, 50, and 150 kDa. Specific adsorptive capacity of the emulsion enhanced gradually after emulsion injection and reached its maximum (3.5-5 mg of proteins per 1 ml of the emulsion) after 24 hours.

  19. Plasma urocortin in human systolic heart failure.

    Science.gov (United States)

    Ng, Leong L; Loke, Ian W; O'Brien, Russell J; Squire, Iain B; Davies, Joan E

    2004-04-01

    Urocortin (UCN), a member of the corticotrophin-releasing factor family, is expressed in heart, brain and gut. UCN has potent cardiostimulatory, cardioprotective, vasodilator and diuretic/natriuretic effects, and cardiac UCN expression is increased in heart failure (HF). In the present study, we investigated plasma levels of UCN in 119 patients with HF and 212 age- and gender-matched controls to clarify its relationship with gender and disease severity. UCN was elevated in HF [normal males, 19.5 (3.9-68.8) pmol/l and HF males, 50.2 (6.9-108.2) pmol/l, P fall in UCN levels with increasing NYHA class was reinforced by a significant correlation between UCN and ejection fraction ( r(s) = 0.45, P < 0.0005) in HF patients. Although receiver operating characteristic (ROC) curves for diagnosis of all HF cases yielded an area under the curve (AUC) of 0.76, ROC AUCs for patients with early HF (NYHA class I and II) were better (0.91). ROC AUCs for logistic models incorporating N-terminal probrain natriuretic peptide (N-BNP) and UCN were better than either peptide alone. In conclusion, plasma UCN is elevated in HF, especially in its early stages. Its decline with increasing HF severity may expedite disease progression due to diminished cardioprotective/anti-inflammatory effects. UCN measurement may also complement N-BNP in the diagnosis of early HF.

  20. Radioimmunoassay of human β-lipotropin in unextracted plasma

    International Nuclear Information System (INIS)

    Wiedemann, E.; Saito, T.; Linfoot, J.A.; Li, C.H.

    1977-01-01

    A sensitive and specific radioimmunoassay for human β-lipotropin (β/sub h/-LPH) in unextracted plasma was developed using pure β/sub h/-LPH as tracer and standard and an antiserum not cross-reacting with human β-MSH and hACTH. In healthy volunteers plasma β/sub h/-LPH ranged from <20 to 150 pg/ml at 8:00 a.m. and rose after metyrapone administration. β/sub h/-LPH was very low in panhypopituitarism, normal in most patients with untreated Cushing's disease, elevated in acromegaly and extremely high in Nelson's syndrome

  1. Cow's milk proteins in human milk.

    Science.gov (United States)

    Coscia, A; Orrù, S; Di Nicola, P; Giuliani, F; Rovelli, I; Peila, C; Martano, C; Chiale, F; Bertino, E

    2012-01-01

    Cow's milk proteins (CMPs) are among the best characterized food allergens. Cow's milk contains more than twenty five different proteins, but only whey proteins alpha-lactalbumin, beta-lactoglobulin, bovine serum albumin (BSA), and lactoferrin, as well as the four caseins, have been identified as allergens. Aim of this study was to investigate by proteomics techniques cow's milk allergens in human colostrum of term and preterm newborns' mothers, not previously detected, in order to understand if such allergens could be cause of sensitization during lactation. Term colostrum samples from 62 healthy mothers and preterm colostrum samples from 11 healthy mothers were collected for this purpose. The most relevant finding was the detection of the intact bovine alpha-S1-casein in both term and preterm colostrum. Using this method, which allows direct proteins identification, beta-lactoglobulin was not detected in any of colostrum samples. According to our results bovine alpha 1 casein that is considered a major cow's milk allergen is readily secreted in human milk: further investigations are needed in order to clarify if alpha-1-casein has a major role in sensitization or tolerance to cow's milk of exclusively breastfed predisposed infants.

  2. Radioimmunoassay for platelet activation specific protein GMP-140 on the platelet surface and in plasma

    International Nuclear Information System (INIS)

    Wu Guoxin; Li Jianyong; Ruan Changgeng

    1991-08-01

    Using monoclonal antibody (McAb) SZ-51 which is specific for an alpha-granule membrane protein (GMP-140) on the surface of human activated platelets, the platelet GMP-140 expression in fixed whole blood was measured by direct radioimmunoassay and GMP-140 microparticles in plasma was measured by sandwich method. The GMP-140 molecules per platelet or milliliter (mL) were calculated for the following subjects; acute myocardial infarction; cerebro thrombosis; diabetic mellitus; asthma attack; epidemic hemorrhagic fever etc.. By comparing with the concentration of thromboxane B 2 (TXB 2 ) and von Willebrand factor (vWF) in plasma, it is confirmed that the measurement of GMP-140 molecules is better than that of TXB 2 and vWF. It is a sensitive and specific method for evaluating the platelet activation degree in vivo. The establishment of this method will be useful to diagnosing the thrombotic disorders and studying the pathogenesis of some other diseases

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

    Science.gov (United States)

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

    2007-08-17

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

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

    Science.gov (United States)

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

    2018-06-01

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

  5. Palmitoylation of Sindbis Virus TF Protein Regulates Its Plasma Membrane Localization and Subsequent Incorporation into Virions.

    Science.gov (United States)

    Ramsey, Jolene; Renzi, Emily C; Arnold, Randy J; Trinidad, Jonathan C; Mukhopadhyay, Suchetana

    2017-02-01

    Palmitoylation is a reversible, posttranslational modification that helps target proteins to cellular membranes. The alphavirus small membrane proteins 6K and TF have been reported to be palmitoylated and to positively regulate budding. 6K and TF are isoforms that are identical in their N termini but unique in their C termini due to a -1 ribosomal frameshift during translation. In this study, we used cysteine (Cys) mutants to test differential palmitoylation of the Sindbis virus 6K and TF proteins. We modularly mutated the five Cys residues in the identical N termini of 6K and TF, the four additional Cys residues in TF's unique C terminus, or all nine Cys residues in TF. Using these mutants, we determined that TF palmitoylation occurs primarily in the N terminus. In contrast, 6K is not palmitoylated, even on these shared residues. In the C-terminal Cys mutant, TF protein levels increase both in the cell and in the released virion compared to the wild type. In viruses with the N-terminal Cys residues mutated, TF is much less efficiently localized to the plasma membrane, and it is not incorporated into the virion. The three Cys mutants have minor defects in cell culture growth but a high incidence of abnormal particle morphologies compared to the wild-type virus as determined by transmission electron microscopy. We propose a model where the C terminus of TF modulates the palmitoylation of TF at the N terminus, and palmitoylated TF is preferentially trafficked to the plasma membrane for virus budding. Alphaviruses are a reemerging viral cause of arthritogenic disease. Recently, the small 6K and TF proteins of alphaviruses were shown to contribute to virulence in vivo Nevertheless, a clear understanding of the molecular mechanisms by which either protein acts to promote virus infection is missing. The TF protein is a component of budded virions, and optimal levels of TF correlate positively with wild-type-like particle morphology. In this study, we show that the

  6. Human Cementum Protein 1 induces expression of bone and cementum proteins by human gingival fibroblasts

    International Nuclear Information System (INIS)

    Carmona-Rodriguez, Bruno; Alvarez-Perez, Marco Antonio; Narayanan, A. Sampath; Zeichner-David, Margarita; Reyes-Gasga, Jose; Molina-Guarneros, Juan; Garcia-Hernandez, Ana Lilia; Suarez-Franco, Jose Luis; Chavarria, Ivet Gil; Villarreal-Ramirez, Eduardo; Arzate, Higinio

    2007-01-01

    We recently presented evidence showing that a human cementoblastoma-derived protein, named Cementum Protein 1 (CEMP1) may play a role as a local regulator of cementoblast differentiation and cementum-matrix mineralization. This protein was shown to be expressed by cementoblasts and progenitor cells localized in the periodontal ligament. In this study we demonstrate that transfection of CEMP1 into human gingival fibroblasts (HGF) induces mineralization and expression of bone and cementum-matrix proteins. The transfected HGF cells had higher alkaline phosphatase activity and proliferation rate and they expressed genes for alkaline phosphatase, bone sialoprotein, osteocalcin, osteopontin, the transcription factor Runx2/Cbfa1, and cementum attachment protein (CAP). They also produced biological-type hydroxyapatite. These findings indicate that the CEMP1 might participate in differentiation and mineralization of nonosteogenic cells, and that it might have a potential function in cementum and bone formation

  7. Delipidation of Plasma Has Minimal Effects on Human Butyrylcholinesterase

    Directory of Open Access Journals (Sweden)

    Seda Onder

    2018-02-01

    Full Text Available Human butyrylcholinesterase (BChE is purified in large quantities from Cohn fraction IV-4 to use for protection against the toxicity of chemical warfare agents. Small scale preliminary experiments use outdated plasma from the American Red Cross as the starting material for purifying BChE (P06276. Many of the volunteer donor plasma samples are turbid with fat, the donor having eaten fatty food before the blood draw. The turbid fat interferes with enzyme assays performed in the spectrophotometer and with column chromatography. Our goal was to find a method to remove fat from plasma without loss of BChE activity. Satisfactory delipidation was achieved by adding a solution of 10% dextran sulfate and calcium chloride to fatty plasma, followed by centrifugation, and filtration through a 0.8 μm filter. Treatment with Aerosil also delipidated fatty plasma, but was accompanied by loss of 50% of the plasma volume. BChE activity and the BChE isozyme pattern on nondenaturing gel electrophoresis were unaffected by delipidation. BChE in delipidated plasma was efficiently captured by immobilized monoclonal antibodies B2 18-5 and mAb2. The immunopurified BChE was released from antibody binding with acid and visualized as a highly enriched, denatured BChE preparation by SDS gel electrophoresis. In conclusion, delipidation with dextran sulfate/CaCl2 preserves BChE activity and the tetramer structure of BChE.

  8. Human parvovirus PARV4 in plasma pools of Chinese origin.

    Science.gov (United States)

    Ma, Y-Y; Guo, Y; Zhao, X; Wang, Z; Lv, M-M; Yan, Q-P; Zhang, J-G

    2012-10-01

    Human parvovirus 4 (PARV4) is present in blood and blood products. As the presence and levels of PARV4 in Chinese source plasma pools have never been determined, we implemented real-time quantitative PCR to investigate the presence of PARV4 in source plasma pools in China. Results showed that 26·15% (51/195) of lots tested positive for PARV4. The amounts of DNA ranged from 2·83 × 10(3) copies/ml to 2·35×10(7) copies/ml plasma. The high level of PARV4 in plasma pools may pose a potential risk to recipients. Further studies on the pathogenesis of PARV4 are urgently required. © 2012 The Author(s). Vox Sanguinis © 2012 International Society of Blood Transfusion.

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

    Science.gov (United States)

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

    1993-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Dorota CYGAN-SZCZEGIELNIAK

    2015-09-01

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

  11. Radioimmunoassay of cholecystokinin in human tissue and plasma

    International Nuclear Information System (INIS)

    Jansen, J.B.M.J.; Lamers, C.B.H.W.

    1983-01-01

    A highly sensitive radioimmunoassay for cholecystokinin (CCK) without any cross-reactivity with gastrin is described. The antibody was raised in a rabbit by immunisation with 30% CCK and bound to all COOH-terminal CCK-peptides containing at least 14 amino acid residues. The affinity constant of the antibody was 59.4 x 10 10 l/mol. CCK 33 conjugated to [ 125 I]hydroxyphenylpropionic acid-succinimide ester was used as label. The binding between label and antibody was inhibited by 50% (ID 50 ) at a concentration of 2.8 pmol/l cholecystokinin 33. The detection limit of the assay was between 0.5 and 1.0 pmol/l plasma. Concentrations of CCK in aqueous acid extracts of human upper small intestine were 36.5 +- 9.8 pmol/g and of human cerebral cortex 28.2 +- 2.5 pmol/g tissue. Plasma samples were extracted in 96% ethanol prior to assay. No advantage was obtained by adding aprotinin to the tubes. When frozen at -20 0 C plasma CCK was stable for at least 6 months. Basal plasma CCK concentrations in 30 normal subjects were very low, 0.9 +- 0.1 pmol/l, range 0.5 to 3.1 pmol/l. Intraduodenal administration of fat induced significant increases in plasma CCK from 1.1 +- 0.1 to 8.2 +- 1.3 pmol/l (p = 0.01). Infusion of exogenous CCK, resulting in plasma CCK levels slightly lower than those measured during administration of fat, induced pancreatic enzyme secretion and gallbladder contraction. The reliability of this radioimmunoassay for measurements of CCK in human plasma was extensively evaluated. (Auth.)

  12. Plasma Surface Modification for Immobilization of Bone Morphogenic Protein-2 on Polycaprolactone Scaffolds

    Science.gov (United States)

    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.

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

    International Nuclear Information System (INIS)

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

    1987-01-01

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

  14. A scored human protein-protein interaction network to catalyze genomic interpretation

    DEFF Research Database (Denmark)

    Li, Taibo; Wernersson, Rasmus; Hansen, Rasmus B

    2017-01-01

    Genome-scale human protein-protein interaction networks are critical to understanding cell biology and interpreting genomic data, but challenging to produce experimentally. Through data integration and quality control, we provide a scored human protein-protein interaction network (InWeb_InBioMap,......Genome-scale human protein-protein interaction networks are critical to understanding cell biology and interpreting genomic data, but challenging to produce experimentally. Through data integration and quality control, we provide a scored human protein-protein interaction network (In...

  15. Diffusion of protein through the human cornea.

    Science.gov (United States)

    Charalel, Resmi A; Engberg, Kristin; Noolandi, Jaan; Cochran, Jennifer R; Frank, Curtis; Ta, Christopher N

    2012-01-01

    To determine the rate of diffusion of myoglobin and bovine serum albumin (BSA) through the human cornea. These small proteins have hydrodynamic diameters of approximately 4.4 and 7.2 nm, and molecular weights of 16.7 and 66 kDa, for myoglobin and BSA, respectively. Diffusion coefficients were measured using a diffusion chamber where the protein of interest and balanced salt solution were in different chambers separated by an ex vivo human cornea. Protein concentrations in the balanced salt solution chamber were measured over time. Diffusion coefficients were calculated using equations derived from Fick's law and conservation of mass in a closed system. Our experiments demonstrate that the diffusion coefficient of myoglobin is 5.5 ± 0.9 × 10(-8) cm(2)/s (n = 8; SD = 1.3 × 10(-8) cm(2)/s; 95% CI: 4.6 × 10(-8) to 6.4 × 10(-8) cm(2)/s) and the diffusion coefficient of BSA is 3.1 ± 1.0 × 10(-8) cm(2)/s (n = 8; SD = 1.4 × 10(-8) cm(2)/s; 95% CI: 2.1 × 10(-8) to 4.1 × 10(-8) cm(2)/s). Our study suggests that molecules as large as 7.2 nm may be able to passively diffuse through the human cornea. With applications in pharmacotherapy and the development of an artificial cornea, further experiments are warranted to fully understand the limits of human corneal diffusion and its clinical relevance. Copyright © 2012 S. Karger AG, Basel.

  16. HPLC Method for Determination of Rifaximin in Human Plasma ...

    African Journals Online (AJOL)

    The present study was aimed at developing a simple, sensitive, and specific liquid chromatography–tandem mass spectrometry method for the quantification of rifaximin in human plasma using rifaximin D6 as internal standard. Chromatographic separation was performed on Zorbax SB C18, 4.6 x 75 mm, 3.5 μm column with ...

  17. Radioimmunoassay of follicle stimulating hornone in human plasma

    International Nuclear Information System (INIS)

    Akande, E.O.

    1976-01-01

    A radioimmunoassay method for Follicle Stimulating Hormone (fsh) in human plasma is described. The method proved to be reliable in determining fsh levels in normally menstruating women as well as women in varying clinical states. The patterns and levels of fsh obtained from 16 menstrual cycles in 12 normally menstruating women showed agreement with previous results of Franchimont, Faiman and Ryan, etc

  18. Inferring modules from human protein interactome classes

    Directory of Open Access Journals (Sweden)

    Chaurasia Gautam

    2010-07-01

    Full Text Available Abstract Background The integration of protein-protein interaction networks derived from high-throughput screening approaches and complementary sources is a key topic in systems biology. Although integration of protein interaction data is conventionally performed, the effects of this procedure on the result of network analyses has not been examined yet. In particular, in order to optimize the fusion of heterogeneous interaction datasets, it is crucial to consider not only their degree of coverage and accuracy, but also their mutual dependencies and additional salient features. Results We examined this issue based on the analysis of modules detected by network clustering methods applied to both integrated and individual (disaggregated data sources, which we call interactome classes. Due to class diversity, we deal with variable dependencies of data features arising from structural specificities and biases, but also from possible overlaps. Since highly connected regions of the human interactome may point to potential protein complexes, we have focused on the concept of modularity, and elucidated the detection power of module extraction algorithms by independent validations based on GO, MIPS and KEGG. From the combination of protein interactions with gene expressions, a confidence scoring scheme has been proposed before proceeding via GO with further classification in permanent and transient modules. Conclusions Disaggregated interactomes are shown to be informative for inferring modularity, thus contributing to perform an effective integrative analysis. Validation of the extracted modules by multiple annotation allows for the assessment of confidence measures assigned to the modules in a protein pathway context. Notably, the proposed multilayer confidence scheme can be used for network calibration by enabling a transition from unweighted to weighted interactomes based on biological evidence.

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

    Science.gov (United States)

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

    2013-01-01

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

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

    Science.gov (United States)

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

    2012-06-01

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

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

    Science.gov (United States)

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

    1984-08-01

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

  2. Mitochondrial Fusion Proteins and Human Diseases

    Directory of Open Access Journals (Sweden)

    Michela Ranieri

    2013-01-01

    Full Text Available Mitochondria are highly dynamic, complex organelles that continuously alter their shape, ranging between two opposite processes, fission and fusion, in response to several stimuli and the metabolic demands of the cell. Alterations in mitochondrial dynamics due to mutations in proteins involved in the fusion-fission machinery represent an important pathogenic mechanism of human diseases. The most relevant proteins involved in the mitochondrial fusion process are three GTPase dynamin-like proteins: mitofusin 1 (MFN1 and 2 (MFN2, located in the outer mitochondrial membrane, and optic atrophy protein 1 (OPA1, in the inner membrane. An expanding number of degenerative disorders are associated with mutations in the genes encoding MFN2 and OPA1, including Charcot-Marie-Tooth disease type 2A and autosomal dominant optic atrophy. While these disorders can still be considered rare, defective mitochondrial dynamics seem to play a significant role in the molecular and cellular pathogenesis of more common neurodegenerative diseases, for example, Alzheimer’s and Parkinson’s diseases. This review provides an overview of the basic molecular mechanisms involved in mitochondrial fusion and focuses on the alteration in mitochondrial DNA amount resulting from impairment of mitochondrial dynamics. We also review the literature describing the main disorders associated with the disruption of mitochondrial fusion.

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

    Science.gov (United States)

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

    2017-08-15

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

  4. ADIPONECTIN AND C-REACTIVE PROTEIN RELATIONSHIP IN PLASMA AND ADIPOSE TISSUE (STUDY AMONG HEALTHY OBESE EGYPTIAN FEMALES)

    International Nuclear Information System (INIS)

    SOLIMAN, S.E.T.

    2008-01-01

    The adipokine, adiponectin inhibits vascular inflammation and acts as an endogenous modulator of obesity - linked diseases. High - sensitive C-reactive protein (hs-CRP) is recently debated as a risk factor and mediator for atherosclerosis. The present study investigated the association between adiponectin and hs-CRP in plasma and adipose tissue, and their relation to body composition and insulin sensitivity in a cohort of normal (30 subjects), obese (30 subjects) and morbidly - obese females (10 subjects). Messenger RNA (mRNA) expression of CRP and adiponectin in human adipose tissue were measured using real-time polymerase chain reaction. Plasma adiponectin and insulin were measured using radioimmunoassay methods, while, plasma hs-CRP was measured using ultrasensitive latex method.Results showed that adiponectin was negatively correlated with weight, BMI and insulin sensitivity index, and positively correlated with HDLc. The plasma hs-CRP levels were negatively correlated with plasma adiponectin. The plasma adiponectin levels being significantly lower and plasma hs-CRP being significantly higher in obese than normal females. Real- Time PCR analysis revealed the expression of CRP m-RNA in human adipose tissue and this was inversely correlated to adiponectin m RNA. These results suggest that elevation of CRP and reduction of adiponectin could emerge as mediators of atherogenesis and insulin resistance

  5. Lipidomics reveals a remarkable diversity of lipids in human plasma.

    Science.gov (United States)

    Quehenberger, Oswald; Armando, Aaron M; Brown, Alex H; Milne, Stephen B; Myers, David S; Merrill, Alfred H; Bandyopadhyay, Sibali; Jones, Kristin N; Kelly, Samuel; Shaner, Rebecca L; Sullards, Cameron M; Wang, Elaine; Murphy, Robert C; Barkley, Robert M; Leiker, Thomas J; Raetz, Christian R H; Guan, Ziqiang; Laird, Gregory M; Six, David A; Russell, David W; McDonald, Jeffrey G; Subramaniam, Shankar; Fahy, Eoin; Dennis, Edward A

    2010-11-01

    The focus of the present study was to define the human plasma lipidome and to establish novel analytical methodologies to quantify the large spectrum of plasma lipids. Partial lipid analysis is now a regular part of every patient's blood test and physicians readily and regularly prescribe drugs that alter the levels of major plasma lipids such as cholesterol and triglycerides. Plasma contains many thousands of distinct lipid molecular species that fall into six main categories including fatty acyls, glycerolipids, glycerophospholipids, sphingolipids, sterols, and prenols. The physiological contributions of these diverse lipids and how their levels change in response to therapy remain largely unknown. As a first step toward answering these questions, we provide herein an in-depth lipidomics analysis of a pooled human plasma obtained from healthy individuals after overnight fasting and with a gender balance and an ethnic distribution that is representative of the US population. In total, we quantitatively assessed the levels of over 500 distinct molecular species distributed among the main lipid categories. As more information is obtained regarding the roles of individual lipids in health and disease, it seems likely that future blood tests will include an ever increasing number of these lipid molecules.

  6. Transferrin coated nanoparticles: study of the bionano interface in human plasma.

    Directory of Open Access Journals (Sweden)

    Andrzej S Pitek

    Full Text Available It is now well established that the surface of nanoparticles (NPs in a biological environment is immediately modified by the adsorption of biomolecules with the formation of a protein corona and it is also accepted that the protein corona, rather than the original nanoparticle surface, defines a new biological identity. Consequently, a methodology to effectively study the interaction between nanomaterials and the biological corona encountered within an organism is a key objective in nanoscience for understanding the impact of the nanoparticle-protein interactions on the biological response in vitro and in vivo. Here, we outline an integrated methodology to address the different aspects governing the formation and the function of the protein corona of polystyrene nanoparticles coated with Transferrin by different strategies. Protein-NP complexes are studied both in situ (in human plasma, full corona FC and after washing (hard corona, HC in terms of structural properties, composition and second-order interactions with protein microarrays. Human protein microarrays are used to effectively study NP-corona/proteins interactions addressing the growing demand to advance investigations of the extrinsic function of corona complexes. Our data highlight the importance of this methodology as an analysis to be used in advance of the application of engineered NPs in biological environments.

  7. Effect of storage duration on cytokine stability in human serum and plasma.

    Science.gov (United States)

    Vincent, Fabien B; Nim, Hieu T; Lee, Jacinta P W; Morand, Eric F; Harris, James

    2018-06-14

    Quantification of analytes such as cytokines in serum samples is intrinsic to translational research in immune diseases. Optimising pre-analytical conditions is critical for ensuring study quality, including evaluation of cytokine stability. We aimed to evaluate the effect on cytokine stability of storage duration prior to freezing of serum, and compare to plasma samples obtained from patients with systemic lupus erythematosus (SLE). Protein stability was analysed by simultaneously quantifying 18 analytes using a custom multi-analyte profile in SLE patient serum and plasma samples that had been prospectively stored at 4 °C for pre-determined periods between 0 and 30 days, prior to freezing. Six analytes were excluded from analysis, because most tested samples were above or below the limit of detection. Amongst the 12 analysed proteins, 11 did not show significant signal degradation. Significant signal degradation was observed from the fourth day of storage for a single analyte, CCL19. Proteins levels were more stable in unseparated serum compared to plasma for most analytes, with the exception of IL-37 which appears slightly more stable in plasma. Based on this, a maximum 3 days of storage at 4 °C for unseparated serum samples is recommended for biobanked samples intended for cytokine analysis in studies of human immune disease. Copyright © 2018 Elsevier Ltd. All rights reserved.

  8. Increased Plasma Levels of Heme Oxygenase-1 in Human Brucellosis.

    Science.gov (United States)

    Chen, Zhe; Zhang, Yu-Xue; Fu, Dong-Wei; Gao, Qing-Feng; Ge, Feng-Xia; Liu, Wei-Hua

    2016-08-01

    Brucellosis is associated with inflammation and the oxidative stress response. Heme oxygenase-1 (HO-1) is a cytoprotective stress-responsive enzyme that has anti-inflammatory and anti-oxidant effects. Nevertheless, the role of HO-1 in human brucellosis has not yet been studied. The aim of this study was to examine the plasma levels of HO-1 in patients with brucellosis and to evaluate the ability of plasma HO-1 levels as an auxiliary diagnosis, a severity predictor, and a monitor for brucellosis treatments. A total of 75 patients with brucellosis were divided into the acute, subacute, chronic active, and chronic stable groups. An additional 20 volunteers were included as the healthy control group. The plasma HO-1 levels and other laboratory parameters were measured in all groups. Furthermore, the plasma levels of HO-1 in the acute group were compared before and after treatment. The plasma HO-1 levels were considerably increased in the acute (4.97 ± 3.55), subacute (4.98 ± 3.23), and chronic active groups (4.43 ± 3.00) with brucellosis compared to the healthy control group (1.03 ± 0.63) (p brucellosis (r = 0.707, p brucellosis status and may be used as a supplementary plasma marker for diagnosing brucellosis and monitoring its treatment.

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

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    Jana Ferdin

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

  10. Simultaneous Quantification of Antidiabetic Agents in Human Plasma by a UPLC-QToF-MS Method.

    Directory of Open Access Journals (Sweden)

    Mariana Millan Fachi

    Full Text Available An ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry method for the simultaneous quantification of chlorpropamide, glibenclamide, gliclazide, glimepiride, metformin, nateglinide, pioglitazone, rosiglitazone, and vildagliptin in human plasma was developed and validated, using isoniazid and sulfaquinoxaline as internal standards. Following plasma protein precipitation using acetonitrile with 1% formic acid, chromatographic separation was performed on a cyano column using gradient elution with water and acetonitrile, both containing 0.1% formic acid. Detection was performed in a quadrupole time-of-flight analyzer, using electrospray ionization operated in the positive mode. Data from validation studies demonstrated that the new method is highly sensitive, selective, precise (RSD 0.99, free of matrix and has no residual effects. The developed method was successfully applied to volunteers' plasma samples. Hence, this method was demonstrated to be appropriate for clinical monitoring of antidiabetic agents.

  11. Ascorbic acid protects lipids in human plasma and low-density lipoprotein against oxidative damage

    Energy Technology Data Exchange (ETDEWEB)

    Frei, B. (Department of Nutrition, Harvard School of Public Health, Boston, MA (Unites States))

    1991-12-01

    The authors exposed human blood plasma and low-density lipoprotein (LDL) to many different oxidative challenges and followed the temporal consumption of endogenous antioxidants in relation to the initiation of oxidative damage. Under all types of oxidizing conditions, ascorbic acid completely protects lipids in plasma and LDL against detectable peroxidative damage as assessed by a specific and highly sensitive assay for lipid peroxidation. Ascorbic acid proved to be superior to the other water-soluble plasma antioxidants bilirubin, uric acid, and protein thiols as well as to the lipoprotein-associated antioxidants alpha-tocopherol, ubiquinol-10, lycopene, and beta-carotene. Although these antioxidants can lower the rate of detectable lipid peroxidation, they are not able to prevent its initiation. Only ascorbic acid is reactive enough to effectively intercept oxidants in the aqueous phase before they can attack and cause detectable oxidative damage to lipids.

  12. Trace elements and protein in human milk

    International Nuclear Information System (INIS)

    Abusamra, Y.I.H.

    1995-01-01

    The trace elements Zn, Fe, Cu, Mn, Ni and Pb and some related major elements which are Ca, Cl K and total protein contents of human samples from ninety mothers were examined in this study. Samples were collected from Khartoum, Khartoum North and Omdurman, from the second day of delivery up to the third month where the milk reaches a relatively stable levels. These samples representing different stages of lactation which are colostrum ( 1-3 days ), tranitional ( up to 14 days ) and mature milk. The principle aim of this study is to measure the trace elements and protein contents in relation to stage of lactation and to compare with the literature. Atomic absorption spectroscopy and X-ray fluorescence were used to measure trace elements in the samples. The methods were found to be quite reliable as proved by the analysis of the standard reference material HM-1. Whereas neutron activation analysis was used for measurements of total protein. Colostrum was found to have the highest amounts of trace elements and protein. Fe mean concentration was 273 g/dm 3 at colostrum stage and it decreased to 146 g/dm 3 in mature milk ( 49% ). Zn decreased from 6000 g/dm 3 in colostrum to 1300 g/dm 3 in mature stage ( 78% ). Mn was 12g/dm 3 in colostrum, and it decreased to 2.9 g/dm 3 in mature milk ( 75% ). Cu decreased from 370 g/dm 3 to 117 g/dm 3 ( 68% ). Ni decreased from 24 g/dm 3 to 8.8 g/dm 3 ( 63% ) and Pb from 12 g/dm 3 to 2.6 g/dm 3 ( 76% ). Total protein was 37.3% of the dry milk in colostrum and it was 12.2% in mature milk. (author). 75 refs., 25 tabs., 30 figs

  13. Up-Regulation of Antioxidant Proteins in the Plasma Proteome during Saturation Diving: Unique Coincidence under Hypobaric Hypoxia.

    Science.gov (United States)

    Domoto, Hideharu; Iwaya, Keiichi; Ikomi, Fumitaka; Matsuo, Hirotaka; Tadano, Yutaka; Fujii, Shigenori; Tachi, Kazuyoshi; Itoh, Yoshiyuki; Sato, Michiya; Inoue, Kimitoshi; Shinomiya, Nariyoshi

    2016-01-01

    Saturation diving (SD) is one of the safest techniques for tolerating hyperbaric conditions for long durations. However, the changes in the human plasma protein profile that occur during SD are unknown. To identify differential protein expression during or after SD, 65 blood samples from 15 healthy Japanese men trained in SD were analyzed by two-dimensional fluorescence difference gel electrophoresis. The expression of two proteins, one 32.4 kDa with an isoelectric point (pI) of 5.8 and the other 44.8 kDa with pI 4.0, were elevated during SD to 60, 100, and 200 meters sea water (msw). The expression of these proteins returned to pre-diving level when the SD training was completed. The two proteins were identified using in-gel digestion and mass spectrometric analysis; the 32.4 kDa protein was transthyretin and the 44.8 kDa protein was alpha-1-acid glycoprotein 1. Oxidation was detected at methionine 13 of transthyretin and at methionine 129 of alpha-1-acid glycoprotein 1 by tandem mass spectrometry. Moreover, haptoglobin was up-regulated during the decompression phase of 200 msw. These plasma proteins up-regulated during SD have a common function as anti-oxidants. This suggests that by coordinating their biological effects, these proteins activate a defense mechanism to counteract the effects of hyperbaric-hyperoxic conditions during SD.

  14. Up-Regulation of Antioxidant Proteins in the Plasma Proteome during Saturation Diving: Unique Coincidence under Hypobaric Hypoxia.

    Directory of Open Access Journals (Sweden)

    Hideharu Domoto

    Full Text Available Saturation diving (SD is one of the safest techniques for tolerating hyperbaric conditions for long durations. However, the changes in the human plasma protein profile that occur during SD are unknown. To identify differential protein expression during or after SD, 65 blood samples from 15 healthy Japanese men trained in SD were analyzed by two-dimensional fluorescence difference gel electrophoresis. The expression of two proteins, one 32.4 kDa with an isoelectric point (pI of 5.8 and the other 44.8 kDa with pI 4.0, were elevated during SD to 60, 100, and 200 meters sea water (msw. The expression of these proteins returned to pre-diving level when the SD training was completed. The two proteins were identified using in-gel digestion and mass spectrometric analysis; the 32.4 kDa protein was transthyretin and the 44.8 kDa protein was alpha-1-acid glycoprotein 1. Oxidation was detected at methionine 13 of transthyretin and at methionine 129 of alpha-1-acid glycoprotein 1 by tandem mass spectrometry. Moreover, haptoglobin was up-regulated during the decompression phase of 200 msw. These plasma proteins up-regulated during SD have a common function as anti-oxidants. This suggests that by coordinating their biological effects, these proteins activate a defense mechanism to counteract the effects of hyperbaric-hyperoxic conditions during SD.

  15. Inhibition of phospholipase A2 from human plasma by sodium bisulfite

    International Nuclear Information System (INIS)

    Wiggins, C.W.; Franson, R.C.

    1987-01-01

    The anti-oxidant sodium bisulfite has been shown to inhibit acid active(lysosomal), non-Ca ++ -dependent phospholipase A 2 (PLA 2 ), and to interact reversibly with unsaturated fatty acids, altering their chromatographic mobility. The authors examined the effect of bisulfite on neutral active, Ca ++ -dependent PLA 2 from human plasma. Using [1- 14 C]oleate-labelled autoclaved E. coli as substrate, PLA 2 activity was inhibited in a dose-dependent manner by bisulfite. Maximal inhibition occurred at 100μM bisulfite. Preincubation of plasma for 0-30 minutes with bisulfite resulted in a time-dependent increase in PLA 2 inhibition. Preincubation of substrate with bisulfite had no such effect. When the plasma PLA 2 was purified 25-fold by SP-Sephadex chromatography it was no longer inhibited by bisulfite. The SP-Sephadex wash through fraction, which contained greater than 95% of the applied protein but not PLA 2 activity, did not inhibit the purified enzyme. When incubated with bisulfite however, the SP-wash through fraction produced dose-dependent inhibition of the purified enzyme. These results indicate that sodium bisulfite inhibits human plasma PLA 2 , in vitro, indirectly by interaction with a factor(s) present in plasma and suggests that anti-oxidants may similarly influence expression of extracellular PLA 2 in vivo

  16. Calcium-binding proteins from human platelets

    International Nuclear Information System (INIS)

    Gogstad, G.O.; Krutnes, M.B.; Solum, N.O.

    1983-01-01

    Calcium-binding platelet proteins were examined by crossed immunoelectrophoresis of solubilized platelets against antibodies to whole platelets followed by incubation of the immunoplates with 45 Ca 2 + and autoradiography. When the immunoplates had been pretreated with EDTA at pH 9.0 in order to remove divalent cations, three immunoprecipitates were markedly labelled with 45 Ca 2 + . These corresponded to the glycoprotein IIb-IIIa complex, glycoprotein Ia and a presently unidentified antigen termed G18. These antigens were membrane-bound and surface-oriented. When an excess of EDTA was introduced in the incubation media the results revealed that the glycoprotein IIb-IIIa complex and antigen G18, but not glycoprotein Ia, contained sites with a stronger affinity for calcium than has EDTA at pH 7.4 Immunoprecipitates of the separate glycoproteins IIb and IIIa both bound calcium in the same manner as the glycoprotein IIb-IIIa complex. As another approach, platelet-rich plasma was incubated with 45 Ca 2 + prior to crossed immunoelectrophoresis of the solubilized platelets. A single immunoprecipitate was wekly labelled. This did not correspond to any of the immunoprecipitates which were visible after staining with Coomassie blue. The labelling of this antigen was markedly increased when the platelt-rich plasma had been preincubated with EDTA and in this case a weak labelling of the glycoprotein IIB-IIIa precipitate also became apparent. No increased incorporation of calcium occured in any of these immunoprecipitates when the platelets were aggregated with ADP in the presence of 45 Ca 2 + . (orig.)

  17. Translational analysis of mouse and human placental protein and mRNA reveals distinct molecular pathologies in human preeclampsia.

    Science.gov (United States)

    Cox, Brian; Sharma, Parveen; Evangelou, Andreas I; Whiteley, Kathie; Ignatchenko, Vladimir; Ignatchenko, Alex; Baczyk, Dora; Czikk, Marie; Kingdom, John; Rossant, Janet; Gramolini, Anthony O; Adamson, S Lee; Kislinger, Thomas

    2011-12-01

    Preeclampsia (PE) adversely impacts ~5% of pregnancies. Despite extensive research, no consistent biomarkers or cures have emerged, suggesting that different molecular mechanisms may cause clinically similar disease. To address this, we undertook a proteomics study with three main goals: (1) to identify a panel of cell surface markers that distinguish the trophoblast and endothelial cells of the placenta in the mouse; (2) to translate this marker set to human via the Human Protein Atlas database; and (3) to utilize the validated human trophoblast markers to identify subgroups of human preeclampsia. To achieve these goals, plasma membrane proteins at the blood tissue interfaces were extracted from placentas using intravascular silica-bead perfusion, and then identified using shotgun proteomics. We identified 1181 plasma membrane proteins, of which 171 were enriched at the maternal blood-trophoblast interface and 192 at the fetal endothelial interface with a 70% conservation of expression in humans. Three distinct molecular subgroups of human preeclampsia were identified in existing human microarray data by using expression patterns of trophoblast-enriched proteins. Analysis of all misexpressed genes revealed divergent dysfunctions including angiogenesis (subgroup 1), MAPK signaling (subgroup 2), and hormone biosynthesis and metabolism (subgroup 3). Subgroup 2 lacked expected changes in known preeclampsia markers (sFLT1, sENG) and uniquely overexpressed GNA12. In an independent set of 40 banked placental specimens, GNA12 was overexpressed during preeclampsia when co-incident with chronic hypertension. In the current study we used a novel translational analysis to integrate mouse and human trophoblast protein expression with human microarray data. This strategy identified distinct molecular pathologies in human preeclampsia. We conclude that clinically similar preeclampsia patients exhibit divergent placental gene expression profiles thus implicating divergent

  18. Serum Amyloid P Component (SAP) Interactome in Human Plasma Containing Physiological Calcium Levels.

    Science.gov (United States)

    Poulsen, Ebbe Toftgaard; Pedersen, Kata Wolff; Marzeda, Anna Maria; Enghild, Jan J

    2017-02-14

    The pentraxin serum amyloid P component (SAP) is secreted by the liver and found in plasma at a concentration of approximately 30 mg/L. SAP is a 25 kDa homopentamer known to bind both protein and nonprotein ligands, all in a calcium-dependent manner. The function of SAP is unclear but likely involves the humoral innate immune system spanning the complement system, inflammation, and coagulation. Also, SAP is known to bind to the generic structure of amyloid deposits and possibly to protect them against proteolysis. In this study, we have characterized the SAP interactome in human plasma containing the physiological Ca 2+ concentration using SAP affinity pull-down and co-immunoprecipitation experiments followed by mass spectrometry analyses. The analyses resulted in the identification of 33 proteins, of which 24 were direct or indirect interaction partners not previously reported. The SAP interactome can be divided into categories that include apolipoproteins, the complement system, coagulation, and proteolytic regulation.

  19. Protein selectivity with immobilized metal ion-tacn sorbents: chromatographic studies with human serum proteins and several other globular proteins.

    Science.gov (United States)

    Jiang, W; Graham, B; Spiccia, L; Hearn, M T

    1998-01-01

    The chromatographic selectivity of the immobilized chelate system, 1,4,7-triazocyclononane (tacn), complexed with the borderline metal ions Cu2+, Cr3+, Mn2+, Co2+, Zn2+, and Ni2+ has been investigated with hen egg white lysozyme, horse heart cytochrome c, and horse skeletal muscle myoglobin, as well as proteins present in partially fractionated preparations of human plasma. The effects of ionic strength and pH of the loading and elution buffers on protein selectivities of these new immobilized metal ion affinity chromatographic (IMAC) systems have been examined. The results confirm that immobilized Mn;pl-tacn sorbents exhibit a novel type of IMAC behavior with proteins. In particular, the chromatographic properties of these immobilized M(n+)-tacn ligand systems were significantly different compared to the IMAC behavior observed with other types of immobilized tri- and tetradentate chelating ligands, such as iminodiacetic acid, O-phosphoserine, or nitrilotriacetic acid, when complexed with borderline metal ions. The experimental results have consequently been evaluated in terms of the additional contributions to the interactive processes mediated by effects other than solely the conventional lone pair Lewis soft acid-Lewis soft base coordination interactions, typically found for the IMAC of proteins with borderline and soft metal ions, such as Cu2+ or Ni2+.

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

    Science.gov (United States)

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

    2009-06-01

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

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

    Directory of Open Access Journals (Sweden)

    Alexandre eMartinière

    2013-12-01

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

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

    Science.gov (United States)

    Martinière, Alexandre; Runions, John

    2013-01-01

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

  3. A feeder-free, human plasma-derived hydrogel for maintenance of a human embryonic stem cell phenotype in vitro

    Directory of Open Access Journals (Sweden)

    Lewis Fiona C

    2012-08-01

    Full Text Available Abstract Background Human embryonic stem cells (hESCs represent a tremendous resource for cell therapies and the study of human development; however to maintain their undifferentiated state in vitro they routinely require the use of mouse embryonic fibroblast (MEF feeder-layers and exogenous protein media supplementation. Results These well established requirements can be overcome and in this study, it will be demonstrated that phenotypic stability of hESCs can be maintained using a novel, human plasma protein-based hydrogel as an extracellular culture matrix without the use of feeder cell co-culture. hESCs were resuspended in human platelet poor plasma (PPP, which was gelled by the addition of calcium containing DMEM-based hESC culture medium. Phenotypic and genomic expression of the pluripotency markers OCT4, NANOG and SOX2 were measured using immunohistochemistry and qRT-PCR respectively. Typical hESC morphology was demonstrated throughout in vitro culture and both viability and phenotypic stability were maintained throughout extended culture, up to 25 passages. Conclusions PPP-derived hydrogel has demonstrated to be an efficacious alternative to MEF co-culture with its hydrophilicity allowing for this substrate to be delivered via minimally invasive procedures in a liquid phase with polymerization ensuing in situ. Together this provides a novel technique for the study of this unique group of stem cells in either 2D or 3D both in vitro and in vivo.

  4. Plasma protein carbonyl responses to anaerobic exercise in female cyclists

    Directory of Open Access Journals (Sweden)

    M.E Afzalpour

    2016-03-01

    Full Text Available Single bouts of aerobic exercise may leads to oxidative stress due to the use of oxygen for metabolism and the generation of reactive oxygen. In athletes, oxidative stress can lead to several deleterious performance effects, such as muscular oxidative damage, muscle soreness, loss of skeletal muscle force production and/or inflammation. However, little is known regarding the severity and duration of oxidative stress arising from intensive anaerobic modes of exercise in aerobically-trained athletes. The purpose of this study was to investigate the effect of a single bout of intensive anaerobic exercise on plasma protein carbonyl (PC in aerobically-trained women. Aerobically-trained, provincial female cyclists [n = 18, age: 24.2±2.7 years; stature: 163.6±4.6 cm; body mass: 53.4±4.2 kg] were randomly assigned into either a non-exercising control (CON; n = 9 or experimental (EXP; n = 9 group that underwent a 30-second anaerobic (Wingate cycle ergometer exercise session. Blood sampling took place before exercise, immediately after the exercise (IE, and 24 hours following the exercise (24HR bout. In the EXP, results indicated significant (P ≤ 0.05 differences in PC levels between the pre-test and IE (0.010±0.0124 to 0.0149±0.0420 mmol/milt; P = 0.010, and IE and 24HR (0.0149±0.0420 to 0.0111±0.0183 mmol/milt; P = 0.013. No significant differences were observed between pre-test and 24HR (0.010±0.0124 to 0.0111±0.0183 mmol/milt; P = 0.371. These results indicate that oxidative protein damage, as indicated by PC levels, rises immediately with the onset of anaerobic exercise, but returns to resting levels within 24 hours following exercise in aerobically-trained women.

  5. Radioimmunoassay for human plasma 8-arginine-vasopressin

    International Nuclear Information System (INIS)

    Conte-Devolx, B.; Rougon-Rapuzzi, G.; Millet, Y.

    1977-01-01

    A radioimmunoassay for human plasma vasopressin (AVP) which permits the estimation of antidiuretic hormone (ADH) level as low as 0.8pg/ml, was developed. The average plasma level of AVP after overnight water restriction was found to be 14.3pg/ml (sd=4.4pg/ml) in normal subjects. They provoked a hypersecretion of ADH by the intravenous injection of 1-2mg of nicotine. In 11 volunteer normal subjects this stimulation by nicotine provoked ADH hypersecretion which reached a maximum between 2nd and 15th minutes after injection. In 3 cases of diabetes insipidus, nicotine injection did not induce ADH hypersecretion: in 1 case of potomania this response was weak; in 2 cases of syndrome of inappropriate ADH secretion, AVP plasma levels were elevated and the response after nicotine stimulation was exaggerated [fr

  6. Radioimmunoassay for human plasma 8-arginine-vasopressin

    Energy Technology Data Exchange (ETDEWEB)

    Conte-Devolx, B [Hopital de la Conception, 13 - Marseille (France); Rougon-Rapuzzi, G [Centre National de la Recherche Scientifique, 13 - Marseille (France); Millet, Y [Aix-Marseille-2 Univ., 13 - Marseille (France)

    1977-01-01

    A radioimmunoassay for human plasma vasopressin (AVP) which permits the estimation of antidiuretic hormone (ADH) level as low as 0.8pg/ml, was developed. The average plasma level of AVP after overnight water restriction was found to be 14.3pg/ml (sd=4.4pg/ml) in normal subjects. They provoked a hypersecretion of ADH by the intravenous injection of 1-2mg of nicotine. In 11 volunteer normal subjects this stimulation by nicotine provoked ADH hypersecretion which reached a maximum between 2nd and 15th minutes after injection. In 3 cases of diabetes insipidus, nicotine injection did not induce ADH hypersecretion: in 1 case of potomania this response was weak; in 2 cases of syndrome of inappropriate ADH secretion, AVP plasma levels were elevated and the response after nicotine stimulation was exaggerated.

  7. Protein precipitation: an expedient procedure for the routine analysis of the plasma metabolites of [123I]IBZM

    International Nuclear Information System (INIS)

    Zea-Ponce, Yolanda; Laruelle, Marc

    1999-01-01

    Plasma metabolite analysis of the single photon emission computed tomography (SPECT) D 2 /D 3 receptor radiotracer (S)(-)-N-[(1-ethyl-2-pyrrolidinyl)methyl]-2-hydroxy-3-[ 123 I] iodo-6-methoxyb enzamide ([ 123 I]IBZM) is needed for the equilibrium analysis of the SPECT data, in brain imaging studies involving bolus plus constant infusion paradigm. The purpose of these experiments was to find an appropriate procedure to expedite this analysis during routine determinations. The procedure was applied to the plasma analysis of 22 human subjects. Each plasma sample was subjected to acetonitrile protein precipitation. After separation of the pellet, the acetonitrile fraction contained 91%±2% (n=88) of the mixture of labeled metabolites and parent compound. The recovery coefficient of unmetabolized [ 123 I]IBZM determined with an standard plasma sample was 95%±2% (n=22). The percent parent compound present in the extracted fraction, measured by high performance liquid chromatography, was 16%±9% (n=85) and the percent metabolites was 84%±9% (n=85). Free fraction determination (f 1 , fraction of radiotracer unbound to protein), was 4%±0.8% (n=22). Free fraction of parent was 15%±8% (n=85). The results indicate that acetonitrile protein precipitation is an adequate method for the analysis of the [ 123 I]IBZM plasma metabolites

  8. STIM proteins and the endoplasmic reticulum-plasma membrane junctions.

    Science.gov (United States)

    Carrasco, Silvia; Meyer, Tobias

    2011-01-01

    Eukaryotic organelles can interact with each other through stable junctions where the two membranes are kept in close apposition. The junction that connects the endoplasmic reticulum to the plasma membrane (ER-PM junction) is unique in providing a direct communication link between the ER and the PM. In a recently discovered signaling process, STIM (stromal-interacting molecule) proteins sense a drop in ER Ca(2+) levels and directly activate Orai PM Ca(2+) channels across the junction space. In an inverse process, a voltage-gated PM Ca(2+) channel can directly open ER ryanodine-receptor Ca(2+) channels in striated-muscle cells. Although ER-PM junctions were first described 50 years ago, their broad importance in Ca(2+) signaling, as well as in the regulation of cholesterol and phosphatidylinositol lipid transfer, has only recently been realized. Here, we discuss research from different fields to provide a broad perspective on the structures and unique roles of ER-PM junctions in controlling signaling and metabolic processes.

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

    Science.gov (United States)

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

    2013-06-01

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

  10. DETECTION AND ISOLATION OF CD59 FROM HUMAN SEMINAL PLASMA

    Directory of Open Access Journals (Sweden)

    A REZAIE

    2002-06-01

    Full Text Available Introduction. CD59 is one of the complement regulatory proteins (CRPs which exert inhibitory function by blocking the formation of C5b-9 complex or Membrane Attacks complex (MAC. Regarding the therapeutic role of CD59 in treatment of pathological effects in uncontrolled activation of complements system and its efficiency to overcome the hyper-acute rejection, CD59 was suggested for maintenance of transplanted organ. In this study We determined and isolated CD59 from seminal plasma. Methods. Six normospermic sample according to WHO standards were chosen. Plasma of samples was separated and to remove the postasomes, the seminal plama was ultra centrifuged. CD59 was detected by Dot-Blot using CD59 mAb (MEM43. The molecular weight and purity of protein was detected by SOS-PAGE method follwed by Westerm Blot. Results. Protein was present in the 6.5 ml and 15ml of gel fitration fractions. Molecular weight based on marker size in these two fractions was 65 and 21KD respectively. Discussion. CD59 had previously beem purified by lysis of erythrocyte cell membrane. Because of use of detergent and preservative agents, this method decreased physiologic effects of the protein. In this study the isolation was performed from prostasome granules" without using of any detergent and preservative agents.

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

    Science.gov (United States)

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

    2017-11-09

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

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

    International Nuclear Information System (INIS)

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

    1988-01-01

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

  13. Atomic force microscopy on plasma membranes from Xenopus laevis oocytes containing human aquaporin 4.

    Science.gov (United States)

    Orsini, Francesco; Santacroce, Massimo; Cremona, Andrea; Gosvami, Nitya N; Lascialfari, Alessandro; Hoogenboom, Bart W

    2014-11-01

    Atomic force microscopy (AFM) is a unique tool for imaging membrane proteins in near-native environment (embedded in a membrane and in buffer solution) at ~1 nm spatial resolution. It has been most successful on membrane proteins reconstituted in 2D crystals and on some specialized and densely packed native membranes. Here, we report on AFM imaging of purified plasma membranes from Xenopus laevis oocytes, a commonly used system for the heterologous expression of membrane proteins. Isoform M23 of human aquaporin 4 (AQP4-M23) was expressed in the X. laevis oocytes following their injection with AQP4-M23 cRNA. AQP4-M23 expression and incorporation in the plasma membrane were confirmed by the changes in oocyte volume in response to applied osmotic gradients. Oocyte plasma membranes were then purified by ultracentrifugation on a discontinuous sucrose gradient, and the presence of AQP4-M23 proteins in the purified membranes was established by Western blotting analysis. Compared with membranes without over-expressed AQP4-M23, the membranes from AQP4-M23 cRNA injected oocytes showed clusters of structures with lateral size of about 10 nm in the AFM topography images, with a tendency to a fourfold symmetry as may be expected for higher-order arrays of AQP4-M23. In addition, but only infrequently, AQP4-M23 tetramers could be resolved in 2D arrays on top of the plasma membrane, in good quantitative agreement with transmission electron microscopy analysis and the current model of AQP4. Our results show the potential and the difficulties of AFM studies on cloned membrane proteins in native eukaryotic membranes. Copyright © 2014 John Wiley & Sons, Ltd.

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

    Science.gov (United States)

    Yokogoshi, Hidehiko; Wurtman, Richard J.

    1986-01-01

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

  15. Protein receptor-independent plasma membrane remodeling by HAMLET

    DEFF Research Database (Denmark)

    Nadeem, Aftab; Sanborn, Jeremy; Gettel, Douglas L.

    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...

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

    Directory of Open Access Journals (Sweden)

    Shao-Pai Wu

    2006-03-01

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

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

    DEFF Research Database (Denmark)

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

    2015-01-01

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

  18. Simultaneous quantification of 21 water soluble vitamin circulating forms in human plasma by liquid chromatography-mass spectrometry.

    Science.gov (United States)

    Meisser Redeuil, Karine; Longet, Karin; Bénet, Sylvie; Munari, Caroline; Campos-Giménez, Esther

    2015-11-27

    This manuscript reports a validated analytical approach for the quantification of 21 water soluble vitamins and their main circulating forms in human plasma. Isotope dilution-based sample preparation consisted of protein precipitation using acidic methanol enriched with stable isotope labelled internal standards. Separation was achieved by reversed-phase liquid chromatography and detection performed by tandem mass spectrometry in positive electrospray ionization mode. Instrumental lower limits of detection and quantification reached water soluble vitamins in human plasma single donor samples. The present report provides a sensitive and reliable approach for the quantification of water soluble vitamins and main circulating forms in human plasma. In the future, the application of this analytical approach will give more confidence to provide a comprehensive assessment of water soluble vitamins nutritional status and bioavailability studies in humans. Copyright © 2015 Elsevier B.V. All rights reserved.

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

    Science.gov (United States)

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

    2014-01-01

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

  20. Caloric Restriction and Exercise Increase Plasma ANGPTL4 Levels in Humans via Elevated Free Fatty Acids

    NARCIS (Netherlands)

    Kersten, A.H.; Lichtenstein, L.L.; Steenbergen, E.; Mudde, C.M.; Hendriks, H.F.J.; Hesselink, M.K.; Schrauwen, P.; Müller, M.R.

    2009-01-01

    Objective - Plasma lipoprotein levels are determined by the balance between lipoprotein production and clearance. Recently, angiopoietin-like protein 4 (ANGPTL4) was uncovered as a novel endocrine factor that potently raises plasma triglyceride levels by inhibiting triglyceride clearance. However,

  1. Caloric restriction and exercise increase plasma ANGPTL4 levels in humans via elevated free fatty acids.

    NARCIS (Netherlands)

    Kersten, S.; Lichtenstein, L.; Steenbergen, E.; Mudde, K.; Hendriks, H.F.; Hesselink, M.K.; Schrauwen, P.; Muller, M

    2009-01-01

    OBJECTIVE: Plasma lipoprotein levels are determined by the balance between lipoprotein production and clearance. Recently, angiopoietin-like protein 4 (ANGPTL4) was uncovered as a novel endocrine factor that potently raises plasma triglyceride levels by inhibiting triglyceride clearance. However,

  2. Caloric restriction and exercise increase plasma ANGPTL4 levels in humans via elevated free fatty acids

    NARCIS (Netherlands)

    Kersten, S.; Lichtenstein, L.; Steenbergen, E.; Mudde, K.; Hendriks, H.F.J.; Hesselink, M.K.; Schrauwen, P.; Müller, M.

    2009-01-01

    OBJECTIVE-: Plasma lipoprotein levels are determined by the balance between lipoprotein production and clearance. Recently, angiopoietin-like protein 4 (ANGPTL4) was uncovered as a novel endocrine factor that potently raises plasma triglyceride levels by inhibiting triglyceride clearance. However,

  3. Compact conformations of human protein disulfide isomerase.

    Directory of Open Access Journals (Sweden)

    Shang Yang

    Full Text Available Protein disulfide isomerase (PDI composed of four thioredoxin-like domains a, b, b', and a', is a key enzyme catalyzing oxidative protein folding in the endoplasmic reticulum. Large scale molecular dynamics simulations starting from the crystal structures of human PDI (hPDI in the oxidized and reduced states were performed. The results indicate that hPDI adopts more compact conformations in solution than in the crystal structures, which are stabilized primarily by inter-domain interactions, including the salt bridges between domains a and b' observed for the first time. A prominent feature of the compact conformations is that the two catalytic domains a and a' can locate close enough for intra-molecular electron transfer, which was confirmed by the characterization of an intermediate with a disulfide between the two domains. Mutations, which disrupt the inter-domain interactions, lead to decreased reductase activity of hPDI. Our molecular dynamics simulations and biochemical experiments reveal the intrinsic conformational dynamics of hPDI and its biological impact.

  4. Hippophae rhamnoides L. Fruits Reduce the Oxidative Stress in Human Blood Platelets and Plasma

    Directory of Open Access Journals (Sweden)

    Beata Olas

    2016-01-01

    Full Text Available Effects of the phenolic fraction from Hippophae rhamnoides fruits on the production of thiobarbituric acid reactive substances (TBARS, a marker of lipid peroxidation and the generation of superoxide anion (O2-∙ in human blood platelets (resting platelets and platelets stimulated by a strong physiological agonist, thrombin were studied in vitro. We also examined antioxidant properties of this fraction against human plasma lipid peroxidation and protein carbonylation induced by a strong biological oxidant, hydrogen peroxide (H2O2 or H2O2/Fe (a donor of hydroxyl radicals. The tested fraction of H. rhamnoides (0.5– 50 µg/mL; the incubation time: 15 and 60 min inhibited lipid peroxidation induced by H2O2 or H2O2/Fe. The H. rhamnoides phenolic fraction inhibited not only plasma lipid peroxidation, but also plasma protein carbonylation stimulated by H2O2 or H2O2/Fe. Moreover, the level of O2-∙ in platelets significantly decreased. In comparative experiments, the H. rhamnoides fraction was a more effective antioxidant than aronia extract or grape seed extract (at the highest tested concentration, 50 µg/mL. The obtained results suggest that H. rhamnoides fruits may be a new, promising source of natural compounds with antioxidant and antiplatelet activity beneficial not only for healthy people, but also for those with oxidative stress-associated diseases.

  5. Proteomic Biomarker Discovery in 1000 Human Plasma Samples with Mass Spectrometry.

    Science.gov (United States)

    Cominetti, Ornella; Núñez Galindo, Antonio; Corthésy, John; Oller Moreno, Sergio; Irincheeva, Irina; Valsesia, Armand; Astrup, Arne; Saris, Wim H M; Hager, Jörg; Kussmann, Martin; Dayon, Loïc

    2016-02-05

    The overall impact of proteomics on clinical research and its translation has lagged behind expectations. One recognized caveat is the limited size (subject numbers) of (pre)clinical studies performed at the discovery stage, the findings of which fail to be replicated in larger verification/validation trials. Compromised study designs and insufficient statistical power are consequences of the to-date still limited capacity of mass spectrometry (MS)-based workflows to handle large numbers of samples in a realistic time frame, while delivering comprehensive proteome coverages. We developed a highly automated proteomic biomarker discovery workflow. Herein, we have applied this approach to analyze 1000 plasma samples from the multicentered human dietary intervention study "DiOGenes". Study design, sample randomization, tracking, and logistics were the foundations of our large-scale study. We checked the quality of the MS data and provided descriptive statistics. The data set was interrogated for proteins with most stable expression levels in that set of plasma samples. We evaluated standard clinical variables that typically impact forthcoming results and assessed body mass index-associated and gender-specific proteins at two time points. We demonstrate that analyzing a large number of human plasma samples for biomarker discovery with MS using isobaric tagging is feasible, providing robust and consistent biological results.

  6. High-performance liquid chromatographic quantification of rifampicin in human plasma: method for Therapecutic drug monitoring

    International Nuclear Information System (INIS)

    Sameh, T.; Hanene, E.; Jebali, N.

    2013-01-01

    A high performance liquid chromatography (HPLC) method has been developed that allows quantification of Rifampicin in human plasma. The method is based on the precipitation of proteins in human plasma with methanol. Optimal assay conditions were found with a C18 column and a simple mobile phase consisting of 0.05 M dipotassic hydrogen phosphate buffer and acetonitrile (53/47, V/V) with 0.086 % diethylamin, pH = 4.46. The flow-rate was 0.6 ml /mm and the drug was monitored at 340 nm. Results from the HPLC analyses showed that the assay method is linear in the concentration range of 1-40 micro g/ml, (r2 >0.99). The limit of quantification and limit of detection of Rifampicin were 0.632 micro g/ml and 0.208 micro g/ml, respectively. Intraday and interday coefficient of variation and bias were below 10% for all samples, suggesting good precision and accuracy of the method. Recoveries were greater than 90% in a plasma sample volume of 100 micro l. The method is being successfully applied to therapeutic drug monitoring of Rifapicin in plasma samples of tuberculosis and staphylococcal infections patients. (author)

  7. Quantification of pharmaceutical peptides in human plasma by LC-ICP-MS sulfur detection

    DEFF Research Database (Denmark)

    Møller, Laura Hyrup; Macherius, André; Hansen, Thomas Hesselhøj

    2016-01-01

    A method for quantification of a pharmaceutical peptide in human plasma was developed using gradient elution LC-ICP-MS. A membrane desolvation (MD) system was applied to remove organic solvents from the eluent prior to the detection as SO+ in the dynamic reaction cell (DRC) of the ICP-DRC-MS inst......A method for quantification of a pharmaceutical peptide in human plasma was developed using gradient elution LC-ICP-MS. A membrane desolvation (MD) system was applied to remove organic solvents from the eluent prior to the detection as SO+ in the dynamic reaction cell (DRC) of the ICP......-DRC-MS instrument and subsequent quantification by post-column isotope dilution (IDA). Plasma proteins were precipitated prior to analysis. Analytical figures of merit including linearity, precision, LOD, LOQ and accuracy were considered satisfactory for analysis of plasma samples. The selectivity of the developed...... method was demonstrated for five pharmaceutically relevant peptides: desmopressin, penetratin, substance P, PTH (1-34) and insulin. Preliminary experiments on an ICP-MS/MS system using oxygen to reduce the effect of organic solvents were also performed to compare sensitivity. The results of the study...

  8. Proliferation-promoting effect of platelet-rich plasma on human adipose-derived stem cells and human dermal fibroblasts.

    Science.gov (United States)

    Kakudo, Natsuko; Minakata, Tatsuya; Mitsui, Toshihito; Kushida, Satoshi; Notodihardjo, Frederik Zefanya; Kusumoto, Kenji

    2008-11-01

    This study evaluated changes in platelet-derived growth factor (PDGF)-AB and transforming growth factor (TGF)-beta1 release from platelets by platelet-rich plasma activation, and the proliferation potential of activated platelet-rich plasma and platelet-poor plasma on human adipose-derived stem cells and human dermal fibroblasts. Platelet-rich plasma was prepared using a double-spin method, with the number of platelets counted in each preparation stage. Platelet-rich and platelet-poor plasma were activated with autologous thrombin and calcium chloride, and levels of platelet-released PDGF-AB and TGF-beta1 were determined by enzyme-linked immunosorbent assay. Cells were cultured for 1, 4, or 7 days in serum-free Dulbecco's Modified Eagle Medium supplemented with 5% whole blood plasma, nonactivated platelet-rich plasma, nonactivated platelet-poor plasma, activated platelet-rich plasma, or activated platelet-poor plasma. In parallel, these cells were cultured for 1, 4, or 7 days in serum-free Dulbecco's Modified Eagle Medium supplemented with 1%, 5%, 10%, or 20% activated platelet-rich plasma. The cultured human adipose-derived stem cells and human dermal fibroblasts were assayed for proliferation. Platelet-rich plasma contained approximately 7.9 times as many platelets as whole blood, and its activation was associated with the release of large amounts of PDGF-AB and TGF-beta1. Adding activated platelet-rich or platelet-poor plasma significantly promoted the proliferation of human adipose-derived stem cells and human dermal fibroblasts. Adding 5% activated platelet-rich plasma to the medium maximally promoted cell proliferation, but activated platelet-rich plasma at 20% did not promote it. Platelet-rich plasma can enhance the proliferation of human adipose-derived stem cells and human dermal fibroblasts. These results support clinical platelet-rich plasma application for cell-based, soft-tissue engineering and wound healing.

  9. Acrylonitrile exposure: the effect on p53 and p21WAF1 protein levels in the blood plasma of occupationally exposed workers and in vitro in human diploid lung fibroblasts

    Czech Academy of Sciences Publication Activity Database

    Rössner ml., Pavel; Binková, Blanka; Chvátalová, Irena; Šrám, Radim

    č. 517 (2002), s. 239-250 ISSN 0027-5107 R&D Projects: GA MŽP SI/340/1/97 Institutional research plan: CEZ:AV0Z5039906 Keywords : Acrylonitrile * p53 protein * p21WAF1 protein Subject RIV: DN - Health Impact of the Environment Quality Impact factor: 3.158, year: 2002

  10. Comparative study of the Ar and He atmospheric pressure plasmas on E-cadherin protein regulation for plasma-mediated transdermal drug delivery

    Science.gov (United States)

    Lee, Hyun Young; Hae Choi, Jeong; Hong, Jin Woo; Kim, Gyoo Cheon; Lee, Hae June

    2018-05-01

    The effects of argon plasma (ArP) and helium plasma (HeP) jets on E-cadherin protein function have been tested in order to choose the working gas for a better plasma-mediated transdermal drug delivery. The plasma-mediated changes of the E-cadherin function and the skin penetration efficacies of epidermal growth factor (EGF) were monitored in vitro using HaCaT human keratinocytes and in vivo using hairless mice. The ArP showed higher efficacy for E-cadherin regulation and EGF absorption than HeP under the same applied voltage and the same gas flow rate. The ArP generates higher volume power density, higher discharge current peak, and more reactive species than HeP, especially for OH with the same operating parameters. Moreover, the effect of ArP on E-cadherin function was blocked by the use of a grounded metal mesh. Taken together, this study presents the possibility that the synergetic effect of negative charges with radicals plays an important role in plasma-mediated E-cadherin regulation, which leads to enhanced transdermal drug delivery.

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

    Science.gov (United States)

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

    2018-03-01

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

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

    DEFF Research Database (Denmark)

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

    2008-01-01

    We have developed a simple method for isolating and purifying plasma membrane proteins from various cell types. This one-step affinity-chromatography method uses the property of the lectin concanavalin A (ConA) and the technique of magnetic bead separation to obtain highly purified plasma membrane...... proteins from crude membrane preparations or cell lines. ConA is immobilized onto magnetic beads by binding biotinylated ConA to streptavidin magnetic beads. When these ConA magnetic beads were used to enrich plasma membranes from a crude membrane preparation, this procedure resulted in 3.7-fold enrichment...... of plasma membrane marker 5'-nucleotidase activity with 70% recovery of the activity in the crude membrane fraction of rat liver. In agreement with the results of 5'-nucleotidase activity, immunoblotting with antibodies specific for a rat liver plasma membrane protein, CEACAM1, indicated that CEACAM1...

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

    DEFF Research Database (Denmark)

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

    2015-01-01

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

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

    KAUST Repository

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

    2010-01-01

    Plasma protein adsorption is regarded as a key factor in the in vivo organ distribution of intravenously administered drug carriers, and strongly depends on vector surface characteristics. The present study aimed to characterize the "protein corona" absorbed onto DC-Chol-DOPE cationic liposomes. This system was chosen because it is one of the most efficient and widely used non-viral formulations in vitro and a potential candidate for in vivo transfection of genetic material. After incubation of human plasma with cationic liposomes, nanoparticle-protein complex was separated from plasma by centrifugation. An integrated approach based on protein separation by one-dimensional 12% polyacrylamide gel electrophoresis followed by the automated HPLC-Chip technology coupled to a high-resolution mass spectrometer was employed for protein corona characterization. Thirty gel lanes, approximately 2 mm, were cut, digested and analyzed by HPLC-MS/MS. Fifty-eight human plasma proteins adsorbed onto DC-Chol-DOPE cationic liposomes were identified. The knowledge of the interactions of proteins with liposomes can be exploited for future controlled design of colloidal drug carriers and possibly in the controlled creation of biocompatible surfaces of other devices that come into contact with proteins in body fluids. © 2010 Springer-Verlag.

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

    KAUST Repository

    Capriotti, Anna Laura

    2010-09-22

    Plasma protein adsorption is regarded as a key factor in the in vivo organ distribution of intravenously administered drug carriers, and strongly depends on vector surface characteristics. The present study aimed to characterize the "protein corona" absorbed onto DC-Chol-DOPE cationic liposomes. This system was chosen because it is one of the most efficient and widely used non-viral formulations in vitro and a potential candidate for in vivo transfection of genetic material. After incubation of human plasma with cationic liposomes, nanoparticle-protein complex was separated from plasma by centrifugation. An integrated approach based on protein separation by one-dimensional 12% polyacrylamide gel electrophoresis followed by the automated HPLC-Chip technology coupled to a high-resolution mass spectrometer was employed for protein corona characterization. Thirty gel lanes, approximately 2 mm, were cut, digested and analyzed by HPLC-MS/MS. Fifty-eight human plasma proteins adsorbed onto DC-Chol-DOPE cationic liposomes were identified. The knowledge of the interactions of proteins with liposomes can be exploited for future controlled design of colloidal drug carriers and possibly in the controlled creation of biocompatible surfaces of other devices that come into contact with proteins in body fluids. © 2010 Springer-Verlag.

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

    DEFF Research Database (Denmark)

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

    2017-01-01

    The effect of experimentally increasing the postpartum protein supply on plasma protein synthesis, rumen tissue proliferation, and immune homeostasis was studied using 8 periparturient Holstein cows in a complete randomized design. At calving, cows were assigned to abomasal infusion of water (CTRL......) or casein (CAS) in addition to a lactation diet. Casein infusion was gradually decreased from 696 ± 1 g/d at +2 d relative to calving (DRTC) to 212 ± 10 g/d at +29 DRTC to avoid excessive supply. Synthesis rate of plasma proteins was measured at –14, +4, +15, and +29 DRTC by measuring [13C]Phe isotopic...... enrichment in arterial plasma free Phe, total plasma proteins, and albumin after 3, 5, and 7 h of jugular ring[13C]Phe infusion. Plasma volume was determined at +4 and +29 DRTC by dilution of a [125I]BSA dose. Synthesis rate of tissue protein in biopsied rumen papillae was determined by measuring [13C...

  17. Specific radioimmunoassay of human. beta. -endorphin in unextracted plasma

    Energy Technology Data Exchange (ETDEWEB)

    Wiedemann, E. (Univ. of California, Berkeley); Saito, T.; Linfoot, J.A.; Li, C.H.

    1979-09-01

    With an antiserum against human ..beta..-endorphin (..beta..-EP) crossreacting <2% with human ..beta..-lipotropin (..beta..-LPH) by weight we have developed a radioimmunoassay that can detect 1 pg ..beta..-EP in diluted raw plasma. In a.m. fasting plasma of 14 normal subjects ..beta..-EP ranged from <5 to 45 pg/ml. ..beta..-EP was elevated in untreated, but normal in successfully treated Cushing's disease; undetectable in a patient with adrenal adenoma; extremely high in Nelson's syndrome; and elevated in a patient with bronchogenic carcinoma before, but undetectable after tumor resection. In subjects with intact hypothalamic-pituitary-adrenal axis, ..beta..-EP was undectectable after dexamethasone and increased after metyrapone administration and insulin-induced hypoglycemia. ..beta..-EP concentration was considerably lower in serum than in simultaneously collected plasma, but increased in serum left unfrozen for several hours after clot removal. Thus, ..beta..-EP behaves like a hormone responding to the same stimuli as ACTH and ..beta..-LPH and blood appears to contain enzymes both generating and destroying immunoreactive ..beta..-EP.

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

    OpenAIRE

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

    2010-01-01

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

  19. Development of human protein reference database as an initial platform for approaching systems biology in humans

    DEFF Research Database (Denmark)

    Peri, Suraj; Navarro, J Daniel; Amanchy, Ramars

    2003-01-01

    Human Protein Reference Database (HPRD) is an object database that integrates a wealth of information relevant to the function of human proteins in health and disease. Data pertaining to thousands of protein-protein interactions, posttranslational modifications, enzyme/substrate relationships...

  20. Decreased Bacterial Attachment and Protein Adsorption to Coatings Produced by Low Enegy Plasma Polymerization

    DEFF Research Database (Denmark)

    Andersen, T.E.; Kingshott, Peter; Benter, M.

    .figure) .and E. coli grown on uncoated silicone compared to PP-PVP coated silicone (right figure). Results from the flow chamber analysis shows PP-PVP to be very good at preventing E. coli colonization during prolonged growth in flow chamber. At this point other surfaces and bacteria remains to be tested...... adsorption and bacteria attachment/colonization. This is emphasized by the fact that long dwelling urinary catheters, which is a typical silicone medical device, causes 5% per day incidence of urinary tract infection [1,2]. A demand therefore exists for surface modifications providing the silicone material......-coated crystals were then treated with one of the plasma polymerized coatings. Adsorption of fibrinogen, human serum albumin or immunoglobulin G was measured using a QCM-D instrument [5] (model E4, Q-Sense AB, Vastra Frolunda, Sweden) using a solution of 50llg/1 protein in PBS buffer. Results and Discussion: Our...

  1. Mistimed food intake and sleep alters 24-hour time-of-day patterns of the human plasma proteome.

    Science.gov (United States)

    Depner, Christopher M; Melanson, Edward L; McHill, Andrew W; Wright, Kenneth P

    2018-06-05

    Proteomics holds great promise for understanding human physiology, developing health biomarkers, and precision medicine. However, how much the plasma proteome varies with time of day and is regulated by the master circadian suprachiasmatic nucleus brain clock, assessed here by the melatonin rhythm, is largely unknown. Here, we assessed 24-h time-of-day patterns of human plasma proteins in six healthy men during daytime food intake and nighttime sleep in phase with the endogenous circadian clock (i.e., circadian alignment) versus daytime sleep and nighttime food intake out of phase with the endogenous circadian clock (i.e., circadian misalignment induced by simulated nightshift work). We identified 24-h time-of-day patterns in 573 of 1,129 proteins analyzed, with 30 proteins showing strong regulation by the circadian cycle. Relative to circadian alignment, the average abundance and/or 24-h time-of-day patterns of 127 proteins were altered during circadian misalignment. Altered proteins were associated with biological pathways involved in immune function, metabolism, and cancer. Of the 30 circadian-regulated proteins, the majority peaked between 1400 hours and 2100 hours, and these 30 proteins were associated with basic pathways involved in extracellular matrix organization, tyrosine kinase signaling, and signaling by receptor tyrosine-protein kinase erbB-2. Furthermore, circadian misalignment altered multiple proteins known to regulate glucose homeostasis and/or energy metabolism, with implications for altered metabolic physiology. Our findings demonstrate the circadian clock, the behavioral wake-sleep/food intake-fasting cycle, and interactions between these processes regulate 24-h time-of-day patterns of human plasma proteins and help identify mechanisms of circadian misalignment that may contribute to metabolic dysregulation.

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

    Science.gov (United States)

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

    2014-07-01

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

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

    DEFF Research Database (Denmark)

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

    2013-01-01

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

  4. Pregnancy-associated plasma protein-A and the vulnerable plaque

    DEFF Research Database (Denmark)

    Jespersen, Camilla H B; Vestergaard, Kirstine R; Schou, Morten

    2014-01-01

    For more than a decade, pregnancy-associated plasma protein-A (PAPP-A) has been examined for its relation to acute coronary syndrome (ACS) and the vulnerable plaque. This review summarizes the current knowledge of plasma PAPP-A in relation to nonpregnant individuals focusing on patients with ACS...

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

    NARCIS (Netherlands)

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

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

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

    International Nuclear Information System (INIS)

    Stender, S.; Zilversmit, D.B.

    1981-01-01

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

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

    DEFF Research Database (Denmark)

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

    2014-01-01

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

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

    Science.gov (United States)

    2009-07-01

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

  9. A Physical Interaction Network of Dengue Virus and Human Proteins*

    Science.gov (United States)

    Khadka, Sudip; Vangeloff, Abbey D.; Zhang, Chaoying; Siddavatam, Prasad; Heaton, Nicholas S.; Wang, Ling; Sengupta, Ranjan; Sahasrabudhe, Sudhir; Randall, Glenn; Gribskov, Michael; Kuhn, Richard J.; Perera, Rushika; LaCount, Douglas J.

    2011-01-01

    Dengue virus (DENV), an emerging mosquito-transmitted pathogen capable of causing severe disease in humans, interacts with host cell factors to create a more favorable environment for replication. However, few interactions between DENV and human proteins have been reported to date. To identify DENV-human protein interactions, we used high-throughput yeast two-hybrid assays to screen the 10 DENV proteins against a human liver activation domain library. From 45 DNA-binding domain clones containing either full-length viral genes or partially overlapping gene fragments, we identified 139 interactions between DENV and human proteins, the vast majority of which are novel. These interactions involved 105 human proteins, including six previously implicated in DENV infection and 45 linked to the replication of other viruses. Human proteins with functions related to the complement and coagulation cascade, the centrosome, and the cytoskeleton were enriched among the DENV interaction partners. To determine if the cellular proteins were required for DENV infection, we used small interfering RNAs to inhibit their expression. Six of 12 proteins targeted (CALR, DDX3X, ERC1, GOLGA2, TRIP11, and UBE2I) caused a significant decrease in the replication of a DENV replicon. We further showed that calreticulin colocalized with viral dsRNA and with the viral NS3 and NS5 proteins in DENV-infected cells, consistent with a direct role for calreticulin in DENV replication. Human proteins that interacted with DENV had significantly higher average degree and betweenness than expected by chance, which provides additional support for the hypothesis that viruses preferentially target cellular proteins that occupy central position in the human protein interaction network. This study provides a valuable starting point for additional investigations into the roles of human proteins in DENV infection. PMID:21911577

  10. A physical interaction network of dengue virus and human proteins.

    Science.gov (United States)

    Khadka, Sudip; Vangeloff, Abbey D; Zhang, Chaoying; Siddavatam, Prasad; Heaton, Nicholas S; Wang, Ling; Sengupta, Ranjan; Sahasrabudhe, Sudhir; Randall, Glenn; Gribskov, Michael; Kuhn, Richard J; Perera, Rushika; LaCount, Douglas J

    2011-12-01

    Dengue virus (DENV), an emerging mosquito-transmitted pathogen capable of causing severe disease in humans, interacts with host cell factors to create a more favorable environment for replication. However, few interactions between DENV and human proteins have been reported to date. To identify DENV-human protein interactions, we used high-throughput yeast two-hybrid assays to screen the 10 DENV proteins against a human liver activation domain library. From 45 DNA-binding domain clones containing either full-length viral genes or partially overlapping gene fragments, we identified 139 interactions between DENV and human proteins, the vast majority of which are novel. These interactions involved 105 human proteins, including six previously implicated in DENV infection and 45 linked to the replication of other viruses. Human proteins with functions related to the complement and coagulation cascade, the centrosome, and the cytoskeleton were enriched among the DENV interaction partners. To determine if the cellular proteins were required for DENV infection, we used small interfering RNAs to inhibit their expression. Six of 12 proteins targeted (CALR, DDX3X, ERC1, GOLGA2, TRIP11, and UBE2I) caused a significant decrease in the replication of a DENV replicon. We further showed that calreticulin colocalized with viral dsRNA and with the viral NS3 and NS5 proteins in DENV-infected cells, consistent with a direct role for calreticulin in DENV replication. Human proteins that interacted with DENV had significantly higher average degree and betweenness than expected by chance, which provides additional support for the hypothesis that viruses preferentially target cellular proteins that occupy central position in the human protein interaction network. This study provides a valuable starting point for additional investigations into the roles of human proteins in DENV infection.

  11. Antibodies to lactalbumin interfere with its radioimmunoassay in human plasma

    Energy Technology Data Exchange (ETDEWEB)

    Stevens, U; Laurence, D J.R. [Royal Marsden Hospital, London (UK); Ormerod, M G [Institute of Cancer Research, Sutton (UK). Surrey Branch

    1978-01-01

    Two radioimmunoassays for human lactalbumin have been established using a rabbit antiserum. One assay uses a second antibody to separate bound from free label; the other uses polyethylene glycol to precipitate gamma globulin non-specifically. It is confirmed that about half the normal human population have a substance in their blood which inhibits the binding of lactalbumin to the rabbit antibody. Comparison of the two assays has demonstrated that this material is not lactalbumin but a naturally occurring antibody. It is shown that it is in the IgG fraction of human plasma and is probably a cross-reacting antibody to bovine lactalbumin. None out of fifteen males and fourteen out of fifty eight non-pregnant, non-lacatating females had low levels of lactalbumin in their blood (0.6-2.0 ng/ml). The assay could not detect a statistically significant difference between normal women and those with either benign breast disease or metastatic mammary carcinoma.

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

    Science.gov (United States)

    Tellier, Julie; Shi, Wei; Minnich, Martina; Liao, Yang; Crawford, Simon; Smyth, Gordon K; Kallies, Axel; Busslinger, Meinrad; Nutt, Stephen L

    2015-01-01

    Plasma cell differentiation requires silencing of B cell transcription, while establishing antibody-secretory function and long-term survival. The transcription factors Blimp-1 and IRF4 are essential for plasma cell generation, however their function in mature plasma cells has remained elusive. We have found that while IRF4 was essential for plasma cell survival, Blimp-1 was dispensable. Blimp-1-deficient plasma cells retained their transcriptional identity, but lost the ability to secrete antibody. Blimp-1 regulated many components of the unfolded protein response (UPR), including XBP-1 and ATF6. The overlap of Blimp-1 and XBP-1 function was restricted to the UPR, with Blimp-1 uniquely regulating mTOR activity and plasma cell size. Thus, Blimp-1 is required for the unique physiological capacity of plasma cells that enables the secretion of protective antibody. PMID:26779600

  13. Convective transport of highly plasma protein bound drugs facilitates direct penetration into deep tissues after topical application

    Science.gov (United States)

    Dancik, Yuri; Anissimov, Yuri G; Jepps, Owen G; Roberts, Michael S

    2012-01-01

    AIMS To relate the varying dermal, subcutaneous and muscle microdialysate concentrations found in man after topical application to the nature of the drug applied and to the underlying physiology. METHODS We developed a physiologically based pharmacokinetic model in which transport to deeper tissues was determined by tissue diffusion, blood, lymphatic and intersitial flow transport and drug properties. The model was applied to interpret published human microdialysis data, estimated in vitro dermal diffusion and protein binding affinity of drugs that have been previously applied topically in vivo and measured in deep cutaneous tissues over time. RESULTS Deeper tissue microdialysis concentrations for various drugs in vivo vary widely. Here, we show that carriage by the blood to the deeper tissues below topical application sites facilitates the transport of highly plasma protein bound drugs that penetrate the skin, leading to rapid and significant concentrations in those tissues. Hence, the fractional concentration for the highly plasma protein bound diclofenac in deeper tissues is 0.79 times that in a probe 4.5 mm below a superficial probe whereas the corresponding fractional concentration for the poorly protein bound nicotine is 0.02. Their corresponding estimated in vivo lag times for appearance of the drugs in the deeper probes were 1.1 min for diclofenac and 30 min for nicotine. CONCLUSIONS Poorly plasma protein bound drugs are mainly transported to deeper tissues after topical application by tissue diffusion whereas the transport of highly plasma protein bound drugs is additionally facilitated by convective blood, lymphatic and interstitial transport to deep tissues. PMID:21999217

  14. Hemocompatible control of sulfobetaine-grafted polypropylene fibrous membranes in human whole blood via plasma-induced surface zwitterionization.

    Science.gov (United States)

    Chen, Sheng-Han; Chang, Yung; Lee, Kueir-Rarn; Wei, Ta-Chin; Higuchi, Akon; Ho, Feng-Ming; Tsou, Chia-Chun; Ho, Hsin-Tsung; Lai, Juin-Yih

    2012-12-21

    In this work, the hemocompatibility of zwitterionic polypropylene (PP) fibrous membranes with varying grafting coverage of poly(sulfobetaine methacrylate) (PSBMA) via plasma-induced surface polymerization was studied. Charge neutrality of PSBMA-grafted layers on PP membrane surfaces was controlled by the low-pressure and atmospheric plasma treatment in this study. The effects of grafting composition, surface hydrophilicity, and hydration capability on blood compatibility of the membranes were determined. Protein adsorption onto the different PSBMA-grafted PP membranes from human fibrinogen solutions was measured by enzyme-linked immunosorbent assay (ELISA) with monoclonal antibodies. Blood platelet adhesion and plasma clotting time measurements from a recalcified platelet-rich plasma solution were used to determine if platelet activation depends on the charge bias of the grafted PSBMA layer. The charge bias of PSBMA layer deviated from the electrical balance of positively and negatively charged moieties can be well-controlled via atmospheric plasma-induced interfacial zwitterionization and was further tested with human whole blood. The optimized PSBMA surface graft layer in overall charge neutrality has a high hydration capability and keeps its original blood-inert property of antifouling, anticoagulant, and antithrmbogenic activities when it comes into contact with human blood. This work suggests that the hemocompatible nature of grafted PSBMA polymers by controlling grafting quality via atmospheric plasma treatment gives a great potential in the surface zwitterionization of hydrophobic membranes for use in human whole blood.

  15. Plasma immersion ion implantation of polyurethane shape memory polymer: Surface properties and protein immobilization

    Science.gov (United States)

    Cheng, Xinying; Kondyurin, Alexey; Bao, Shisan; Bilek, Marcela M. M.; Ye, Lin

    2017-09-01

    Polyurethane-type shape memory polymers (SMPU) are promising biomedical implant materials due to their ability to recover to a predetermined shape from a temporary shape induced by thermal activation close to human body temperature and their advantageous mechanical properties including large recovery strains and low recovery stresses. Plasma Immersion Ion Implantation (PIII) is a surface modification process using energetic ions that generates radicals in polymer surfaces leading to carbonisation and oxidation and the ability to covalently immobilise proteins without the need for wet chemistry. Here we show that PIII treatment of SMPU significantly enhances its bioactivity making SMPU suitable for applications in permanent implantable biomedical devices. Scanning Electron Microscopy (SEM), contact angle measurements, surface energy measurements, attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy and X-ray photoelectron spectroscopy (XPS) were used to characterise the PIII modified surface, including its after treatment aging kinetics and its capability to covalently immobilise protein directly from solution. The results show a substantial improvement in wettability and dramatic changes of surface chemical composition dependent on treatment duration, due to the generation of radicals and subsequent oxidation. The SMPU surface, PIII treated for 200s, achieved a saturated level of covalently immobilized protein indicating that a full monolayer coverage was achieved. We conclude that PIII is a promising and efficient surface modification method to enhance the biocompatibility of SMPU for use in medical applications that demand bioactivity for tissue integration and stability in vivo.

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

    International Nuclear Information System (INIS)

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

    2011-01-01

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

  17. Human Serum Protein-Bound iodine and Protein Fractions at ...

    African Journals Online (AJOL)

    Iodine profile of Nigerians at different ages in both sexes and in pregnant women, and under narcotic influence, such as alcoholism, cigarette smoking and marijuana addiction were studied. Their serum total protein, albumin and globulin concentrations were also determined. Results of the study showed that serum protein ...

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

    Science.gov (United States)

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

    2014-09-03

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

  19. Differential Impact of Plasma Proteins on the Adhesion Efficiency of Vascular-Targeted Carriers (VTCs) in Blood of Common Laboratory Animals.

    Science.gov (United States)

    Namdee, Katawut; Sobczynski, Daniel J; Onyskiw, Peter J; Eniola-Adefeso, Omolola

    2015-12-16

    Vascular-targeted carrier (VTC) interaction with human plasma is known to reduce targeted adhesion efficiency in vitro. However, the role of plasma proteins on the adhesion efficiency of VTCs in laboratory animals remains unknown. Here, in vitro blood flow assays are used to explore the effects of plasma from mouse, rabbit, and porcine on VTC adhesion. Porcine blood exhibited a strong negative plasma effect on VTC adhesion while no significant plasma effect was found with rabbit and mouse blood. A brush density poly(ethylene glycol) (PEG) on VTCs was effective at improving adhesion of microsized, but not nanosized, VTCs in porcine blood. Overall, the results suggest that porcine models, as opposed to mouse, can serve as better models in preclinical research for predicting the in vivo functionality of VTCs for use in humans. These considerations hold great importance for the design of various pharmaceutical products and development of reliable drug delivery systems.

  20. The glycemic, insulinemic and plasma amino acid responses to equi-carbohydrate milk meals, a pilot- study of bovine and human milk

    DEFF Research Database (Denmark)

    Gunnerud, Ulrika; Holst, Jens Juul; Östman, Elin

    2012-01-01

    Dairy proteins, in particular the whey fraction, exert insulinogenic properties and facilitate glycemic regulation through a mechanism involving elevation of certain plasma amino acids, and stimulation of incretins. Human milk is rich in whey protein and has not been investigated in this respect....

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

    Science.gov (United States)

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

    2017-03-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2012-06-29

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

  3. The antibacterial activity of peptides derived from human beta-2 glycoprotein I is inhibited by protein H and M1 protein from Streptococcus pyogenes

    NARCIS (Netherlands)

    Nilsson, Maria; Wasylik, Sylwia; Mörgelin, Matthias; Olin, Anders I.; Meijers, Joost C. M.; Derksen, Ronald H. W. M.; de Groot, Philip G.; Herwald, Heiko

    2008-01-01

    During the last years, the importance of antibacterial peptides has attracted considerable attention. We report here that peptides derived from the fifth domain of beta-2 glycoprotein I (beta(2)GPI), a human heparin binding plasma protein, have antibacterial activities against Gram-positive and

  4. Protein Detection Using the Multiplexed Proximity Extension Assay (PEA) from Plasma and Vaginal Fluid Applied to the Indicating FTA Elute Micro Card™

    Science.gov (United States)

    Berggrund, Malin; Ekman, Daniel; Gustavsson, Inger; Sundfeldt, Karin; Olovsson, Matts; Enroth, Stefan; Gyllensten, Ulf

    2016-01-01

    The indicating FTA elute micro card™ has been developed to collect and stabilize the nucleic acid in biological samples and is widely used in human and veterinary medicine and other disciplines. This card is not recommended for protein analyses, since surface treatment may denature proteins. We studied the ability to analyse proteins in human plasma and vaginal fluid as applied to the indicating FTA elute micro card™ using the sensitive proximity extension assay (PEA). Among 92 proteins in the Proseek Multiplex Oncology Iv2 panel, 87 were above the limit of detection (LOD) in liquid plasma and 56 among 92 above LOD in plasma applied to FTA cards. Washing and protein elution protocols were compared to identify an optimal method. Liquid-based cytology samples showed a lower number of proteins above LOD than FTA cards with vaginal fluid samples applied. Our results demonstrate that samples applied to the indicating FTA elute micro card™ are amendable to protein analyses, given that a sensitive protein detection assay is used. The results imply that biological samples applied to FTA cards can be used for DNA, RNA and protein detection. PMID:28936257

  5. Protein Detection Using the Multiplexed Proximity Extension Assay (PEA from Plasma and Vaginal Fluid Applied to the Indicating FTA Elute Micro Card™

    Directory of Open Access Journals (Sweden)

    Malin Berggrund

    2016-01-01

    Full Text Available The indicating FTA elute micro card™ has been developed to collect and stabilize the nucleic acid in biological samples and is widely used in human and veterinary medicine and other disciplines. This card is not recommended for protein analyses, since surface treatment may denature proteins. We studied the ability to analyse proteins in human plasma and vaginal fluid as applied to the indicating FTA elute micro card™ using the sensitive proximity extension assay (PEA. Among 92 proteins in the Proseek Multiplex Oncology Iv2 panel, 87 were above the limit of detection (LOD in liquid plasma and 56 among 92 above LOD in plasma applied to FTA cards. Washing and protein elution protocols were compared to identify an optimal method. Liquid-based cytology samples showed a lower number of proteins above LOD than FTA cards with vaginal fluid samples applied. Our results demonstrate that samples applied to the indicating FTA elute micro card™ are amendable to protein analyses, given that a sensitive protein detection assay is used. The results imply that biological samples applied to FTA cards can be used for DNA, RNA and protein detection.

  6. Protein Detection Using the Multiplexed Proximity Extension Assay (PEA) from Plasma and Vaginal Fluid Applied to the Indicating FTA Elute Micro Card™.

    Science.gov (United States)

    Berggrund, Malin; Ekman, Daniel; Gustavsson, Inger; Sundfeldt, Karin; Olovsson, Matts; Enroth, Stefan; Gyllensten, Ulf

    2016-01-01

    The indicating FTA elute micro card™ has been developed to collect and stabilize the nucleic acid in biological samples and is widely used in human and veterinary medicine and other disciplines. This card is not recommended for protein analyses, since surface treatment may denature proteins. We studied the ability to analyse proteins in human plasma and vaginal fluid as applied to the indicating FTA elute micro card™ using the sensitive proximity extension assay (PEA). Among 92 proteins in the Proseek Multiplex Oncology Iv2 panel, 87 were above the limit of detection (LOD) in liquid plasma and 56 among 92 above LOD in plasma applied to FTA cards. Washing and protein elution protocols were compared to identify an optimal method. Liquid-based cytology samples showed a lower number of proteins above LOD than FTA cards with vaginal fluid samples applied. Our results demonstrate that samples applied to the indicating FTA elute micro card™ are amendable to protein analyses, given that a sensitive protein detection assay is used. The results imply that biological samples applied to FTA cards can be used for DNA, RNA and protein detection.

  7. Culture Medium Supplements Derived from Human Platelet and Plasma: Cell Commitment and Proliferation Support

    Directory of Open Access Journals (Sweden)

    Anita Muraglia

    2017-11-01

    Full Text Available Present cell culture medium supplements, in most cases based on animal sera, are not fully satisfactory especially for the in vitro expansion of cells intended for human cell therapy. This paper refers to (i an heparin-free human platelet lysate (PL devoid of serum or plasma components (v-PL and (ii an heparin-free human serum derived from plasma devoid of PL components (Pl-s and to their use as single components or in combination in primary or cell line cultures. Human mesenchymal stem cells (MSC primary cultures were obtained from adipose tissue, bone marrow, and umbilical cord. Human chondrocytes were obtained from articular cartilage biopsies. In general, MSC expanded in the presence of Pl-s alone showed a low or no proliferation in comparison to cells grown with the combination of Pl-s and v-PL. Confluent, growth-arrested cells, either human MSC or human articular chondrocytes, treated with v-PL resumed proliferation, whereas control cultures, not supplemented with v-PL, remained quiescent and did not proliferate. Interestingly, signal transduction pathways distinctive of proliferation were activated also in cells treated with v-PL in the absence of serum, when cell proliferation did not occur, indicating that v-PL could induce the cell re-entry in the cell cycle (cell commitment, but the presence of serum proteins was an absolute requirement for cell proliferation to happen. Indeed, Pl-s alone supported cell growth in constitutively activated cell lines (U-937, HeLa, HaCaT, and V-79 regardless of the co-presence of v-PL. Plasma- and plasma-derived serum were equally able to sustain cell proliferation although, for cells cultured in adhesion, the Pl-s was more efficient than the plasma from which it was derived. In conclusion, the cells expanded in the presence of the new additives maintained their differentiation potential and did not show alterations in their karyotype.

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

    Science.gov (United States)

    Leser, George P; Lamb, Robert A

    2017-05-01

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

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

    International Nuclear Information System (INIS)

    Hau, D.M.

    1984-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

    Hau, D.M.

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

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

    DEFF Research Database (Denmark)

    Jessen, Flemming; Wulff, Tune

    2015-01-01

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

  12. Altered plasma apolipoprotein modifications in patients with pancreatic cancer: protein characterization and multi-institutional validation.

    Directory of Open Access Journals (Sweden)

    Kazufumi Honda

    Full Text Available BACKGROUND: Among the more common human malignancies, invasive ductal carcinoma of the pancreas has the worst prognosis. The poor outcome seems to be attributable to difficulty in early detection. METHODS: We compared the plasma protein profiles of 112 pancreatic cancer patients with those of 103 sex- and age-matched healthy controls (Cohort 1 using a newly developed matrix-assisted laser desorption/ionization (oMALDI QqTOF (quadrupole time-of-flight mass spectrometry (MS system. RESULTS: We found that hemi-truncated apolipoprotein AII dimer (ApoAII-2; 17252 m/z, unglycosylated apolipoprotein CIII (ApoCIII-0; 8766 m/z, and their summed value were significantly decreased in the pancreatic cancer patients [P = 1.36×10(-21, P = 4.35×10(-14, and P = 1.83×10(-24 (Mann-Whitney U-test; area-under-curve values of 0.877, 0.798, and 0.903, respectively]. The significance was further validated in a total of 1099 plasma/serum samples, consisting of 2 retrospective cohorts [Cohort 2 (n = 103 and Cohort 3 (n = 163] and a prospective cohort [Cohort 4 (n = 833] collected from 8 medical institutions in Japan and Germany. CONCLUSIONS: We have constructed a robust quantitative MS profiling system and used it to validate alterations of modified apolipoproteins in multiple cohorts of patients with pancreatic cancer.

  13. Isolation and characterization of gelatin-binding proteins from goat seminal plasma

    Directory of Open Access Journals (Sweden)

    Lazure Claude

    2003-04-01

    Full Text Available Abstract A family of proteins designated BSP-A1, BSP-A2, BSP-A3 and BSP-30 kDa (collectively called BSP proteins for Bovine Seminal Plasma proteins constitute the major protein fraction in the bull seminal plasma. These proteins interact with choline phospholipids on the sperm surface and play a role in the membrane stabilization (decapacitation and destabilization (capacitation process. Homologous proteins have been isolated from boar and stallion seminal plasma. In the current study we report the isolation and preliminary characterization of homologous proteins from goat seminal plasma. Frozen semen (-80°C was thawed and centrifuged to remove sperm. The proteins in the supernatant were precipitated by the addition of cold ethanol. The precipitates were dissolved in ammonium bicarbonate and lyophilised. The lyophilised proteins were dissolved in phosphate buffer and loaded onto a gelatin-agarose column, which was previously equilibrated with the same buffer. The column was successively washed with phosphate buffer, with phosphate buffer saline and with 0.5 M urea in phosphate buffer saline to remove unadsorbed proteins, and the adsorbed proteins were eluted with 5 M urea in phosphate buffer saline. Analysis of pooled, dialysed and lyophilised gelatin-agarose adsorbed protein fraction by SDS-PAGE indicated the presence of four protein bands that were designated GSP-14 kDa, GSP-15 kDa, GSP-20 kDa and GSP-22 kDa (GSP, Goat Seminal Plasma proteins. Heparin-affinity chromatography was then used for the separation of GSP-20 and -22 kDa from GSP-14 and -15 kDa. Finally, HPLC separation permitted further isolation of each one from the other. Amino acid sequence analysis of these proteins indicated that they are homologous to BSP proteins. In addition, these BSP homologs bind to hen's egg-yolk low-density lipoproteins. These results together with our previous data indicate that BSP family proteins are ubiquitous in mammalian seminal plasma, exist in

  14. The human protein disulfide isomerase gene family

    Directory of Open Access Journals (Sweden)

    Galligan James J

    2012-07-01

    Full Text Available Abstract Enzyme-mediated disulfide bond formation is a highly conserved process affecting over one-third of all eukaryotic proteins. The enzymes primarily responsible for facilitating thiol-disulfide exchange are members of an expanding family of proteins known as protein disulfide isomerases (PDIs. These proteins are part of a larger superfamily of proteins known as the thioredoxin protein family (TRX. As members of the PDI family of proteins, all proteins contain a TRX-like structural domain and are predominantly expressed in the endoplasmic reticulum. Subcellular localization and the presence of a TRX domain, however, comprise the short list of distinguishing features required for gene family classification. To date, the PDI gene family contains 21 members, varying in domain composition, molecular weight, tissue expression, and cellular processing. Given their vital role in protein-folding, loss of PDI activity has been associated with the pathogenesis of numerous disease states, most commonly related to the unfolded protein response (UPR. Over the past decade, UPR has become a very attractive therapeutic target for multiple pathologies including Alzheimer disease, Parkinson disease, alcoholic and non-alcoholic liver disease, and type-2 diabetes. Understanding the mechanisms of protein-folding, specifically thiol-disulfide exchange, may lead to development of a novel class of therapeutics that would help alleviate a wide range of diseases by targeting the UPR.

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

    Science.gov (United States)

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

    2017-04-01

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

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

    Science.gov (United States)

    Qiao, Zhenzhen; Libault, Marc

    2017-10-03

    Plasma membrane microdomains are plasma membrane sub-compartments enriched in sphingolipids and sterols, and composed by a specific set of proteins. They are involved in recognizing signal molecules, transducing these signals, and controlling endocytosis and exocytosis processes. In a recent study, applying biochemical and microscopic methods, we characterized the soybean GmFWL1 protein, a major regulator of soybean nodulation, as a new membrane microdomain-associated protein. Interestingly, upon rhizobia inoculation of the soybean root system, GmFWL1 and one of its interacting partners, GmFLOT2/4, both translocate to the root hair cell tip, the primary site of interaction and infection between soybean and Rhizobium. The role of GmFWL1 as a plasma membrane microdomain-associated protein is also supported by immunoprecipitation assays performed on soybean nodules, which revealed 178 GmFWL1 protein partners including a large number of microdomain-associated proteins such as GmFLOT2/4. In this addendum, we provide additional information about the identity of the soybean proteins repetitively identified as GmFWL1 protein partners. Their function is discussed especially in regard to plant-microbe interactions and microbial symbiosis. This addendum will provide new insights in the role of plasma membrane microdomains in regulating legume nodulation.

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

    NARCIS (Netherlands)

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

    1996-01-01

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

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

    International Nuclear Information System (INIS)

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

    1976-01-01

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

  19. Determination of moxifloxacin in human plasma, plasma ultrafiltrate, and cerebrospinal fluid by a rapid and simple liquid chromatography- tandem mass spectrometry method.

    Science.gov (United States)

    Pranger, Arianna D; Alffenaar, Jan-Willem C; Wessels, A Mireille A; Greijdanus, Ben; Uges, Donald R A

    2010-04-01

    Moxifloxacin (MFX) is a useful agent in the treatment of multi-drug-resistant tuberculosis (MDR-TB). At Tuberculosis Centre Beatrixoord, a referral center for tuberculosis in the Netherlands, approximately 36% of the patients have received MFX as treatment. Based on the variability of MFX AUC, the variability of in vitro susceptibility to MFX of M. tuberculosis, and the variability of penetration into sanctuary sites, measuring the concentration of MFX in plasma and cerebrospinal fluid (CSF) could be recommended. Therefore, a rapid and validated liquid chromatography-tandem mass spectrometry (LC-MS-MS) analyzing method with a simple pretreatment procedure was developed for therapeutic drug monitoring of MFX in human plasma and CSF. Because of the potential influence of protein binding on efficacy, we decided to determine both bound and unbound (ultrafiltrate) fraction of MFX. The calibration curves were linear in the therapeutic range of 0.05 to 5.0 mg/L plasma and CSF with CV in the range of -5.4% to 9.3%. MFX ultrafiltrate samples could be determined with the same method setup for analysis of MFX in CSF. The LC-MS-MS method developed in this study is suitable for monitoring MFX in human plasma, plasma ultrafiltrate, and CSF.

  20. High-throughput sequencing of human plasma RNA by using thermostable group II intron reverse transcriptases

    Science.gov (United States)

    Qin, Yidan; Yao, Jun; Wu, Douglas C.; Nottingham, Ryan M.; Mohr, Sabine; Hunicke-Smith, Scott; Lambowitz, Alan M.

    2016-01-01

    Next-generation RNA-sequencing (RNA-seq) has revolutionized transcriptome profiling, gene expression analysis, and RNA-based diagnostics. Here, we developed a new RNA-seq method that exploits thermostable group II intron reverse transcriptases (TGIRTs) and used it to profile human plasma RNAs. TGIRTs have higher thermostability, processivity, and fidelity than conventional reverse transcriptases, plus a novel template-switching activity that can efficiently attach RNA-seq adapters to target RNA sequences without RNA ligation. The new TGIRT-seq method enabled construction of RNA-seq libraries from RNA in RNA in 1-mL plasma samples from a healthy individual revealed RNA fragments mapping to a diverse population of protein-coding gene and long ncRNAs, which are enriched in intron and antisense sequences, as well as nearly all known classes of small ncRNAs, some of which have never before been seen in plasma. Surprisingly, many of the small ncRNA species were present as full-length transcripts, suggesting that they are protected from plasma RNases in ribonucleoprotein (RNP) complexes and/or exosomes. This TGIRT-seq method is readily adaptable for profiling of whole-cell, exosomal, and miRNAs, and for related procedures, such as HITS-CLIP and ribosome profiling. PMID:26554030

  1. Exploring the Human Plasma Proteome for Humoral Mediators of Remote Ischemic Preconditioning - A Word of Caution

    Science.gov (United States)

    Helgeland, Erik; Breivik, Lars Ertesvåg; Vaudel, Marc; Svendsen, Øyvind Sverre; Garberg, Hilde; Nordrehaug, Jan Erik; Berven, Frode Steingrimsen; Jonassen, Anne Kristine

    2014-01-01

    Despite major advances in early revascularization techniques, cardiovascular diseases are still the leading cause of death worldwide, and myocardial infarctions contribute heavily to this. Over the past decades, it has become apparent that reperfusion of blood to a previously ischemic area of the heart causes damage in and of itself, and that this ischemia reperfusion induced injury can be reduced by up to 50% by mechanical manipulation of the blood flow to the heart. The recent discovery of remote ischemic preconditioning (RIPC) provides a non-invasive approach of inducing this cardioprotection at a distance. Finding its endogenous mediators and their operative mode is an important step toward increasing the ischemic tolerance. The release of humoral factor(s) upon RIPC was recently demonstrated and several candidate proteins were published as possible mediators of the cardioprotection. Before clinical applicability, these potential biomarkers and their efficiency must be validated, a task made challenging by the large heterogeneity in reported data and results. Here, in an attempt to reproduce and provide more experimental data on these mediators, we conducted an unbiased in-depth analysis of the human plasma proteome before and after RIPC. From the 68 protein markers reported in the literature, only 28 could be mapped to manually reviewed (Swiss-Prot) protein sequences. 23 of them were monitored in our untargeted experiment. However, their significant regulation could not be reproducibly estimated. In fact, among the 394 plasma proteins we accurately quantified, no significant regulation could be confidently and reproducibly assessed. This indicates that it is difficult to both monitor and reproduce published data from experiments exploring for RIPC induced plasma proteomic regulations, and suggests that further work should be directed towards small humoral factors. To simplify this task, we made our proteomic dataset available via ProteomeXchange, where

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

    DEFF Research Database (Denmark)

    Liaset, Bjørn; Madsen, Lise; Hao, Qin

    2009-01-01

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

  3. Comparison of human plasma low- and high-density lipoproteins as substrates for lecithin: cholesterol acyltransferase.

    Science.gov (United States)

    Barter, P J; Hopkins, G J; Gorjatschko, L

    1984-01-17

    A recent observation that lecithin: cholesterol acyltransferase (EC 2.3.1.43) interacts with both low-density lipoproteins (LDL) and high-density lipoproteins (HDL) in human plasma is in apparent conflict with an earlier finding that the purified enzyme, while highly reactive with isolated HDL, was only minimally reactive with LDL. There is evidence, however, that lecithin: cholesterol acyltransferase may exist physiologically as a component of a complex with other proteins and that studies with the isolated enzyme may therefore provide misleading results. Consequently, interactions of the enzyme with isolated human lipoproteins have been re-examined in incubations containing lecithin: cholesterol acyltransferase as a component of human lipoprotein-free plasma in which a physiologically active complex of the enzyme with other proteins may have been preserved. In this system there was a ready esterification of the free cholesterol associated with both LDL and HDL-subfraction 3 (HDL3) in reactions that obeyed typical enzyme-saturation kinetics. For a given preparation of lipoprotein-free plasma the Vmax values with LDL and with HDL3 were virtually identical. The apparent Km for free cholesterol associated with HDL3 was 5.6 X 10(-5) M, while for that associated with LDL it was 4.1 X 10(-4) M. This implied that, in terms of free cholesterol concentration, the affinity of HDL3 for lecithin: cholesterol acyltransferase was about 7-times greater than that of LDL. When expressed in terms of lipoprotein particle concentration, however, it was apparent that the affinity of LDL for the enzyme was considerably greater than that of HDL3. When the lipoprotein fractions were equated in terms of lipoprotein surface area, the apparent affinities of the two fractions for the enzyme were found to be comparable.

  4. Intracellular Delivery of Proteins with Cell-Penetrating Peptides for Therapeutic Uses in Human Disease.

    Science.gov (United States)

    Dinca, Ana; Chien, Wei-Ming; Chin, Michael T

    2016-02-22

    Protein therapy exhibits several advantages over small molecule drugs and is increasingly being developed for the treatment of disorders ranging from single enzyme deficiencies to cancer. Cell-penetrating peptides (CPPs), a group of small peptides capable of promoting transport of molecular cargo across the plasma membrane, have become important tools in promoting the cellular uptake of exogenously delivered proteins. Although the molecular mechanisms of uptake are not firmly established, CPPs have been empirically shown to promote uptake of various molecules, including large proteins over 100 kiloDaltons (kDa). Recombinant proteins that include a CPP tag to promote intracellular delivery show promise as therapeutic agents with encouraging success rates in both animal and human trials. This review highlights recent advances in protein-CPP therapy and discusses optimization strategies and potential detrimental effects.

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

    Science.gov (United States)

    Isono, Erika; Kalinowska, Kamila

    2017-12-01

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

  6. A human protein interaction network shows conservation of aging processes between human and invertebrate species.

    Directory of Open Access Journals (Sweden)

    Russell Bell

    2009-03-01

    Full Text Available We have mapped a protein interaction network of human homologs of proteins that modify longevity in invertebrate species. This network is derived from a proteome-scale human protein interaction Core Network generated through unbiased high-throughput yeast two-hybrid searches. The longevity network is composed of 175 human homologs of proteins known to confer increased longevity through loss of function in yeast, nematode, or fly, and 2,163 additional human proteins that interact with these homologs. Overall, the network consists of 3,271 binary interactions among 2,338 unique proteins. A comparison of the average node degree of the human longevity homologs with random sets of proteins in the Core Network indicates that human homologs of longevity proteins are highly connected hubs with a mean node degree of 18.8 partners. Shortest path length analysis shows that proteins in this network are significantly more connected than would be expected by chance. To examine the relationship of this network to human aging phenotypes, we compared the genes encoding longevity network proteins to genes known to be changed transcriptionally during aging in human muscle. In the case of both the longevity protein homologs and their interactors, we observed enrichments for differentially expressed genes in the network. To determine whether homologs of human longevity interacting proteins can modulate life span in invertebrates, homologs of 18 human FRAP1 interacting proteins showing significant changes in human aging muscle were tested for effects on nematode life span using RNAi. Of 18 genes tested, 33% extended life span when knocked-down in Caenorhabditis elegans. These observations indicate that a broad class of longevity genes identified in invertebrate models of aging have relevance to human aging. They also indicate that the longevity protein interaction network presented here is enriched for novel conserved longevity proteins.

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

    Science.gov (United States)

    Roy, Jahnabi; Pondenis, Holly; Fan, Timothy M; Das, Aditi

    2015-10-20

    Mammalian plasma membrane proteins make up the largest class of drug targets yet are difficult to study in a cell free system because of their intransigent nature. Herein, we perform direct encapsulation of plasma membrane proteins derived from mammalian cells into a functional nanodisc library. Peptide fingerprinting was used to analyze the proteome of the incorporated proteins in nanodiscs and to further demonstrate that the lipid composition of the nanodiscs directly affects the class of protein that is incorporated. Furthermore, the functionality of the incorporated membrane proteome was evaluated by measuring the activity of membrane proteins: Na(+)/K(+)-ATPase and receptor tyrosine kinases. This work is the first report of the successful establishment and characterization of a cell free functional library of mammalian membrane proteins into nanodiscs.

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

    Science.gov (United States)

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

    2017-07-05

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

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

    Science.gov (United States)

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

    2010-12-03

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

  10. In vitro studies on the distribution of probucol among human plasma lipoproteins

    International Nuclear Information System (INIS)

    Urien, S.; Riant, P.; Albengres, E.; Brioude, R.; Tillement, J.P.

    1984-01-01

    The role of human plasma lipoproteins as carriers in the blood transport of the cholesterol-lowering and water-insoluble drug, probucol, was investigated in in vitro studies. [ 14 C]Probucol was incubated in whole human blood, a serum pool, individual diluted sera, and isolated protein and lipoprotein fractions. In whole blood, about 90% partitioned in plasma. Following ultracentrifugal fractionation of the serum, it was found that less than 5% distributed in the d greater than 1.20 protein fraction (albumin-rich fraction) and more than 95% in the lipoprotein fractions. The distribution of probucol in the lipoprotein fractions correlated with the lipoprotein total lipid volume under saturation conditions (incubation of isolated lipoprotein fractions) as well as nonsaturation conditions (fractionation of serum exposed to [ 14 C]probucol). Incubation of the albumin-rich fraction and of apolipoproteins originating from the isolated lipoprotein fractions showed that they account for a negligible part in the interaction of probucol with blood components. The probucol uptake of individual sera was shown to be correlated to the lipid content of the serum. When probucol was incubated in erythrocyte suspensions containing variable amounts of lipoproteins, probucol partitioned less in erythrocytes as the lipoprotein concentration increased in the suspension

  11. Effect of pregnancy-associated plasma protein-A (PAPP-A) single-nucleotide polymorphisms on the level and activity of PAPP-A and the hormone profile in fluid from normal human small antral follicles

    DEFF Research Database (Denmark)

    Bøtkjær, Jane Alrø; Borgbo, Tanni; Kløverpris, Søren

    2016-01-01

    Objective: To reveal a possible relationship between two single nucleotide polymorphisms (SNPs) in PAPP-A—1224 (rs7020782) and 327 (rs12375498)—and the level and activity of PAPP-A in follicular fluid (FF) of human small antral follicles, and to analyze the intrafollicular hormone levels. Design:...

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

    Science.gov (United States)

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

    2017-07-15

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

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

    Czech Academy of Sciences Publication Activity Database

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

    2004-01-01

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

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

    Science.gov (United States)

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

    2017-05-01

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

  15. Plasma Renin Activity in Children with Protein Energy Malnutrition ...

    African Journals Online (AJOL)

    Plasma renin activity was measured by bio-assay in 100 children with kwashiorkor and in 20 healthy children, and also by radio-immunoassay in another 26 children with kwashiorkor and in another 20 healthy children. Both methods showed that (compared with healthy children) renin activity was significantly increased in ...

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

    International Nuclear Information System (INIS)

    Li Jinping; Ma Liren

    1986-01-01

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

  17. Effects of atorvastatin on human c reactive protein metabolism

    Science.gov (United States)

    Statins are known to reduce plasma C-reactive protein (CRP) concentrations. Our goals were to define the mechanisms by which CRP was reduced by maximal dose atorvastatin. Eight subjects with combined hyperlipidemia (5 men and 3 postmenopausal women) were enrolled in a randomized, placebo-controlled...

  18. Obesity and Low-Grade Inflammation Increase Plasma Follistatin-Like 3 in Humans

    DEFF Research Database (Denmark)

    Brandt, Claus; Pedersen, Maria; Rinnov, Anders

    2014-01-01

    , plasma leptin, fasting insulin, and HOMA B and negatively with HOMA S. Furthermore plasma fstl3 correlated positively with plasma TNF-α and IL-6 levels. Infusion of LPS and TNF-α, but not IL-6 and insulin, increased plasma fstl3 in humans. CONCLUSION: Plasma fstl3 is increased in obese subjects......BACKGROUND: Rodent models suggest that follistatin-like 3 (fstl3) is associated with diabetes and obesity. In humans, plasma fstl3 is reduced with gestational diabetes. In vitro, TNF-α induces fstl3 secretion, which suggests a link to inflammation. OBJECTIVE: To elucidate the association between...... plasma fstl3 and obesity, insulin resistance, and low-grade inflammation in humans. STUDY DESIGN: Plasma fstl3 levels were determined in a cross-sectional study including three groups: patients with type 2 diabetes, impaired glucose tolerance, and healthy controls. In addition, lipopolysaccharide (LPS...

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

    International Nuclear Information System (INIS)

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

    2006-01-01

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

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

    Science.gov (United States)

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

    2006-08-01

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

  1. Semen quality and concentration of soluble proteins in the seminal plasma of Alpine bucks Semen quality and concentration of soluble proteins in the seminal plasma of Alpine bucks

    Directory of Open Access Journals (Sweden)

    Simone Eliza Facione Guimarães

    2010-06-01

    Full Text Available It was aimed to study the in vitro seminal quality analyzed by complementary tests and to compare them with physical, morphological and biochemical aspects of male goat semen of the Alpine breed. This experiment took place at the Federal University of Viçosa, situated at 20º45’ S latitude and 42º51’ W longitude, Southwest of Brazil. It was done during the summer months of January and February, and three adult male goats of the Alpine breed were used in intensive conditions. The semen was collected by artificial vagina method. In all semen samples (45 ejaculates, after the physical and morphological analysis, the hiposmotic test was done. In 24 ejaculates, it were done thermo-resistance test, and in 21 ejaculates it were determined the concentration of total soluble proteins in seminal plasma. The male goats presented difference in the semen physical and morphological aspects, in the hiposmotic test and thermo-resistance test, but they did not presented difference in total soluble proteins concentration in seminal plasma. Results of the slow thermo-resistance test and hiposmotic test were positively correlated (r = 0.60. It was concluded, according to our results, that the concentration of total soluble proteins in seminal plasma can not be used as a parameter to predict the seminal quality of Alpine bucks.It was aimed to study the in vitro seminal quality analyzed by complementary tests and to compare them with physical, morphological and biochemical aspects of male goat semen of the Alpine breed. This experiment took place at the Federal University of Viçosa, situated at 20º45’ S latitude and 42º51’ W longitude, Southwest of Brazil. It was done during the summer months of January and February, and three adult male goats of the Alpine breed were used in intensive conditions. The semen was collected by artificial vagina method. In all semen samples (45 ejaculates, after the physical and morphological analysis, the hiposmotic test

  2. Protein kinase CK2 in human diseases

    DEFF Research Database (Denmark)

    Guerra, Barbara; Issinger, Olaf-Georg

    2008-01-01

    Protein kinase CK2 (formerly referred to as casein kinase II) is an evolutionary conserved, ubiquitous protein kinase. There are two paralog catalytic subunits, i.e. alpha (A1) and alpha' (A2). The alpha and alpha' subunits are linked to two beta subunits to produce a heterotetrameric structure...

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

    Science.gov (United States)

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

    2016-02-01

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

  4. The preparation of albumin as a biological drug from human plasma by fiber filtration

    Directory of Open Access Journals (Sweden)

    Mousavi Hosseini K

    2011-08-01

    Full Text Available "nBackground: In recent years, consumption of whole-blood for the treatment of patients has decreased but use of biological plasma-derived medicines such as albumin, immunoglobulin and coagulation factors have increased instead. Paying attention to albumin molecular structure is important for its isolation from human plasma. Albumin is a single-chain protein consisting of about 585 amino acids and a molecular weight of 66500 Daltons. Albumin is a stable molecule and it is spherical in shape. There are different methods for human albumin preparation. Considering the large consumption of this biological drug in clinical settings, methods with fewer steps in production line are of big advantage in saving time and manufacturing more products."n "nMethods: In this project, we prepared human albumin using hollow fiber cartridges in order to omit the rework on fraction V+VI. Human albumin is usually produced by the application of cold ethanol method, where albumin is obtained from fraction V by doing a rework on fraction V+VI to separate fraction V."n "nResults: In the current work, human albumin was prepared from fraction V+VI by the help of hollow fiber cartridges. With a concentration of 20%, the obtained albumin had 96.5% of monomer and 3.5% of polymer and polymer aggregate."n "nConclusion: Comparing the obtained human albumin with a number of commercial human albumin samples by the use of SDS-page, the results were satisfactory regarding the 3.5 percent polymer and aggregate rate for the prepared albumin.

  5. Deoxyribonucleic-binding homeobox proteins are augmented in human cancer

    DEFF Research Database (Denmark)

    Wewer, U M; Mercurio, A M; Chung, S Y

    1990-01-01

    Homeobox genes encode sequence-specific DNA-binding proteins that are involved in the regulation of gene expression during embryonic development. In this study, we examined the expression of homeobox proteins in human cancer. Antiserum was obtained against a synthetic peptide derived from...... was then isolated and used to elicit a rabbit antiserum. In immunostaining, both antisera reacted with the nuclei of cultured tumor cells. In tissue sections of human carcinoma, nuclear immunoreactivity was observed in the tumor cells in 40 of 42 cases examined. Adjacent normal epithelial tissue obtained from......, the presence of the homeobox transcript in human carcinoma was documented by in situ hybridization and RNase protection mapping. These results demonstrate that human cancer is associated with the expression of homeobox proteins. Such homeobox proteins, as well as other regulatory proteins, could be involved...

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

    Science.gov (United States)

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

    2015-10-14

    Plasma membrane intrinsic proteins (PIPs) are one subfamily of aquaporins that mediate the transmembrane transport of water. To reveal their function in poplar, we generated transgenic poplar plants in which the translation of PIP genes was downregulated by RNA interference investigated these plants with a comprehensive leaf plasma membrane proteome and physiome analysis. First, inhibition of PIP synthesis strongly altered the leaf plasma membrane protein composition. Strikingly, several signaling components and transporters involved in the regulation of stomatal movement were differentially regulated in transgenic poplars. Furthermore, hormonal crosstalk related to abscisic acid, auxin and brassinosteroids was altered, in addition to cell wall biosynthesis/cutinization, the organization of cellular structures and membrane trafficking. A physiological analysis confirmed the proteomic results. The leaves had wider opened stomata and higher net CO2 assimilation and transpiration rates as well as greater mesophyll conductance for CO2 (gm) and leaf hydraulic conductance (Kleaf). Based on these results, we conclude that PIP proteins not only play essential roles in whole leaf water and CO2 flux but have important roles in the regulation of stomatal movement. Copyright © 2015. Published by Elsevier B.V.

  7. Protein Translation and Signaling in Human Eosinophils

    Directory of Open Access Journals (Sweden)

    Stephane Esnault

    2017-09-01

    Full Text Available We have recently reported that, unlike IL-5 and GM-CSF, IL-3 induces increased translation of a subset of mRNAs. In addition, we have demonstrated that Pin1 controls the activity of mRNA binding proteins, leading to enhanced mRNA stability, GM-CSF protein production and prolonged eosinophil (EOS survival. In this review, discussion will include an overview of cap-dependent protein translation and its regulation by intracellular signaling pathways. We will address the more general process of mRNA post-transcriptional regulation, especially regarding mRNA binding proteins, which are critical effectors of protein translation. Furthermore, we will focus on (1 the roles of IL-3-driven sustained signaling on enhanced protein translation in EOS, (2 the mechanisms regulating mRNA binding proteins activity in EOS, and (3 the potential targeting of IL-3 signaling and the signaling leading to mRNA binding activity changes to identify therapeutic targets to treat EOS-associated diseases.

  8. Human Cells as Platform to Produce Gamma-Carboxylated Proteins.

    Science.gov (United States)

    de Sousa Bomfim, Aline; de Freitas, Marcela Cristina Corrêa; Covas, Dimas Tadeu; de Sousa Russo, Elisa Maria

    2018-01-01

    The gamma-carboxylated proteins belong to a family of proteins that depend on vitamin K for normal biosynthesis. The major representative gamma-carboxylated proteins are the coagulation system proteins, for example, factor VII, factor IX, factor X, prothrombin, and proteins C, S, and Z. These molecules have harbored posttranslational modifications, such as glycosylation and gamma-carboxylation, and for this reason they need to be produced in mammalian cell lines. Human cells lines have emerged as the most promising alternative to the production of gamma-carboxylated proteins. In this chapter, the methods to generate human cells as a platform to produce gamma-carboxylated proteins, for example the coagulation factors VII and IX, are presented. From the cell line modification up to the vitamin K adaptation of the produced cells is described in the protocols presented in this chapter.

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

    KAUST Repository

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

    2012-01-01

    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

  10. Plasma membrane proteomics of human breast cancer cell lines identifies potential targets for breast cancer diagnosis and treatment.

    Directory of Open Access Journals (Sweden)

    Yvonne S Ziegler

    Full Text Available The use of broad spectrum chemotherapeutic agents to treat breast cancer results in substantial and debilitating side effects, necessitating the development of targeted therapies to limit tumor proliferation and prevent metastasis. In recent years, the list of approved targeted therapies has expanded, and it includes both monoclonal antibodies and small molecule inhibitors that interfere with key proteins involved in the uncontrolled growth and migration of cancer cells. The targeting of plasma membrane proteins has been most successful to date, and this is reflected in the large representation of these proteins as targets of newer therapies. In view of these facts, experiments were designed to investigate the plasma membrane proteome of a variety of human breast cancer cell lines representing hormone-responsive, ErbB2 over-expressing and triple negative cell types, as well as a benign control. Plasma membranes were isolated by using an aqueous two-phase system, and the resulting proteins were subjected to mass spectrometry analysis. Overall, each of the cell lines expressed some unique proteins, and a number of proteins were expressed in multiple cell lines, but in patterns that did not always follow traditional clinical definitions of breast cancer type. From our data, it can be deduced that most cancer cells possess multiple strategies to promote uncontrolled growth, reflected in aberrant expression of tyrosine kinases, cellular adhesion molecules, and structural proteins. Our data set provides a very rich and complex picture of plasma membrane proteins present on breast cancer cells, and the sorting and categorizing of this data provides interesting insights into the biology, classification, and potential treatment of this prevalent and debilitating disease.

  11. Radioimmunoassay of human eosinophil cationic protein

    International Nuclear Information System (INIS)

    Venge, P.; Roxin, L.E.; Olsson, I.

    1977-01-01

    A radioimmunosorbent assay has been developed which allows the detection in serum of a cationic protein derived from eosinophil granulocytes. In 34 healthy individuals the mean level was 31 μg/l. with a range of 5 to 55 μg/l. The serum concentration of 'eosinophil' cationic protein was correlated (P<0.001) to the number of eosinophil granulocytes in peripheral blood. Quantitiation of 'eosinophil' cationic protein in serum might be useful in the study of eosinophil granulocyte turnover and function in vivo. (author)

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

    Science.gov (United States)

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

    2014-01-01

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

  13. Pathogen inactivation in fresh frozen plasma using riboflavin and ultraviolet light: Effects on plasma proteins and coagulation factor VIII

    Directory of Open Access Journals (Sweden)

    Stanojković Zoran

    2011-01-01

    Full Text Available Background/Aim. Riboflavin (vitamin B2 activated by ultraviolet (UV light, produces active oxygen which damages cell membrane and prevents replication of the carrier of diseases (viruses, bacteria, protozoa in all blood products. The aim of this study was to establish the influence of the process of photo inactivation in pathogens using riboflavin and UV rays on the concentration of coagulation factor VIII:C (FVIII:C and proteins in plasma that were treated before freezing. Methods. The examination included 20 units of plasma, separated from whole blood donated by voluntary blood donors around 6 hours from the moment of collection. The units were pooled and separated in to two groups: one consisted of 10 control units and the other of 10 experimental units. Experimental units of the plasma were treated by riboflavin (35 mL and UV rays (6.24 J/mL, 265-370 nm on Mirasol aparature (Caridian BCT Biotechnologies, USA in approximate duration of 6 minutes. Furthermore, 35 mL of saline solution was added to the control plasma. One sample for examining was taken from the control plasma (KG and two residual were taken from experimental plasma after the addition of riboflavin either before (EG1 or post illumination (EG2. Results. Comparing the mean values of FVIII:C (% we noticed statistically significantly higher level in the EG1 group than in the EG2 group (65.00 ± 4.52 vs 63.20 ± 4.73; t = 4.323, p = 0.002, while between the KG and experimental groups (EG1 and EG2 there was no statistically significant difference in the concentration of FVIII:C. There was a statistically significant decrease of albumin concentration (g/L in the EG2 group comparing to the KG (33.35 ± 0.94 vs 31.94 ± 0.84; t = 3.534, p = 0.002, but there was no mentioned difference in albumin concentration between the KG and the EG1, so as between the EG1 and the EG2. Conclusion. Plasma inactivated by riboflavin and UV rays (Mirasol PRT sistem, Caridian BCT, USA keeps all the

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

    Science.gov (United States)

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

    2017-09-01

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

  15. Glycine uptake by microvillous and basal plasma membrane vesicles from term human placentae.

    Science.gov (United States)

    Dicke, J M; Verges, D; Kelley, L K; Smith, C H

    1993-01-01

    Like most amino acids, glycine is present in higher concentrations in the fetus than in the mother. Unlike most amino acids, animal studies suggest fetal concentrations of glycine are minimally in excess of those required for protein synthesis. Abnormal glycine utilization has also been demonstrated in small-for-gestational age human fetuses. The mechanism(s) of glycine uptake in the human placenta are unknown. In other mammalian cells glycine is a substrate for the A, ASC and Gly amino acid transport systems. In this study human placental glycine uptake was characterized using microvillous and basal plasma membrane vesicles each prepared from the same placenta. In both membranes glycine uptake was mediated predominantly by the sodium-dependent A system. Competitive inhibition studies suggest that in microvillous vesicles the small percentage of sodium-dependent glycine uptake not inhibited by methylaminoisobutyric acid (MeAIB) shares a transport system with glycine methyl ester and sarcosine, substrates of the Gly system in other tissues. In addition there are mediated sodium-independent and non-selective transport mechanisms in both plasma membranes. If fetal glycine availability is primarily contingent upon the common and highly regulated A system, glycine must compete with many other substrates potentially resulting in marginal fetal reserves, abnormal utilization and impaired growth.

  16. Identification and characterization of riboflavin-binding proteins in human circulation

    International Nuclear Information System (INIS)

    Innis-Whitehouse, W.S.A.

    1988-01-01

    Riboflavin binding by plasma proteins from healthy human subjects was examined by equilibrium dialysis and binding was observed to vary over a greater than 10-fold range. 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, although FMN and photo-chemical degradation products were more tightly bound. Most of the binding occurred in the gamma-globulin fraction and was attributed to immunoglobulins because the binding proteins and immunoglobulins copurified using various methods, were removed by treatment of plasma with protein A-agarose, and were coincident upon immuno-electrophoresis followed by autoradiography to detect [2- 14 C]-riboflavin. Binding differences among plasma samples were reflected in the binding recovered with the immunoglobulin fractions; however, there was not a direct relationship between the amount of immunoglobulin and the amount of [2- 14 C]riboflavin bound. Hence, it appeared that the binding was due to a subfraction of immunoglobulins

  17. Protein receptor-independent plasma membrane remodeling by HAMLET: a tumoricidal protein-lipid complex.

    Science.gov (United States)

    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.

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

    OpenAIRE

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

    2015-01-01

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

  19. Temporal expression profiling of plasma proteins reveals oxidative stress in early stages of Type 1 Diabetes progression

    International Nuclear Information System (INIS)

    Liu, Chih-Wei; Bramer, Lisa; Computational Modeling); Webb-Robertson, Bobbie-Jo; Computational Modeling); Waugh, Kathleen; Rewers, Marian J.; Zhang, Qibin; Biochemistry)

    2017-01-01

    We report that blood markers other than islet autoantibodies are greatly needed to indicate the pancreatic beta cell destruction process as early as possible, and more accurately reflect the progression of Type 1 Diabetes Mellitus (T1D). To this end, a longitudinal proteomic profiling of human plasma using TMT-10plex-based LC-MS/MS analysis was performed to track temporal proteomic changes of T1D patients (n = 11) across 9 serial time points, spanning the period of T1D natural progression, in comparison with those of the matching healthy controls (n = 10). To our knowledge, the current study represents the largest (> 2000 proteins measured) longitudinal expression profiles of human plasma proteome in T1D research. By applying statistical trend analysis on the temporal expression patterns between T1D and controls, and Benjamini-Hochberg procedure for multiple-testing correction, 13 protein groups were regarded as having statistically significant differences during the entire follow-up period. Moreover, 16 protein groups, which play pivotal roles in response to oxidative stress, have consistently abnormal expression trend before seroconversion to islet autoimmunity. Importantly, the expression trends of two key reactive oxygen species-decomposing enzymes, Catalase and Superoxide dismutase were verified independently by ELISA.

  20. Cellulose membranes are more effective in holding back vital proteins and exhibit less interaction with plasma proteins during hemodialysis.

    Science.gov (United States)

    Pešić, Ivana; Müller, Gerhard A; Baumann, Cosima; Dihazi, Gry H; Koziolek, Michael J; Eltoweissy, Marwa; Bramlage, Carsten; Asif, Abdul R; Dihazi, Hassan

    2013-04-01

    The vast majority of patients with end-stage renal disease are treated with intermittent hemodialysis as a form of renal replacement therapy. To investigate the impact of hemodialysis membrane material on vital protein removal, dialysates from 26 well-characterized hemodialysis patients were collected 5 min after beginning, during 5h of treatment, as well as 5 min before ending of the dialysis sessions. Dialysis sessions were performed using either modified cellulose (n=12) (low-flux and high flux) or synthetic Polyflux (n=14) (low-flux and high-flux) dialyzer. Protein removal during hemodialysis was quantified and the dialysate proteome patterns were analyzed by 2-DE, MS and Western blot. There was a clear correlation between the type of membrane material and the amount of protein removed. Synthetic Polyflux membranes exhibit strong interaction with plasma proteins resulting in a significantly higher protein loss compared to modified cellulosic membrane. Moreover, the proteomics analysis showed that the removed proteins represented different molecular weight range and different functional groups: transport proteins, protease inhibitors, proteins with role in immune response and regulations, constructive proteins and as a part of HLA immune complex. The effect of this protein removal on hemodialysis treatment outcome should be investigated in further studies. Copyright © 2013 Elsevier B.V. All rights reserved.

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

    International Nuclear Information System (INIS)

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

    1988-01-01

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

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

    Science.gov (United States)

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

    2015-11-01

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

  3. Arabidopsis protein kinase PKS5 inhibits the plasma membrane H+ -ATPase by preventing interaction with 14-3-3 protein

    DEFF Research Database (Denmark)

    Fuglsang, Anja Thoe; Guo, Yan; Cuin, Tracey A.

    2007-01-01

    Regulation of the trans-plasma membrane pH gradient is an important part of plant responses to several hormonal and environmental cues, including auxin, blue light, and fungal elicitors. However, little is known about the signaling components that mediate this regulation. Here, we report...... that an Arabidopsis thaliana Ser/Thr protein kinase, PKS5, is a negative regulator of the plasma membrane proton pump (PM Hþ-ATPase). Loss-of-function pks5 mutant plants are more tolerant of high external pH due to extrusion of protons to the extracellular space. PKS5 phosphorylates the PM Hþ-ATPase AHA2 at a novel...

  4. The study of the proteome of healthy human blood plasma under conditions of long-term confinement in an isolation chamber.

    Science.gov (United States)

    Trifonova, O P; Pastushkova, L Kh; Samenkova, N F; Chernobrovkin, A L; Karuzina, I I; Lisitsa, A V; Larina, I M

    2013-05-01

    We identified changes in the proteome of healthy human blood plasma caused by exposure to 105-day confinement in an isolation chamber. After removal of major proteins and concentration of minor proteins, plasma fractions were analyzed by two-dimensional electrophoresis followed by identification of significantly different protein spots by mass spectrometric analysis of the peptide fragments. The levels of α- and β-chains of fibrinogen, a fragment of complement factor C4, apolipoproteins AI and E, plasminogen factor C1 complement, and immunoglobulin M changed in participants during the isolation period. These changes probably reflect the adaptive response to altered conditions of life.

  5. Comparison of Three Sample Preparation Procedures for the Quantification of L-Arginine, Asymmetric Dimethylarginine, and Symmetric Dimethylarginine in Human Plasma Using HPLC-FLD

    DEFF Research Database (Denmark)

    Schou-Pedersen, Anne Marie Voigt; Lykkesfeldt, Jens

    2018-01-01

    Increased asymmetric dimethylarginine (ADMA) in human plasma has been associated with reduced generation of nitric oxide, leading to atherosclerotic diseases. ADMA may therefore be an important biomarker for cardiovascular disease. In the present study, three sample preparation techniques were co...... sample preparation of human plasma samples before HPLC-FLD in providing important information regarding elevated ADMA concentrations.......Increased asymmetric dimethylarginine (ADMA) in human plasma has been associated with reduced generation of nitric oxide, leading to atherosclerotic diseases. ADMA may therefore be an important biomarker for cardiovascular disease. In the present study, three sample preparation techniques were...... compared regarding the quantification of L-arginine and ADMA in human plasma: (A) protein precipitation (PP) based on aqueous trichloroacetic acid (TCA), (B) PP using a mixture of ammonia and acetonitrile, and (C) solid-phase extraction (SPE). The samples were analysed by using high-performance liquid...

  6. Further insight into the roles of the glycans attached to human blood protein C inhibitor

    DEFF Research Database (Denmark)

    Sun, Wei; Parry, Simon; Ubhayasekera, Wimal

    2010-01-01

    Protein C inhibitor (PCI) is a 57-kDa glycoprotein that exists in many tissues and secretions in human. As a member of the serpin superfamily of proteins it displays unusually broad protease specificity. PCI is implicated in the regulation of a wide range of processes, including blood coagulation......, fertilization, prevention of tumors and pathogen defence. It has been reported that PCI isolated from human blood plasma is highly heterogeneous, and that this heterogeneity is caused by differences in N-glycan structures, N-glycosylation occupancy, and the presence of two forms that differ by the presence...... or absence of 6 amino acids at the amino-terminus. In this study we have verified that such heterogeneity exists in PCI purified from single individuals, and that individuals of two different ethnicities possess a similar PCI pattern, verifying that the micro-heterogeneity is conserved among humans...

  7. Quantitation of glial fibrillary acidic protein in human brain tumours

    DEFF Research Database (Denmark)

    Rasmussen, S; Bock, E; Warecka, K

    1980-01-01

    The glial fibrillary acidic protein (GFA) content of 58 human brain tumours was determined by quantitative immunoelectrophoresis, using monospecific antibody against GFA. Astrocytomas, glioblastomas, oligodendrogliomas, spongioblastomas, ependymomas and medulloblastomas contained relatively high...

  8. Determination of flomoxef in human plasma by liquid chromatography/electrospray ionization tandem mass spectrometry.

    Science.gov (United States)

    Kravtsova, Oxana Yu; Paramonov, Sergey A; Vasilevich, Natalya I; Kazyulkin, Denis N; Vlasova, Ekaterina; Engsig, Michael

    2013-12-01

    A specific, sensitive, rapid and reproducible method for the determination of flomoxef in human plasma using high-performance liquid chromatography-tandem mass spectrometry was developed and validated. Flomoxef was detected using an electrospay ionization method operated in negative-ion mode. Chromatographic separation was performed in gradient elution mode on a Luna® C18(2) column (3 μM, 20 × 4.0 mm) at a flow rate of 1 mL/min and runtime 3.5 min. The mobile phase consisted of acetonitrile and water containing 0.1% formic acid as additive. Extraction of flomoxef from plasma and precipitation of plasma proteins was performed with acetonitrile with an absolute recovery of 86.4 ± 1.6%. The calibration curve was linear with a correlation coefficient of 0.999 over the concentration range 10-5000 ng/mL and the lower limit of quantification was 10 ng/mL. The intra- and inter-day precisions were flomoxef revealed that it could be successfully analyzed at 4 ºС over 24 h, but it was unstable in solutions at room temperature during short-term storage (4 h) and several freeze-thaw cycles. Copyright © 2013 John Wiley & Sons, Ltd.

  9. Plasma protein carbonyl levels and breast cancer risk

    Czech Academy of Sciences Publication Activity Database

    Rössner ml., Pavel; Terry, M. B.; Gammon, M. D.; Agrawal, M.; Zhang, F. F.; Ferris, J.S.; Teitelbaum, S. L.; Eng, S. M.; Gaudet, M. M.; Neugut, A. I.; Santella, R. M.

    2007-01-01

    Roč. 11, č. 5 (2007), s. 1138-1148 ISSN 1582-1838 Institutional research plan: CEZ:AV0Z50390512 Keywords : oxidative stress * protein carbonyl * breast cancer Subject RIV: DN - Health Impact of the Environment Quality Impact factor: 6.807, year: 2007

  10. Modeling of human factor Va inactivation by activated protein C

    Directory of Open Access Journals (Sweden)

    Bravo Maria

    2012-05-01

    Full Text Available Abstract Background Because understanding of the inventory, connectivity and dynamics of the components characterizing the process of coagulation is relatively mature, it has become an attractive target for physiochemical modeling. Such models can potentially improve the design of therapeutics. The prothrombinase complex (composed of the protease factor (FXa and its cofactor FVa plays a central role in this network as the main producer of thrombin, which catalyses both the activation of platelets and the conversion of fibrinogen to fibrin, the main substances of a clot. A key negative feedback loop that prevents clot propagation beyond the site of injury is the thrombin-dependent generation of activated protein C (APC, an enzyme that inactivates FVa, thus neutralizing the prothrombinase complex. APC inactivation of FVa is complex, involving the production of partially active intermediates and “protection” of FVa from APC by both FXa and prothrombin. An empirically validated mathematical model of this process would be useful in advancing the predictive capacity of comprehensive models of coagulation. Results A model of human APC inactivation of prothrombinase was constructed in a stepwise fashion by analyzing time courses of FVa inactivation in empirical reaction systems with increasing number of interacting components and generating corresponding model constructs of each reaction system. Reaction mechanisms, rate constants and equilibrium constants informing these model constructs were initially derived from various research groups reporting on APC inactivation of FVa in isolation, or in the presence of FXa or prothrombin. Model predictions were assessed against empirical data measuring the appearance and disappearance of multiple FVa degradation intermediates as well as prothrombinase activity changes, with plasma proteins derived from multiple preparations. Our work integrates previously published findings and through the cooperative

  11. Protein buffering in model systems and in whole human saliva.

    Directory of Open Access Journals (Sweden)

    Andreas Lamanda

    Full Text Available The aim of this study was to quantify the buffer attributes (value, power, range and optimum of two model systems for whole human resting saliva, the purified proteins from whole human resting saliva and single proteins. Two model systems, the first containing amyloglucosidase and lysozyme, and the second containing amyloglucosidase and alpha-amylase, were shown to provide, in combination with hydrogencarbonate and di-hydrogenphosphate, almost identical buffer attributes as whole human resting saliva. It was further demonstrated that changes in the protein concentration as small as 0.1% may change the buffer value of a buffer solution up to 15 times. Additionally, it was shown that there was a protein concentration change in the same range (0.16% between saliva samples collected at the time periods of 13:00 and others collected at 9:00 am and 17:00. The mode of the protein expression changed between these samples corresponded to the change in basic buffer power and the change of the buffer value at pH 6.7. Finally, SDS Page and Ruthenium II tris (bathophenantroline disulfonate staining unveiled a constant protein expression in all samples except for one 50 kDa protein band. As the change in the expression pattern of that 50 kDa protein band corresponded to the change in basic buffer power and the buffer value at pH 6.7, it was reasonable to conclude that this 50 kDa protein band may contain the protein(s belonging to the protein buffer system of human saliva.

  12. Protein biosynthesis in isolated human scalp hair follicles.

    Science.gov (United States)

    Vermorken, A J; Weterings, P J; Bloemendal, H

    1979-02-15

    The present study demonstrates that protein biosynthesis can be studied in single isolated human scalp hair follicles. The matrix and the sheath are the main regions where amino acids are built in. Incorporation is linear for at least five hours. The newly synthesized proteins can be separated into a water-soluble, a urea-soluble and a urea-insoluble fraction. Product analysis has been performed on the first two fractions, revealing different protein patterns.

  13. Complete cDNA sequence coding for human docking protein

    Energy Technology Data Exchange (ETDEWEB)

    Hortsch, M; Labeit, S; Meyer, D I

    1988-01-11

    Docking protein (DP, or SRP receptor) is a rough endoplasmic reticulum (ER)-associated protein essential for the targeting and translocation of nascent polypeptides across this membrane. It specifically interacts with a cytoplasmic ribonucleoprotein complex, the signal recognition particle (SRP). The nucleotide sequence of cDNA encoding the entire human DP and its deduced amino acid sequence are given.

  14. Small GTP-binding proteins in human endothelial cells

    NARCIS (Netherlands)

    de Leeuw, H. P.; Koster, P. M.; Calafat, J.; Janssen, H.; van Zonneveld, A. J.; van Mourik, J. A.; Voorberg, J.

    1998-01-01

    Small GTP-binding proteins of the Ras superfamily control an extensive number of intracellular events by alternating between GDP- and GTP-bound conformation. The presence of members of this protein family was examined in human umbilical vein endothelial cells employing RT-PCR. Sequence analysis of

  15. In vivo extracellular matrix protein expression by human periodontal ...

    African Journals Online (AJOL)

    It is well known that the orthodontic force applied to teeth generates a series of events that remodel the periodontal ligament (PDL). Extracellular matrix proteins (ECM) are described as molecular regulators of these events. However, the exact contribution of these proteins in human PDL modeling by orthodontic force ...

  16. G-protein activity in Percoll-purified plasma membranes, bulk plasma membranes, and low-density plasma membranes isolated from rat cerebral cortex

    Czech Academy of Sciences Publication Activity Database

    Bouřová, Lenka; Stöhr, Jiří; Lisý, Václav; Rudajev, Vladimír; Novotný, Jiří; Svoboda, Petr

    2009-01-01

    Roč. 15, č. 4 (2009), BR111-BR122 ISSN 1234-1010 R&D Projects: GA MŠk(CZ) LC554; GA MŠk(CZ) LC06063; GA ČR(CZ) GA309/06/0121; GA AV ČR(CZ) IAA500110606 Institutional research plan: CEZ:AV0Z50110509 Keywords : rat cerebral cortex * plasma membrane * G-protein activity Subject RIV: CE - Biochemistry Impact factor: 1.543, year: 2009

  17. Comparative N-glycoproteomic and phosphoproteomic profiling of human placental plasma membrane between normal and preeclampsia pregnancies with high-resolution mass spectrometry.

    Directory of Open Access Journals (Sweden)

    Fuqiang Wang

    Full Text Available Preeclampsia is a serious complication of pregnancy, which affects 2-8% of all pregnancies and is one of the leading causes of maternal and perinatal mortality and morbidity worldwide. To better understand the molecular mechanisms involved in pathological development of placenta in preeclampsia, we used high-resolution LC-MS/MS technologies to construct a comparative N-glycoproteomic and phosphoproteomic profiling of human placental plasma membrane in normal and preeclamptic pregnancies. A total of 1027 N-glyco- and 2094 phospho- sites were detected in human placental plasma membrane, and 5 N-glyco- and 38 phospho- proteins, respectively, with differentially expression were definitively identified between control and preeclamptic placental plasma membrane. Further bioinformatics analysis indicated that these differentially expressed proteins correlate with several specific cellular processes occurring during pathological changes of preeclamptic placental plasma membrane.

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

    International Nuclear Information System (INIS)

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

    1991-01-01

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

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

    Science.gov (United States)

    Golan, Yonatan; Sherman, Eilon

    2017-06-01

    The plasma membrane is a complex medium where transmembrane proteins diffuse and interact to facilitate cell function. Membrane protein mobility is affected by multiple mechanisms, including crowding, trapping, medium elasticity and structure, thus limiting our ability to distinguish them in intact cells. Here we characterize the mobility and organization of a short transmembrane protein at the plasma membrane of live T cells, using single particle tracking and photoactivated-localization microscopy. Protein mobility is highly heterogeneous, subdiffusive and ergodic-like. Using mobility characteristics, we segment individual trajectories into subpopulations with distinct Gaussian step-size distributions. Particles of low-to-medium mobility consist of clusters, diffusing in a viscoelastic and fractal-like medium and are enriched at the centre of the cell footprint. Particles of high mobility undergo weak confinement and are more evenly distributed. This study presents a methodological approach to resolve simultaneous mixed subdiffusion mechanisms acting on polydispersed samples and complex media such as cell membranes.

  20. Effects of plant proteins on postprandial, free plasma amino acid concentrations in rainbow trout (Oncorhynchus mykiss)

    DEFF Research Database (Denmark)

    Larsen, Bodil Katrine; Dalsgaard, Anne Johanne Tang; Pedersen, Per Bovbjerg

    2012-01-01

    proteins from wheat, peas, field beans, sunflower and soybean. Blood samples were obtained from the caudal vein of 7 fish in each dietary treatment group prior to feeding, as well as: 2, 4, 6, 8, 12, 24, 48 and 72 h after feeding (sampling 7 new fish at each time point), and plasma amino acid......Postprandial patterns in plasma free amino acid concentrations were investigated in juvenile rainbow trout (Oncorhynchus mykiss) fed either a fish meal based diet (FM) or a diet (VEG) where 59% of fish meal protein (corresponding to 46% of total dietary protein) was replaced by a matrix of plant...... the two dietary treatment groups correlated largely with the amino acid content of the two diets except for methionine, lysine and arginine, where the differences were more extreme than what would be expected from differences in dietary concentrations. The apparent protein digestibility coefficient...

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

    International Nuclear Information System (INIS)

    Nosseir, N.M.

    2011-01-01

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

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

    Science.gov (United States)

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

    2017-10-01

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

  3. Protein dynamics in individual human cells: experiment and theory.

    Directory of Open Access Journals (Sweden)

    Ariel Aharon Cohen

    Full Text Available A current challenge in biology is to understand the dynamics of protein circuits in living human cells. Can one define and test equations for the dynamics and variability of a protein over time? Here, we address this experimentally and theoretically, by means of accurate time-resolved measurements of endogenously tagged proteins in individual human cells. As a model system, we choose three stable proteins displaying cell-cycle-dependant dynamics. We find that protein accumulation with time per cell is quadratic for proteins with long mRNA life times and approximately linear for a protein with short mRNA lifetime. Both behaviors correspond to a classical model of transcription and translation. A stochastic model, in which genes slowly switch between ON and OFF states, captures measured cell-cell variability. The data suggests, in accordance with the model, that switching to the gene ON state is exponentially distributed and that the cell-cell distribution of protein levels can be approximated by a Gamma distribution throughout the cell cycle. These results suggest that relatively simple models may describe protein dynamics in individual human cells.

  4. Gadolinium-based magnetic resonance contrast agents at 7 Tesla: in vitro T1 relaxivities in human blood plasma.

    Science.gov (United States)

    Noebauer-Huhmann, Iris M; Szomolanyi, Pavol; Juras, Vladimír; Kraff, Oliver; Ladd, Mark E; Trattnig, Siegfried

    2010-09-01

    PURPOSE/INTRODUCTION: The aim of this study was to determine the T1 relaxivities (r1) of 8 gadolinium (Gd)-based MR contrast agents in human blood plasma at 7 Tesla, compared with 3 Tesla. Eight commercially available Gd-based MR contrast agents were diluted in human blood plasma to concentrations of 0, 0.25, 0.5, 1, and 2 mmol/L. In vitro measurements were performed at 37 degrees C, on a 7 Tesla and on a 3 Tesla whole-body magnetic resonance imaging scanner. For the determination of T1 relaxation times, Inversion Recovery Sequences with inversion times from 0 to 3500 ms were used. The relaxivities were calculated. The r1 relaxivities of all agents, diluted in human blood plasma at body temperature, were lower at 7 Tesla than at 3 Tesla. The values at 3 Tesla were comparable to those published earlier. Notably, in some agents, a minor negative correlation of r1 with a concentration of up to 2 mmol/L could be observed. This was most pronounced in the agents with the highest protein-binding capacity. At 7 Tesla, the in vitro r1 relaxivities of Gd-based contrast agents in human blood plasma are lower than those at 3 Tesla. This work may serve as a basis for the application of Gd-based MR contrast agents at 7 Tesla. Further studies are required to optimize the contrast agent dose in vivo.

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

    Czech Academy of Sciences Publication Activity Database

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

    2001-01-01

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

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

    OpenAIRE

    Siddiqui, F A; Lian, E C

    1985-01-01

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

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

    OpenAIRE

    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 ...

  8. Quantification of rifampicin in human plasma and cerebrospinal fluid by a highly sensitive and rapid liquid chromatographic–tandem mass spectrometric method

    OpenAIRE

    Srivastava, Abhishek; Waterhouse, David; Ardrey, Alison; Ward, Stephen A.

    2012-01-01

    A highly sensitive and rapid liquid chromatography tandem mass spectrometry (LC–MS/MS) method has been developed to measure the levels of the antitubercular drug rifampicin (RIF) in human plasma and cerebrospinal fluid (CSF). The analyte and internal standard (IS) were isolated from plasma and CSF by a simple organic solvent based precipitation of proteins followed by centrifugation. Detection was carried out by electrospray positive ionization mass spectrometry in the multiple-reaction monit...

  9. Human-specific protein isoforms produced by novel splice sites in the human genome after the human-chimpanzee divergence

    Directory of Open Access Journals (Sweden)

    Kim Dong Seon

    2012-11-01

    Full Text Available Abstract Background Evolution of splice sites is a well-known phenomenon that results in transcript diversity during human evolution. Many novel splice sites are derived from repetitive elements and may not contribute to protein products. Here, we analyzed annotated human protein-coding exons and identified human-specific splice sites that arose after the human-chimpanzee divergence. Results We analyzed multiple alignments of the annotated human protein-coding exons and their respective orthologous mammalian genome sequences to identify 85 novel splice sites (50 splice acceptors and 35 donors in the human genome. The novel protein-coding exons, which are expressed either constitutively or alternatively, produce novel protein isoforms by insertion, deletion, or frameshift. We found three cases in which the human-specific isoform conferred novel molecular function in the human cells: the human-specific IMUP protein isoform induces apoptosis of the trophoblast and is implicated in pre-eclampsia; the intronization of a part of SMOX gene exon produces inactive spermine oxidase; the human-specific NUB1 isoform shows reduced interaction with ubiquitin-like proteins, possibly affecting ubiquitin pathways. Conclusions Although the generation of novel protein isoforms does not equate to adaptive evolution, we propose that these cases are useful candidates for a molecular functional study to identify proteomic changes that might bring about novel phenotypes during human evolution.

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

    Science.gov (United States)

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

    2018-06-01

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

  11. Hybrid Synthetic Receptors on MOSFET Devices for Detection of Prostate Specific Antigen in Human Plasma.

    Science.gov (United States)

    Tamboli, Vibha K; Bhalla, Nikhil; Jolly, Pawan; Bowen, Chris R; Taylor, John T; Bowen, Jenna L; Allender, Chris J; Estrela, Pedro

    2016-12-06

    The study reports the use of extended gate field-effect transistors (FET) for the label-free and sensitive detection of prostate cancer (PCa) biomarkers in human plasma. The approach integrates for the first time hybrid synthetic receptors comprising of highly selective aptamer-lined pockets (apta-MIP) with FETs for sensitive detection of prostate specific antigen (PSA) at clinically relevant concentrations. The hybrid synthetic receptors were constructed by immobilizing an aptamer-PSA complex on gold and subjecting it to 13 cycles of dopamine electropolymerization. The polymerization resulted in the creation of highly selective polymeric cavities that retained the ability to recognize PSA post removal of the protein. The hybrid synthetic receptors were subsequently used in an extended gate FET setup for electrochemical detection of PSA. The sensor was reported to have a limit of detection of 0.1 pg/mL with a linear detection range from 0.1 pg/mL to 1 ng/mL PSA. Detection of 1-10 pg/mL PSA was also achieved in diluted human plasma. The present apta-MIP sensor developed in conjunction with FET devices demonstrates the potential for clinical application of synthetic hybrid receptors for the detection of clinically relevant biomarkers in complex samples.

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

    Science.gov (United States)

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

    2018-05-04

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

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

    Science.gov (United States)

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

    2018-02-14

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

  14. Prediction of protein-protein interactions between viruses and human by an SVM model

    Directory of Open Access Journals (Sweden)

    Cui Guangyu

    2012-05-01

    Full Text Available Abstract Background Several computational methods have been developed to predict protein-protein interactions from amino acid sequences, but most of those methods are intended for the interactions within a species rather than for interactions across different species. Methods for predicting interactions between homogeneous proteins are not appropriate for finding those between heterogeneous proteins since they do not distinguish the interactions between proteins of the same species from those of different species. Results We developed a new method for representing a protein sequence of variable length in a frequency vector of fixed length, which encodes the relative frequency of three consecutive amino acids of a sequence. We built a support vector machine (SVM model to predict human proteins that interact with virus proteins. In two types of viruses, human papillomaviruses (HPV and hepatitis C virus (HCV, our SVM model achieved an average accuracy above 80%, which is higher than that of another SVM model with a different representation scheme. Using the SVM model and Gene Ontology (GO annotations of proteins, we predicted new interactions between virus proteins and human proteins. Conclusions Encoding the relative frequency of amino acid triplets of a protein sequence is a simple yet powerful representation method for predicting protein-protein interactions across different species. The representation method has several advantages: (1 it enables a prediction model to achieve a better performance than other representations, (2 it generates feature vectors of fixed length regardless of the sequence length, and (3 the same representation is applicable to different types of proteins.

  15. Micro patterning of cell and protein non-adhesive plasma polymerized coatings for biochip applications

    DEFF Research Database (Denmark)

    Bouaidat, Salim; Berendsen, C.; Thomsen, P.

    2004-01-01

    Micro scale patterning of bioactive surfaces is desirable for numerous biochip applications. Polyethyleneoxide-like (PEO-like) coating with non-fouling functionality has been deposited using low frequency AC plasma polymerization. The non-fouling properties of the coating were tested with human c...... and versatility of the plasma-polymerized coatings, make this technology highly suitable for bio-MEMS and biochip applications, where patterned high contrast non-fouling surfaces are needed....

  16. Human neuroglobin protein in cerebrospinal fluid

    Directory of Open Access Journals (Sweden)

    Whalen Gail

    2005-02-01

    Full Text Available Abstract Background Neuroglobin is a hexacoordinated member of the globin family of proteins. It is predominantly localized to various brain regions and retina where it may play a role in protection against ischemia and nitric oxide-induced neural injury. Cerebrospinal fluid was collected from 12 chronic regional or systemic pain and 5 control subjects. Proteins were precipitated by addition of 50% 0.2 N acetic acid, 50% ethanol, 0.02% sodium bisulfite. The pellet was extensively digested with trypsin. Peptides were separated by capillary liquid chromatography using a gradient from 95% water to 95% acetonitrile in 0.2% formic acid, and eluted through a nanoelectrospray ionization interface into a quadrapole – time-of-flight dual mass spectrometer (QToF2, Waters, Milford, MA. Peptides were sequenced (PepSeq, MassLynx v3.5 and proteins identified using MASCOT ®. Results Six different neuroglobin peptides were identified in various combinations in 3 of 9 female pain subjects, but none in male pain, or female or male control subjects. Conclusion This is the first description of neuroglobin in cerebrospinal fluid. The mechanism(s leading to its release in chronic pain states remain to be defined.

  17. Determination of human muscle protein fractional synthesis rate

    DEFF Research Database (Denmark)

    Bornø, Andreas; Hulston, Carl J; van Hall, Gerrit

    2014-01-01

    In the present study, different MS methods for the determination of human muscle protein fractional synthesis rate (FSR) using [ring-(13)C6 ]phenylalanine as a tracer were evaluated. Because the turnover rate of human skeletal muscle is slow, only minute quantities of the stable isotopically...

  18. Guidelines for the nomenclature of the human heat shock proteins

    NARCIS (Netherlands)

    Kampinga, Harm H.; Hageman, Jurre; Vos, Michel J.; Kubota, Hiroshi; Tanguay, Robert M.; Bruford, Elspeth A.; Cheetham, Michael E.; Chen, B.; Hightower, Lawrence E.

    The expanding number of members in the various human heat shock protein (HSP) families and the inconsistencies in their nomenclature have often led to confusion. Here, we propose new guidelines for the nomenclature of the human HSP families, HSPH (HSP110), HSPC (HSP90), HSPA (HSP70), DNAJ (HSP40),

  19. Time- and radiation-dose dependent changes in the plasma proteome after total body irradiation of non-human primates: Implications for biomarker selection.

    Directory of Open Access Journals (Sweden)

    Stephanie D Byrum

    Full Text Available Acute radiation syndrome (ARS is a complex multi-organ disease resulting from total body exposure to high doses of radiation. Individuals can be exposed to total body irradiation (TBI in a number of ways, including terrorist radiological weapons or nuclear accidents. In order to determine whether an individual has been exposed to high doses of radiation and needs countermeasure treatment, robust biomarkers are needed to estimate radiation exposure from biospecimens such as blood or urine. In order to identity such candidate biomarkers of radiation exposure, high-resolution proteomics was used to analyze plasma from non-human primates following whole body irradiation (Co-60 at 6.7 Gy and 7.4 Gy with a twelve day observation period. A total of 663 proteins were evaluated from the plasma proteome analysis. A panel of plasma proteins with characteristic time- and dose-dependent changes was identified. In addition to the plasma proteomics study reported here, we recently identified candidate biomarkers using urine from these same non-human primates. From the proteomic analysis of both plasma and urine, we identified ten overlapping proteins that significantly differentiate both time and dose variables. These shared plasma and urine proteins represent optimal candidate biomarkers of radiation exposure.

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

    Science.gov (United States)

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

    2017-04-20

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

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

    Science.gov (United States)

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

    2013-04-01

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

  2. Differential dissociation micromethod for the investigation of binding of metandrostenolone (Nerobol) to plasma proteins

    International Nuclear Information System (INIS)

    Bojadzsieva, Milka; Kocsar, Laszlo; Kremmer, Tibor

    1985-01-01

    A micromethod was developed to determine the binding of anabolic streoids to plasma proteins. The new procedure combines precipitation with ammonium sulphate and differential dissociation. The binding parameters (association constant, specific binding capacity) are calculated on the basis of dissociation curves of sup(3)H-metandrostenolone from the precipitated sexual binding globuline. (L.E.)

  3. Differential dissociation micromethod for the investigation of binding of metandrostenolone (Nerobol) to plasma proteins

    Energy Technology Data Exchange (ETDEWEB)

    Bojadzsieva, M.; Kocsar, L. (Orszagos Frederic Joliot-Curie Sugarbiologiai es Sugaregeszseguegyi Kutato Intezet, Budapest (Hungary)); Kremmer, T. (Orszagos Onkologiai Intezet, Budapest (Hungary))

    1985-01-01

    A micromethod was developed to determine the binding of anabolic steroids to plasma proteins. The new procedure combines precipitation with ammonium sulphate and differential dissociation. The binding parameters (association constant, specific binding capacity) are calculated on the basis of dissociation curves of sup(3)H-metandrostenolone from the precipitated sexual binding globuline.

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

    NARCIS (Netherlands)

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

    1991-01-01

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

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

    NARCIS (Netherlands)

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

    2018-01-01

    The plasma membrane (PM) of Saccharomyces cerevisiae contains membrane compartments, MCC/eisosomes and MCPs, named after the protein residents Can1 and Pma1, respectively. Using high-resolution fluorescence microscopy techniques we show that Can1 and the homologous transporter Lyp1 are able to

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

    Science.gov (United States)

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

    2015-06-01

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

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

    African Journals Online (AJOL)

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

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

    International Nuclear Information System (INIS)

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

    1986-01-01

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

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

    Czech Academy of Sciences Publication Activity Database

    Grossmann, G.; Malínský, Jan; Stahlschmidt, W.; Loibl, M.; Weig-Meckl, I.; Frommer, W.B.; Opekarová, Miroslava; Tanner, W.

    2008-01-01

    Roč. 183, č. 6 (2008), s. 1075-1088 ISSN 0021-9525 R&D Projects: GA ČR GA204/06/0009; GA ČR GA204/07/0133; GA ČR GC204/08/J024 Institutional research plan: CEZ:AV0Z50390703; CEZ:AV0Z50200510 Keywords : Lithium acetate * Membrane compartment of Can1 * Monomeric red fluorescent protein Subject RIV: EA - Cell Biology Impact factor: 9.120, year: 2008

  10. Nuclear pore complex protein mediated nuclear localization of dicer protein in human cells.

    Directory of Open Access Journals (Sweden)

    Yoshinari Ando

    Full Text Available Human DICER1 protein cleaves double-stranded RNA into small sizes, a crucial step in production of single-stranded RNAs which are mediating factors of cytoplasmic RNA interference. Here, we clearly demonstrate that human DICER1 protein localizes not only to the cytoplasm but also to the nucleoplasm. We also find that human DICER1 protein associates with the NUP153 protein, one component of the nuclear pore complex. This association is detected predominantly in the cytoplasm but is also clearly distinguishable at the nuclear periphery. Additional characterization of the NUP153-DICER1 association suggests NUP153 plays a crucial role in the nuclear localization of the DICER1 protein.

  11. Leptin level in plasma of lactating buffaloes fed two diets with different energy and protein concentrations

    Directory of Open Access Journals (Sweden)

    A. Parmeggiani

    2011-03-01

    Full Text Available Leptin, a protein mainly secreted from the white adipocytes, has been shown to contribute to the regulation of energy metabolism, feeding behaviour and whole body energy balance. Moreover, leptin gene activity and leptin secretion are correlated with body adiposity and changes in food intake. Furthermore, leptin could also modulate endocrine response to changes in nutritional status and/or tissue sensitivity to hormones (Houseknecht et al., 1998; Romsos, 1998. Several factors are known to influence plasma leptin in rodents and humans: particularly it increases by body fatness, insulin, glucocorticoids, estrogens and decreases by food deprivation (Saladin et al., 1995; Ahima et al., 1996; Shimizu et al., 1997. These ones and several other observations have led to the hypothesis that leptin is a signal arising from adipose tissue, linked to the level of fat reserves and/or the nutritional status. This signal directly influences the central nervous system and peripheral organs, resulting in a better adaptation of body metabolism and physiological functions to the availability of metabolic energy...........

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

    DEFF Research Database (Denmark)

    Iversen, Kasper; Teisner, Ane; Dalager, Soren

    2011-01-01

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

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

    DEFF Research Database (Denmark)

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

    2010-01-01

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

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

    NARCIS (Netherlands)

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

    2005-01-01

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

  15. Determination of protein carbonyls in plasma, cell extracts, tissue homogenates, isolated proteins

    DEFF Research Database (Denmark)

    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...

  16. A plasma coagulation assay for an activated protein C-independent anticoagulant activity of protein S

    NARCIS (Netherlands)

    van Wijnen, M.; van 't Veer, C.; Meijers, J. C.; Bertina, R. M.; Bouma, B. N.

    1998-01-01

    To study the physiological importance of the activated protein C (APC)-independent anticoagulant activity of protein S, we developed an assay specific for this activity. The ability of protein S to prolong the clotting time in an APC-independent way was expressed as the ratio of the clotting time in

  17. Human neuronal cell protein responses to Nipah virus infection

    Directory of Open Access Journals (Sweden)

    Hassan Sharifah

    2007-06-01

    Full Text Available Abstract Background Nipah virus (NiV, a recently discovered zoonotic virus infects and replicates in several human cell types. Its replication in human neuronal cells, however, is less efficient in comparison to other fully susceptible cells. In the present study, the SK-N-MC human neuronal cell protein response to NiV infection is examined using proteomic approaches. Results Method for separation of the NiV-infected human neuronal cell proteins using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE was established. At least 800 protein spots were resolved of which seven were unique, six were significantly up-regulated and eight were significantly down-regulated. Six of these altered proteins were identified using mass spectrometry (MS and confirmed using MS/MS. The heterogenous nuclear ribonucleoprotein (hnRNP F, guanine nucleotide binding protein (G protein, voltage-dependent anion channel 2 (VDAC2 and cytochrome bc1 were present in abundance in the NiV-infected SK-N-MC cells in contrast to hnRNPs H and H2 that were significantly down-regulated. Conclusion Several human neuronal cell proteins that are differentially expressed following NiV infection are identified. The proteins are associated with various cellular functions and their abundance reflects their significance in the cytopathologic responses to the infection and the regulation of NiV replication. The potential importance of the ratio of hnRNP F, and hnRNPs H and H2 in regulation of NiV replication, the association of the mitochondrial protein with the cytopathologic responses to the infection and induction of apoptosis are highlighted.

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

    Science.gov (United States)

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

    2016-11-01

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

  19. SDS-binding assay based on tyrosine fluorescence as a tool to determine binding properties of human serum albumin in blood plasma

    Science.gov (United States)

    Zhdanova, Nadezda; Shirshin, Evgeny; Fadeev, Victor; Priezzhev, Alexander

    2016-04-01

    Among all plasma proteins human serum albumin (HSA) is the most studied one as it is the main transport protein and can bind a wide variety of ligands especially fatty acids (FAs). The concentration of FAs bound to HSA in human blood plasma differs by three times under abnormal conditions (fasting, physical exercises or in case of social important diseases). In the present study a surfactant sodium dodecyl sulfate (SDS) was used to simulate FAs binding to HSA. It was shown that the increase of Tyr fluorescence of human blood plasma due to SDS addition can be completely explained by HSA-SDS complex formation. Binding parameters of SDS-HSA complex (average number of sites and apparent constant of complex formation) were determined from titration curves based on tyrosine (Tyr) fluorescence.

  20. Prolactin-inducible proteins in human breast cancer cells

    International Nuclear Information System (INIS)

    Shiu, R.P.; Iwasiow, B.M.

    1985-01-01

    The mechanism of action of prolactin in target cells and the role of prolactin in human breast cancer are poorly understood phenomena. The present study examines the effect of human prolactin (hPRL) on the synthesis of unique proteins by a human breast cancer cell line, T-47D, in serum-free medium containing bovine serum albumin. [ 35 S]Methionine-labeled proteins were analysed by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis and fluorography. Treatment of cells with hPRL (1-1000 ng/ml) and hydrocortisone (1 microgram/ml) for 36 h or longer resulted in the synthesis and secretion of three proteins having molecular weights of 11,000, 14,000, and 16,000. Neither hPRL nor hydrocortisone alone induced these proteins. Of several other peptide hormones tested, only human growth hormone, a hormone structurally and functionally similar to hPRL, could replace hPRL in causing protein induction. These three proteins were, therefore, referred to as prolactin-inducible proteins (PIP). Each of the three PIPs was purified to homogeneity by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and specific antibodies were generated to them in rabbits. By immunoprecipitation and immunoblotting (Western blot) of proteins secreted by T-47D cells, it was demonstrated that the three PIPs were immunologically identical to one another. In addition, the 16-kDa and 14-kDa proteins (PIP-16 and PIP-14), and not the 11-kDa protein (PIP-11), incorporated [ 3 H]glycosamine. Furthermore, 2-deoxyglucose (2 mM) and tunicamycin (0.5 micrograms/ml), two compounds known to inhibit glycosylation, blocked the production of PIP-16 and PIP-14, with a concomitant increase in the accumulation of PIP-11

  1. Bioanalytical method development and validation for determination of metoprolol tartarate and hydrochlorothiazide using HPTLC in human plasma

    Directory of Open Access Journals (Sweden)

    Ambadas Ranganath Rote

    2013-12-01

    Full Text Available A simple, sensitive, rapid and economic chromatographic method has been developed for determination of metoprolol tartarate and hydrochlorothiazide in human plasma using paracetamol as an internal standard. The analytical technique used for method development was high-performance thin-layer chromatography. HPTLC Camag with precoated silica gel Plate 60F254 (20 cm×10 cm at 250 µm thicknesses (E. Merck, Darmstadt, Germany was used as the stationary phase. The mobile phase used consisted of chloroform: methanol: ammonia (9:1:0.5v/v/v. Densitometric analysis was carried out at a wavelength of 239 nm. The rf values for hydrochlorothiazide, paracetamol and metoprolol tartarate were 0.13±0.04, 0.28±0.05, 0.48±0.04, respectively. Plasma samples were extracted by protein precipitation with methanol. Concentration ranges of 200, 400, 600, 800, 1000, 1200 ng/mL and 2000, 4000, 6000, 8000, 10000, 12000 ng/mL of hydrochlorothiazide and metoprolol tartarate, respectively, were used with plasma for the calibration curves. The percent recovery of metoprolol tartarate and hydrochlorothiazide was found to be 77.30 and 77.02 %, respectively. The stability of metoprolol tartarate and hydrochlorothiazide in plasma were confirmed during three freeze-thaw cycles (-20 ºC on a bench for 24 hours and post-preparatively for 48 hours. The proposed method was validated statistically and proved suitable for determination of metoprolol tartarate and hydrochlorothiazide in human plasma.

  2. Identification of proteins that may directly interact with human RPA.

    Science.gov (United States)

    Nakaya, Ryou; Takaya, Junichiro; Onuki, Takeshi; Moritani, Mariko; Nozaki, Naohito; Ishimi, Yukio

    2010-11-01

    RPA, which consisted of three subunits (RPA1, 2 and 3), plays essential roles in DNA transactions. At the DNA replication forks, RPA binds to single-stranded DNA region to stabilize the structure and to assemble other replication proteins. Interactions between RPA and several replication proteins have been reported but the analysis is not comprehensive. We systematically performed the qualitative analysis to identify RPA interaction partners to understand the protein-protein interaction at the replication forks. We expressed in insect cells the three subunits of human RPA, together with one replication protein, which is present at the forks under normal conditions and/or under the replication stress conditions, to examine the interaction. Among 30 proteins examined in total, it was found that at least 14 proteins interacted with RPA. RPA interacted with MCM3-7, MCM-BP and CDC45 proteins among the proteins that play roles in the initiation and the elongation of the DNA replication. RPA bound with TIPIN, CLASPIN and RAD17, which are involved in the DNA replication checkpoint functions. RPA also bound with cyclin-dependent kinases and an amino-terminal fragment of Rb protein that negatively regulates DNA replication. These results suggest that RPA interacts with the specific proteins among those that play roles in the regulation of the replication fork progression.

  3. Organization of the gene coding for human protein C inhibitor (plasminogen activator inhibitor-3). Assignment of the gene to chromosome 14

    NARCIS (Netherlands)

    Meijers, J. C.; Chung, D. W.

    1991-01-01

    Protein C inhibitor (plasminogen activator inhibitor-3) is a plasma glycoprotein and a member of the serine proteinase inhibitor superfamily. In the present study, the human gene for protein C inhibitor was isolated and characterized from three independent phage that contained overlapping inserts

  4. Analysis of plasma membrane Ca(2+)-ATPase expression in control and SV40-transformed human fibroblasts.

    Science.gov (United States)

    Reisner, P D; Brandt, P C; Vanaman, T C

    1997-01-01

    It has been long known that neoplastic transformation is accompanied by a lowered requirement for extracellular Ca2+ for growth. The studies presented here demonstrate that human fibroblastic cell lines produce the two commonly found 'housekeeping' isoforms of the plasma membrane Ca(2+)-ATPase (PMCA), PMCA1b and 4b, and at the expression of both is demonstrably lower in cell lines neoplastically transformed by SV40 than in the corresponding parental cell lines. Western blot analyses of lysates from control (GM00037) and SV40-transformed (GM00637) skin fibroblasts revealed a 138 kDa PMCA whose level was significantly lower in the SV40-transformed cells relative to either total cellular protein or alpha-tubulin. Similar analyses of plasma membrane preparations from control WI-38) and SV40-transformed (WI-38VA13) lung fibroblasts revealed 3-4-fold lower levels of PMCA in the SV40-transformed cells. Competitive ELISAs performed on detergent solubilized plasma membrane preparations indicated at least 3-4-fold lower levels of PMCA in the SV40-transformed cell lines compared to controls. Reverse transcriptase coupled-PCR analyses showed that PMCA1b and PMCA4b were the only isoforms expressed in all four cell lines. The PMCA4b mRNA level detected by Northern analysis also was substantially lower in SV40 transformed skin fibroblasts than in non-transformed fibroblasts. Quantitative RT-PCR analyses showed levels of PMCA1b and 4b mRNAs to be 5 and 10-fold lower, respectively, in GM00637 than in GM00037 when the levels of PCR products were normalized to glyceraldehyde-3-phosphate dehydrogenase (G3PDH) mRNA. These results demonstrate that the expression of these distinct PMCA genes is substantially lower in SV40 transformed human skin and lung fibroblasts and may be coordinately regulated in these cells.

  5. Human serum protein and C-reactive protein levels among HIV ...

    African Journals Online (AJOL)

    Human serum protein and C-reactive protein levels were determined among HIV patients visiting St Camillus Hospital, Uromi, Edo State, Nigeria, between January to March, 2013. Fifty (50) HIV patients (20 males; 30 females) and 50 control subjects (24 males; 26 females) were enrolled for this study. The clinical status of ...

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

    Science.gov (United States)

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

    2015-08-01

    Protein oxidation is involved in regulatory physiological events as well as in damage to tissues and is thought to play a key role in the pathophysiology of diseases and in the aging process. Protein-bound carbonyls represent a marker of global protein oxidation, as they are generated by multiple different reactive oxygen species in blood, tissues and cells. Sample preparation and stabilization are key steps in the accurate quantification of oxidation-related products and examination of physiological/pathological processes. This review therefore focuses on the sample preparation processes used in the most relevant methods to detect protein carbonyls after derivatization with 2,4-dinitrophenylhydrazine with an emphasis on measurement in plasma, cells, organ homogenates, isolated proteins and organelles. Sample preparation, derivatization conditions and protein handling are presented for the spectrophotometric and HPLC method as well as for immunoblotting and ELISA. An extensive overview covering these methods in previously published articles is given for researchers who plan to measure protein carbonyls in different samples. © 2015 Published by Elsevier Ltd.

  7. A method for studies on interactions between a gold-based drug and plasma proteins based on capillary electrophoresis with inductively coupled plasma mass spectrometry detection

    DEFF Research Database (Denmark)

    Nguyen, Tam T T N; Østergaard, Jesper; Gammelgaard, Bente

    2015-01-01

    An analytical method based on capillary electrophoresis (CE) and inductively coupled plasma mass spectrometry (ICP-MS) detection was developed for studies on the interaction of gold-containing drugs and plasma proteins using auranofin as example. A detection limit of 18 ng/mL of auranofin corresp...

  8. Human Proteinpedia enables sharing of human protein data

    Energy Technology Data Exchange (ETDEWEB)

    Mathivanan, Suresh; Ahmed, Mukhtar; Ahn, Natalie G.; Alexandre, Hainard; Amanchy, Ramars; Andrews, Philip C.; Bader, Joel S.; Balgley, Brian M.; Bantscheff, Marcus; Bennett, Keiryn; Bjorling, Erik; Blagoev, Blagoy; Bose , Ron; Brahmachari, Samir K.; Burlingame, Alma S.; Bustelo, Xos R.; Cagney, Gerard; Cantin, Greg T; Cardasis, Helene L; Celis, Julio E; Chaerkady, Raghothama; Chu, Feixia; Cole, Phillip A.; Costello, Catherine E; Cotter , Robert J.; Crockett, David; DeLany , James P.; De Marzo, Angelo M; DeSouza, Leroi V; Deutsch, Eric W.; Dransfield , Eric; Drewes , Gerard; Droit , Arnaud; Dunn, Michael; Elenitoba-Johnson, Kojo; Ewing, Rob M.; Van Eyk , Jennifer; Faca , Vitor; Falkner , Jayson; Fang, Xiangming; Fenselau , Catherine; Figeys , Daniel; Gagne , Pierre; Gelfi , Cecilia; Gevaert , Kris; Gimble , Jeffrey; Gnad , Florian; Goel, Renu; Gromov , Pavel; Hanash, Samir M.; Hancock, William S.; Harsha , HC; Hart , Gerald; Faith , Hays; He , Fuchu; Hebbar , Prashantha; Helsens , Kenny; Hermeking , Heiko; Hide , Winston; Hjerno, Karin; Hochstrasser, Denis F.; Hofmann, Oliver; Horn , David M.; Hruban , Ralph H.; Ibarrola , Nieves; James , Peter; Jensen , Ole N.; Jensen, Pia H.; Jung , Peter; Kandasamy, Kumaran; Kheterpal , Indu; Kikuno , Reiko; Korf, Ulrike; Korner, Roman; Kuster, Bernhard; Kwon , Min-Seok; Lee , Hyoung-Joo; Lee , Young - Jin; Lefevre , Michael; Lehvaslaiho, Minna; Lescuyer, Pierre; Levander, Fredrik; Lim, Megan S.; Lobke, Christian; Loo, Joseph; Mann, Matthias; Martens , Lennart; Martinez-Heredia, Juan; McComb, Mark E.; McRedmond , James; Mehrle, Alexander; Menon, Rajasree; Miller, Christine A.; Mischak, Harald; Mohan, S Sujatha; Mohmood , Riaz; Molina , Henrik; Moran , Michael F.; Morgan, James D.; Moritz , Robert; Morzel, Martine; Muddiman, David C.; Nalli , Anuradha; Navarro, J. D.; Neubert , Thomas A.; Ohara , Osamu; Oliva, Rafael; Omenn, Gilbert; Oyama , Masaaki; Paik, Young-Ki; Pennington , Kyla; Pepperkok, Rainer; Periaswamy, Balamurugan; Petricoin, Emanuel F.; Poirier, Guy G.; Prasad, T S Keshava; Purvine, Samuel O.; Rahiman , B Abdul; Ramachandran, Prasanna; Ramachandra , Y L; Rice, Robert H.; Rick , Jens; Ronnholm , Ragna H.; Salonen , Johanna; Sanchez , Jean - Charles; Sayd , Thierry; Seshi, Beerelli; Shankari, Kripa; Sheng , Shi Jun; Shetty , Vivekananda; Shivakumar, K.; Simpson, Richard J.; Sirdeshmukh, Ravi; Siu , K W Michael; Smith, Jeffrey C.; Smith, Richard D.; States, David J.; Sugano, Sumio; Sullivan , Matthew; Superti - Furga, Giulio; Takatalo , Maarit; Thongboonkerd , Visith; Trinidad , Jonathan C.; Uhlen , Mathias; Vandekerckhove, Joel; Vasilescu , Julian; Veenstra, Timothy D.; Vidal - Taboada, Jose - Manuel; Vihinen, Mauno; Wait , Robin; Wang, Xiaoyue; Wiemann, Stefan; Wu , Billy; Xu, Tao; Yates, John R.; Zhong, Jun; Zhou, Ming; Zhu, Yunping; Zurbig, Petra; Pandey, Akhilesh

    2008-02-01

    Proteomic technologies, such as yeast twohybrid, mass spectrometry (MS), protein/ peptide arrays and fluorescence microscopy, yield multi-dimensional data sets, which are often quite large and either not published or published as supplementary information that is not easily searchable. Without a system in place for standardizing and sharing data, it is not fruitful for the biomedical community to contribute these types of data to centralized repositories. Even more difficult is the annotation and display of pertinent information in the context of the corresponding proteins. Wikipedia, an online encyclopedia that anyone can edit, has already proven quite successful1 and can be used as a model for sharing biological data. However, the need for experimental evidence, data standardization and ownership of data creates scientific obstacles.

  9. Analysis of Alpha-2 Macroglobulin from the Long-Lived and Cancer-Resistant Naked Mole-Rat and Human Plasma.

    Science.gov (United States)

    Thieme, René; Kurz, Susanne; Kolb, Marlen; Debebe, Tewodros; Holtze, Susanne; Morhart, Michaela; Huse, Klaus; Szafranski, Karol; Platzer, Matthias; Hildebrandt, Thomas B; Birkenmeier, Gerd

    2015-01-01

    The naked mole-rat (NMR) is a long-lived and cancer resistant species. Identification of potential anti-cancer and age related mechanisms is of great interest and makes this species eminent to investigate anti-cancer strategies and understand aging mechanisms. Since it is known that the NMR expresses higher liver mRNA-levels of alpha 2-macroglobulin than mice, nothing is known about its structure, functionality or expression level in the NMR compared to the human A2M. Here we show a comprehensive analysis of NMR- and human plasma-A2M, showing a different prediction in glycosylation of NMR-A2M, which results in a higher molecular weight compared to human A2M. Additionally, we found a higher concentration of A2M (8.3±0.44 mg/mL vs. and 4.4±0.20 mg/mL) and a lower total plasma protein content (38.7±1.79 mg/mL vs. 61.7±3.20 mg/mL) in NMR compared to human. NMR-A2M can be transformed by methylamine and trypsin resulting in a conformational change similar to human A2M. NMR-A2M is detectable by a polyclonal antibody against human A2M. Determination of tryptic and anti-tryptic activity of NMR and human plasma revealed a higher anti-tryptic activity of the NMR plasma. On the other hand, less proteolytic activity was found in NMR plasma compared to human plasma. We found transformed NMR-A2M binding to its specific receptor LRP1. We could demonstrate lower protein expression of LRP1 in the NMR liver tissue compared to human but higher expression of A2M. This was accompanied by a higher EpCAM protein expression as central adhesion molecule in cancer progression. NMR-plasma was capable to increase the adhesion in human fibroblast in vitro most probably by increasing CD29 protein expression. This is the first report, demonstrating similarities as well as distinct differences between A2M in NMR and human plasma. This might be directly linked to the intriguing phenotype of the NMR and suggests that A2M might probably play an important role in anti-cancer and the anti

  10. Analysis of Alpha-2 Macroglobulin from the Long-Lived and Cancer-Resistant Naked Mole-Rat and Human Plasma.

    Directory of Open Access Journals (Sweden)

    René Thieme

    Full Text Available The naked mole-rat (NMR is a long-lived and cancer resistant species. Identification of potential anti-cancer and age related mechanisms is of great interest and makes this species eminent to investigate anti-cancer strategies and understand aging mechanisms. Since it is known that the NMR expresses higher liver mRNA-levels of alpha 2-macroglobulin than mice, nothing is known about its structure, functionality or expression level in the NMR compared to the human A2M.Here we show a comprehensive analysis of NMR- and human plasma-A2M, showing a different prediction in glycosylation of NMR-A2M, which results in a higher molecular weight compared to human A2M. Additionally, we found a higher concentration of A2M (8.3±0.44 mg/mL vs. and 4.4±0.20 mg/mL and a lower total plasma protein content (38.7±1.79 mg/mL vs. 61.7±3.20 mg/mL in NMR compared to human. NMR-A2M can be transformed by methylamine and trypsin resulting in a conformational change similar to human A2M. NMR-A2M is detectable by a polyclonal antibody against human A2M. Determination of tryptic and anti-tryptic activity of NMR and human plasma revealed a higher anti-tryptic activity of the NMR plasma. On the other hand, less proteolytic activity was found in NMR plasma compared to human plasma.We found transformed NMR-A2M binding to its specific receptor LRP1. We could demonstrate lower protein expression of LRP1 in the NMR liver tissue compared to human but higher expression of A2M. This was accompanied by a higher EpCAM protein expression as central adhesion molecule in cancer progression. NMR-plasma was capable to increase the adhesion in human fibroblast in vitro most probably by increasing CD29 protein expression. This is the first report, demonstrating similarities as well as distinct differences between A2M in NMR and human plasma. This might be directly linked to the intriguing phenotype of the NMR and suggests that A2M might probably play an important role in anti-cancer and the

  11. Proteomic characterization of the human centrosome by protein correlation profiling

    DEFF Research Database (Denmark)

    Andersen, Jens S; Wilkinson, Christopher J; Mayor, Thibault

    2003-01-01

    chromosomes between dividing cells. Despite the importance of this organelle to cell biology and more than 100 years of study, many aspects of its function remain enigmatic and its structure and composition are still largely unknown. We performed a mass-spectrometry-based proteomic analysis of human...... centrosomes in the interphase of the cell cycle by quantitatively profiling hundreds of proteins across several centrifugation fractions. True centrosomal proteins were revealed by both correlation with already known centrosomal proteins and in vivo localization. We identified and validated 23 novel...... components and identified 41 likely candidates as well as the vast majority of the known centrosomal proteins in a large background of nonspecific proteins. Protein correlation profiling permits the analysis of any multiprotein complex that can be enriched by fractionation but not purified to homogeneity....

  12. Enhanced human bone marrow mesenchymal stem cell functions on cathodic arc plasma-treated titanium

    Directory of Open Access Journals (Sweden)

    Zhu W

    2015-12-01

    Full Text Available Wei Zhu,1 George Teel,1 Christopher M O’Brien,1 Taisen Zhuang,1 Michael Keidar,1 Lijie Grace Zhang1–3 1Department of Mechanical and Aerospace Engineering, 2Department of Biomedical Engineering, 3Department of Medicine, The George Washington University, Washington, DC, USA Abstract: Surface modification of titanium for use in orthopedics has been explored for years; however, an ideal method of integrating titanium with native bone is still required to this day. Since human bone cells directly interact with nanostructured extracellular matrices, one of the most promising methods of improving titanium’s osseointegration involves inducing biomimetic nanotopography to enhance cell–implant interaction. In this regard, we explored an approach to functionalize the surface of titanium by depositing a thin film of textured titanium nanoparticles via a cathodic arc discharge plasma. The aim is to improve human bone marrow mesenchymal stem cell (MSC attachment and differentiation and to reduce deleterious effects of more complex surface modification methods. Surface functionalization was analyzed by scanning electron microscopy, atomic force microscopy, contact angle testing, and specific protein adsorption. Scanning electron microscopy and atomic force microscopy examination demonstrate the deposition of titanium nanoparticles and the surface roughness change after coating. The specific fibronectin adsorption was enhanced on the modified titanium surface that associates with the improved hydrophilicity. MSC adhesion and proliferation were significantly promoted on the nanocoated surface. More importantly, compared to bare titanium, greater production of total protein, deposition of calcium mineral, and synthesis of alkaline phosphatase were observed from MSCs on nanocoated titanium after 21 days. The method described herein presents a promising alternative method for inducing more cell favorable nanosurface for improved orthopedic applications

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

    International Nuclear Information System (INIS)

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

    2008-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2008-05-07

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

  15. Quantitative determination of polysulfide in albumins, plasma proteins and biological fluid samples using a novel combined assays approach.

    Science.gov (United States)

    Ikeda, Mayumi; Ishima, Yu; Shibata, Akitomo; Chuang, Victor T G; Sawa, Tomohiro; Ihara, Hideshi; Watanabe, Hiroshi; Xian, Ming; Ouchi, Yuya; Shimizu, Taro; Ando, Hidenori; Ukawa, Masami; Ishida, Tatsuhiro; Akaike, Takaaki; Otagiri, Masaki; Maruyama, Toru

    2017-05-29

    Hydrogen sulfide (H 2 S) signaling involves polysulfide (RSS n SR') formation on various proteins. However, the current lack of sensitive polysulfide detection assays poses methodological challenges for understanding sulfane sulfur homeostasis and signaling. We developed a novel combined assay by modifying Sulfide Antioxidant Buffer (SAOB) to produce an "Elimination Method of Sulfide from Polysulfide" (EMSP) treatment solution that liberates sulfide, followed with methylene blue (MB) sulfide detection assay. The combined EMSP-MB sulfide detection assay performed on low molecular weight sulfur species showed that sulfide was produced from trisulfide compounds such as glutathione trisulfide and diallyl trisulfide, but not from the thiol compounds such as cysteine, cystine and glutathione. In the case of plasma proteins, this novel combined detection assay revealed that approximately 14.7, 1.7, 3.9, 3.7 sulfide mol/mol released from human serum albumin, α 1 -anti-trypsin, α 1 -acid glycoprotein and ovalbumin, respectively, suggesting that serum albumin is a major pool of polysulfide in human blood circulation. Taken together with the results of albumins of different species, the liberated sulfide has a good correlation with cysteine instead of methionine, indicating the site of incorporation of polysulfide is cysteine. With this novel sulfide detention assay, approximately 8,000, 120 and 1100 μM of polysulfide concentrations was quantitated in human healthy plasma, saliva and tear, respectively. Our promising polysulfide specific detection assay can be a very important tool because quantitative determination of polysulfide sheds light on the functional consequence of protein-bound cysteine polysulfide and expands the research area of reactive oxygen to reactive polysulfide species. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. Mining the human tissue proteome for protein citrullination.

    Science.gov (United States)

    Lee, Chien-Yun; Wang, Dongxue; Wilhelm, Mathias; Zolg, Daniel Paul; Schmidt, Tobias; Schnatbaum, Karsten; Reimer, Ulf; Pontén, Fredrik; Uhlén, Mathias; Hahne, Hannes; Kuster, Bernhard

    2018-04-02

    Citrullination is a post-translational modification of arginine catalyzed by five peptidylarginine deiminases (PADs) in humans. The loss of a positive charge may cause structural or functional alterations and while the modification has been linked to several diseases including rheumatoid arthritis and cancer, its physiological or pathophysiological roles remain largely unclear. In part this is owing to limitations in available methodology able to robustly enrich, detect and localize the modification. As a result, only few citrullination sites have been identified on human proteins with high confidence. In this study, we mined data from mass spectrometry-based deep proteomic profiling of 30 human tissues to identify citrullination sites on endogenous proteins. Database searching of ~70 million tandem mass spectra yielded ~13,000 candidate spectra which were further triaged by spectrum quality metrics and the detection of the specific neutral loss of isocyanic acid from citrullinated peptides to reduce false positives. Because citrullination is easily confused with deamidation, we synthetized ~2,200 citrullinated and 1,300 deamidated peptides to build a library of reference spectra. This led to the validation of 375 citrullination sites on 209 human proteins. Further analysis showed that >80% of the identified modifications sites were new and for 56% of the proteins, citrullination was detected for the first time. Sequence motif analysis revealed a strong preference for Asp and Gly, residues around the citrullination site. Interestingly, while the modification was detected in 26 human tissues with the highest levels found in brain and lung, citrullination levels did not correlate well with protein expression of the PAD enzymes. Even though the current work represents the largest survey of protein citrullination to date, the modification was mostly detected on high abundant proteins arguing that the development of specific enrichment methods would be required in order

  17. Plasma dihydroxyphenylalanine (DOPA) is independent of sympathetic activity in humans

    DEFF Research Database (Denmark)

    Eldrup, E; Christensen, N J; Andreasen, J

    1989-01-01

    in diabetic patients with autonomic neuropathy compared to diabetics without neuropathy, whereas baseline plasma DOPA concentrations were similar in the three groups investigated: 6.55 (5.03-7.26, median [interquartile range], n = 8) nmol l-1 in diabetics with neuropathy, 7.41 (5.79-7.97, n = 8) nmol l-1...... in diabetics without neuropathy, and 6.85 (5.58-7.36, n = 8) nmol l-1 in controls. No relationship was obtained between baseline values of plasma NE and plasma DOPA. Plasma DOPA did not change in the upright position, whereas plasma NE increased significantly. Our results indicate that plasma DOPA...... is not related to sympathetic activity and may be of non-neuronal origin....

  18. Comparative plasma lipidome between human and cynomolgus monkey: are plasma polar lipids good biomarkers for diabetic monkeys?

    Directory of Open Access Journals (Sweden)

    Guanghou Shui

    Full Text Available BACKGROUND: Non-human primates (NHP are now being considered as models for investigating human metabolic diseases including diabetes. Analyses of cholesterol and triglycerides in plasma derived from NHPs can easily be achieved using methods employed in humans. Information pertaining to other lipid species in monkey plasma, however, is lacking and requires comprehensive experimental analysis. METHODOLOGIES/PRINCIPAL FINDINGS: We examined the plasma lipidome from 16 cynomolgus monkey, Macaca fascicularis, using liquid chromatography coupled with mass spectrometry (LC/MS. We established novel analytical approaches, which are based on a simple gradient elution, to quantify polar lipids in plasma including (i glycerophospholipids (phosphatidylcholine, PC; phosphatidylethanolamine, PE; phosphatidylinositol, PI; phosphatidylglycerol, PG; phosphatidylserine, PS; phosphatidic acid, PA; (ii sphingolipids (sphingomyelin, SM; ceramide, Cer; Glucocyl-ceramide, GluCer; ganglioside mannoside 3, GM3. Lipidomic analysis had revealed that the plasma of human and cynomolgus monkey were of similar compositions, with PC, SM, PE, LPC and PI constituting the major polar lipid species present. Human plasma contained significantly higher levels of plasmalogen PE species (p<0.005 and plasmalogen PC species (p<0.0005, while cynomolgus monkey had higher levels of polyunsaturated fatty acyls (PUFA in PC, PE, PS and PI. Notably, cynomolgus monkey had significantly lower levels of glycosphingolipids, including GluCer (p<0.0005 and GM(3 (p<0.0005, but higher level of Cer (p<0.0005 in plasma than human. We next investigated the biochemical alterations in blood lipids of 8 naturally occurring diabetic cynomolgus monkeys when compared with 8 healthy controls. CONCLUSIONS: For the first time, we demonstrated that the plasma of human and cynomolgus monkey were of similar compositions, but contained different mol distribution of individual molecular species. Diabetic monkeys

  19. Isolation of low-molecular-weight lead-binding protein from human erythrocytes

    International Nuclear Information System (INIS)

    Raghavan, S.R.V.; Gonick, H.C.

    1977-01-01

    In blood, lead is mainly associated with erythrocytes and only a very small amount is found in plasma. Previously it was thought that the lead was bound to the erythrocyte cell membrane but more recently it has been observed that lead is bound primarily to the cell contents, ostensibly hemoglobin. In examining the lead-binding properties of normal human erythrocytes and those of lead-exposed industrial workers, we have found that, whereas lead binds only to hemoglobin in normal erythrocytes, there is also appreciable binding of lead to a low-molecular weight-protein in erythrocytes from lead-exposed workers. The synthesis of this protein may be induced by lead exposure. The 10,000 molecular weight protein may act as a storage site and mechanism for segregating lead in a non-toxic form

  20. Amino acid sequence and posttranslational modifications of human factor VIIa from plasma and transfected baby hamster kidney cells

    International Nuclear Information System (INIS)

    Thim, L.; Bjoern, S.; Christensen, M.; Nicolaisen, E.M.; Lund-Hansen, T.; Pedersen, A.H.; Hedner, U.

    1988-01-01

    Blood coagulation factor VII is a vitamin K dependent glycoprotein which in its activated form, factor VII a , participates in the coagulation process by activating factor X and/or factor IX in the presence of Ca 2+ and tissue factor. Three types of potential posttranslational modifications exist in the human factor VII a molecule, namely, 10 γ-carboxylated, N-terminally located glutamic acid residues, 1 β-hydroxylated aspartic acid residue, and 2 N-glycosylated asparagine residues. In the present study, the amino acid sequence and posttranslational modifications of recombinant factor VII a as purified from the culture medium of a transfected baby hamster kidney cell line have been compared to human plasma factor VII a . By use of HPLC, amino acid analysis, peptide mapping, and automated Edman degradation, the protein backbone of recombinant factor VII a was found to be identical with human factor VII a . Asparagine residues 145 and 322 were found to be fully N-glycosylated in human plasma factor VII a . In the recombinant factor VII a , asparagine residue 322 was fully glycosylated whereas asparagine residue 145 was only partially (approximately 66%) glycosylated. Besides minor differences in the sialic acid and fucose contents, the overall carbohydrate compositions were nearly identical in recombinant factor VII a and human plasma factor VII a . These results show that factor VII a as produced in the transfected baby hamster kidney cells is very similar to human plasma factor VII a and that this cell line thus might represent an alternative source for human factor VII a

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

    Science.gov (United States)

    Cihil, Kristine M; Swiatecka-Urban, Agnieszka

    2013-12-13

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

  2. Structural History of Human SRGAP2 Proteins.

    Science.gov (United States)

    Sporny, Michael; Guez-Haddad, Julia; Kreusch, Annett; Shakartzi, Sivan; Neznansky, Avi; Cross, Alice; Isupov, Michail N; Qualmann, Britta; Kessels, Michael M; Opatowsky, Yarden

    2017-06-01

    In the development of the human brain, human-specific genes are considered to play key roles, conferring its unique advantages and vulnerabilities. At the time of Homo lineage divergence from Australopithecus, SRGAP2C gradually emerged through a process of serial duplications and mutagenesis from ancestral SRGAP2A (3.4-2.4 Ma). Remarkably, ectopic expression of SRGAP2C endows cultured mouse brain cells, with human-like characteristics, specifically, increased dendritic spine length and density. To understand the molecular mechanisms underlying this change in neuronal morphology, we determined the structure of SRGAP2A and studied the interplay between SRGAP2A and SRGAP2C. We found that: 1) SRGAP2A homo-dimerizes through a large interface that includes an F-BAR domain, a newly identified F-BAR extension (Fx), and RhoGAP-SH3 domains. 2) SRGAP2A has an unusual inverse geometry, enabling associations with lamellipodia and dendritic spine heads in vivo, and scaffolding of membrane protrusions in cell culture. 3) As a result of the initial partial duplication event (∼3.4 Ma), SRGAP2C carries a defective Fx-domain that severely compromises its solubility and membrane-scaffolding ability. Consistently, SRGAP2A:SRAGP2C hetero-dimers form, but are insoluble, inhibiting SRGAP2A activity. 4) Inactivation of SRGAP2A is sensitive to the level of hetero-dimerization with SRGAP2C. 5) The primal form of SRGAP2C (P-SRGAP2C, existing between ∼3.4 and 2.4 Ma) is less effective in hetero-dimerizing with SRGAP2A than the modern SRGAP2C, which carries several substitutions (from ∼2.4 Ma). Thus, the genetic mutagenesis phase contributed to modulation of SRGAP2A's inhibition of neuronal expansion, by introducing and improving the formation of inactive SRGAP2A:SRGAP2C hetero-dimers, indicating a stepwise involvement of SRGAP2C in human evolutionary history. © The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  3. Characterization of the B-chain of human plasma α2HS-glycoprotein. The complete amino acid sequence and primary structure of its heteroglycan

    NARCIS (Netherlands)

    Vliegenthart, J.F.G.; Gejyo, F.; Chang, J.-L.; Bürgi, W.; Schmid, K.; Offner, G.D.; Troxler, R.F.; Halbeek, H. van

    1983-01-01

    α2HS-Glycoprotein, a normal human plasma protein, was recently shown to consist of two polypeptide chains. In the present study, we have separated these two chains from one another and have elucidated the complete primary structure of the B-chain. Employing automated Edman degradation, the

  4. Increased MMP-9 and TIMP-1 in mouse neonatal brain and plasma and in human neonatal plasma after hypoxia-ischemia: a potential marker of neonatal encephalopathy.

    Science.gov (United States)

    Bednarek, Nathalie; Svedin, Pernilla; Garnotel, Roselyne; Favrais, Géraldine; Loron, Gauthier; Schwendiman, Leslie; Hagberg, Henrik; Morville, Patrice; Mallard, Carina; Gressens, Pierre

    2012-01-01

    To implement neuroprotective strategies in newborns, sensitive and specific biomarkers are needed for identifying those who are at risk for brain damage. We evaluated the effectiveness of matrix metalloproteinases (MMPs) and their naturally occurring tissue inhibitors of metalloproteinases (TIMPs) in predicting neonatal encephalopathy (NE) damage in newborns. Plasma MMP-9 and TIMP-1 levels were upregulated as early as 1 h after the HI insult but not did not show such elevations after other types of injury (ibotenate-induced excitotoxicity, hypoxia, lipopolysaccharide-induced inflammation), and brain levels reflected this increase soon thereafter. We confirmed these results by carrying out plasma MMP-9 and TIMP-1 measurements in human newborns with NE. In these infants, protein levels of MMP-9 and TIMP-1 were found to be elevated during a short window up to 6 h after birth. This feature is particularly useful in identifying newborns in need of neuroprotection. A second peak observed 72 h after birth is possibly related to the second phase of energy failure after a HI insult. Our data, although preliminary, support the use of MMP-9 and TIMP-1 as early biomarkers for the presence and extent of perinatal brain injury in human term newborns. We first used a mouse model of neonatal HI injury to explore mechanistic aspects such as the time course of these markers after the hypoxia-ischemia event, and the correlation between the levels of these candidate markers in brain and plasma.

  5. Effect of Bovine Plasma Protein on Autolysis and Gelation of Protein Extracted from Giant Squid (Dosidicus gigas Mantle

    Directory of Open Access Journals (Sweden)

    Laura Raquel Marquez-Alvarez

    2015-01-01

    Full Text Available The effect of bovine plasma protein (BPP on the inhibition of autolytic activity and its effect on the gelling properties of a protein concentrate (PC obtained from jumbo squid (Dosidicus gigas mantle were investigated. Sols and gels were prepared from the PC by adding different amounts of BPP (0, 1, and 2%. Dynamic oscillatory measurements indicated that systems with 1% BPP had a higher elastic modulus (G′, in which hydrophobic interactions were favored. Concerning the technological and textural quality of the gels, BPP caused a greater water holding capacity (WHC, force, cohesiveness, and elasticity, probably due to improvement of the electrostatic and hydrophobic interactions during gel formation. Scanning electron microscopy (SEM allowed visualization of the formation of more rigid and ordered gels with less porosity when BPP was added