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Sample records for por sds-page nivel

  1. SDS -PAGE and Western Blotting Techniques.

    PubMed

    Blancher, C; Jones, A

    2001-01-01

    The goal of Western blotting, or more correctly, immunoblotting, is to identify with a specific antibody a particular antigen within a complex mixture of proteins that has been fractionated in a polyacrylamide gel and immobilized onto a membrane. Immunoblotting can be used to determine a number of important characteristics of protein antigens-the presence and quantity of an antigen, the relative molecular weight of the polypeptide chain, and the efficiency of extraction of the antigen.Immunoblotting occurs in six stages: (1) extraction and quantification of protein samples; (2) resolution of the protein sample in sodium dodecyl sulfatepolyacrylamide denaturing gel electrophoresis (SDS-PAGE); (3) transfer of the separated polypeptides to a membrane support; (4) blocking nonspecific binding sites on the membrane; (5) addition of antibodies; and (6) detection.Sample preparation is important for obtaining accurate separation of the proteins on the basis of molecular weight. Depending on whether an antigen is primarily extracellular, cytoplasmic, or membrane-associated different procedures might be required to prepare the sample initially. Although there are exceptions, many soluble nuclear and cytoplasmic proteins can be solubilized by lysis buffers that contain the nonionic detergent Nonidet P-40 (NP-40) and either no salt at all or relatively high concentrations of salt (e.g., 0.5 M NaCl). However, the efficiency of extraction is often greatly affected by pH of the buffer and the presence or absence of chelating agents such EDTA.

  2. Determination of the Subunit Molecular Mass and Composition of Alcohol Dehydrogenase by SDS-PAGE

    ERIC Educational Resources Information Center

    Nash, Barbara T.

    2007-01-01

    SDS-PAGE is a simple, rapid technique that has many uses in biochemistry and is readily adaptable to the undergraduate laboratory. It is, however, a technique prone to several types of procedural pitfalls. This article describes the use of SDS-PAGE to determine the subunit molecular mass and composition of yeast alcohol dehydrogenase employing…

  3. Determination of the Subunit Molecular Mass and Composition of Alcohol Dehydrogenase by SDS-PAGE

    ERIC Educational Resources Information Center

    Nash, Barbara T.

    2007-01-01

    SDS-PAGE is a simple, rapid technique that has many uses in biochemistry and is readily adaptable to the undergraduate laboratory. It is, however, a technique prone to several types of procedural pitfalls. This article describes the use of SDS-PAGE to determine the subunit molecular mass and composition of yeast alcohol dehydrogenase employing…

  4. Preparative SDS PAGE as an Alternative to His-Tag Purification of Recombinant Amelogenin

    PubMed Central

    Gabe, Claire M.; Brookes, Steven J.; Kirkham, Jennifer

    2017-01-01

    Recombinant protein technology provides an invaluable source of proteins for use in structure-function studies, as immunogens, and in the development of therapeutics. Recombinant proteins are typically engineered with “tags” that allow the protein to be purified from crude host cell extracts using affinity based chromatography techniques. Amelogenin is the principal component of the developing enamel matrix and a frequent focus for biomineralization researchers. Several groups have reported the successful production of recombinant amelogenins but the production of recombinant amelogenin free of any tags, and at single band purity on silver stained SDS PAGE is technically challenging. This is important, as rigorous structure-function research frequently demands a high degree of protein purity and fidelity of protein sequence. Our aim was to generate His-tagged recombinant amelogenin at single band purity on silver stained SDS PAGE for use in functionality studies after His-tag cleavage. An acetic acid extraction technique (previously reported to produce recombinant amelogenin at 95% purity directly from E. coli) followed by repeated rounds of nickel column affinity chromatography, failed to generate recombinant amelogenin at single band purity. This was because following an initial round of nickel column affinity chromatography, subsequent cleavage of the His-tag was not 100% efficient. A second round of nickel column affinity chromatography, used in attempts to separate the cleaved His-tag free recombinant from uncleaved His-tagged contaminants, was still unsatisfactory as cleaved recombinant amelogenin exhibited significant affinity for the nickel column. To solve this problem, we used preparative SDS PAGE to successfully purify cleaved recombinant amelogenins to single band purity on silver stained SDS PAGE. The resolving power of preparative SDS PAGE was such that His-tag based purification of recombinant amelogenin becomes redundant. We suggest that acetic

  5. Preparative SDS PAGE as an Alternative to His-Tag Purification of Recombinant Amelogenin.

    PubMed

    Gabe, Claire M; Brookes, Steven J; Kirkham, Jennifer

    2017-01-01

    Recombinant protein technology provides an invaluable source of proteins for use in structure-function studies, as immunogens, and in the development of therapeutics. Recombinant proteins are typically engineered with "tags" that allow the protein to be purified from crude host cell extracts using affinity based chromatography techniques. Amelogenin is the principal component of the developing enamel matrix and a frequent focus for biomineralization researchers. Several groups have reported the successful production of recombinant amelogenins but the production of recombinant amelogenin free of any tags, and at single band purity on silver stained SDS PAGE is technically challenging. This is important, as rigorous structure-function research frequently demands a high degree of protein purity and fidelity of protein sequence. Our aim was to generate His-tagged recombinant amelogenin at single band purity on silver stained SDS PAGE for use in functionality studies after His-tag cleavage. An acetic acid extraction technique (previously reported to produce recombinant amelogenin at 95% purity directly from E. coli) followed by repeated rounds of nickel column affinity chromatography, failed to generate recombinant amelogenin at single band purity. This was because following an initial round of nickel column affinity chromatography, subsequent cleavage of the His-tag was not 100% efficient. A second round of nickel column affinity chromatography, used in attempts to separate the cleaved His-tag free recombinant from uncleaved His-tagged contaminants, was still unsatisfactory as cleaved recombinant amelogenin exhibited significant affinity for the nickel column. To solve this problem, we used preparative SDS PAGE to successfully purify cleaved recombinant amelogenins to single band purity on silver stained SDS PAGE. The resolving power of preparative SDS PAGE was such that His-tag based purification of recombinant amelogenin becomes redundant. We suggest that acetic acid

  6. Staining of proteins for 2D SDS-PAGE using Coomassie Blue--speed versus sensitivity?

    PubMed

    Májek, Pavel; Riedelová-Reicheltová, Zuzana; Suttnar, Jirí; Dyr, Jan E

    2013-07-01

    Several new fast staining protocols for the visualization of proteins separated by SDS-PAGE utilizing Coomassie Blue staining (CBS) have been described in literature. The sensitivity of a newly designed staining protocol is usually estimated using 1D SDS-PAGE of serially diluted protein samples. However, this approach is not predictive and satisfactory for 2D SDS-PAGE capable of resolving hundreds or thousands of different proteins in a single analysis. In this work, a new fast staining protocol recently introduced by Dong et al. (PLoS One 2011, 6, e22394) was compared to colloidal CBS. The number of detectable spots in 2D SDS-PAGE of identical blood plasma samples in repeated runs was chosen as a sensitivity criterion. Further, the influence of gel boiling on the subsequent protein identification by MS was investigated. In spite of its advantages, the staining protocol according to Dong et al. (PLoS One 2011, 6, e22394) seems to be less sensitive than colloidal Coomassie staining when the number of detected spots is the evaluating criterion. No obvious influence of gel boiling on the protein identification was observed.

  7. One-dimensional SDS-polyacrylamide gel electrophoresis (1D SDS-PAGE).

    PubMed

    Brunelle, Julie L; Green, Rachel

    2014-01-01

    This protocol describes a denaturing polyacrylamide gel system utilizing sodium dodecyl sulfate (SDS) to separate protein molecules based on size as first described by Laemmli (1970). SDS-PAGE can be used to monitor protein purifications, check the purity of samples, and to estimate molecular weights for unknown proteins.

  8. Teaching the structure of immunoglobulins by molecular visualization and SDS-PAGE analysis.

    PubMed

    Rižner, Tea Lanišnik

    2014-01-01

    This laboratory class combines molecular visualization and laboratory experimentation to teach the structure of the immunoglobulins (Ig). In the first part of the class, the three-dimensional structures of the human IgG and IgM molecules available through the RCSB PDB database are visualized using freely available software. In the second part, IgG and IgM are studied using electrophoretic methods. Through SDS-PAGE analysis under reducing conditions, the students determine the number and molecular masses of the polypeptide chains, while through SDS-PAGE under nonreducing conditions, the students assess the oligomerization of these Ig molecules. The aims of this class are to expand upon the knowledge and understanding of the Ig structure that the students have gained from classroom lectures. The combination of this molecular visualization of the Ig molecules and the SDS-PAGE experimentation ensures variety in the teaching techniques, while the implication of the Ig molecules in human disease promotes interest for biomedical students.

  9. Desialylation improves the detection of recombinant erythropoietins in urine samples analyzed by SDS-PAGE.

    PubMed

    Desharnais, Philippe; Naud, Jean-François; Ayotte, Christiane

    2013-01-01

    Recombinant erythropoietin (rhEPO) has been misused for over two decades by athletes, mainly but not only in endurance sports. A direct rhEPO detection method in urine by isoelectric focusing (IEF) was introduced in 2000, but the emergence of third-generation erythropoiesis-stimulating agents and so-called biosimilar rhEPOs, together with the sensitivity of human endogenous EPO (huEPO) pattern to enzymatic activities and its modification following short strenuous exercise, prompted the development of a complementary test based on SDS-PAGE analysis. While Mircera and NESP are easily detected with the existing IEF and SDS-PAGE methods, some samples containing both epoetin-α/β and huEPO present profiles that are still difficult to interpret. As doping practices have moved to micro-dosing, these mixed patterns are more frequently observed. We investigated the impact of enzymatic desialylation on the urinary and serum EPO profiles obtained by SDS-PAGE with the aim of improving the separation of the bands in these mixed EPO populations. We observed that the removal with neuraminidase of the sialic acid moieties from the different EPOs studied reduced their apparent molecular weight (MW) and increased the migration distance between huEPO and rhEPO centroids, therefore eliminating the size overlaps between them and improving the detection of rhEPO.

  10. Improved Coomassie Blue Dye-Based Fast Staining Protocol for Proteins Separated by SDS-PAGE

    PubMed Central

    Májek, Pavel; Riedelová-Reicheltová, Zuzana; Pecánková, Klára; Dyr, Jan E.

    2013-01-01

    The time required to visualize proteins using Coomassie Blue dye has been significantly reduced with the introduction of fast staining protocols based on staining with a Coomassie Blue dye solution at boiling temperatures. However, fast stainings suffer from high gel backgrounds, reducing the signal-to-noise ratio and limiting the number of detectable spots in the case of 2D SDS-PAGE. The aim of this work was to eliminate the high gel background, and thus improve fast staining protocols based on Coomassie Blue dye. We show that merely replacing water with a 4 mM EDTA washing solution at boiling temperatures, results in a transparent gel background within 50 to 60 minutes of destaining. Moreover, when a combination of imidazole-zinc reverse staining and Coomassie Blue-based fast staining is used the sensitivity is improved significantly; nanogram amounts of proteins can be detected using 1D SDS-PAGE, and about 30% to 60% more spots can be detected with 2D SDS-PAGE in plasma, platelet, and rat brain tissue samples. This work represents an optimized fast staining protocol with improved sensitivity, requiring between 60 to 75 minutes to complete protein visualization. PMID:24278455

  11. Improved coomassie blue dye-based fast staining protocol for proteins separated by SDS-PAGE.

    PubMed

    Májek, Pavel; Riedelová-Reicheltová, Zuzana; Pecánková, Klára; Dyr, Jan E

    2013-01-01

    The time required to visualize proteins using Coomassie Blue dye has been significantly reduced with the introduction of fast staining protocols based on staining with a Coomassie Blue dye solution at boiling temperatures. However, fast stainings suffer from high gel backgrounds, reducing the signal-to-noise ratio and limiting the number of detectable spots in the case of 2D SDS-PAGE. The aim of this work was to eliminate the high gel background, and thus improve fast staining protocols based on Coomassie Blue dye. We show that merely replacing water with a 4 mM EDTA washing solution at boiling temperatures, results in a transparent gel background within 50 to 60 minutes of destaining. Moreover, when a combination of imidazole-zinc reverse staining and Coomassie Blue-based fast staining is used the sensitivity is improved significantly; nanogram amounts of proteins can be detected using 1D SDS-PAGE, and about 30% to 60% more spots can be detected with 2D SDS-PAGE in plasma, platelet, and rat brain tissue samples. This work represents an optimized fast staining protocol with improved sensitivity, requiring between 60 to 75 minutes to complete protein visualization.

  12. SDS-PAGE separation of dermatophyte antigens, and western immunoblotting in feline dermatophytosis.

    PubMed

    Sparkes, A H; Stokes, C R; Gruffydd-Jones, T J

    1994-11-01

    Water-soluble antigens liberated from the disrupted mycelium of nine dermatophytes (seven isolates of Microsporum canis, one each of Microsporum gypseum and Trichophyton mentagrophytes) were compared by analytical slab SDS-PAGE. No substantial differences were observed between the protein bands of the M. Canis isolates, but certain distinctive bands were apparent in the other two species examined. Western immunoblotting using M. canis-derived antigens separated by SDS-PAGE was used to investigate the humoral immune response in 79 cats with naturally-occurring dermatophytosis (72 with M. canis, six with M. gypseum and one with T. mentagrophytes) and this information was compared to results of immunoblots from 46 control (non-dermatophyte exposed) cats. Seven dominant bands (bands which occurred frequently and stained heavily) were identified in immunoblots from the dermatophyte-infected cats with apparent molecular weights varying between 39 and 120 kD. None of these bands were totally specific markers for dermatophytosis as a variable proportion of the control cats showed reactivity to all these proteins. However, most (73%) of the dermatophyte-infected cats showed reactivity to six or seven of the identified bands whereas most (80%) of the control cats showed reactivity to between zero and three of these bands (p < 0.005). Western immunoblotting could be used to select individual immunodominant antigens for further evaluation of protective (cell-mediated) immunity.

  13. Identification of Neisseria meningitidis outer membrane vesicle complexes using 2-D high resolution clear native/SDS-PAGE.

    PubMed

    Marzoa, Juan; Sánchez, Sandra; Ferreirós, Carlos M; Criado, María Teresa

    2010-01-01

    The identification and characterization of meningococcal outer membrane vesicle complexes can be important for gaining an in-depth understaining of their structure and functionality. Analysis of the vesicle complexome by 'traditional' 2-D analysis, in which isoelectrofocusing is used for separation in the first dimension, is hampered by the high hydrophobicity and extreme isoelectric points of many relevant proteins. Analysis of the meningococcal outer membrane vesicle complexome using Blue Native (nondenaturing) electrophoresis instead of isoelectrofocusing in the first dimension showed several porin complexes, but their composition could not be clearly resolved after separation by SDS-PAGE in the second dimension. In this work, using a recently described native separation technique -high resolution Clear Native Electrophoresis-and different bidimensional approaches, we were able to demonstrate the presence of relevant outer membrane complexes which could be resolved with a higher resolution than in previous analysis. The most relevant were nine porin complexes formed by different combinations of the meningococcal PorA, PorB and RmpM proteins, and comparison with the complexes formed in specific knockout mutants allowed us to infer the relevance of each porin in the formation of each complex.

  14. Sensitive method to identify and characterize proteinases in situ after SDS-PAGE.

    PubMed

    Williams, J; McGrath, W J; Mangel, W F

    2000-11-01

    Cells and body fluids contain numerous, different proteinases; to identify and characterize them are both important and difficult tasks. Especially difficult to identify and characterize are highly specific proteinases. Here, we present an extremely sensitive and quantitative method to characterize proteinases fractionated by SDS-PAGE that cleave specific rhodamine-based fluorogenic substrates. To test the sensitivity of the technique, we used trypsin as our model system. Filter paper impregnated with rhodamine-based fluorogenic substrates was placed on a gel, and bands of fluorescence originating from specific proteinases were visualized in real time. The method is very sensitive; picogram amounts of trypsin can be detected. The method should be very general, in that even proteinases whose substrates require amino acids C-terminal to the cleavage site may be identified and characterized. The results allow one to obtain not only information on the substrate specificity of a specific enzyme but also information about its molecular weight.

  15. SDS-PAGE of recombinant and endogenous erythropoietins: benefits and limitations of the method for application in doping control.

    PubMed

    Reichel, Christian; Kulovics, Ronald; Jordan, Veronika; Watzinger, Martina; Geisendorfer, Thomas

    2009-01-01

    Doping of athletes with recombinant and genetically modified erythropoietins (EPO) is currently detected by isoelectric focusing (IEF). The application of these drugs leads to a significant change in the isoform profile of endogenous urinary erythropoietin (uhEPO). Dynepo, MIRCERA, biosimilars with variable IEF-profiles as well as active urines and effort urines have made additional testing strategies necessary. The new generation of small molecule EPO-receptor stimulating agents like Hematide will also challenge the analytical concept of detecting the abuse of erythropoiesis stimulating agents (ESA). By determining their apparent molecular masses with SDS-PAGE a clear differentiation between endogenous and exogenous substances also concerning new EPO modifications is possible. Due to the orthogonal character of IEF- and SDS-PAGE both methods complement each other. The additional benefits of SDS-PAGE especially in relation to active and effort urines as well as the detection of Dynepo were investigated. Due to significant differences between the apparent molecular masses of uhEPO/serum EPO (shEPO) and recombinant, genetically or chemically modified erythropoietins the presence of active or effort urines was easily revealed. The characteristic band shape and apparent molecular mass of Dynepo on SDS-PAGE additionally evidenced the presence of this substance in urine. A protocol for the detection of EPO-doping in serum and plasma by SDS-PAGE was developed. Blood appears to be the ideal matrix for detecting all forms ESA-doping in the future.

  16. Nuclear proteasomes carry a constitutive posttranslational modification which derails SDS-PAGE (but not CTAB-PAGE).

    PubMed

    Pitcher, David S; de Mattos-Shipley, Kate; Wang, Ziming; Tzortzis, Konstantinos; Goudevenou, Katerina; Flynn, Helen; Bohn, Georg; Rahemtulla, Amin; Roberts, Irene; Snijders, Ambrosius P; Karadimitris, Anastasios; Kleijnen, Maurits F

    2014-12-01

    We report that subunits of human nuclear proteasomes carry a previously unrecognised, constitutive posttranslational modification. Subunits with this modification are not visualised by SDS-PAGE, which is used in almost all denaturing protein gel electrophoresis. In contrast, CTAB-PAGE readily visualises such modified subunits. Thus, under most experimental conditions, with identical samples, SDS-PAGE yielded gel electrophoresis patterns for subunits of nuclear proteasomes which were misleading and strikingly different from those obtained with CTAB-PAGE. Initial analysis indicates a novel modification of a high negative charge with some similarity to polyADP-ribose, possibly explaining compatibility with (positively-charged) CTAB-PAGE but not (negatively-charged) SDS-PAGE and providing a mechanism for how nuclear proteasomes may interact with chromatin, DNA and other nuclear components.

  17. Detection of the end point temperature of thermal denatured protein in fish and chicken meat through SDS-PAGE electrophoresis

    NASA Astrophysics Data System (ADS)

    Gao, Hongwei; Mao, Mao; Liang, Chengzhu; Lin, Chao; Xiang, Jianhai

    2009-03-01

    Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was applied in the detection of the end point temperature (EPT) of thermal denatured protein in fish and meat in this study. It was also used in studying the thermal denatured temperature range of proteins in salmon and chicken meat. The results show that the temperature ranges of denatured proteins were from 65°C to 75°C, and these temperature ranges were influenced by the processing methods. Through SDS-PAGE, the features of repeated heating thermal denatured proteins under the same temperature and processing time were studied. The electrophoresis patterns of thermal denatured proteins determined through repeated heating at the same temperature did not exhibit any change. For the detection of cooked fish and meat samples, they were subjected to applying the SDS-PAGE method, which revealed an EPT ranging from 60°C to 80°C.

  18. Optimized sample preparation of endoscopic collected pancreatic fluid for SDS-PAGE analysis.

    PubMed

    Paulo, Joao A; Lee, Linda S; Wu, Bechien; Repas, Kathryn; Banks, Peter A; Conwell, Darwin L; Steen, Hanno

    2010-07-01

    The standardization of methods for human body fluid protein isolation is a critical initial step for proteomic analyses aimed to discover clinically relevant biomarkers. Several caveats have hindered pancreatic fluid proteomics, including the heterogeneity of samples and protein degradation. We aim to optimize sample handling of pancreatic fluid that has been collected using a safe and effective endoscopic collection method (endoscopic pancreatic function test). Using SDS-PAGE protein profiling, we investigate (i) precipitation techniques to maximize protein extraction, (ii) auto-digestion of pancreatic fluid following prolonged exposure to a range of temperatures, (iii) effects of multiple freeze-thaw cycles on protein stability, and (iv) the utility of protease inhibitors. Our experiments revealed that TCA precipitation resulted in the most efficient extraction of protein from pancreatic fluid of the eight methods we investigated. In addition, our data reveal that although auto-digestion of proteins is prevalent at 23 and 37 degrees C, incubation on ice significantly slows such degradation. Similarly, when the sample is maintained on ice, proteolysis is minimal during multiple freeze-thaw cycles. We have also determined the addition of protease inhibitors to be assay-dependent. Our optimized sample preparation strategy can be applied to future proteomic analyses of pancreatic fluid.

  19. Optimized Sample Preparation of Endoscopic (ePFT) Collected Pancreatic Fluid for SDS-PAGE Analysis

    PubMed Central

    Paulo, Joao A.; Lee, Linda S.; Wu, Bechien; Repas, Kathryn; Banks, Peter A.; Conwell, Darwin L.; Steen, Hanno

    2011-01-01

    The standardization of methods for human body fluid protein isolation is a critical initial step for proteomic analyses aimed to discover clinically-relevant biomarkers. Several caveats have hindered pancreatic fluid proteomics, including the heterogeneity of samples and protein degradation. We aim to optimize sample handling of pancreatic fluid that has been collected using a safe and effective endoscopic collection method (ePFT). Using SDS-PAGE protein profiling, we investigate (1) precipitation techniques to maximize protein extraction, (2) auto-digestion of pancreatic fluid following prolonged exposure to a range of temperatures, (3) effects of multiple freeze-thaw cycles on protein stability, and (4) the utility of protease inhibitors. Our experiments revealed that trichloroacetic acid (TCA) precipitation resulted in the most efficient extraction of protein from pancreatic fluid of the eight methods we investigated. In addition, our data reveal that although auto-digestion of proteins is prevalent at 23°C and 37°C, incubation on ice significantly slows such degradation. Similarly, when the sample is maintained on ice, proteolysis is minimal during multiple freeze-thaw cycles. We have also determined the addition of protease inhibitors to be assay-dependent. Our optimized sample preparation strategy can be applied to future proteomic analyses of pancreatic fluid. PMID:20589857

  20. Artifactual formation of disulfide bonds during SDS-PAGE analysis of type I copper proteins

    SciTech Connect

    Kumar, M.A.; Davidson, V.L. )

    1991-03-11

    Amicyanin is a monomeric Type I blue' copper protein, which possesses a single cysteine that serves as one of the ligands to copper. Amicyanin denatured by heating in SDS in the presence of {beta}-mercaptoethanol ({beta}ME) migrated during SDS-PAGE with an M{sub r} = 15,000. When heated in SDS in the absence of {beta}ME it exhibited an M{sub r} = 30,000. If treated with {beta}ME, but not heated it exhibited an M{sub r} = 15,000. Similar data were obtained with the small blue copper protein, azurin. Ascorbate oxidase is a multicopper enzyme which is composed of two identical non-covalently bound subunits, each of which possesses a Type I copper center. When this blue' oxidase was denatured by heating in SDS with {beta}ME, or incubated with {beta}ME without heating, a single band of M{sub r} = 70,000 was observed. When heated in SDS without {beta}ME, a species twice that size was observed. Thus, exposure during denaturation of the free sulfhydryl of the Type I copper binding site can cause formation of disulfide bonds between otherwise unlinked polypeptides.

  1. Real-time Fluorescence Polarization Microscopy of the Moving Boundary in Cross-Gradient SDS-PAGE

    NASA Astrophysics Data System (ADS)

    Hwang, Jeeseong; Giulian, Gary

    2003-03-01

    Real-time Fluorescence Polarization Microscopy of the Moving Boundary in Cross-Gradient SDS-PAGE Jeeseong Hwang, Jeffrey R. Krogmeier, Angela M. Bardo, Scott N. Goldie, Lori S. Goldner; Optical Technology Division, National Institute of Standards and Technology, Gaithersburg, MD 20899 Gary G. Giulian, Carl R. Merril; National Institute of Mental Health, National Institutes of Health, Bethesda, MD 20892 Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) is a popular method to separate proteins by their apparent molecular weight. However, it is a limited technique due, in part, to its low spatial resolution. In order to improve the resolution and to enhance the detection sensitivity of proteins separated by SDS-PAGE we are studying the detergent properties at the moving boundary of precast Tris-Tricine-Acetate cross-gradient gels using fluorescent cationic and pH indicating dyes. We have developed real-time full-field fluorescence polarization microscopy to monitor the dynamic fluorescence anisotropy from the cationic tetramethylindocarbocyanine dyes localized in the "extended stack", a concentrated detergent zone. We will present quantitative results of the fluorescence anisotropy. Our system is capable of analyzing local structures of the detergent molecules in the moving boundary of SDS-PAGE and the microenvironment(s) near the boundary. We will discuss the significance of these results and their potential role in enhanced protein separation.

  2. Improved Method for Reliable HMW-GS Identification by RP-HPLC and SDS-PAGE in Common Wheat Cultivars.

    PubMed

    Jang, You-Ran; Beom, Hye-Rang; Altenbach, Susan B; Lee, Min-Ki; Lim, Sun-Hyung; Lee, Jong-Yeol

    2017-06-24

    The accurate identification of alleles for high-molecular weight glutenins (HMW-GS) is critical for wheat breeding programs targeting end-use quality. RP-HPLC methods were optimized for separation of HMW-GS, resulting in enhanced resolution of 1By and 1Dx subunits. Statistically significant differences in retention times (RTs) for subunits corresponding to HMW-GS alleles were determined using 16 standard wheat cultivars with known HMW-GS compositions. Subunits that were not identified unambiguously by RP-HPLC were distinguished by SDS-PAGE or inferred from association with linked subunits. The method was used to verify the allelic compositions of 32 Korean wheat cultivars previously determined using SDS-PAGE and to assess the compositions of six new Korean cultivars. Three cultivars contained subunits that were identified incorrectly in the earlier analysis. The improved RP-HPLC method combined with conventional SDS-PAGE provides for accurate, efficient and reliable identification of HMW-GS and will contribute to efforts to improve wheat end-use quality.

  3. Combination of SDS-PAGE and intact mass analysis for rapid determination of heterogeneities in monoclonal antibody therapeutics.

    PubMed

    Yamada, Hideaki; Matsumura, Chiemi; Yamada, Keita; Teshima, Koichiro; Hiroshima, Takashi; Kinoshita, Mitsuhiro; Suzuki, Shigeo; Kakehi, Kazuaki

    2017-05-01

    mAbs are currently mainstream in biopharmaceuticals, and their market has been growing due to their high target specificity. Characterization of heterogeneities in mAbs is performed to secure their quality and safety by physicochemical analyses. However, they require time-consuming task, which often strain the resources of drug development in pharmaceuticals. Rapid and direct method to determine the heterogeneities should be a powerful tool for pharmaceutical analysis. Considering the advantages of electrophoresis and MS, this study addresses the combination of SDS-PAGE and intact mass analysis, which provides direct, rapid, and orthogonal determination of heterogeneities in mAb therapeutics. mAb therapeutics that migrated in SDS-PAGE were recovered from gel by treatment with SDC-containing buffer. Usage of SDC-containing buffer as extraction solvent and ethanol-based staining solution enhanced the recovery of intact IgG from SDS-PAGE gels. Recovery of mAbs reached more than 86% with 0.2% SD. The heterogeneities, especially N-glycan variants in the recovered mAb therapeutics, were clearly determined by intact mass analysis. We believe that the study is important in pharmaceuticals‧ perspective since orthogonal combination of gel electrophoresis and intact mass analysis should be pivotal role for rapid and precise characterization of mAbs. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. Purification of recombinant Aβ(1-42) and pGlu-Aβ(3-42) using preparative SDS-PAGE.

    PubMed

    Spahn, Claudia; Wermann, Michael; Eichentopf, Rico; Hause, Gerd; Schlenzig, Dagmar; Schilling, Stephan

    2017-08-01

    Recombinant expression and purification of amyloid peptides represents a common basis for investigating the molecular mechanisms of amyloid formation and toxicity. However, the isolation of the recombinant peptides is hampered by inefficient separation from contaminants such as the fusion protein required for efficient expression in E. coli. Here, we present a new approach for the isolation of highly purified Aβ(1-42) and pGlu-Aβ(3-42), which is based on a separation using preparative SDS-PAGE. The method relies on the purification of the Aβ fusion protein by affinity chromatography followed by preparative SDS-PAGE under reducing conditions and subsequent removal of detergents by precipitation. The application of preparative SDS-PAGE represents the key step to isolate highly pure recombinant Aβ, which has been applied for characterization of aggregation and toxicity. Thereby, the yield of the purification strategy was  >60%. To the best of our knowledge, this is the first description of an electrophoresis-based method for purification of a recombinant Aβ peptide. Therefore, the method might be of interest for isolation of other amyloid peptides, which are critical for conventional purification strategies due to their aggregation propensity. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. SDS-PAGE and IR spectroscopy to evaluate modifications in the viral protein profile induced by a cationic porphyrinic photosensitizer.

    PubMed

    Costa, Liliana; Esteves, Ana Cristina; Correia, António; Moreirinha, Catarina; Delgadillo, Ivonne; Cunha, Ângela; Neves, Maria G P S; Faustino, Maria A F; Almeida, Adelaide

    2014-12-01

    Reactive oxygen species can be responsible for microbial photodynamic inactivation due to its toxic effects, which include severe damage to proteins, lipids and nucleic acids. In this study, the photo-oxidative modifications of the proteins of a non-enveloped T4-like bacteriophage, induced by the cationic porphyrin 5,10,15-tris(1-methylpyridinium-4-yl)-20-(pentafluorophenyl)porphyrin tri-iodide were evaluated. Two methods were used: sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and infrared spectroscopy. SDS-PAGE analysis showed that the phage protein profile was considerably altered after photodynamic treatment. Seven protein bands putatively corresponding to capsid and tail tube proteins were attenuated and two other were enhanced. Infrared spectroscopy confirmed the time-dependent alteration on the phage protein profile detected by SDS-PAGE, indicative of a response to oxidative damage. Infrared analysis showed to be a promising and rapid screening approach for the analysis of the modifications induced on viral proteins by photosensitization. In fact, one single infrared spectrum can highlight the changes induced to all viral molecular structures, overcoming the delays and complex protocols of the conventional methods, in a much simple and cost effective way.

  6. TCEP-based rSDS-PAGE AND nLC-ESI-LTQ-MS/MS for oxaliplatin metalloproteomic analysis.

    PubMed

    Mena, M L; Moreno-Gordaliza, E; Gómez-Gómez, M M

    2013-11-15

    In this work, the reactivity of the citostatic drugs such as oxaliplatin, cisplatin and carboplatin towards proteins and the stability of Pt-protein complexes along their storage were evaluated. Neither native-PAGE nor nrSDS-PAGE seems to be suitable for the separation of carboplatin-binding proteins. A reducing electrophoretic separation procedure able to maintain the integrity of oxaliplatin-protein complexes has been developed. The method is based on SDS-PAGE under conditions provided by the thiol-free reducing agent tris (2-carboxyethyl) phosphine (TCEP), which allowed the separation of oxaliplatin-binding proteins in narrow bands with almost quantitative recoveries. Different amounts of platinum-bound protein bands covering the range 0.3-2.0 μg were excised and mineralised for platinum determination, showing good linearity. Limits of detection for a mixture of five standard proteins (transferrin, albumin, carbonic anhydrase, myoglobin and cytochrome c) incubated with oxaliplatin were within the range 11.0-44.0 pg of platinum, which were satisfactory for their application to biological samples. The suitability of the TCEP-based SDS-PAGE for the separation of platinum-enriched protein fractions of a kidney cytosol from a rat treated with oxaliplatin was demonstrated. The identification of high Pt to protein ratio cytosolic fractions was carried out by separating the cytosolic platinum-binding proteins by SEC-ICP-MS. Several cytosolic renal proteins were identified in those gel bands containing platinum-enriched protein fractions using nLC-ESI-LTQ-MS/MS after in-gel digested with trypsin. In addition, fractions containing platinum-enriched proteins with lower theorical molecular weight were directly analysed by nLC-ESI-LTQ-MS/MS after in-solution tryptic digestion allowing protein identification.

  7. Bifunctional glass membrane designed to interface SDS-PAGE separations of proteins with the detection of peptides by mass spectrometry.

    PubMed

    Hattan, Stephen J; Du, Jie; Parker, Kenneth C

    2015-04-07

    We describe the construction and characterization of a novel membrane designed to allow proteins separated by gel electrophoresis (SDS-PAGE) to be detected as peptides by mass spectrometry in an efficient and comprehensive manner. The key attribute of the membrane is a bifunctional design that allows for the digestion of protein(s) and retention of the resulting peptides with minimal lateral diffusion. Silane chemistries are used to differentially treat the opposing surfaces of a glass filter paper to enable this unique capability.

  8. Separation of thylakoid membrane proteins by sucrose gradient ultracentrifugation or blue native-SDS-PAGE two-dimensional electrophoresis.

    PubMed

    D'Amici, Gian Maria; Huber, Christian G; Zolla, Lello

    2009-01-01

    Generally, a combination of two or more chromatographic and/or electrophoretic methods is conducted to separate membrane protein complexes. Here we describe how thylakoid membrane protein complexes from the photosynthetic apparatus can be successfully separated by two main steps: preparative methods that enable purification of membrane protein complexes in the native (intact) form, and analytical methods that allow resolution of each membrane protein. Thus, separation of intact supercomplexes was achieved by solubilisation of the sample using mild detergents followed either by sucrose gradient ultracentrifugation or by blue native gel (BNG) electrophoresis. Complexes, thus, recovered were then resolved further using either reversed phase liquid chromatography or SDS-PAGE respectively.

  9. Mobility shift detection of phosphorylation on large proteins using a Phos-tag SDS-PAGE gel strengthened with agarose.

    PubMed

    Kinoshita, Eiji; Kinoshita-Kikuta, Emiko; Ujihara, Hiromi; Koike, Tohru

    2009-08-01

    We describe a novel technique of phosphate-affinity SDS-PAGE using Phos-tag to analyze large phosphoproteins with molecular masses of more than 200 kDa. The protein phosphoisotypes were clearly separated as up-shifted migration bands in a 3% w/v polyacrylamide gel containing 20 microM Phos-tag and 0.5% w/v agarose. In subsequent immunoblotting, the procedure permitted the determination of the phosphoisotypes of high-molecular-mass proteins, such as mTOR (289 kDa), ATM kinase (350 kDa), and 53BP1 (213 kDa).

  10. Comparison of Strong Cation Exchange and SDS/PAGE Fractionation for Analysis of Multi-Protein Complexes

    PubMed Central

    Das, Sudipto; Bosley, Allen D.; Ye, Xiaoying; Chan, King C.; Chu, Isabel; Green, Jeffery E.; Issaq, Haleem J.; Veenstra, Timothy D.; Andresson, Thorkell

    2013-01-01

    Affinity purification of protein complexes followed by identification using liquid chromatography/mass spectrometry (LC-MS/MS) is a robust method to study the fundamental process of protein interaction. While affinity isolation reduces the complexity of the sample, fractionation prior to LC-MS/MS analysis is still necessary to maximize protein coverage. In this study, we compared the protein coverage obtained via LC-MS/MS analysis of protein complexes pre-fractionated using two commonly employed methods, SDS-PAGE and strong cation exchange chromatography (SCX). The two complexes analyzed focused on the nuclear proteins Bmi-1 and GATA3 that were expressed within the cells at low and high levels, respectively. Pre-fractionation of the complexes at the peptide level using SCX consistently resulted in the identification of approximately 3-fold more proteins compared to separation at the protein level using SDS-PAGE. The increase in the number of identified proteins was especially pronounced for the Bmi-1 complex, where the target protein was expressed at a low level. The data shows that pre-fractionation of affinity isolated protein complexes using SCX prior to LC-MS/MS analysis significantly increases the number of identified proteins and individual protein coverage, particularly for target proteins expressed at low levels. PMID:20968308

  11. 16-BAC/SDS-PAGE analysis of membrane proteins of yeast mitochondria purified by free flow electrophoresis.

    PubMed

    Braun, Ralf J; Kinkl, Norbert; Zischka, Hans; Ueffing, Marius

    2009-01-01

    Mitochondria are essential organelles in cellular metabolism. These organelles are bounded by two membranes, the outer and inner membrane. Especially the inner membrane comprises a high content of proteins, for example, the protein complexes of the respiratory chain. High-resolution separation and analysis of such membrane proteins, for example, by two-dimensional gel electrophoresis (2-DE), is hampered by their hydrophobicity and tendency for aggregation. Here, we describe the separation of mitochondrial membrane proteins of Saccharomyces cerevisiae by 16-benzyldimethyl-n-hexadecylammonium chloride/sodium dodecyl sulfate polyacrylamide gel electrophoresis (16-BAC/SDS-PAGE). This method enables the separation of membrane proteins owing to the solubilizing power of the ionic detergents 16-BAC and SDS, respectively. Mitochondria were isolated from yeast cultures by differential centrifugation and were further purified by free flow electrophoresis (FFE) in zone-electrophoretic mode (ZE). Subsequently, membrane proteins from ZE-FFE-purified mitochondria were enriched by carbonate extraction and subjected to 16-BAC/SDS-PAGE. The resulting protein spot patterns were visualized by a highly sensitive fluorescence stain with ruthenium-II-bathophenantroline disulfonate chelate (RuBP), and by colloidal Coomassie staining. Proteins were identified by Maldi-Tof mass spectrometry and peptide mass fingerprinting.

  12. Quantitative determination of titin and nebulin in poultry meat by SDS-PAGE with an internal standard.

    PubMed

    Tomaszewska-Gras, Jolanta; Kijowski, Jacek; Schreurs, Franciscus J G

    2002-09-01

    The method of quantitative determination of titin and nebulin in chicken meat by SDS-PAGE electrophoresis technique was developed by application of β-galactosidase as the internal standard. The method was tested first on marker protein samples of known concentrations (myosin, transferrin, glutamic dehydrogenase) and then the method was used in the determination of titin and nebulin content in chicken meat. The method demonstrated high accuracy, confirmed by correlation coefficient 0.91÷0.99. Two gel analysis techniques, i.e. scanning and densitometry were also compared. By the use of marker proteins as well as titin and nebulin, higher accuracy and precision were achieved in scanning than in the densitometric technique. Recoveries of three marker proteins were between 93 and 102% for the technique of the scanning of the gel and between 99 and 116% for the densitometry.

  13. [Identification of extrinsic proteins in boneless cooked ham by SDS-PAGE: detection level in model systems].

    PubMed

    López, Laura Beatríz; Greco, Carola Beatriz; Ronayne de Ferrer, Patricia; Valencia, Mirta Eva

    2006-09-01

    Protein extraction and separation in polyacrylamide slab gel electrophoresis with Laemmli system (SDS-PAGE) were used to establish the detection level of protein raw materials in mixtures with porcine meat in boneless cooked ham. Model systems of boneless cooked ham with soy protein isolates, caseinate, skim powdered milk, bovine plasma, porcine plasma and whey proteins were studied. The quantification level of this method was 0.5% for soy protein isolates, caseinate and bovine plasma and 1.0% for porcine plasma, milk powder and whey proteins in boneless cooked ham. The electrophoretic method proved to be useful to identify some proteinic raw materials in porcine meat products and verify compliance with Argentine legislation. It may be used as a control methodology.

  14. Investigation of relationships between barley stress peptides and beer gushing using SDS-PAGE and MS screening.

    PubMed

    Hégrová, Blanka; Farková, Marta; Macuchová, Simona; Havel, Josef; Preisler, Jan

    2009-12-01

    The relationship between gushing and antifungal peptides in barley and malt kernels was examined for five barley varieties produced in the Czech Republic with four conditions of infection and treatment. Proteome changes during pathogen-seed interaction were observed with SDS-PAGE and MALDI-TOF MS. These methods were applied as a fast screening for observing the relationship between gushing and peptides/proteins. It was found that the presence of basic peptides, presumably hordothionins and non-specific lipid transfer protein type 1, did not correlate with the degree of gushing for malt (/r/ in <0.07, 0.34>), (/r/ in <0.01, 0.49>), respectively, as detected by both methods.

  15. The antimicrobial mechanism of electrochemically activated water against Pseudomonas aeruginosa and Escherichia coli as determined by SDS-PAGE analysis.

    PubMed

    Cloete, T E; Thantsha, M S; Maluleke, M R; Kirkpatrick, R

    2009-08-01

    To expose bacteria to anolyte and subsequently investigate the effect of anolyte on the protein profiles of treated bacteria. Proteins were extracted from bacteria treated with different concentrations of anolyte and analysed using SDS-PAGE. Fewer and more faint protein bands were observed for concentrated halide anolyte treated bacteria when compared to untreated bacteria while extra protein bands were observed for bacteria exposed to dilute concentrations. The undiluted and the 10(-1) dilution of halide derived anolyte was effective in killing the test bacteria. Anolyte caused bacterial death by complete destruction of proteins or by causing oxidative stress which resulted in protein fragmentation. The results of this study provide information on the antimicrobial mechanism of anolyte on other bacteria for which the information is currently unavailable.

  16. Pre-staining of glycoprotein in SDS-PAGE by the synthesis of a new hydrazide derivative.

    PubMed

    Zhou, Ayi; Zhou, Tieli; Yu, Dongdong; Shen, Yingjie; Shen, Jiayi; Zhu, Zhongxin; Jin, Litai; Zhang, Huajie; Wang, Yang

    2015-11-01

    In this study, a new hydrazide derivative (UGF202) was synthesized and introduced as a highly sensitive and selective fluorescent probe to pre-stain glycoproteins in 1D and 2D SDS-PAGE. As low as 0.5-1 ng glycoproteins (transferrin, α1-acid glycoprotein, avidin) could be selectively detected, which is comparable to that of Pro-Q Emerald 300 stain, one of the most sensitive and commonly used glycoprotein staining kit. In addition, the specificity of the newly developed method was confirmed by the study of de-glycosylation, glycoproteins affinity enrichment and LC-MS/MS, respectively. According to the results, it is concluded that UGF202 pre-stain can provide an alternative for the visualization of gel-separated glycoproteins.

  17. Aggregation of frog rhodopsin to oligomers and their dissociation to monomer: application of BN- and SDS-PAGE.

    PubMed

    Shukolyukov, S A

    2009-06-01

    After solubilization of frog rod outer segments (ROS) with mild detergents (digitonin, n-dodecyl-beta-D-maltoside, Chaps, Triton X-100) and subsequent one-dimensional blue native polyacrylamide gel electrophoresis (1D BN-PAGE), the position of rhodopsin (Rh) on the gradient gel does not match the monomer with molecular weight of 40 kDa but appears self-associated into aggregate of Rh (RhA) with molecular mass varying in different detergents from 85 to 125 kDa. Short-term treatment (~2 h) of the excised BN-PAGE strip containing RhA by denaturing detergent mixture (10% SDS + 1 mM dithiothreitol (DTT)) followed by 2D SDS-PAGE revealed dissociation of the RhA into opsin monomer and unidentified proteins. Long-term treatment (approximately 2 days) of RhA that included extraction, denaturation, concentration, and electrophoresis induced, along with dissociation of RhA into opsin monomer + unidentified proteins, also formation of opsin dimers, trimers, and higher oligomers owing to a secondary aggregation of opsin. Direct solubilization of the ROS by harsh SDS + DTT detergent mixture followed by 1D SDS-PAGE revealed only opsin monomer that upon heating disappeared, transforming into higher oligomers owing to secondary aggregation. The data show that degree of Rh oligomerization depends on specific conditions in which it stays. In the native state in the photoreceptor membrane as well as in mild detergents frog Rh exists mainly as dimers or higher oligomers. After solubilization with denaturing detergents, RhA can dissociate into monomers that then spontaneously self-associate into higher oligomers under the influence of various factors (for example, heating).

  18. Lab-on-a-chip and SDS-PAGE analysis of hemolymph protein profile from Rhipicephalus microplus (Acari: Ixodidae) infected with entomopathogenic nematode and fungus.

    PubMed

    Golo, Patrícia Silva; Dos Santos, Alessa Siqueira de Oliveira; Monteiro, Caio Marcio Oliveira; Perinotto, Wendell Marcelo de Souza; Quinelato, Simone; Camargo, Mariana Guedes; de Sá, Fillipe Araujo; Angelo, Isabele da Costa; Martins, Marta Fonseca; Prata, Marcia Cristina de Azevedo; Bittencourt, Vânia Rita Elias Pinheiro

    2016-09-01

    In the present study, lab-on-a-chip electrophoresis (LoaC) was suggested as an alternative method to the conventional polyacrylamide gel electrophoresis under denaturing conditions (SDS-PAGE) to analyze raw cell-free tick hemolymph. Rhipicephalus microplus females were exposed to the entomopathogenic fungus Metarhizium anisopliae senso latu IBCB 116 strain and/or to the entomopathogenic nematode Heterorhabditis indica LPP1 strain. Hemolymph from not exposed or exposed ticks was collected 16 and 24 h after exposure and analyze by SDS-PAGE or LoaC. SDS-PAGE yielded 15 bands and LoaC electrophoresis 17 bands. Despite the differences in the number of bands, when the hemolymph protein profiles of exposed or unexposed ticks were compared in the same method, no suppressing or additional bands were detected among the treatments regardless the method (i.e., SDS-PAGE or chip electrophoresis using the Protein 230 Kit®). The potential of LoaC electrophoresis to detect protein bands from tick hemolymph was considered more efficient in comparison to the detection obtained using the traditional SDS-PAGE method, especially when it comes to protein subunits heavier than 100 KDa. LoaC electrophoresis provided a very good reproducibility, and is much faster than the conventional SDS-PAGE method, which requires several hours for one analysis. Despite both methods can be used to analyze tick hemolymph composition, LoaC was considered more suitable for cell-free hemolymph protein separation and detection. LoaC hemolymph band percent data reported changes in key proteins (i.e., HeLp and vitellogenin) exceptionally important for tick embryogenesis. This study reported, for the first time, tick hemolymph protein profile using LoaC.

  19. Regional heterogeneity of glycoconjugate distribution in the glomerulus revealed by lectin-gold cytochemistry and SDS-PAGE.

    PubMed Central

    Brown, D.; Vassalli, J. D.; Kunz, A.; Mühlhauser, J.; Orci, L.; Mulhauser, J.

    1986-01-01

    The authors have used SDS-PAGE and lectin overlay analysis in parallel with lectin-gold cytochemistry to identify Helix pomatia lectin (HPL) binding glycoconjugates in rat kidney glomeruli. Previous work revealed HPL binding sites only beneath podocyte foot process bases, where they contact the glomerular basement membrane. It is shown here that after neuraminidase digestion of thin sections of glomeruli before incubation with HPL-gold complexes, the number of HPL binding sites is markedly increased. These new sites are mainly associated with the podocyte free surface (adjacent to the urinary space) and with capillary endothelial cells. By lectin overlays, this neuraminidase-dependent HPL binding was shown to be due to reaction of the lectin with desialylated podocalyxin. In contrast, HPL binding sites detected prior to neuraminidase digestion are associated with a novel glycoconjugate having a lower electrophoretic mobility than podocalyxin. Although any role for this glycoconjugate is at present speculative, it is strategically positioned at the site of interaction between foot process bases and the glomerular basement membrane. Its presence correlates with normal podocyte architecture, as shown by our previous studies on developmental and aminonucleoside nephrosis-associated changes in HPL binding to podocytes. Images Figure 1 Figure 3 Figure 4 Figure 5 Figure 6 PMID:2432793

  20. SDS-PAGE analysis of M. leprae protein antigens reacting with antibodies from sera from lepromatous patients and infected armadillos.

    PubMed Central

    Chakrabarty, A K; Maire, M A; Lambert, P H

    1982-01-01

    Studies have been conducted to characterize M. leprae bacilli derived from infected armadillos. First, the proteins of the mycobacterial extracts were fractionated by SDS-PAGE. Subsequently, the proteins in the gel were electrophoretically transferred on a strip of nitrocellulose paper by the technique of 'electrophoretic blotting'. The separated bacterial protein bands, thus immobilized on the nitrocellulose paper were made to react immunologically with sera from the lepromatous patients, infected armadillo sera and other experimental mycobacterial antisera. It was observed that a majority of M. leprae proteins contained antigenic determinants also present on proteins of BCG. In addition, only two specific antigen bands of 33KD and 12KD were conspicuously detected by the patients' sera and the infected armadillo sera. These substances were further identified as polysaccharides or glycoproteins since they could only be stained by Schiff's reagent or alcian blue. Only 12KD glycoprotein band reacted with concanavalin A, whereas wheat germ agglutinin (WGA) did not show any reaction with them. These 33KD and 12KD glycoprotein antigens were found to lose their antigenicity after pepsin treatment and can be considered as glycoproteins. Further, radiolabelling experiments showed that 12KD antigen underwent radioiodination under usual conditions, but 33KD glycoprotein failed to be similarly radiolabelled. It is suggested that these protein antigens have M. leprae specific determinants on a cross-reacting component. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 PMID:6184189

  1. Two dimensional blue native/SDS-PAGE to identify mitochondrial complex I subunits modified by 4-hydroxynonenal (HNE).

    PubMed

    Wu, Jinzi; Luo, Xiaoting; Yan, Liang-Jun

    2015-01-01

    The lipid peroxidation product 4-hydroxynonenal (HNE) can form protein-linked HNE adducts, thereby impacting protein structure and function. Mitochondrial complex I (NADH-ubiquinone oxidoreductase), containing at least 45 subunits in mammalian cells, sits in a lipid-rich environment and is thus very susceptible to HNE modifications. In this paper, a procedure for the identification of HNE-modified complex I subunits is described. Complex I was isolated by first dimensional non-gradient blue native polyacrylamide gel electrophoresis (BN-PAGE). The isolated complex I band, visualized by either Coomassie blue staining or silver staining, was further analyzed by second dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). HNE-modified proteins were visualized by Western blotting probed with anti-HNE antibodies. HNE-positive bands were then excised and the proteins contained in them were identified by mass spectrometric peptide sequencing. The method was successfully applied for the identification of two complex I subunits that showed enhanced HNE-modifications in diabetic kidney mitochondria.

  2. Polymorphism of omega-gliadins in durum wheat as revealed by the two-step APAGE/SDS-PAGE technique.

    PubMed

    Nieto-Taladriz, M T; Branlard, G; Dardevet, M

    1994-03-01

    Polymorphism of omega-gliadins was studied in 243 durum wheats from 27 countries using the two-step one-dimensional APAGE/SDS-PAGE technique. A total of 12 bands of different mobility were observed, and four of them were found to be different from those previously detected by Khelifi et al. (1992) in bread wheat. Fifteen alleles, six coded by the Gli-A1 locus and nine coded by the Gli-B1 locus, were identified, accounting for 19 different electrophoretic patterns. Seven new alleles were detected: two at the Gli-A1 locus and five at the Gli-B1 locus. The polymorphism found at the Gli-A1 and Gli-B1 loci was slightly greater than that found in bread wheat. Allelic differences between both species were higher at the Gli-B1 locus. A comparison of the frequencies of alleles in both species was carried out. The null allele, Gli-A1e, was more common in durum wheat than in bread wheat. The Gli-B1b allele, present in 60% of the bread wheats, was found in only 2% of the durum wheats and Gli-B1e, very common in durum wheat (45%), was rare in bread wheat (4%). The Gli-B1IV allele, common in durum wheat (28%), was not detected in bread wheat.

  3. Proteomic study of muscle sarcoplasmic proteins using AUT-PAGE/SDS-PAGE as two-dimensional gel electrophoresis.

    PubMed

    Picariello, Gianluca; De Martino, Alessandra; Mamone, Gianfranco; Ferranti, Pasquale; Addeo, Francesco; Faccia, Michele; Spagnamusso, Salvatore; Di Luccia, Aldo

    2006-03-20

    In the present study, an alternative procedure for two-dimensional (2D) electrophoretic analysis in proteomic investigation of the most represented basic muscle water-soluble proteins is suggested. Our method consists of Acetic acid-Urea-Triton polyacrylamide gel (AUT-PAGE) analysis in the first dimension and standard sodium dodecyl sulphate polyacrylamide gel (SDS-PAGE) in the second dimension. Although standard two-dimensional Immobilized pH Gradient-Sodium Dodecyl-Sulphate (2D IPG-SDS) gel electrophoresis has been successfully used to study these proteins, most of the water-soluble proteins are spread on the alkaline part of the 2D map and are poorly focused. Furthermore, the similarity in their molecular weights impairs resolution of the classical approach. The addition of Triton X-100, a non-ionic detergent, into the gel induces a differential electrophoretic mobility of proteins as a result of the formation of mixed micelles between the detergent and the hydrophobic moieties of polypeptides, separating basic proteins with a criterion similar to reversed phase chromatography based on their hydrophobicity. The acid pH induces positive net charges, increasing with the isoelectric point of proteins, thus allowing enhanced resolution in the separation. By using 2D AUT-PAGE/SDS electrophoresis approach to separate water-soluble proteins from fresh pork and from dry-cured products, we could spread proteins over a greater area, achieving a greater resolution than that obtained by IPG in the pH range 3-10 and 6-11. Sarcoplasmic proteins undergoing proteolysis during the ripening of products were identified by Matrix Assisted Laser Desorption/Ionization-Time of Flight (MALDI-ToF) mass spectrometry peptide mass fingerprinting in a easier and more effective way. Two-dimensional AUT-PAGE/SDS electrophoresis has allowed to simplify separation of sarcoplasmic protein mixtures making this technique suitable in the defining of quality of dry-cured pork products by immediate

  4. The effect of the biological variability of samples on Coomassie blue dye based fast staining for SDS-PAGE in nonfixed gels.

    PubMed

    Májek, Pavel; Pecankova, Klara; Dyr, Jan E

    2014-10-01

    Fast-staining protocols based on the use of Coomassie blue dye for SDS-PAGE separated proteins, represent a quick and simple solution for protein visualization. It has been shown however, that in some cases a phenomenon of missing spots or spot discoloration may be observed in the proteome pattern when the standard fast-staining protocol is used. In this work, it is demonstrated that this occurrence is affected by the biological variability of samples, and therefore, cannot be observed in all samples. Moreover, it is demonstrated that the phenomenon is manifested exclusively in nonfixed gels, and that including a fixation step into the fast-staining protocol prevented this phenomenon. In conclusion, it has been demonstrated that standard Coomassie blue dye based fast staining for SDS-PAGE resolved proteins is affected by the biological variability of samples in nonfixed gels. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. SDS-PAGE analysis of Aβ oligomers is disserving research into Alzheimer´s disease: appealing for ESI-IM-MS

    PubMed Central

    Pujol-Pina, Rosa; Vilaprinyó-Pascual, Sílvia; Mazzucato, Roberta; Arcella, Annalisa; Vilaseca, Marta; Orozco, Modesto; Carulla, Natàlia

    2015-01-01

    The characterization of amyloid-beta peptide (Aβ) oligomer forms and structures is crucial to the advancement in the field of Alzheimer´s disease (AD). Here we report a critical evaluation of two methods used for this purpose, namely sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), extensively used in the field, and ion mobility coupled to electrospray ionization mass spectrometry (ESI-IM-MS), an emerging technique with great potential for oligomer characterization. To evaluate their performance, we first obtained pure cross-linked Aβ40 and Aβ42 oligomers of well-defined order. Analysis of these samples by SDS-PAGE revealed that SDS affects the oligomerization state of Aβ42 oligomers, thus providing flawed information on their order and distribution. In contrast, ESI-IM-MS provided accurate information, while also reported on the chemical nature and on the structure of the oligomers. Our findings have important implications as they challenge scientific paradigms in the AD field built upon SDS-PAGE characterization of Aβ oligomer samples. PMID:26450154

  6. Molecular weight determination of high molecular mass (glyco)proteins using CGE-on-a-chip, planar SDS-PAGE and MALDI-TOF-MS.

    PubMed

    Müller, Roland; Marchetti-Deschmann, Martina; Elgass, Helmut; Breiteneder, Heimo; Kratzmeier, Martin; Allmaier, Günter

    2010-12-01

    The molecular weights (MW) of seven (glyco)proteins, of which five were plasma-derived, with MWs higher than 200 kDa were determined with three techniques: CGE-on-a-chip, SDS-PAGE and MALDI-TOF-MS. While the analysis of medium to high MW proteins with SDS-PAGE was an already well-established technique, the usefulness of MALDI-TOF-MS for the exact MW determination of high mass proteins was only partly described in literature so far. CGE-on-a-chip is the newest of all three applied techniques and was so far not applicable. Therefore, it was not evaluated for high MW (glyco)proteins. All proteins were analyzed under nonreducing as well as reducing conditions. In this work, it was demonstrated that all three described techniques were capable of determining the MW of all high molecular weight (glyco)proteins. The noncommercial CGE-on-a-chip assay allowed for the first time the electrophoretic separation of proteins in the MW range from 14 to 1000 kDa. MW assignment was limited to 500 kDa in the case of SDS-PAGE and 660 kDa in the case of the high MW CGE-on-a-chip assay. With the proper matrix and sample preparation, analysis with a standard MALDI-TOF-MS provided accurate MWs for all high MW proteins up to 1 MDa.

  7. Characterization of lower molecular weight artifact bands of recombinant monoclonal IgG1 antibodies on non-reducing SDS-PAGE.

    PubMed

    Liu, Hongcheng; Gaza-Bulseco, Georgeen; Chumsae, Chris; Newby-Kew, Abigail

    2007-11-01

    SDS-PAGE under non-reducing conditions is one of the most commonly used techniques for recombinant monoclonal antibody purity and stability indicating assay. On non-reducing SDS-PAGE, bands with a lower molecular weight than the intact antibody are routinely observed and is a common feature of IgG molecules. These fragments were analyzed by in-gel digestion followed by matrix-assisted-laser-desorption-ionization time-of-flight (MALDI-TOF) mass spectrometry, Western blot and by comparing the banding pattern of sample prepared in the presence of a reducing reagent. The fragments bands were identified as antibody lacking one light chain, two heavy chains, one light chain and one heavy chain, free heavy chain and free light chain. Sensitivity of fragmentation to sample buffer pH, incubation time, reducing reagent and alkylation reagents indicated that fragments were formed during sample preparation, but not present in the samples analyzed. Disulfide bond scrambling and beta-elimination are the two major mechanisms of the formation antibody fragments. Mass spectrometry analysis suggested that disulfide bond scrambling can be prevented by specifically modifying free sulhydryl using alkylation and thus reduced the amount of artifacts on non-reducing SDS-PAGE. Breakage of disulfide bonds by beta-elimination was evidenced by the detection of dehydroalanine using mass spectrometry.

  8. SDS-PAGE analysis of Aβ oligomers is disserving research into Alzheimer´s disease: appealing for ESI-IM-MS.

    PubMed

    Pujol-Pina, Rosa; Vilaprinyó-Pascual, Sílvia; Mazzucato, Roberta; Arcella, Annalisa; Vilaseca, Marta; Orozco, Modesto; Carulla, Natàlia

    2015-10-09

    The characterization of amyloid-beta peptide (Aβ) oligomer forms and structures is crucial to the advancement in the field of Alzheimer´s disease (AD). Here we report a critical evaluation of two methods used for this purpose, namely sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), extensively used in the field, and ion mobility coupled to electrospray ionization mass spectrometry (ESI-IM-MS), an emerging technique with great potential for oligomer characterization. To evaluate their performance, we first obtained pure cross-linked Aβ40 and Aβ42 oligomers of well-defined order. Analysis of these samples by SDS-PAGE revealed that SDS affects the oligomerization state of Aβ42 oligomers, thus providing flawed information on their order and distribution. In contrast, ESI-IM-MS provided accurate information, while also reported on the chemical nature and on the structure of the oligomers. Our findings have important implications as they challenge scientific paradigms in the AD field built upon SDS-PAGE characterization of Aβ oligomer samples.

  9. SDS-PAGE analysis of Aβ oligomers is disserving research into Alzheimer´s disease: appealing for ESI-IM-MS

    NASA Astrophysics Data System (ADS)

    Pujol-Pina, Rosa; Vilaprinyó-Pascual, Sílvia; Mazzucato, Roberta; Arcella, Annalisa; Vilaseca, Marta; Orozco, Modesto; Carulla, Natàlia

    2015-10-01

    The characterization of amyloid-beta peptide (Aβ) oligomer forms and structures is crucial to the advancement in the field of Alzheimer´s disease (AD). Here we report a critical evaluation of two methods used for this purpose, namely sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), extensively used in the field, and ion mobility coupled to electrospray ionization mass spectrometry (ESI-IM-MS), an emerging technique with great potential for oligomer characterization. To evaluate their performance, we first obtained pure cross-linked Aβ40 and Aβ42 oligomers of well-defined order. Analysis of these samples by SDS-PAGE revealed that SDS affects the oligomerization state of Aβ42 oligomers, thus providing flawed information on their order and distribution. In contrast, ESI-IM-MS provided accurate information, while also reported on the chemical nature and on the structure of the oligomers. Our findings have important implications as they challenge scientific paradigms in the AD field built upon SDS-PAGE characterization of Aβ oligomer samples.

  10. Quantification of tear proteins by SDS-PAGE with an internal standard protein: a new method with special reference to small volume tears.

    PubMed

    Li, Kaijun; Chen, Ziyan; Duan, Fang; Liang, Jingwen; Wu, Kaili

    2010-06-01

    Quantitative determination of tear proteins is critical to our understanding of those ocular diseases with tear protein changes, but remains technically complex due to the small sample volumes available from patients. The aim of this study was to efficiently quantify the tear proteins by SDS-PAGE with an internal standard protein in small tear volumes. Schirmer test paper and capillary tubes were used to collect tear samples. Soybean trypsin inhibitor (SBTI) was used as an external standard or an internal standard to analyze tear samples in 15% SDS-PAGE gel. The total tear protein and its major components were quantified by band densitometry. Total tear protein concentrations were also measured by Bradford assay. Using this internal standard method, we compared differences between tear samples collected by the Schirmer test paper and capillary tube, and then examined the differences detected between tear samples obtained from young and elderly people. Using SBTI as an internal standard in SDS-PAGE, the total tear protein concentrations were determined to be 12.03 +/- 0.45 mg/ml, showing no difference from the result determined by the Bradford assay (P > 0.05). The quantities of the eight major tear protein bands were 2.26 +/- 0.07(18.8%), 0.10 +/- 0.01(0.8%), 0.63 +/- 0.13(5.3%), 0.52 +/- 0.07(4.3%), 1.14 +/- 0.18(9.5%), 1.33 +/- 0.21(11.1%), 2.76 +/- 0.16(23.0%), and 2.95 +/- 0.13 mg/ml (24.5%), similar to the result obtained by using SBTI as an external standard (P > 0.05). Comparing the methods of collection, we identified that the Schirmer test paper sampling induced increased concentrations of band 2 (mainly HSA) in tears, and decreased five other major tear protein bands. Comparing total protein concentrations among different age groups, we noted a higher total protein concentration in young people (P < 0.05). This high protein level was contributed equally by all major tear protein components. Using SBTI as an internal standard, we can simultaneously

  11. Isoelectric focusing of human hair keratins: correlation with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) patterns and effect of cosmetic treatments.

    PubMed

    Rodriguez-Calvo, M S; Carracedo, A; Muñoz, I; Concheiro, L

    1992-03-01

    A new isoelectric focusing (IEF) technique in polyacrylamide gels with 6M urea and 1.5% Nonidet P40 has been developed to characterize human hair samples. The phenotypes demonstrated with this procedure has been correlated with the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) patterns described by other authors. The method described can be applied in the forensic science analysis of a single human hair. Using the same IEF technique we have studied the changes in electrophoretic patterns of cosmetically treated hair. The characteristics of the modifications observed and its utility in forensic science work are also discussed in this paper.

  12. In Vitro Antibacterial Mechanism of Action of Crude Garlic (Allium sativum) Clove Extract on Selected Probiotic Bifidobacterium Species as Revealed by SEM, TEM, and SDS-PAGE Analysis.

    PubMed

    Booyens, J; Labuschagne, M C; Thantsha, M S

    2014-06-01

    There has been much research on the effects of garlic (Allium sativum) on numerous pathogens, but very few, if any, studies on its effect on beneficial, probiotic bifidobacteria. We have recently shown that garlic exhibits antibacterial activity against bifidobacteria. The mechanism by which garlic kills bifidobacteria is yet to be elucidated. This study sought to determine the mechanism of action of garlic clove extract on selected Bifidobacterium species using scanning and transmission electron microscopy and SDS-PAGE analysis. SEM micrographs revealed unusual morphological changes such as cell elongation, cocci-shaped cells with cross-walls, and distorted cells with bulbous ends. With TEM, observed changes included among others, condensation of cytoplasmic material, disintegration of membranes, and loss of structural integrity. SDS-PAGE analysis did not reveal any differences in whole-cell protein profiles of untreated and garlic clove extract-treated cells. The current study is the first to reveal the mechanism of action of garlic clove extract on probiotic Bifidobacterium species. The results indicate that garlic affects these beneficial bacteria in a manner similar to that exhibited in pathogens. These results therefore further highlight that caution should be taken especially when using raw garlic and probiotic bifidobacteria simultaneously as viability of these bacteria could be reduced by allicin released upon crushing of garlic cloves, thereby limiting the health benefits that the consumer anticipate to gain from probiotics.

  13. Characterization of antigenic property of Toxocara canis and Toxascaris leonina adults and larvae through immunodiagnostic electrophoresis (SDS-PAGE) and western blot technique.

    PubMed

    el-Massry, A A

    1999-08-01

    Differential molecular studies were performed by sodium dodecyle sulphate polyacrylamide gel electrophoresis--SDS-PAGE--between somatic antigen of Toxocara canis and Toxascaris leonina, adults and larvae, recovered from dogs. SDS-PAGE of both adults somatic antigen showed two closely similar bands (90.00, 91.95 KDa and 69.25-70.56 KDa). Each parasite had characteristic bands clustered in different molecular weights. While for their larval antigen, T. canis showed a very different antigenic profile when analysed in comparison to T. leonina antigen except at one band (66.85-66.89 KDa). The Western blot analysis showed four prominent bands represented immunoreaction between the separated somatic antigen of T. canis adults and experimentally immunized rabbit with the corresponding parasite (125.37, 117.73, 90.00 and 69.25 KDa). While separated antigen of T. leonina adults immune reacted with the corresponding hyperimmune rabbit sera at 119.04, 91.95 and 70.56 KDa. The Western blot showed cross reactive immune bands between T. canis and T. leonina adults somatic antigen at two bands (90.00, 91.95 KDa and 69.25-70.56 KDa). The polypeptide bands reacted at 125.37 KDa and 117.73 KDa can be used as specific finger print for T. canis adults while that at 119.04 KDa was specific for T. leonina adult worm.

  14. Protein identification and quantification from riverbank grape, Vitis riparia: Comparing SDS-PAGE and FASP-GPF techniques for shotgun proteomic analysis.

    PubMed

    George, Iniga S; Fennell, Anne Y; Haynes, Paul A

    2015-09-01

    Protein sample preparation optimisation is critical for establishing reproducible high throughput proteomic analysis. In this study, two different fractionation sample preparation techniques (in-gel digestion and in-solution digestion) for shotgun proteomics were used to quantitatively compare proteins identified in Vitis riparia leaf samples. The total number of proteins and peptides identified were compared between filter aided sample preparation (FASP) coupled with gas phase fractionation (GPF) and SDS-PAGE methods. There was a 24% increase in the total number of reproducibly identified proteins when FASP-GPF was used. FASP-GPF is more reproducible, less expensive and a better method than SDS-PAGE for shotgun proteomics of grapevine samples as it significantly increases protein identification across biological replicates. Total peptide and protein information from the two fractionation techniques is available in PRIDE with the identifier PXD001399 (http://proteomecentral.proteomexchange.org/dataset/PXD001399). © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. Genetic variation of jointed goatgrass (Aegilops cylindrica Host.) from Iran using RAPD-PCR and SDS-PAGE of seed proteins.

    PubMed

    Farkhari, M; Naghavi, M R; Pyghambari, S A; Sabokdast

    2007-09-01

    Genetic variation of 28 populations of jointed goatgrass (Aegilops cylindrica Host.), collected from different parts of Iran, were evaluated using both RAPD-PCR and SDS-PAGE of seed proteins. The diversity within and between populations for the three-band High Molecular Weight (HMW) subunits of glutenin pattern were extremely low. Out of 15 screened primers of RAPD, 14 primers generated 133 reproducible fragments which among them 92 fragments were polymorphic (69%). Genetic similarity calculated from the RAPD data ranged from 0.64 to 0.98. A dendrogram was prepared on the basis of a similarity matrix using the UPGMA algorithm and separated the 28 populations into two groups. Confusion can happen between populations with the same origin as well as between populations of very diverse geographical origins. Our results show that compare to seed storage protein, RAPD is suitable for genetic diversity assessment in Ae. cylindrica populations.

  16. Comparison of low molecular weight glutenin subunits identified by SDS-PAGE, 2-DE, MALDI-TOF-MS and PCR in common wheat

    PubMed Central

    2010-01-01

    Background Low-molecular-weight glutenin subunits (LMW-GS) play a crucial role in determining end-use quality of common wheat by influencing the viscoelastic properties of dough. Four different methods - sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), two-dimensional gel electrophoresis (2-DE, IEF × SDS-PAGE), matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and polymerase chain reaction (PCR), were used to characterize the LMW-GS composition in 103 cultivars from 12 countries. Results At the Glu-A3 locus, all seven alleles could be reliably identified by 2-DE and PCR. However, the alleles Glu-A3e and Glu-A3d could not be routinely distinguished from Glu-A3f and Glu-A3g, respectively, based on SDS-PAGE, and the allele Glu-A3a could not be differentiated from Glu-A3c by MALDI-TOF-MS. At the Glu-B3 locus, alleles Glu-B3a, Glu-B3b, Glu-B3c, Glu-B3g, Glu-B3h and Glu-B3j could be clearly identified by all four methods, whereas Glu-B3ab, Glu-B3ac, Glu-B3ad could only be identified by the 2-DE method. At the Glu-D3 locus, allelic identification was problematic for the electrophoresis based methods and PCR. MALDI-TOF-MS has the potential to reliably identify the Glu-D3 alleles. Conclusions PCR is the simplest, most accurate, lowest cost, and therefore recommended method for identification of Glu-A3 and Glu-B3 alleles in breeding programs. A combination of methods was required to identify certain alleles, and would be especially useful when characterizing new alleles. A standard set of 30 cultivars for use in future studies was chosen to represent all LMW-GS allelic variants in the collection. Among them, Chinese Spring, Opata 85, Seri 82 and Pavon 76 were recommended as a core set for use in SDS-PAGE gels. Glu-D3c and Glu-D3e are the same allele. Two new alleles, namely, Glu-D3m in cultivar Darius, and Glu-D3n in Fengmai 27, were identified by 2-DE. Utilization of the suggested standard cultivar set, seed of

  17. Prestaining of glycoproteins in SDS-PAGE via 4H-[1]-Benzopyrano[4,3-b]thiophene-2-carboxylic acid hydrazide with weak influence on protein mobility.

    PubMed

    Zhu, Zhongxin; Zhou, Xuan; Wang, Yang; Yu, Qing; Zhu, Xinliang; Niu, Chao; Cong, Weitao; Jin, Litai

    2014-12-01

    A new fluorescent prestaining method for gel-separated glycoproteins in 1D and 2D SDS-PAGE was developed by using 4H-[1]-Benzopyrano[4,3-b]thiophene-2-carboxylic acid hydrazide (BH). The prestained gels were readily imaged after electrophoresis without any time-consuming steps needed for poststain. As low as 4-8 ng glycoproteins (transferrin, α1-acid glycoprotein) could be selectively detected, which is comparable to the most commonly used Pro-Q Emerald 488 glycoprotein stain. In addition, subsequent study of deglycosylation, glycoprotein affinity chromatography, and LC-MS/MS analysis were performed to confirm the specificity of the newly developed method. As a result, BH prestain provides a new choice for quick, sensitive, specific, economical, and MS compatible visualization of gel-separated glycoproteins.

  18. Iron stabilizes thylakoid protein-pigment complexes in Indian mustard during Cd-phytoremediation as revealed by BN-SDS-PAGE and ESI-MS/MS.

    PubMed

    Qureshi, M Irfan; D'Amici, Gian Maria; Fagioni, Marco; Rinalducci, Sara; Zolla, Lello

    2010-07-01

    Two-dimensional BN-SDS-PAGE, ESI-MS/MS and electron microscopy (EM) were used to study the role of iron (Fe) under cadmium (Cd) stress in retention of thylakoidal multiprotein complexes (MPCs) and chloroplast ultrastructure of Indian mustard, a moderate hyperaccumulator plant. Mustard was grown hydroponically with or without iron for 17 days and then exposed to CdCl2 for 3 days. Fe deficiency led to an increase in oxidative stress and damage to chloroplast/thylakoids accompanied by a decrease in chlorophyll content; exposure of plants to Cd further enhanced the oxidative stress and Cd accumulation (more in -Fe plants). However, the presence of iron aided plants in the suppression of oxidative stress and retention of chloroplasts and chlorophylls under Cd stress. Proteomic analyses by 2D BN-SDS-PAGE and mass spectrometry showed that Fe deficiency considerably decreased the amount of LHCII trimer, ATPase-F1 portion, cyt b6/f and RuBisCO. No or less reduction, was observed for PSI(RCI+LHCI), the PSII-core monomer, and the PSII subcomplex, while an increase in the LHCII monomer was noted. Under iron deficiency, Cd proved to be very deleterious to MPCs, except for the PSII subcomplex, the LHCII monomer and free proteins which were increased. Iron proved to be very protective in retaining almost all the complexes. MPCs showed greater susceptibility to Cd than Fe deficiency, mainly at the level of RuBisCO and cyt b6/f; an increase in the amount of the PSII subcomplex, LHCII monomer and free proteins indicates differences in the mechanisms affected by Fe deficiency and Cd stress when compared to Fe-fed plants. This study furthers our understanding of the sites actually damaged in MPCs under Fe deficiency and Cd stress. A role emerges for iron in the protection of MPCs and, hence, of the chloroplast. The present study also indicates the importance of iron for efficient phytoextraction/phytoremediation.

  19. Evaluation of a computer-assisted method of analysing SDS-PAGE protein profiles in tracing a hospital outbreak of Serratia marcescens.

    PubMed

    Arzese, A; Botta, G A; Gesu, G P; Schito, G

    1988-07-01

    Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) profiles of bacterial proteins have been successfully used for taxonomical purposes. More recently this technique has been applied to epidemiological investigations in respect of various micro-organisms including Neisseria meningitidis, Staphylococcus aureus and Clostridium difficile. The main limitations of the methods so far described are lack of standardisation in extraction and separation as well as in the analysis of results. Although reproducibility in the same laboratory has been shown to be satisfactory, comparison of results among laboratories is still difficult. Moreover, assessment of differences and/or similarities among chromatograms or autoradiographs showing many bands depends upon qualitative descriptions. Interpretation of densitometric scannings is laborious and time-consuming. In this paper we present our experience of a completely standardised, fully computer-controlled procedure for SDS-PAGE (AMBIS System) in analysing 35S-methionine-labelled total proteins. The methodology proved very useful in monitoring a hospital outbreak of Serratia marcescens. It allowed us to make quantitative comparison in a shorter time as well as to handle easily a great amount of data and usefully integrate it with those obtained with other systems such as serotyping. Furthermore, when the two systems are used together, more precise information can be gained. In this epidemic, serotyping indicated the presence of two groups which would have been missed by PAGE analysis alone. Electrophoretotyping, however, focused on similarities of cellular proteins among the epidemic strains. This allowed us to distinguish them from epidemiologically unrelated strains of the same serogroup.(ABSTRACT TRUNCATED AT 250 WORDS)

  20. Defining the mitochondrial proteomes from five rat organs in a physiologically significant context using 2D blue-native/SDS-PAGE.

    PubMed

    Reifschneider, Nicole H; Goto, Sataro; Nakamoto, Hideko; Takahashi, Ryoya; Sugawa, Michiru; Dencher, Norbert A; Krause, Frank

    2006-05-01

    In accordance with their manifold tasks, various dysfunctions of mitochondria are critically involved in a large number of diseases and the aging process. This has inspired considerable efforts to identify all the mitochondrial proteins by denaturing approaches, notably, the standard gel-based method employing isoelectric focusing. Because a significant part of the mitochondrial proteome is membrane-associated and/or functions as homo- or heterooligomeric protein complexes, there is an urgent need to detect and identify mitochondrial proteins, both membranous and soluble ones, under conditions preserving protein-protein interactions. Here, we investigated mitochondria of five different rat organs (kidney, liver, heart, skeletal muscle, and brain) solubilized with digitonin, enabling the quantitative extraction of the five oxidative phosphorylation (OXPHOS) complexes. The analysis by blue-native (BN)-PAGE recovered the OXPHOS complexes to a large extent as supercomplexes and separated many other protein complexes and individual proteins which were resolved by subsequent 2D SDS-PAGE revealing the tissue-diverse mitochondrial proteomes. Using MS peptide mass fingerprinting, we identified in all five organs 92 nonredundant soluble and membrane-embedded non-OXPHOS proteins, among them, many as constituents of known mitochondrial protein complexes as well as novel ones such as the putative "stomatin-like protein 2 complex" with an apparent mass of ca. 1800 kDa. Interestingly, the identification list included 36 proteins known or presumed to be localized to nonmitochondrial compartments, for example, glycolytic enzymes, clathrin heavy chain, valosin-containing protein/p97, VoV1-ATPase, and Na,K-ATPase. We expect that more than 200 distinct non-OXPHOS proteins of digitonin-solubilized rat mitochondria separated by 2D BN/SDS-PAGE, representing a partial "protein interactome" map, can be identified.

  1. Protein quantification and its tolerance for different interfering reagents using the BCA-method with regard to 2D SDS PAGE.

    PubMed

    Krieg, Rene C; Dong, Yan; Schwamborn, Kristina; Knuechel, Ruth

    2005-10-31

    Measuring the protein content of a sample is a mandatory and frequently practiced procedure in the lab. Although the procedure is quite simple and convenient to perform with commercially available kits, incompatible reagents in the lysate can cause problems in the quality of measurement. Unfortunately these reagents are cornerstones of high efficiency lysing buffers, e.g. high amounts of urea or beta-mercaptoethanol. In this study we addressed the tolerance of the well-known BCA-assay (bicinchoninic acid) to various reagents in different concentrations, with special regard to a subsequent 2D-gelelectrophoresis. As a result, the kit is incompatible with the recipes of regular 2D-buffers. Also, when mixing two different reagents interfering effects will occur in a non-predictable way. Therefore we established a new method to quantify protein content in lysates ready for 2D-gelelectrophoresis: by mixing an aliquot with SDS, an equilibration is performed to that the sample can be run on a regular 1D SDS PAGE. Image analysis following fluorescence staining (SYPRO Ruby) reveals the absolute protein content in comparison to a BSA dilution curve processed accordingly.

  2. Comparison of lipopolysaccharide and protein immunogens from pathogenic Yersinia enterocolitica bio-serotype 1B/O:8 and 2/O:9 using SDS-PAGE.

    PubMed

    Gu, Wen Peng; Wang, Xin; Qiu, Hai Yan; Luo, Xia; Xiao, Yu Chun; Tang, Liu Ying; Kan, Biao; Xu, Jian Guo; Jing, Huai Qi

    2012-06-01

    Yersinia enterocolitica is an extracellular pathogen and its related antigens interact with the host immune system. We investigated the difference in immunological characteristics between a highly pathogenic and poorly pathogenic strain of Y. enterocolitica. We used SDS-PAGE and western blotting to characterize lipopolysaccharide (LPS), Yersinia outer membrane proteins (Yops), membrane proteins, and whole-cell proteins from poorly pathogenic Y. enterocolitica bio-serotype 2/O:9, isolated from China, and highly pathogenic bio-serotype 1B/O:8, isolated from Japan. These two strains of Y. enterocolitica had different LPS immune response patterns. Comparison of their Yops also showed differences that could have accounted for their differences in pathogenicity. The membrane and whole-cell proteins of both strains were similar; immunoblottting showed that the 35 kD and perhaps the 10 kD proteins were immunogens in both strains. The major antigens of the two strains eliciting the host immune response were the LPS and membrane proteins, as shown by comparing protein samples with reference and purified preparations. Copyright © 2012 The Editorial Board of Biomedical and Environmental Sciences. Published by Elsevier B.V. All rights reserved.

  3. Analysis by two-dimensional Blue Native/SDS-PAGE of membrane protein alterations in rat soleus muscle after hindlimb unloading.

    PubMed

    Basco, Davide; Nicchia, Grazia Paola; Desaphy, Jean-François; Camerino, Diana Conte; Frigeri, Antonio; Svelto, Maria

    2010-12-01

    Muscle atrophy occurring in several pathophysiological conditions determines decreases in muscle protein synthesis, increases in the rate of proteolysis and changes in muscle fiber composition. To determine the effect of muscle atrophy induced by hindlimb unloading (HU) on membrane proteins from rat soleus, a proteomic approach based on two-dimensional Blue Native/SDS-PAGE was performed. Proteomic analysis of normal and HU soleus muscle demonstrates statistically significant changes in the relative level of 36 proteins. Among the proteins identified by mass spectrometry, most are involved in pathways associated with muscle fuel utilization, indicating a shift in metabolism from oxidative to glycolytic. Moreover, immunoblotting analysis revealed an increase in aquaporin-4 (AQP4) water channel and an alteration of proteins belonging to the dystrophin-glycoprotein complex (DGC). AQP4 and DGC are regulated in soleus muscle subjected to simulated microgravity in response to compensatory mechanisms induced by muscle atrophy, and they parallel the slow-to-fast twitch conversion that occurs in soleus fibers during HU. In conclusion, the alterations of soleus muscle membrane proteome may play a pivotal role in the mechanisms involved in disuse-induced muscle atrophy.

  4. Quantitative assessment of multi-laboratory reproducibility of SDS-PAGE assays: Digestion pattern of beta-casein and beta-lactoglobulin under simulated conditions.

    PubMed

    Defernez, Marianne; Mandalari, Giuseppina; Mills, Elizabeth Noami Clare

    2010-08-01

    A digestion protocol was applied in triplicate by ten laboratories, simulating in vivo gastric and duodenal conditions. The intra- and inter-laboratory variability in the kinetics of protein degradation was quantified, focussing on the digestion of beta-casein under gastric conditions, and of beta-lactoglobulin (beta-Lg) under duodenal conditions. The addition of surfactants such as phosphatidylcholine (PC) in the digestion mix was also evaluated. Identification and quantification of peptide bands on SDS-PAGE gels formed the basis for analysis. An average intensity loss of 69% (SD=13.5) at 5 min (89% at 10 min, with SD=5.5) was observed for beta-casein, whereas the beta-Lg duodenal digestion showed an 82% loss at 30 min (SD=14.2). Constant rates of first-order reactions showed that for fast reactions, inaccuracies in the time of first sampling contributed to the variability, which were also affected by image quality, saturation, and the splitting of time courses across gels. Breakdown products for beta-casein included ten other polypeptides, with four detected in all and two in most gels, and for beta-Lg ten polypeptides, with five detected in most, and two in two-third of the cases. Addition of PC in the gastric phase led to beta-Lg intensity loss only a quarter as large as without PC and altered beta-Lg proteolysis in the duodenal compartment.

  5. Phos-tag SDS-PAGE systems for phosphorylation profiling of proteins with a wide range of molecular masses under neutral pH conditions.

    PubMed

    Kinoshita, Eiji; Kinoshita-Kikuta, Emiko; Koike, Tohru

    2012-01-01

    We have previously reported a neutral-pH gel system buffered with Bis-Tris hydrochloride (Bis-Tris-HCl) in Zn(2+)-Phos-tag SDS-PAGE for advanced profiling of phosphoproteins with molecular masses of 10-200 kDa. In the current work, we describe characteristics of two neutral-pH gel systems, Bis-Tris-HCl and Tris-acetic acid (Tris-AcOH), based on comparative studies of the separation of a wide range of proteins with molecular masses from 10 to 350 kDa. For 10-200 kDa cellular proteins, the Bis-Tris-HCl system showed a higher resolving power in a 2-D fluorescence DIGE analysis of certain phosphoproteins, e.g. histone H3 (15 kDa) and elongation factor 2 (95 kDa). Furthermore, there was a large difference in the 1-D migration patterns of phosphorylated species of extracellular signal-regulated kinases 1 and 2 (ERK1/2, 44/42 kDa), which arise from changes in the phosphorylation status of the Thr-202 and Tyr-204, in the two buffer systems at the same concentration of Zn(2+)-Phos-tag. In contrast, shifts in the mobility of various phosphorylated species of a high-molecular-mass protein, ataxia telangiectasia-mutated kinase (ATM, 350 kDa), could only be detected in the Tris-AcOH system with a 3% w/v polyacrylamide gel strengthened with 0.5% w/v agarose. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. Evaluation and standardization of different purification procedures for fish bile and liver metallothionein quantification by spectrophotometry and SDS-PAGE analyses.

    PubMed

    Tenório-Daussat, Carolina Lyrio; Resende, Marcia Carolina Martinho; Ziolli, Roberta L; Hauser-Davis, Rachel Ann; Schaumloffel, Dirk; Saint'Pierre, Tatiana D

    2014-03-01

    Fish bile metallothioneins (MT) have been recently reported as biomarkers for environmental metal contamination; however, no studies regarding standardizations for their purification are available. Therefore, different procedures (varying centrifugation times and heat-treatment temperatures) and reducing agents (DTT, β-mercaptoethanol and TCEP) were applied to purify MT isolated from fish (Oreochromis niloticus) bile and liver. Liver was also analyzed, since these two organs are intrinsically connected and show the same trend regarding MT expression. Spectrophotometrical analyses were used to quantify the resulting MT samples, and SDS-PAGE gels were used to qualitatively assess the different procedure results. Each procedure was then statistically evaluated and a multivariate statistical analysis was then applied. A response surface methodology was also applied for bile samples, in order to further evaluate the responses for this matrix. Heat treatment effectively removes most undesired proteins from the samples, however results indicate that temperatures above 70 °C are not efficient since they also remove MTs from both bile and liver samples. Our results also indicate that the centrifugation times described in the literature can be decreased in order to analyze more samples in the same timeframe, of importance in environmental monitoring contexts where samples are usually numerous. In an environmental context, biliary MT was lower than liver MT, as expected, since liver accumulates MT with slower detoxification rates than bile, which is released from the gallbladder during feeding, and then diluted by water. Therefore, bile MT seems to be more adequate in environmental monitoring scopes regarding recent exposure to xenobiotics that may affect the proteomic and metalloproteomic expression of this biological matrix.

  7. SDS-PAGE Analysis of the Outer Membrane Proteins of Uropathogenic Escherichia coli Isolated from Patients in Different Wards of Nemazee Hospital, Shiraz, Iran

    PubMed Central

    Dehghani, Behzad; Mottamedifar, Mohammad; Khoshkharam-Roodmajani, Hossein; Hassanzadeh, Amir; Zomorrodian, Kamyar; Rahimi, Amir

    2016-01-01

    Background: Outer membrane proteins (OMPs) constitute the main structure and about half of the cell wall of Gram-negative bacteria. The OMPs of Escherichia coli (E. coli) play an important role in its drug resistance. Previous studies have shown that the OMPs of E. coli enhance its pathogenic effects by helping the bacterium to evade the immune defense and promote its adsorption to host cells. We sought to compare E. coli isolates collected from different hospital wards and to perform a primary investigation of the association between the serotypes and profiles of their OMPs. We also aimed to detect the diversity of the E. coli isolates from the hospitalized patients. Methods: A total of 115 isolates of E. coli were collected from patients hospitalized in Nemazee Hospital, Shiraz, Iran. After biochemical and serological tests, OMPs were extracted by using glass beads and N-Lauroylsarcosine sodium. OMP typing was done by 10% SDS-PAGE and Coomassie brilliant blue staining. In terms of the number of protein bands, OMP-I was detected with 2 bands, OMP-α with 3 bands, and OMP-β with1 band. Results: Of the 115 isolates, 103 were OMP-I and 12 were OMP-α; none of the isolates belonged to OMP-β. Our statistical analyses showed a relationship between OMP patterns and other factors, including hospital wards and source of samples. Serotyping showed that the majority of the isolates were O128. Conclusion: Our results demonstrated some similarities between the OMP band patterns of the analyzed groups of E. coli. Of all the OMPs in the isolates from the hospitalized and outpatient department patients, OmpA and OmpC were the most prevalent proteins in the outer membrane of the studied uropathogenic E. coli. PMID:27582589

  8. Analysis of drought-tolerant sugar beet (Beta vulgaris L.) mutants induced with gamma radiation using SDS-PAGE and ISSR markers.

    PubMed

    Sen, Ayse; Alikamanoglu, Sema

    2012-01-01

    Drought is one of the major environmental stresses which greatly affect the plant growth and productivity. In the present study, various doses (0-75Gy) of gamma rays were applied to investigate the effect of radiation on shoot tip explants. It was observed that the regeneration rates and plant fresh weights decreased significantly with an increase in radiation dose. The optimal irradiation doses for mutation induction were determined at 15 and 20Gy. Afterwards, the induction of somatic mutation in sugar beet (Beta vulgaris L.) was investigated by irradiation of shoot tips with 15 and 20Gy gamma rays. Irradiated shoot tips were sub-cultured and M(1)V(1)-M(1)V(3) generations were obtained. Mutants tolerant to drought stress were selected on MS medium, supplemented with 10 and 20gl(-1) PEG6000. Of the M(1)V(3) plantlets, drought-tolerant mutants were selected. Leaf soluble proteins obtained from the control and drought-tolerant mutants were analyzed by SDS-PAGE. A total of 22 protein bands were determined and 2 of them were observed to be drought-tolerant mutants except the control. Polymorphism was also detected among the control and drought-tolerant mutants by DNA fingerprinting using ISSR markers. A total of 106 PCR fragments were amplified with 19 ISSR primers and 91 of them were polymorphic. The dendrograms were separated into two main clusters. First cluster included M8 mutant plant, which was applied 20Gy gamma radiation and regenerated on selective culture media containing 10gl(-1) PEG6000 concentration, and the second cluster was further divided into five sub-clusters.

  9. Impact of various salts on biology, physiology and tissue soluble proteins (SDS-PAGE) of Lymnaea natalensis and their infection with Fasciola gigantica miracidia.

    PubMed

    Mahmoud, Moamena Bayoumi

    2006-08-01

    The effect of sublethal concentrations of magnesium sulphate (MgSo4), sodium chloride (NaCl), potassium chloride (KCl), calcium sulphate (CaSo4) and sodium carbonate (Na2Co3) on survival, fecundity, Lymnaea natalensis infection with Fasciola gigantica miracidia and the activities of certain enzymes in tissue homogenate of these snails were evaluated. Tissue soluble proteins of the snails were analyzed by SDS-PAGE. The results indicated that Na2Co3 was more toxic to L. natalensis than the other salts. Egg laying capacity was declined after two weeks of continuous exposure to Na2Co3 (0.38 eggs/snail/week) compared to (13.85 eggs/snail/week) for controls. The lowest infection rate was among snails exposed to MgSo4 (31.25%) with a significant variation from controls (64.28%). The salts supperssed cercarial production of infected snails (14.6 metacercariae/ snail/stimulant) at MgSo4 compared to (59.3 metacercariae/ snail/stimulant) for controls. The maintenance of L. natalensis in the salts solutions for four successive weeks reduced the total protein content in their tissues (13.2 mg/g) at Na2Co3 (100 ppm) compared to (21.47 mg/g) for controls. The activity of alkaline phosphatase was significantly reduced in treated snails than controls. The electrophoretic data revealed a common band between controls and treated snails with a molecular weight of 79.42 to 80.55 kDa. There were bands in treated snails but not in controls (41.2 & 45 kDa). A band was detected for snails treated with each salt but not with controls or the other treated groups (28.68 kDa at NaCl, 100.75 kDa at CaSo4 & 205 Kda at KCl).

  10. Comparison the effect of three commercial enzymes for enzymatic hydrolysis of two substrates (rice bran protein concentrate and soy-been protein) with SDS-PAGE.

    PubMed

    Ahmadifard, Nasrollah; Murueta, Julio Humberto Cordova; Abedian-Kenari, Abdolmohammad; Motamedzadegan, Ali; Jamali, Hadi

    2016-02-01

    In this research enzymatic hydrolysis of rice bran protein concentrate (RBPC) and soybean Protein (SBP) as control were studied with 3 commercial enzymes (Alcalase®, Papain and acommercial 3-enzyme cocktail containing of 1.6 mg ml(-1) Trypsin, 3.1 mg ml(-1) Chymotrypsin, 1.3 mg ml(-1)Aminopeptidase (SIGMA P7500) and 7.95 mg ml(-1)pronase type XIV (SIGMA P5147) by the pH stat method. The hydrolysis was carried out at temperature of 28 C, 60 min and pH 8.00. Results were showed that RBPC, and SBP had higher Degree hydrolysis (DH %) with Alcalase® enzyme. Alcalase®had stronger capability for hydrolysis compared to the other tested enzymes. After 60 minute of hydrolysis time, the DH% of Alcalase® for RBPC and SBP was 12.69 and 12.50 %, respectively. In contrast, papain enzyme was showed lowest DH% in three substrates that 1.56 and 1.24 % were for SBP and RBPC, respectively.The hydrolysis of the protein fraction performed the three enzymes on the two substrates was followed in SDS-PAGE. RBPC and SBP showed almost complete digestion with Alcalase® enzyme after 60 minutes. 3-enzyme cocktail enzyme hydrolyzed better the RBPC than the SBP. Papain enzyme had less effect on the two substrates than other 2 enzymes. It was found that Alcalase® has highest capability for hydrolysis compared to other enzyme preparations. The high value protein hydrolysates prepared by Alcalase® can be used as value added ingredients in many food formulations. They are also suitable for a broad range of industrial food applications and also for cosmetic and personal care products.

  11. Proteomic analysis of human bladder epithelial cells by 2D blue native SDS-PAGE reveals TCDD-induced alterations of calcium and iron homeostasis possibly mediated by nitric oxide.

    PubMed

    Verma, Nisha; Pink, Mario; Petrat, Frank; Rettenmeier, Albert W; Schmitz-Spanke, Simone

    2015-01-02

    A proteomic analysis of the interaction among multiprotein complexes involved in 2,3,7,8-dibenzo-p-dioxin (TCDD)-mediated toxicity in urinary bladder epithelial RT4 cells was performed using two-dimensional blue native SDS-PAGE (2D BN/SDS-PAGE). To enrich the protein complexes, unexposed and TCDD-exposed cells were fractionated. BN/SDS-PAGE of the resulting fractions led to an effective separation of proteins and protein complexes of various origins, including cell membrane, mitochondria, and other intracellular compartments. Major differences between the proteome of control and exposed cells involved the alteration of many calcium-regulated proteins (calmodulin, protein S100-A2, annexin A5, annexin A10, gelsolin isoform b) and iron-regulated proteins (ferritin, heme-binding protein 2, transferrin). On the basis of these findings, the intracellular calcium concentration was determined, revealing a significant increase after 24 h of exposure to TCDD. Moreover, the concentration of the labile iron pool (LIP) was also significantly elevated in TCDD-exposed cells. This increase was strongly inhibited by the calmodulin (CaM) antagonist W-7, which pointed toward a possible interaction between iron and calcium signaling. Because nitric oxide (NO) production was significantly enhanced in TCDD-exposed cells and was also inhibited by W-7, we hypothesize that alterations in calcium and iron homeostasis upon exposure to TCDD may be linked through NO generated by CaM-activated nitric oxide synthase. In our model, we propose that NO produced upon TCDD exposure interacts with the iron centers of iron-regulatory proteins (IRPs) that modulate the alteration of ferritin and transferrin, resulting in an augmented cellular LIP and, hence, increased toxicity.

  12. Light, scanning electron microscopy and SDS-PAGE studies on the effect of the essential oil, Citrus sinensis var. balady on the embryonic development of camel tick Hyalomma dromedarii (Koch, 1818) (Acari: Ixodidae).

    PubMed

    Salwa, M Habeeb; Abdel-Shafy, Sobhy; Youssef, Abd El-Ghany A

    2007-04-15

    GC-MSE analysis of the essential oil of fresh fruit peel of Citrus sinensis var. balady recognized two main natural toxic compounds, limonene (83.28%) as hydrocarbon compound and linalool (3.97%) as oxygenated compound. Therefore, the objective of this study was to evaluate its effect on different egg-ages of Hyalomma dromedarii at four concentrations of 1:40, 1:30, 1:20 and 1:15 (oil : ethanol 95%) (v/v). The LC50 values were 1:56, 1:34, 1:41, 1:32, 1: 23, 1:23, 1:18, 1:14 and 1:11 for egg-ages of 2, 4, 6, 9, 11, 13, 16, 18 and 20 day, respectively. Histological Examination (HE), Scanning Electron Microscopy (SEM) and Sodium dodecyle sulphate polyacrylamide gel electrophoresis (SDS-PAGE) were done on the 9th day old-eggs treated with the essential oil 1:32 (the LC50 value of 9 day old-egg). HE was done on the 11, 12, 13, 14 and 15th day old eggs; SEM was done on the 11, 15 and 17th day old eggs and SDS-PAGE was done on the 10, 11, 12, 13, 14, 15 and 17th day old eggs and compared each with those of control. In control, HE showed that nuclei migrated to the periphery and became part of the cytoplasmic membrane, blastula appears as a complete ring cells. Germ layer form and the later differentiate to different organelles such as opithosoma, ambulatory segment and chelicera...etc. while incase of treated eggs, HE showed that irregular manner of ectoplasmic membrane formed, blastula gathered on one or two sides, the cells of germ layer gather on one side as small or large mass or ring shape. Cells gathered as small masses or finger shape without forming any organelles. SEM revealed that heavy small bulging wrinkles were observed on egg shells of control. These wrinkles changed into large size in treated eggs on the 11th day and disappeared at the following days to become smooth surfaced. SDS-PAGE exhibited 15, 14, 14, 12, 17, 14 and 15 bands for treated eggs on the 10, 11, 12, 13, 14, 15 and 17th day old-eggs, respectively and 14, 15, 16, 19, 17, 19 and 18 bands for

  13. Analysis of membrane-protein complexes of the marine sulfate reducer Desulfobacula toluolica Tol2 by 1D blue native-PAGE complexome profiling and 2D blue native-/SDS-PAGE.

    PubMed

    Wöhlbrand, Lars; Ruppersberg, Hanna S; Feenders, Christoph; Blasius, Bernd; Braun, Hans-Peter; Rabus, Ralf

    2016-03-01

    Sulfate-reducing bacteria (SRB) obtain energy from cytoplasmic reduction of sulfate to sulfide involving APS-reductase (AprAB) and dissimilatory sulfite reductase (DsrAB). These enzymes are predicted to obtain electrons from membrane redox complexes, i.e. the quinone-interacting membrane-bound oxidoreductase (QmoABC) and DsrMKJOP complexes. In addition to these conserved complexes, the genomes of SRB encode a large number of other (predicted) membrane redox complexes, the function and actual formation of which is unknown. This study reports the establishment of 1D Blue Native-PAGE complexome profiling and 2D BN-/SDS-PAGE for analysis of the membrane protein complexome of the marine sulfate reducer Desulfobacula toluolica Tol2. Analysis of normalized score profiles of >800 proteins in combination with hierarchical clustering and identification of 2D BN-/SDS-PAGE separated spots demonstrated separation of membrane complexes in their native form, e.g. ATP synthase. In addition to the QmoABC and DsrMKJOP complexes, other complexes were detected that constitute the basic membrane complexome of D. toluolica Tol2, e.g. transport proteins (e.g. sodium/sulfate symporters) or redox complexes involved in Na(+) -based bioenergetics (RnfABCDEG). Notably, size estimation indicates dimer and quadruple formation of the DsrMKJOP complex in vivo. Furthermore, cluster analysis suggests interaction of this complex with a rhodanese-like protein (Tol2_C05230) possibly representing a periplasmic electron transfer partner for DsrMKJOP. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  14. Anti-cancer effect of Betulin on a human lung cancer cell line: a pharmacoproteomic approach using 2 D SDS PAGE coupled with nano-HPLC tandem Mass Spectrometry.

    PubMed

    Pyo, Jae Sung; Roh, Si Hun; Kim, Dae Ki; Lee, Jin Gyun; Lee, Yong Yook; Hong, Soon Sun; Kwon, Sung Won; Park, Jeong Hill

    2009-02-01

    Betulin is a representative compound of Betula platyphylla, a tree species belonging to the Betulaceae family. In this investigation, we revealed that betulin showed anticancer activity on human lung cancer A549 cells by inducing apoptosis and changes in protein expression profiles were observed. Upon flow cytometry analysis, the surface of betulin-treated cells was found to be annexin-V positive and propidium iodide (PI) negative, which indicated that the cells were apoptotic. In order to identify the molecular players involved in betulin-induced apoptosis, cellular proteins were applied to two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (2 D SDS PAGE) for differential proteomic analysis. As a result, four downregulated proteins and three upregulated proteins were identified by nano-HPLC MS/MS. The four downregulated proteins were poly(rC)-binding protein 1, isoform 1 of 3-hydroxyacyl-CoA dehydrogenase type 2, heat shock protein 90-alpha 2, and enoyl-CoA hydratase; the three upregulated proteins were aconitate hydratase, malate dehydrogenase, and splicing factor arginine/serine-rich 1. These differentially expressed proteins explained the cytotoxicity of betulin against human lung cancer A549 cells, and the proteomic approach was thus shown to be a potential tool for understanding the pharmacological activities of pharmacophores.

  15. HFIP Extraction Followed by 2D CTAB/SDS-PAGE Separation: A New Methodology for Protein Identification from Tissue Sections after MALDI Mass Spectrometry Profiling for Personalized Medicine Research

    PubMed Central

    Longuespée, Rémi; Tastet, Christophe; Desmons, Annie; Kerdraon, Olivier; Day, Robert

    2014-01-01

    Abstract Matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI) and profiling technology have become the easiest methods for quickly accessing the protein composition of a tissue area. Unfortunately, the demand for the identification of these proteins remains unmet. To overcome this bottleneck, we combined several strategies to identify the proteins detected via MALDI profiling including on-tissue protein extraction using hexafluoroIsopropanol (1,1,1,3,3,3-hexafluoro-2-propanol, HFIP) coupled with two-dimensional cetyl trimethylammonium bromide/sodium dodecyl sulfate–polyacrylamide gel electrophoresis (2D CTAB/SDS-PAGE) for separation followed by trypsin digestion and MALDI-MS analyses for identification. This strategy was compared with an on-tissue bottom-up strategy that we previously developed. The data reflect the complementarity of the approaches. An increase in the number of specific proteins identified has been established. This approach demonstrates the potential of adapted extraction procedures and the combination of parallel identification approaches for personalized medicine applications. The anatomical context provides important insight into identifying biomarkers and may be considered a first step for tissue-based biomarker research, as well as the extemporaneous examination of biopsies during surgery. PMID:24841221

  16. Molecular weight abnormalities of the CTCF transcription factor: CTCF migrates aberrantly in SDS-PAGE and the size of the expressed protein is affected by the UTRs and sequences within the coding region of the CTCF gene.

    PubMed

    Klenova, E M; Nicolas, R H; U, S; Carne, A F; Lee, R E; Lobanenkov, V V; Goodwin, G H

    1997-02-01

    CTCF belongs to the Zn finger transcription factors family and binds to the promoter region of c-myc. CTCF is highly conserved between species, ubiquitous and localised in nuclei. The endogenous CTCF migrates as a 130 kDa (CTCF-130) protein on SDS-PAGE, however, the open reading frame (ORF) of the CTCF cDNA encodes only a 82 kDa protein (CTCF-82). In the present study we investigate this phenomenon and show with mass-spectra analysis that this occurs due to aberrant mobility of the CTCF protein. Another paradox is that our original cDNA, composed of the ORF and 3'-untranslated region (3'-UTR), produces a protein with the apparent molecular weight of 70 kDa (CTCF-70). This paradox has been found to be an effect of the UTRs and sequences within the coding region of the CTCF gene resulting in C-terminal truncation of CTCF-130. The potential attenuator has been identified and point-mutated. This restored the electrophoretic mobility of the CTCF protein to 130 kDa. CTCF-70, the aberrantly migrating CTCF N-terminus per se, is also detected in some cell types and therefore may have some biological implications. In particular, CTCF-70 interferes with CTCF-130 normal function, enhancing transactivation induced by CTCF-130 in COS6 cells. The mechanism of CTCF-70 action and other possible functions of CTCF-70 are discussed.

  17. Molecular weight abnormalities of the CTCF transcription factor: CTCF migrates aberrantly in SDS-PAGE and the size of the expressed protein is affected by the UTRs and sequences within the coding region of the CTCF gene.

    PubMed Central

    Klenova, E M; Nicolas, R H; U, S; Carne, A F; Lee, R E; Lobanenkov, V V; Goodwin, G H

    1997-01-01

    CTCF belongs to the Zn finger transcription factors family and binds to the promoter region of c-myc. CTCF is highly conserved between species, ubiquitous and localised in nuclei. The endogenous CTCF migrates as a 130 kDa (CTCF-130) protein on SDS-PAGE, however, the open reading frame (ORF) of the CTCF cDNA encodes only a 82 kDa protein (CTCF-82). In the present study we investigate this phenomenon and show with mass-spectra analysis that this occurs due to aberrant mobility of the CTCF protein. Another paradox is that our original cDNA, composed of the ORF and 3'-untranslated region (3'-UTR), produces a protein with the apparent molecular weight of 70 kDa (CTCF-70). This paradox has been found to be an effect of the UTRs and sequences within the coding region of the CTCF gene resulting in C-terminal truncation of CTCF-130. The potential attenuator has been identified and point-mutated. This restored the electrophoretic mobility of the CTCF protein to 130 kDa. CTCF-70, the aberrantly migrating CTCF N-terminus per se, is also detected in some cell types and therefore may have some biological implications. In particular, CTCF-70 interferes with CTCF-130 normal function, enhancing transactivation induced by CTCF-130 in COS6 cells. The mechanism of CTCF-70 action and other possible functions of CTCF-70 are discussed. PMID:9016583

  18. Identification and In Silico Analysis of Major Redox Modulated Proteins from Brassica juncea Seedlings Using 2D Redox SDS PAGE (2-Dimensional Diagonal Redox Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis).

    PubMed

    Chaurasia, Satya Prakash; Deswal, Renu

    2017-02-01

    The thiol-disulphide exchange regulates the activity of proteins by redox modulation. Many studies to analyze reactive oxygen species (ROS), particularly, hydrogen peroxide (H2O2) induced changes in the gene expression have been reported, but efforts to detect H2O2 modified proteins are comparatively few. Two-dimensional diagonal redox sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) was used to detect polypeptides which undergo thiol-disulphide exchange in Brassica juncea seedlings following H2O2 (10 mM) treatment for 30 min. Eleven redox responsive polypeptides were identified which included cruciferin, NLI [Nuclear LIM (Lin11, Isl-1 & Mec-3 domains)] interacting protein phosphatase, RuBisCO (ribulose-1,5-bisphosphate carboxylase/oxygenase) large subunit, and myrosinase. Redox modulation of RuBisCO large subunit was further confirmed by western blotting. However, the small subunit of RuBisCO was not affected by these redox changes. All redox modulated targets except NLI interacting protein (although it contains two cysteines) showed oxidation sensitive cysteines by in silico analysis. Interestingly, interactome of cruciferin and myrosinase indicated that they may have additional function(s) beside their well-known roles in the seedling development and abiotic stress respectively. Cruciferin showed interactions with stress associated proteins like defensing-like protein 192 and 2-cys peroxiredoxin. Similarly, myrosinase showed interactions with nitrilase and cytochrome p450 which are involved in nitrogen metabolism and/or hormone biosynthesis. This simple procedure can be used to detect major stress mediated redox changes in other plants.

  19. Comparative SDS-page protein patterns of four ascaridid nematodes.

    PubMed

    Ashour, A A; Taha, H A; Mohammad A el-H

    1995-12-01

    In order to investigate the degree of homogeneity and heterogeneity of the ascaridid nematodes. Toxascaris leonina, Parascaris equorum, Toxocara canis and T. vitulorum, protein extracts from adult worms of the four nematodes were resolved into a number of bands. Comparative analysis of dominant bands showed that 13 bands were common among the four species, but certain unique bands were also found in each species including 4 in T. vitulorum, one in T. leonina, two in T. canis, while P. equorum shares both T. canis and T. leonina in most of their bands. Among the four ascaridid studied, T. vitulorum appears to be the most divergent species.

  20. An SDS-PAGE Examination of Protein Quaternary Structure and Disulfide Bonding for a Biochemistry Laboratory

    ERIC Educational Resources Information Center

    Powers, Jennifer L.; Andrews, Carla S.; St. Antoine, Caroline C.; Jain, Swapan S.; Bevilacqua, Vicky L. H.

    2005-01-01

    Electrophoresis is a valuable tool for biochemists, yet this technique is often not included in biochemistry laboratory curricula owing to time constraints or lack of equipment. Protein structure is also a topic of interest in many disciplines, yet most undergraduate lab experiments focus only on primary structure. In this experiment, students use…

  1. Teaching the Structure of Immunoglobulins by Molecular Visualization and SDS-PAGE Analysis

    ERIC Educational Resources Information Center

    Rižner, Tea Lanišnik

    2014-01-01

    This laboratory class combines molecular visualization and laboratory experimentation to teach the structure of the immunoglobulins (Ig). In the first part of the class, the three-dimensional structures of the human IgG and IgM molecules available through the RCSB PDB database are visualized using freely available software. In the second part, IgG…

  2. Teaching the Structure of Immunoglobulins by Molecular Visualization and SDS-PAGE Analysis

    ERIC Educational Resources Information Center

    Rižner, Tea Lanišnik

    2014-01-01

    This laboratory class combines molecular visualization and laboratory experimentation to teach the structure of the immunoglobulins (Ig). In the first part of the class, the three-dimensional structures of the human IgG and IgM molecules available through the RCSB PDB database are visualized using freely available software. In the second part, IgG…

  3. Accelerated Coomassie Blue staining and destaining of SDS-PAGE gels with application of heat.

    PubMed

    Kurien, Biji T; Scofield, R Hal

    2012-01-01

    Coomassie Brilliant Blue is commonly used for the detection of proteins in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, owing to its reliability and simplicity. Here, we report dramatically decreased protein staining and destaining time, as well as significantly increased detection sensitivity with the application of enhanced heat. The staining time was 5 min at 55, 62.5, or 70°C for a 1.5-mm gel, while it took 45, 45, and 20 min, respectively, for destaining. The staining time could be reduced to 1 min for a 0.8 mm gel stained at 65°C, to 2 min at 60°C and 5 min at 55°C. The destaining of proteins analyzed on a 0.8 mm gel could be accomplished in 8, 15, and 20 min at 65, 60, and 55°C, respectively. Application of heat, thus, enables proteins to be stained and destained rapidly, as well as enhancing detection sensitivity.

  4. Spicy SDS-PAGE gels: curcumin/turmeric as an environment-friendly protein stain.

    PubMed

    Kurien, Biji T; Dorri, Yaser; Scofield, R Hal

    2012-01-01

    Gel proteins are commonly stained with calorimetric/fluorescent dyes. Here, we demonstrate that heat-solubilized curcumin can serve as a nontoxic and environment-friendly fluorescent/colorimetric reversible protein stain. Curcumin, the yellow pigment found in the rhizomes of the perennial herb Curcuma longa (turmeric), is insoluble in aqueous solvents. However, heat (100°C) solubilization in water renders 1.5% of curcumin soluble. Curcumin solubilized by ethanol or alkali is ineffective in staining proteins. Heat solubilized curry spice turmeric stains proteins similarly. Staining is achieved in 30 min, with a sensitivity almost equaling that of Coomassie Brilliant Blue (CBB). Destaining is not required, and excess curcumin/turmeric can be discarded into the sink. Binding of proteins by silver inhibits curcumin binding, suggesting similarity of protein binding by silver and curcumin. It costs $1.5-2.0 to stain a mini-gel with curcumin, while turmeric costs less than 0.005 cent. CBB staining/destaining costs about 2 cents. However, CBB is toxic and its use necessitates specialized disposal efforts. Curcumin/turmeric, thus, can serve as an ideal nontoxic protein stain.

  5. An SDS-PAGE Examination of Protein Quaternary Structure and Disulfide Bonding for a Biochemistry Laboratory

    ERIC Educational Resources Information Center

    Powers, Jennifer L.; Andrews, Carla S.; St. Antoine, Caroline C.; Jain, Swapan S.; Bevilacqua, Vicky L. H.

    2005-01-01

    Electrophoresis is a valuable tool for biochemists, yet this technique is often not included in biochemistry laboratory curricula owing to time constraints or lack of equipment. Protein structure is also a topic of interest in many disciplines, yet most undergraduate lab experiments focus only on primary structure. In this experiment, students use…

  6. Fluorescent Labeling of Proteins and Its Application to SDS-PAGE and Western Blotting.

    PubMed

    Alba, F Javier; Bartolomé, Salvador; Bermúdez, Antonio; Daban, Joan-Ramon

    2015-01-01

    This chapter describes very simple fluorescent methods developed in our laboratory allowing the rapid monitoring of total protein patterns on both sodium dodecyl sulfate (SDS) polyacrylamide gels and western blots. The noncovalent dye Nile red (9-diethylamino-5H-benzo[α]phenoxazine-5-one) is used for the sensitive staining of proteins in SDS gels. This method is compatible with the electroblotting of protein bands and with the staining of the resulting blot with the covalent dye MDPF (2-methoxy-2,4-diphenyl-3(2H)-furanone). These staining procedures are applied sequentially; there is no need to run a duplicate unstained gel for protein blotting. Furthermore, since only the adduct formed by the reaction of MDPF with proteins is fluorescent, there is no need to destain the membrane after protein labeling. In addition, MDPF staining is compatible with further immunodetection of specific bands with polyclonal antibodies. Finally, using the adequate conditions described below, MDPF staining does not preclude the N-terminal sequence analysis of proteins in selected bands.

  7. In silico studies of outer membrane of Neisseria meningitidis por a: its expression and immunogenic properties.

    PubMed

    Behrouzi, Ava; Bouzari, Saeid; Siadat, Seyed Davar; Irani, Shiva

    2014-01-01

    Neisseria meningitidis is a major causative agent of bacterial septicemia and meningitis in humans. Currently, there are no vaccines to prevent disease caused by strains of N.meningitidis serogroup B. The Class 1 Outer Membrane Protein (OMP) has been named porA which is a cation selective transmembrane protein of 45 KDa that forms trimeric pore in the meningococcal outer membrane. PorA from serogroup B N. meningitidis was cloned into prokaryotic expression vector pBAD-gIIIA. Recombinant protein was expressed with arabinose and affinity purified by Ni-NTA agarose, SDS-PAGE and western blotting were performed for protein determination and verification. BALB/c mice were immunized subcutaneously with purified rPorA together with alum adjuvant. Serum antibody responses to serogroups B N.meningitidis were determined by ELISA. Serum IgG response significantly increased in the group immunized with rPorA together with alum adjuvant in comparison with control groups. These results suggest that rPorA can be a potential vaccine candidate for serogroup B N.meningitidis.

  8. In Silico Studies of Outer Membrane of Neisseria Meningitidis Por A: Its Expression and Immunogenic Properties

    PubMed Central

    Behrouzi, Ava; Bouzari, Saeid; Siadat, Seyed Davar; Irani, Shiva

    2014-01-01

    Neisseria meningitidis is a major causative agent of bacterial septicemia and meningitis in humans. Currently, there are no vaccines to prevent disease caused by strains of N.meningitidis serogroup B. The Class 1 Outer Membrane Protein (OMP) has been named porA which is a cation selective transmembrane protein of 45 KDa that forms trimeric pore in the meningococcal outer membrane. PorA from serogroup B N. meningitidis was cloned into prokaryotic expression vector pBAD-gIIIA. Recombinant protein was expressed with arabinose and affinity purified by Ni-NTA agarose, SDS-PAGE and western blotting were performed for protein determination and verification. BALB/c mice were immunized subcutaneously with purified rPorA together with alum adjuvant. Serum antibody responses to serogroups B N.meningitidis were determined by ELISA. Serum IgG response significantly increased in the group immunized with rPorA together with alum adjuvant in comparison with control groups. These results suggest that rPorA can be a potential vaccine candidate for serogroup B N.meningitidis. PMID:25317403

  9. Improved identification of hordeins by cysteine alkylation with 2-bromoethylamine, SDS-PAGE and subsequent in-gel tryptic digestion.

    PubMed

    Rehulková, Helena; Marchetti-Deschmann, Martina; Pittenauer, Ernst; Allmaier, Günter; Rehulka, Pavel

    2009-11-01

    Proteomic-based description of varieties of barley (Hordeum vulgare L.) is a very important task especially in the food and brewing industry. This study is focused on major storage proteins in barley--hordeins--as a group of proteins soluble in alcohol/water mixtures that shows up significant changes in proteomic profiles for different varieties of barley. Unusual amino acid composition of hordeins with low numbers of lysine and arginine in combination with high content of proline and glutamine complicates their identification in a common proteomic workflow, because tryptic digestion produces just a few peptides amenable for successful mass spectrometric analysis. To increase the number of cleavage sites, in this work, cysteines in hordeins were chemically modified with 2-bromoethylamine (BEA) for their conversion into aminoethylcysteines. These mimic lysine residues and are recognized by trypsin as potential cleavage sites (if not followed by a proline residue) on the C-terminal side of the modified cysteine. Small extent of side reactions (towards histidine, N-terminus of the peptide, methionine, and also here the newly discovered reaction towards aminoethylcysteine) during modification with BEA could be observed after a longer period of reaction but this did not hinder the analysis when optimal conditions were used. Application of trypsin for in-gel digestion of hordeins, previously modified chemically with BEA, provided a higher number of short peptides and their subsequent mass spectrometric analysis resulted in an improved identification of hordeins. This approach can also be used for the analysis of other similar protein groups (e.g. gliadins in wheat) or other cysteines containing proteins having a low number of lysine and arginine residues in their primary structure.

  10. A step toward simplified detection of serum albumin on SDS-PAGE using an environment-sensitive flavone sensor.

    PubMed

    Liu, Bin; Pang, Yi; Bouhenni, Rachida; Duah, Ernest; Paruchuri, Sailaja; McDonald, Lucas

    2015-07-14

    In this study, we report a series of novel flavone-based sensors that exhibit a superior fluorescence response when interacting with serum albumin in real serum samples and in acrylamide gels. The detection limit of probe 4 for serum albumin solution is 0.09 μg mL(-1), and the detectable volume for monkey serum reaches as low as 0.03 μL.

  11. A step toward simplified detection of serum albumin on SDS-PAGE using an environment-sensitive flavone sensor

    PubMed Central

    Liu, Bin; Bouhenni, Rachida; Duah, Ernest; Paruchuri, Sailaja; McDonald, Lucas

    2015-01-01

    In this study, we report a series of novel flavone-based sensors that exhibit superior fluorescence response when interacting with serum albumin in real serum samples and in acrylamide gel. The detection limit of probe 4 for serum albumin solution is 0.09 μg/mL, and the detectable volume for monkey serum reaches as low as 0.03 μL. PMID:26068596

  12. Improved method for reliable HMW-GS identification by RP-HPLC and SDS-PAGE in common wheat cultivars

    USDA-ARS?s Scientific Manuscript database

    The accurate identification of alleles for high-molecular weight glutenins (HMW-GS) is critical for wheat breeding programs targeting end-use quality. RP-HPLC methods were optimized for separation of HMW-GS, resulting in enhanced resolution of 1By and 1Dx subunits. Statistically significant differe...

  13. [Cloning and prokaryotic expression of the outer membrane protein gene PorB of Neisseria gonorrhoeae].

    PubMed

    Wang, Yan; Zhang, Lei; Zhang, Li; Wang, Han

    2011-07-01

    To construct a fused expression vector of the outer membrane protein gene PorB of Neisseria gonorrhoeae, express the fusion protein in the prokaryotic system, and obtain a gene recombination protein, for the purpose of preparing the ground for further research on the pathopoiesis and immune protective response of PorB. A pair of primers were designed according to the known sequence of the PorB gene, and the PorB gene was amplified by PCR from the genome of Neisseria gonorrhoeae 29403 and cloned into the prokaryotic expression plasmid pGEX-4T-1 to generate pGEX-4T-PorB recombinants. The recombinant plasmid pGEX4T-PorB was transferred into competent cells E. coli BL21. After confirmed by restriction endonuclease digestion, PCR and DNA sequencing analysis, the recombinant protein was induced to express by isopropyl-beta-D-thiogalactoside (IPTG), and examined by SDS-PAGE and Western blotting. Restriction endonuclease digestion, PCR amplification and DNA sequencing analysis showed that the PorB gene of 1 047 bp was amplified from Neisseria gonorrhoeae DNA, and the recombinant plasmid pGEX-4T-PorB was successfully constructed and highly expressed in E. coli. The prokaryotic expression vector of pGEX-4T-PorB was successfully constructed and efficiently expressed in the prokaryotic system, which has provided a basis for further study on the biological activity of the PorB protein, as well as animal immune experiment and detection of Neisseria gonorrhoeae, and its application as a mucosal immune vaccine.

  14. Isolation of Thylakoid Membrane Complexes from Rice by a New Double-Strips BN/SDS-PAGE and Bioinformatics Prediction of Stromal Ridge Subunits Interaction

    PubMed Central

    Yu, Jianlan; Guo, Lin; Ding, Yi

    2011-01-01

    Thylakoid membrane complexes of rice (Oryza sativa L.) play crucial roles in growth and crop production. Understanding of protein interactions within the complex would provide new insights into photosynthesis. Here, a new “Double-Strips BN/SDS-PAGE” method was employed to separate thylakoid membrane complexes in order to increase the protein abundance on 2D-gels and to facilitate the identification of hydrophobic transmembrane proteins. A total of 58 protein spots could be observed and subunit constitution of these complexes exhibited on 2D-gels. The generality of this new approach was confirmed using thylakoid membrane from spinach (Spinacia oleracea) and pumpkin (Cucurita spp). Furthermore, the proteins separated from rice thylakoid membrane were identified by the mass spectrometry (MS). The stromal ridge proteins PsaD and PsaE were identified both in the holo- and core- PSI complexes of rice. Using molecular dynamics simulation to explore the recognition mechanism of these subunits, we showed that salt bridge interactions between residues R19 of PsaC and E168 of PasD as well as R75 of PsaC and E91 of PsaD played important roles in the stability of the complex. This stromal ridge subunits interaction was also supported by the subsequent analysis of the binding free energy, the intramolecular distances and the intramolecular energy. PMID:21637806

  15. A novel report of metallothioneins in fish bile: SDS-PAGE analysis, spectrophotometry quantification and metal speciation characterization by liquid chromatography coupled to ICP-MS.

    PubMed

    Hauser-Davis, Rachel Ann; Gonçalves, Rodrigo Araújo; Ziolli, Roberta Lourenço; de Campos, Reinaldo Calixto

    2012-07-15

    Metallothioneins (MT) are a validated biomarker for metal exposure. In fish they are usually determined in the liver, while some studies have observed MT in kidney, muscle and gills. There are, however, no reports on MT in fish bile. This study aimed to verify if these proteins are present in fish bile, if they can be used as biomarkers for metal exposure and to characterize the metal speciation present in the different species. Three independent methods to verify MT presence in fish bile were conducted: 1D/2D gel electrophoresis, spectrophotometric analyses and SEC-HPLC-ICP-MS. Results show that all methods verified the presence of these proteins in fish bile, while SEC-HPLC-ICP-MS also characterized also characterized metals responsible for the contamination of the studied sites. Quantification was successfully conducted by two methods, densitometry (1D electrophoresis) and spectrophotometry, and the data indicates that bile MTs follow the same trend as in liver. Therefore, Biliary MT show the potential to be used the same way as MT from other matrices in metal exposure studies, with the advantage of being a simpler matrix and showing the possibility of not sacrificing the animals, as chronic duct cannulation is available. Copyright © 2012 Elsevier B.V. All rights reserved.

  16. Isolation and identification of excretory-secretory and somatic antigens from the Oestrus ovis larvae by SDS-PAGE and immunoblotting.

    PubMed

    Alborzi, Alireza; Jolodar, Abbas; Seyfi Abad Shapouri, Masoudreza; Bagherian Pour, Esmaeil

    2014-01-01

    Oestrus ovis is an economically important parasite of small ruminants and a zoonotic parasite with many reports of ophthalmomyiasis in human from Iran and other countries. The aim of the peresent study was the isolation and identification of excretory-secretory (ES) and somatic (S) antigens of O. ovis second and third stage larvae (L2, L3) collected from Arabi sheep breeds located in southwest of Iran. Positive sera were prepared by marking the sheep, taking blood sample and direct observation of the parasite in the head. Somatic antigens of the larvae (SL2, SL3) were prepared by sonication. Larval excretory-secretory antigens (ESL2, ESL3) were prepared by incubation the larvae in RPMI-1640 RPMI medium. Electrophoretic protein profiles of ESL2 two, ESL3 seven, SL2 eight, SL3 fifteen bands (from 79.0 to below 14.4 KDa) were shown. In immunoblotting with positive sera, four common bands in SL2 and SL3 at 58, 42.0, 29.0 and 28.0 kDa, one specific band in SL3 at 47.0 kDa and one band in ESL2, at 28.0 kDa, and three bands in ESL3 at 58.0, 42.0, 29.0 and 28.0 kDa were recognized. Among the profiles, the 28 kDa protein was the most common antigenic component. Nevertheless, the antigenic proteins 29, 58 kDa were a common protein in electrophoretic patterns of both S and ES proteins of L2 and L3 but, 42.0 kDa antigen the only one detected in immunoblot but not in S and ES protein profiles of the larvae. Therefore, the antigens 29.0, 42.0 and 58.0 kDa can be used for further studies of protective effects and serological diagnostic methods.

  17. Molecular characterization and bio-functional property determination using SDS-PAGE and RP-HPLC of protein fractions from two Nigella species.

    PubMed

    Alu'datt, Muhammad H; Rababah, Taha; Alhamad, Mohammad N; Alodat, Moh'd; Al-Mahasneh, Majdi A; Gammoh, Sana; Ereifej, Khalil; Almajwal, Ali; Kubow, Stan

    2017-09-01

    This study aimed to investigate the molecular and bio-functional properties of protein fractions from Nigella damascena and Nigella arvensis, including the albumin, globulin, glutein-1, glutein-2 and prolamin fractions. Protein subunits were not observed in globulin and prolamin fractions. No peaks appeared in RP-HPLC chromatograms of globulin for either species. Two predominant peaks were observed in the RP-HPLC profiles of all protein fractions. Proteins separated by RP-HPLC have potential inhibitory and antioxidant activities in all fractions. Optimum ACE-inhibitory and antioxidant activities of proteins separated by RP-HPLC were observed in glutein-2 and albumin, respectively, for both species. For pepsin and combined pepsin-trypsin hydrolyses, the highest degree of hydrolysis (DH) was obtained in glutein-2 fraction of Nigella arvensis. Highest ACE-inhibitory activity of hydrolyzed protein fractions was found at 4h via pepsin hydrolysis in globulin fraction of Nigella damascena. Highest antioxidant activities of hydrolyzed protein fractions were found in glutelin-2 for both species. Copyright © 2017 Elsevier Ltd. All rights reserved.

  18. Fluorescence polarization assay and SDS-PAGE confirms matrilysin degrades fibronectin and collagen IV whereas gelatinase A degrades collagen IV but not fibronectin.

    PubMed

    Kraft, P J; Haynes-Johnson, D E; Patel, L; Lenhart, J A; Zivin, R A; Palmer, S S

    2001-10-01

    Matrilysin and gelatinase A are hypothesized to have significant roles in uterine and ovarian function. However, proteolytic activity assays for these enzymes are limited. We describe the development of simple and rapid assays for the proteolysis of fluorescein-labeled full-length substrates, collagen IV (Col-IV) and fibronectin (FN), and demonstrate the selectivity of matrilysin (MMP-7) compared to gelatinase A (MMP-2) for fibronectin. Changes in fluorescence intensity (FIU) and fluorescence polarization (mP) resulting from the protease activity of matrilysin and gelatinase A were measured. These studies show that the fluorescently labeled substrates, Col-IV and FN, are as reliable and amenable to rapid in vitro assay as peptide substrates. In addition, they are easier to use than previously described, non-fluorescent methods. The results demonstrate that assays using full-length, biological matrix proteins are more sensitive indicators of MMP-specific substrate activity than peptide based assays.

  19. Differentiation and numerical analysis of oral yeasts based on SDS-Page profiles. Influence of the culture media on the whole-cell protein extracts.

    PubMed

    Höfling, J F; Rosa, E A; Pereira, C V; Boriollo, M F; Rodrigues, J A

    2001-08-01

    The application of gel electrophoresis and numerical analysis of yeast soluble proteins analysis to the investigation of 12 oral yeast strains belonging to five species is described. It involves one-dimensional electrophoresis of SDS-solubilized whole-cell proteins using different culture media for the cultivation of the cells, integration densitometries in the areas of the gels and percentages of the proteins extraction. These extracts were prepared from four isolates of Candida albicans, two of C. tropicalis, C. guilliermondii, C. parapsilosis and C. krusei. The extracts from whole-cells proteins using different culture media for the cultivation of the cells were fractionated by slab electrophoresis using a discontinuous buffer system. The corresponding patterns showed at least 36 polypeptides in the range of 14.4-200 kDa. Different isolates of each species were clearly different in each of the five species. The data obtained suggest that different nutritional compositions led to the expression of different proteins derived from alternatives metabolic pathways expressed by the electrophoretic profiles. The construction of a database of protein fingerprints and numerical analysis based on such data, may have some implications in the classification and identification of such species with epidemiological, ecological and taxonomic purposes. A well defined or synthetic culture media seems to be much properly.

  20. Sensitive fluorescent staining for proteomic analysis of proteins in 1-D and 2-D SDS-PAGE and its comparison with SYPRO Ruby by PMF.

    PubMed

    Cong, Wei-Tao; Hwang, Sun-Young; Jin, Li-Tai; Choi, Jung-Kap

    2008-11-01

    A novel fluorescence-based method for protein staining on SDS polyacrylamide gel is described. In this method, proteins are stained using counterion (palmatine and SDS) staining solution, which is inexpensive, easy to perform, and does not involve a destaining step. Fixing and staining of proteins using the counterion protocol take less than an hour. As little as 2 ng of protein can be detected. Another interesting feature of the staining protocol described here is the compatibility with MALDI-TOF MS which shows a similar number of identification score and sequence coverage compared with those of SYPRO Ruby.

  1. Analysis of the SDS-PAGE patterns of outer membrane proteins from Escherichia coli strains that have lost the ability to form K1 antigen and varied in the susceptibility to normal human serum.

    PubMed

    Cisowska, Agnieszka; Bugla-Płoskońska, Gabriela

    2014-01-01

    We used SDS-polyacrylamide gel electrophoresis to investigate the outer membrane proteins (OMPs) band composition of 19 Escherichia coli K1 strains that have spontaneously lost the ability to form K1 polysaccharide capsule (E. coli K1-) and demonstrated different degrees of susceptibility to the bactericidal action of normal human serum. Presented results showed that there were differences between E. coli K1- strains in OMPs expressing capacity. The analysis performed on OMPs has not revealed a direct association between the different OMPs band composition and the susceptibility of these strains to the serum.

  2. Portable exhausters POR-004 SKID B, POR-005 SKID C, POR-006 SKID D storage plan

    SciTech Connect

    Nelson, O.D.

    1997-09-04

    This document provides a storage plan for portable exhausters POR-004 SKID B, POR-005 SKID C, AND POR-006 SKID D. The exhausters will be stored until they are needed by the TWRS (Tank Waste Remediation Systems) Saltwell Pumping Program. The storage plan provides criteria for portable exhauster storage, periodic inspections during storage, and retrieval from storage.

  3. Poblaciones de los niveles atómicos en condiciones de no equilibrio

    NASA Astrophysics Data System (ADS)

    Cruzado, A.

    El objetivo de este trabajo es encontrar la distribución de los átomos en los diferentes niveles de energía. Con el propósito de encontrar resultados generales y de amplia aplicación, hemos planteado las ecuaciones de equilibrio estadístico como función del número atómico del elemento a considerar y de las condiciones físicas del medio (temperatura y densidad). Así también se ha intentado tener en cuenta todos los niveles atómicos considerando explícitamente aquellos con un número cuántico principal menor que un cierto valor n y calculando una expresión aproximada para estimar la influencia de los demás.

  4. [Profesionalismo en médicos de un hospital de segundo nivel].

    PubMed

    Prieto-Miranda, Sergio Emilio; Jiménez-Bernardino, Carlos Alberto; Monjaraz-Guzmán, Elizabeth Guadalupe; Esparza-Pérez, Ramiro Israel

    2017-01-01

    Introducción: se requieren años para adquirir las habilidades y destrezas necesarias para un buen desempeño médico y varios más para ser “buen profesionista”. Gran parte de este entrenamiento se fundamenta en esquemas de enseñanza antiguos, siendo que la práctica de la medicina ha cambiado notablemente. No se conocen estudios en nuestro medio que aborden el profesionalismo en médicos. El objetivo de este trabajo fue determinar el nivel de profesionalismo en médicos de un hospital de segundo nivel de atención. Métodos: estudio descriptivo-transversal. Se encuestaron médicos adscritos, residentes e internos en un hospital de segundo nivel. Se elaboró y aplicó un cuestionario sobre profesionalismo, con 18 ítems (3 preguntas demográficas y 15 tipo Likert), que incluyó 4 dimensiones: conocimiento especializado, autonomía en la toma de decisiones, autorregulación y servicio de compromiso social. Se utilizó estadística descriptiva e inferencial. Resultados: se incluyeron 228 médicos, el 57.5% fueron competentes en profesionalismo de manera global. El 93.3% en conocimiento especializado y 60.5% en compromiso social. Los médicos adscritos y residentes obtuvieron las calificaciones más altas, siendo significativas estas diferencias en conocimiento especializado y autonomía (p < 0.001). Los internos obtuvieron puntajes más bajos en todas las categorías. Conclusiones: poco más de la mitad de los médicos presentó un nivel de profesionalismo competente en algunas dimensiones. Se obtuvieron puntajes más altos a mayor grado académico.

  5. La preparacion en ciencia de los candidatos a maestros del nivel elemental primario segun la reforma de la educacion cientifica en Puerto Rico: Una propuesta de secuencia curricular

    NASA Astrophysics Data System (ADS)

    Rodriguez Plaza, Evelyn

    El proposito de esta investigacion fue identificar los componentes de la preparacion en ciencia que deben recibir los estudiantes del Bachillerato en Artes en Educacion Elemental, Nivel Primario, de acuerdo a los documentos que dirigen la reforma de la educacion cientifica en Puerto Rico. Tambien, se identificaron los componentes de los cursos que forman parte de la preparacion en ciencia de estos estudiantes. Se compararon los componentes de la preparacion en ciencia y los componentes de los cursos para determinar congruencias y discrepancias. Con los datos recopilados se identificaron los componentes de los cursos de una secuencia curricular para la preparacion en ciencia de los candidatos a maestros del nivel elemental primario. La secuencia curricular que se propone en esta investigacion incluye cursos de contenido cientifico y de metodologia en la ensenanza de la ciencia disenados para satisfacer las necesidades de los candidatos. Se recomienda que en los procesos para el diseno, la implantacion y la evaluacion de estos cursos participen profesores de ciencia, profesores de educacion y maestros del nivel elemental primario. Todos los cursos de la secuencia curricular deben tener un enfoque constructivista. Las experiencias educativas que se incluyan en los cursos deben aspirar a desarrollar en los candidatos los atributos de la cultura cientifica y actitudes positivas hacia la ciencia y hacia la ensenanza de esta disciplina. El modelaje por parte de los profesores que ensenen los cursos de la secuencia curricular es fundamental en el desarrollo profesional de los candidatos. Se recomienda que en los cursos de contenido cientifico se estudien los conceptos y los conocimientos cientificos que forman parte del curriculo de Kindergarten a tercer grado de forma integrada y con una profundidad universitaria. Estos cursos deben tener un enfoque interdisciplinario e incluir el estudio de la naturaleza de la ciencia y un componente de laboratorio para desarrollar los

  6. Comparacion de modelos de Educacion Sexual en el conocimiento y cambio de actitudes en practicas sexuales por alumnos de nivel superior en la region de Caguas, Puerto Rico

    NASA Astrophysics Data System (ADS)

    Juan, Vallejo Ramos L.

    In opposition to the Sexual Education Traditional Model (SETM) that is used in the state schools of Puerto Rico, the Health Beliefs Model (HBM) appears. It facilitates a curricular design that improves the ability of the students to respond to the group pressure by means of attitudes that stimulate sexual conducts of smaller risk of propagation of the Sexually Transmitted Diseases (STD). In addition, it provides activities to increase the self-esteem, the communication and the decision making. This investigation had the intention to compare the SETM and the HBM in the increase of knowledge and change of attitudes of high risk of propagation of the STD using a validated questionnaire (Agency of the United States for the International-USAID Development), named "Endesa 2007" and, adapted to Puerto Rico by the Dra.Marta Collazo to a sample of students between the 17 and 19 years of 2 state schools of San Lorenzo, as a pretest, and, selected by convenience. Then, a 10 hours training was administered to half of the students using the SETM to STD and condom use lessons. The other half of the students received additional lessons using the HBM. Finally, both groups took the questionnaire again as a posttest. The sample of students, in average, did not reach the knowledge and basic levels of attitudes towards the STD in the pretest. This reflected 2 possible implications on the SETM. In first place, that the way in which the STD is implemented as part of the Sexual Education curriculum is inefficient. Secondly, the possibility that the acquired information or attitudes does not have permanence. Culminated the questionnaire, the HBM increase the knowledge of the STD in 0.41 points (average) over the SETM. There was not a significant difference between both models, in attitudes, implying that both models are equally effective. The findings suggests that the HBM is more effective increasing the knowledge on the STD, but equally effective than the SETM in attitude change for the Puerto Rican youth.

  7. Comparacion de Modelos de Educacion Sexual en El Conocimiento y Cambio de Actitudes en Practicas Sexuales por Alumnos de Nivel Superior en La Region De Caguas, Puerto Rico

    ERIC Educational Resources Information Center

    Juan, Vallejo Ramos L.

    2012-01-01

    In opposition to the Sexual Education Traditional Model (SETM) that is used in the state schools of Puerto Rico, the Health Beliefs Model (HBM) appears. It facilitates a curricular design that improves the ability of the students to respond to the group pressure by means of attitudes that stimulate sexual conducts of smaller risk of propagation of…

  8. Comparacion de Modelos de Educacion Sexual en El Conocimiento y Cambio de Actitudes en Practicas Sexuales por Alumnos de Nivel Superior en La Region De Caguas, Puerto Rico

    ERIC Educational Resources Information Center

    Juan, Vallejo Ramos L.

    2012-01-01

    In opposition to the Sexual Education Traditional Model (SETM) that is used in the state schools of Puerto Rico, the Health Beliefs Model (HBM) appears. It facilitates a curricular design that improves the ability of the students to respond to the group pressure by means of attitudes that stimulate sexual conducts of smaller risk of propagation of…

  9. Evolution of NADPH-cytochrome P450 oxidoreductases (POR) in Apiales - POR 1 is missing.

    PubMed

    Andersen, Trine Bundgaard; Hansen, Niels Bjørn; Laursen, Tomas; Weitzel, Corinna; Simonsen, Henrik Toft

    2016-05-01

    The NADPH-dependent cytochrome P450 oxidoreductase (POR) is the obligate electron donor to eukaryotic microsomal cytochromes P450 enzymes. The number of PORs within plant species is limited to one to four isoforms, with the most common being two PORs per plant. These enzymes provide electrons to a huge number of different cytochromes P450s (from 50 to several hundred within one plant). Within the eudicotyledons, PORs can be divided into two major clades, POR 1 and POR 2. Based on our own sequencing analysis and publicly available data, we have identified 45 PORs from the angiosperm order Apiales. These were subjected to a phylogenetic analysis along with 237 other publicly available (NCBI and oneKP) POR sequences found within the clade Asterids. Here, we show that the order Apiales only harbor members of the POR 2 clade, which are further divided into two distinct subclades. This is in contrast to most other eudicotyledon orders that have both POR 1 and POR 2. This suggests that through gene duplications and one gene deletion, Apiales only contain members of the POR 2 clade. Three POR 2 isoforms from Thapsia garganica L., Apiaceae, were all full-length in an Illumina root transcriptome dataset (available from the SRA at NCBI). All three genes were shown to be functional upon reconstitution into nanodiscs, confirming that none of the isoforms are pseudogenes. Copyright © 2016 Elsevier Inc. All rights reserved.

  10. Portable exhauster POR-007/Skid E and POR-008/Skid F storage plan

    SciTech Connect

    Nelson, O.D.

    1998-07-25

    This document provides storage requirements for 1,000 CFM portable exhausters POR-O07/Skid E and POR-008/Skid F. These requirements are presented in three parts: preparation for storage, storage maintenance and testing, and retrieval from storage. The exhauster component identification numbers listed in this document contain the prefix POR-007 or POR-008 depending on which exhauster is being used.

  11. Consequences of POR mutations and polymorphisms

    PubMed Central

    Miller, Walter L.; Agrawal, Vishal; Sandee, Duanpen; Tee, Meng Kian; Huang, Ningwu; Choi, Ji Ha; Morrissey, Kari; Giacomini, Kathleen M.

    2015-01-01

    P450 oxidoreductase (POR) transports electrons from NADPH to all microsomal cytochrome P450 enzymes, including steroidogenic P450c17, P450c21 and P450aro. Severe POR mutations A287P (in Europeans) and R457H (in Japanese) cause the Antley-Bixler skeletal malformation syndrome (ABS) plus impaired steroidogenesis (causing genital anomalies), but the basis of ABS is unclear. We have characterized the activities of ~40 POR variants, showing that assays based on P450c17 activities, but not cytochrome c assays, correlate with the clinical phenotype. The human POR gene is highly polymorphic: the A503V sequence variant, which decreases P450c17 activities to ~60%, is found on ~28% of human alleles. A promoter polymorphism (~8% of Asians and ~13% of Caucasians) at −152 reduces transcriptional activity by half. Screening of 35 POR variants showed that most mutants lacking activity with P450c17 or cytochrome c also lacked activity to support CYP1A2 and CYP2C19 metabolism of EOMCC (a fluorogenic non-drug substrate), although there were some remarkable differences: Q153R causes ABS and has ~30% of wild-type activity with P450c17 but had 144% of WT activity with CYP1A2 and 284% with CYP2C19. The effects of POR variants on CYP3A4, which metabolizes nearly 50% of clinically used drugs, was examined with multiple, clinically-relevant drug substrates, showing that A287P and R457H dramatically reduce drug metabolism, and that A503V variably impairs drug metabolism. The degree of activity can vary with the drug substrate assayed, as the drugs can influence the conformation of the P450. POR is probably an important contributor to genetic variation in both steroidogenesis and drug metabolism. PMID:21070833

  12. Consequences of POR mutations and polymorphisms.

    PubMed

    Miller, Walter L; Agrawal, Vishal; Sandee, Duanpen; Tee, Meng Kian; Huang, Ningwu; Choi, Ji Ha; Morrissey, Kari; Giacomini, Kathleen M

    2011-04-10

    P450 oxidoreductase (POR) transports electrons from NADPH to all microsomal cytochrome P450 enzymes, including steroidogenic P450c17, P450c21 and P450aro. Severe POR mutations A287P (in Europeans) and R457H (in Japanese) cause the Antley-Bixler skeletal malformation syndrome (ABS) plus impaired steroidogenesis (causing genital anomalies), but the basis of ABS is unclear. We have characterized the activities of ∼40 POR variants, showing that assays based on P450c17 activities, but not cytochrome c assays, correlate with the clinical phenotype. The human POR gene is highly polymorphic: the A503V sequence variant, which decreases P450c17 activities to ∼60%, is found on ∼28% of human alleles. A promoter polymorphism (∼8% of Asians and ∼13% of Caucasians) at -152 reduces transcriptional activity by half. Screening of 35 POR variants showed that most mutants lacking activity with P450c17 or cytochrome c also lacked activity to support CYP1A2 and CYP2C19 metabolism of EOMCC (a fluorogenic non-drug substrate), although there were some remarkable differences: Q153R causes ABS and has ∼30% of wild-type activity with P450c17 but had 144% of WT activity with CYP1A2 and 284% with CYP2C19. The effects of POR variants on CYP3A4, which metabolizes nearly 50% of clinically used drugs, was examined with multiple, clinically relevant drug substrates, showing that A287P and R457H dramatically reduce drug metabolism, and that A503V variably impairs drug metabolism. The degree of activity can vary with the drug substrate assayed, as the drugs can influence the conformation of the P450. POR is probably an important contributor to genetic variation in both steroidogenesis and drug metabolism. Copyright © 2010. Published by Elsevier Ireland Ltd.

  13. Structural Insights into the PorK and PorN Components of the Porphyromonas gingivalis Type IX Secretion System

    PubMed Central

    Gorasia, Dhana G.; Veith, Paul D.; Hanssen, Eric G.; Glew, Michelle D.; Sato, Keiko; Yukitake, Hideharu; Nakayama, Koji; Reynolds, Eric C.

    2016-01-01

    The type IX secretion system (T9SS) has been recently discovered and is specific to Bacteroidetes species. Porphyromonas gingivalis, a keystone pathogen for periodontitis, utilizes the T9SS to transport many proteins including the gingipain virulence factors across the outer membrane and attach them to the cell surface via a sortase-like mechanism. At least 11 proteins have been identified as components of the T9SS including PorK, PorL, PorM, PorN and PorP, however the precise roles of most of these proteins have not been elucidated and the structural organization of these components is unknown. In this study, we purified PorK and PorN complexes from P. gingivalis and using electron microscopy we have shown that PorN and the PorK lipoprotein interact to form a 50 nm diameter ring-shaped structure containing approximately 32–36 subunits of each protein. The formation of these rings was dependent on both PorK and PorN, but was independent of PorL, PorM and PorP. PorL and PorM were found to form a separate stable complex. PorK and PorN were protected from proteinase K cleavage when present in undisrupted cells, but were rapidly degraded when the cells were lysed, which together with bioinformatic analyses suggests that these proteins are exposed in the periplasm and anchored to the outer membrane via the PorK lipid. Chemical cross-linking and mass spectrometry analyses confirmed the interaction between PorK and PorN and further revealed that they interact with the PG0189 outer membrane protein. Furthermore, we established that PorN was required for the stable expression of PorK, PorL and PorM. Collectively, these results suggest that the ring-shaped PorK/N complex may form part of the secretion channel of the T9SS. This is the first report showing the structural organization of any T9SS component. PMID:27509186

  14. Structural Insights into the PorK and PorN Components of the Porphyromonas gingivalis Type IX Secretion System.

    PubMed

    Gorasia, Dhana G; Veith, Paul D; Hanssen, Eric G; Glew, Michelle D; Sato, Keiko; Yukitake, Hideharu; Nakayama, Koji; Reynolds, Eric C

    2016-08-01

    The type IX secretion system (T9SS) has been recently discovered and is specific to Bacteroidetes species. Porphyromonas gingivalis, a keystone pathogen for periodontitis, utilizes the T9SS to transport many proteins including the gingipain virulence factors across the outer membrane and attach them to the cell surface via a sortase-like mechanism. At least 11 proteins have been identified as components of the T9SS including PorK, PorL, PorM, PorN and PorP, however the precise roles of most of these proteins have not been elucidated and the structural organization of these components is unknown. In this study, we purified PorK and PorN complexes from P. gingivalis and using electron microscopy we have shown that PorN and the PorK lipoprotein interact to form a 50 nm diameter ring-shaped structure containing approximately 32-36 subunits of each protein. The formation of these rings was dependent on both PorK and PorN, but was independent of PorL, PorM and PorP. PorL and PorM were found to form a separate stable complex. PorK and PorN were protected from proteinase K cleavage when present in undisrupted cells, but were rapidly degraded when the cells were lysed, which together with bioinformatic analyses suggests that these proteins are exposed in the periplasm and anchored to the outer membrane via the PorK lipid. Chemical cross-linking and mass spectrometry analyses confirmed the interaction between PorK and PorN and further revealed that they interact with the PG0189 outer membrane protein. Furthermore, we established that PorN was required for the stable expression of PorK, PorL and PorM. Collectively, these results suggest that the ring-shaped PorK/N complex may form part of the secretion channel of the T9SS. This is the first report showing the structural organization of any T9SS component.

  15. Aplicación de la metodología Molecular de Orbitales de Defecto Cuántico (MQDO) al cálculo de intensidades vibrónicas y vidas medias de niveles vibracionales

    NASA Astrophysics Data System (ADS)

    María Velasco Sanz, Ana

    Desde que se formuló, en 1996, la metodología Molecular de Orbitales de Defecto Cuántico (MQDO) [1], se han obtenido datos de calidad relativos a intensidades de bandas electrónicas que implican estados Rydberg para una gran variedad de sistemas moleculares [2]. Animados por los buenos resultados obtenidos, recientemente hemos abordado el estudio de transiciones vibrónicas, es decir aquellas que ocurren entre estados vibracionales que pertenecen a distintos estados Rydberg electrónicos. Como prototipo adecuado para nuestros propósitos hemos elegido la molécula de NO, importante en la química de la atmósfera, y para la cual existen en la bibliografía datos experimentales de calidad suficiente para contrastar la validez de nuestros resultados. En concreto, hemos calculado las fuerzas de oscilador y coeficientes de Einstein para transiciones electrónicas y vibrónicas de las principales bandas del NO, al igual que vidas medias radiativas de niveles vibracionales de dicha molécula. Las propiedades estudiadas son esenciales para la comprensión de los aspectos teóricos de los procesos físicos básicos relativos a la dispersión electrónica en moléculas heteronucleares con capas abiertas. Además, valores fiables de probabilidades de transición moleculares tienen importantes aplicaciones en Astrofísica, en la modelización de procesos fotodinámicos moleculares, etc., al igual que para evaluar más profundamente la validez de nuestra metodología teórica.

  16. Concepciones y concepciones alternativas de estudiantes universitarios/as de biologia y futuros maestros/as de Ciencia de escuela secundaria sobre la teoria de evolucion biologica por seleccion natural

    NASA Astrophysics Data System (ADS)

    Morales Ramos, Egda M.

    La teoria de evolucion biologica (TEB) por seleccion natural es uno de los conceptos unificadores mas importantes del curriculo de Biologia. En Puerto Rico se han hecho pocas investigaciones que abunden sobre las concepciones y concepciones alternativas (CA) que tienen los estudiantes universitarios/as de Biologia y los maestros/as de Ciencia del nivel secundario sobre esta teoria. La politica publica educativa actual establece mediante documentos normativos como los Estandares de contenido y Expectativas de grado del Programa de Ciencias [Puerto Rico Core Standards] la ensenanza de esta teoria. Sin embargo, no se encontraron preguntas sobre la seleccion natural en los ejercicios de practica provistos por el Departamento de Educacion para las pruebas estandarizadas lo cual puede influir para que no se ensene adecuadamente. Las preguntas de investigacion fueron 1. ¿Cuales son las concepciones y concepciones alternativas de estudiantes universitarios/as y de los futuros maestros y maestras de Ciencia sobre la TEB? 2. ¿Cuales conceptos que seleccionan los estudiantes universitarios/as y los futuros maestros y maestras de Ciencia sobre la TEB coinciden con lo aceptado como valido por la comunidad cientifica? y 3. ¿Como comparan las respuestas de la prueba original. v. Entendiendo el cambio biologico que mide concepciones y CA sobre la TEB por seleccion natural, con las de la traducida al idioma espanol? Se utilizo el metodo cuantitativo con un diseno de investigacion transversal por encuesta. La tecnica principal para recopilar los datos fue una prueba con doce items, que formo parte de un instrumento para el cual se recopilaron diversas fuentes de evidencia acerca de su validez. Las muestras estuvieron formadas por 69 estudiantes de Ciencias Naturales y por 16 estudiantes futuros maestros y maestras del nivel secundario de la UPR-RP. Se utilizaron estadisticas descriptivas, analisis de Ji cuadrado y se calcularon los coeficientes alfa de Cronbach y de Spearman

  17. Hydrogen-atom transfer in reactions of organic radicals with [Co(II)(por)]* (por = porphyrinato) and in subsequent addition of [Co(H)(por)] to olefins.

    PubMed

    de Bruin, Bas; Dzik, Wojciech I; Li, Shan; Wayland, Bradford B

    2009-01-01

    The mechanisms for hydrogen-atom transfer from the cyanoisopropyl radical (*)C(CH(3))(2)CN to [Co(II)(por)](*) (yielding [Co(III)(H)(por)] and CH(2)=C(CH(3))(CN); por = porphyrinato) and the insertion of vinyl acetate (CH(2)=CHOAc) into the Co-H bond of [Co(H)(por)] (giving [Co(III){CH(OAc)CH(3)}(por)]) were investigated by DFT calculations. The results are compared with experimental data. These reactions are relevant to catalytic chain transfer (CCT) in radical polymerization of olefins mediated by [Co(II)(por)](*), the formation and homolysis of organo-cobalt complexes that mediate living radical polymerization of vinyl acetate, and cobalt-mediated hydrogenation of olefins. Hydrogen transfer from (*)C(CH(3))(2)CN to [Co(II)(por)](*) proceeds via a single transition state that has structural features resembling the products [Co(H)(por)] and CH(2)=C(CH(3))CN. The separated radicals approach to form a close-contact radical pair and then pass through the transition state for hydrogen-atom transfer to form [Co(III)(H)(por)] and CH(2)=C(CH(3))CN. This process provides a very low overall barrier for the hydrogen-atom transfer reaction (DeltaG(double dagger) = +3.8 kcal mol(-1)). The reverse reaction corresponding to the addition of [Co(H)(por)] to CH(2)=C(CH(3))CN has a low barrier (DeltaG(double dagger) = +8.9 kcal mol(-1)) as well. Insertion of vinyl acetate into the Co-H bond of [Co(III)(H)(por)] also proceeds over a low barrier (DeltaG(double dagger) = +11.4 kcal mol(-1)) hydrogen-transfer step from [Co(III)(H)(por)] to a carbon atom of the alkene to produce a close-contact radical pair. Dissociation of the radical pair, reorientation, and radical-radical coupling to form an organo-cobalt complex are the culminating steps in the net insertion of an olefin into the Co-H bond. The computed energies obtained for the hydrogen-atom transfer reactions from (*)C(CH(3))(2)CN to [Co(II)(por)](*) and from [Co(H)(por)] to olefins, as well as the organo-cobalt bond homolysis

  18. Seguridad del paciente en Radioterapia Intraoperatoria: Impacto de los elementos controlados por el Radiofisico

    NASA Astrophysics Data System (ADS)

    Tarjuelo, Juan Lopez

    tambien se uso para estudiar la estabilidad de las camaras de ionizacion mencionadas. Se realizo la dosimetria in vivo en 45 pacientes con MOSFET reforzados mobile TN-502RDM-H, pelicula radiocromica Gafchromic MD-55-2, y se elaboro un modelo teorico para explicar los datos. Por ultimo, al precisarse el uso en RIO de la simulacion virtual y del calculo de la dosis absorbida en el paciente virtual, se ha ilustrado este apartado con la aceptacion y el estado de referencia inicial del planificador de tratamientos modulados con calculo de Monte Carlo Elekta Monaco. Para ello se utilizaron la camara de ionizacion TW31016-0104 y la matriz seven29 de PTW-Freiburg, pelicula radiocromica Gafchromic EBT-2, y diferentes maniquies. Resultados: El FMEA identifico 57 modos de fallo y efectos potenciales. No se experimentaron sucesos relativos a una administracion inadecuada de la dosis absorbida. Se identificaron las revisiones dobles y por un par como claves para reducir los riesgos asociados al equipo de profesionales involucrado en la RIO. Se identificaron tambien oportunidades de mejora con el uso de la automatizacion y el enclavamiento. En cuanto al SPC, los indices de capacidad del proceso abarcaron de 1,6 a 9,3 para un nivel de especificaciones del +/-2%. Las intervenciones simuladas alcanzaron del 2% al 34% de las sesiones de medida. Las camaras de ionizacion Farmer derivaron en direcciones opuestas en un periodo de 6 anos; aunque ello no se aprecio en los informes de calibracion del laboratorio acreditado. No derivo la camara PPC-40. En la dosimetria in vivo, las medidas de los MOSFET no se desviaron significativamente de las medidas con pelicula. Los valores centrales de las dosis absorbidas quedaron entre la dosis absorbida prescrita y la maxima, con lo que indicaron un tratamiento correcto del lecho tumoral. Las anchuras de los intervalos de confianza de las dosis absorbidas esperadas segun el modelo teorico al nivel del 95% abarcaron del 8,6% al 14,7%. Las verificaciones de

  19. The PorX Response Regulator of the Porphyromonas gingivalis PorXY Two-Component System Does Not Directly Regulate the Type IX Secretion Genes but Binds the PorL Subunit.

    PubMed

    Vincent, Maxence S; Durand, Eric; Cascales, Eric

    2016-01-01

    The Type IX secretion system (T9SS) is a versatile multi-protein complex restricted to bacteria of the Bacteriodetes phylum and responsible for the secretion or cell surface exposition of diverse proteins that participate to S-layer formation, gliding motility or pathogenesis. The T9SS is poorly characterized but a number of proteins involved in the assembly of the secretion apparatus in the oral pathogen Porphyromonas gingivalis have been identified based on genome substractive analyses. Among these proteins, PorY, and PorX encode typical two-component system (TCS) sensor and CheY-like response regulator respectively. Although the porX and porY genes do not localize at the same genetic locus, it has been proposed that PorXY form a bona fide TCS. Deletion of porX in P. gingivalis causes a slight decrease of the expression of a number of other T9SS genes, including sov, porT, porP, porK, porL, porM, porN, and porY. Here, we show that PorX and the soluble cytoplasmic domain of PorY interact. Using electrophoretic mobility shift, DNA-protein co-purification and heterologous host expression assays, we demonstrate that PorX does not bind T9SS gene promoters and does not directly regulate expression of the T9SS genes. Finally, we show that PorX interacts with the cytoplasmic domain of PorL, a component of the T9SS membrane core complex and propose that the CheY-like PorX protein might be involved in the dynamics of the T9SS.

  20. The PorX Response Regulator of the Porphyromonas gingivalis PorXY Two-Component System Does Not Directly Regulate the Type IX Secretion Genes but Binds the PorL Subunit

    PubMed Central

    Vincent, Maxence S.; Durand, Eric; Cascales, Eric

    2016-01-01

    The Type IX secretion system (T9SS) is a versatile multi-protein complex restricted to bacteria of the Bacteriodetes phylum and responsible for the secretion or cell surface exposition of diverse proteins that participate to S-layer formation, gliding motility or pathogenesis. The T9SS is poorly characterized but a number of proteins involved in the assembly of the secretion apparatus in the oral pathogen Porphyromonas gingivalis have been identified based on genome substractive analyses. Among these proteins, PorY, and PorX encode typical two-component system (TCS) sensor and CheY-like response regulator respectively. Although the porX and porY genes do not localize at the same genetic locus, it has been proposed that PorXY form a bona fide TCS. Deletion of porX in P. gingivalis causes a slight decrease of the expression of a number of other T9SS genes, including sov, porT, porP, porK, porL, porM, porN, and porY. Here, we show that PorX and the soluble cytoplasmic domain of PorY interact. Using electrophoretic mobility shift, DNA-protein co-purification and heterologous host expression assays, we demonstrate that PorX does not bind T9SS gene promoters and does not directly regulate expression of the T9SS genes. Finally, we show that PorX interacts with the cytoplasmic domain of PorL, a component of the T9SS membrane core complex and propose that the CheY-like PorX protein might be involved in the dynamics of the T9SS. PMID:27630829

  1. PROKARYOTIC EXPRESSION AND BIOACTIVITY EVALUATION OF THE CHIMERIC GENE DERIVED FROM THE GROUP 1 ALLERGENS OF DUST MITES.

    PubMed

    Zhan, Xiaodong; Li, Chaopin; Guo, Wei; Jiang, Yuxin

    2015-12-01

    Antecedentes: se reconstituyó con éxito el gen del grupo 1 alérgenos de los ácaros del polvo, y obtuvo un conjunto de genes barajadas. Con el fin de verificar la predicción en el gen quimérico, hemos clonado tentativamente R8 en el vector que se expresó prokaryoticly, purificó y se evaluó por sus actividades-bio. Métodos: el producto expresado se detectó por SDS-PAGE y la proteína diana se purificó. La proteína purificada R8 se detectó por ELISA. Setenta y cinco ratones BALB/ c se dividieron en 5 grupos, a saber: PBS, rDer f1, rDer p1, R8 y el grupo de asma. Los ratones fueron tratados con alérgenos de ácaros del polvo a los 0, 7, 14 días mediante inyección intraperitoneal y inhaladas desafío como aerosol en día 21 durante 7 días. La inmunoterapia específica para el alérgeno se realizó utilizando rDer f1, rDer p1 y alérgenos R8, respectivamente. El nivel de IFN e IL-4 en BALF se detectó por ELISA. Resultados: el gen quimérico R8 se expresó con una banda de aproximadamente Mr 35000. En comparación con los grupos de rDer f 1 y rDer p 1 [(80,44 ± 15,50) y (90,79 ± 10,38) μg/ml, respectivamente], la capacidad de unión a IgE de la proteína R8 (37,03 ± 12,46) μg/ml fue estadísticamente inferior (P.

  2. Observations of Currents, Temperature, Pressure, and Sea Level in the Gulf of California 1982-1986. A Data Report (Observationes de Corrientes, Temperatura, Presion y Nivel Mar en el Golfo de California 1982-1986. Informe de Datos,

    DTIC Science & Technology

    1986-04-01

    arbitrario). Las noted, the statistics are calculated from the estadisticas de los registros de presibn y nivel del hourly-averaged time series. The...comprendido dentro de [a fase principal del experimento, para fines de comparaci6n. Excepto donde queda indicado, las estadisticas para cada serie

  3. System design description for portable 1,000 CFM exhauster Skids POR-007/Skid E and POR-008/Skid F

    SciTech Connect

    Nelson, O.D.

    1998-07-25

    The primary purpose of the two 1,000 CFM Exhauster Skids, POR-007-SKID E and POR-008-SKID F, is to provide backup to the waste tank primary ventilation systems for tanks 241-C-106 and 241-AY-102, and the AY-102 annulus in the event of a failure during the sluicing of tank 241-C-106 and subsequent transfer of sluiced waste to 241-AY-102. This redundancy is required since both of the tank ventilation systems have been declared as Safety Class systems.

  4. Identification of Porphyromonas gingivalis proteins secreted by the Por secretion system.

    PubMed

    Sato, Keiko; Yukitake, Hideharu; Narita, Yuka; Shoji, Mikio; Naito, Mariko; Nakayama, Koji

    2013-01-01

    The Gram-negative bacterium Porphyromonas gingivalis possesses a number of potential virulence factors for periodontopathogenicity. In particular, cysteine proteinases named gingipains are of interest given their abilities to degrade host proteins and process other virulence factors such as fimbriae. Gingipains are translocated on the cell surface or into the extracellular milieu by the Por secretion system (PorSS), which consists of a number of membrane or periplasmic proteins including PorK, PorL, PorM, PorN, PorO, PorP, PorQ, PorT, PorU, PorV (PG27, LptO), PorW and Sov. To identify proteins other than gingipains secreted by the PorSS, we compared the proteomes of P. gingivalis strains kgp rgpA rgpB (PorSS-proficient strain) and kgp rgpA rgpB porK (PorSS-deficient strain) using two-dimensional gel electrophoresis and peptide-mass fingerprinting. Sixteen spots representing 10 different proteins were present in the particle-free culture supernatant of the PorSS-proficient strain but were absent or faint in that of the PorSS-deficient strain. These identified proteins possessed the C-terminal domains (CTDs), which had been suggested to form the CTD protein family. These results indicate that the PorSS is used for secretion of a number of proteins other than gingipains and that the CTDs of the proteins are associated with the PorSS-dependent secretion. © 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

  5. 'Carbene radicals' in Co(II)(por)-catalyzed olefin cyclopropanation.

    PubMed

    Dzik, Wojciech I; Xu, Xue; Zhang, X Peter; Reek, Joost N H; de Bruin, Bas

    2010-08-11

    The mechanism of cobalt(II)-porphyrin-mediated cyclopropanation of olefins with diazoesters was studied. The first step--reaction of cobalt(II)-porphyrin with ethyl diazoacetate (EDA)--was examined using EPR and ESI-MS techniques. EDA reacts with cobalt(II)-porphyrin to form a 1:1 Co(por)(CHCOOEt) adduct that exists as two isomers: the 'bridging carbene' C' in which the 'carbene' is bound to the metal and the pyrrolic nitrogen of the porphyrin that has a d(7) configuration on the metal, and the 'terminal carbene' C in which the 'carbene' behaves as a redox noninnocent ligand having a d(6) cobalt center and the unpaired electron residing on the 'carbene' carbon atom. The subsequent reactivities of the thus formed 'cobalt carbene radical' with propene, styrene, and methyl acrylate were studied using DFT calculations. The calculations suggest that the formation of the carbene is the rate-limiting step for the unfunctionalized Co(II)(por) and that the cyclopropane ring formation proceeds via a stepwise radical process: Radical addition of the 'carbene radical' C to the C=C double bonds of the olefins results in formation of the gamma-alkyl radical intermediates D. Species D then easily collapse in almost barrierless ring-closure reactions (TS3) to form the cyclopropanes. This radical mechanism readily explains the high activity of Co(II)(por) species in the cyclopropanation of electron-deficient olefins such as methyl acrylate.

  6. Arabidopsis protochlorophyllide oxidoreductase A (PORA) restores bulk chlorophyll synthesis and normal development to a porB porC double mutant.

    PubMed

    Paddock, Troy N; Mason, Mary E; Lima, Daniel F; Armstrong, Gregory A

    2010-03-01

    In angiosperms the strictly light-dependent reduction of protochlorophyllide to chlorophyllide is catalyzed by NADPH:protochlorophyllide oxidoreductase (POR). The Arabidopsis thaliana genome encodes three structurally related but differentially regulated POR genes, PORA, PORB and PORC. PORA is expressed primarily early in development-during etiolation, germination and greening. In contrast, PORB and PORC are not only expressed during seedling development but also throughout the later life of the plant, during which they are responsible for bulk chlorophyll synthesis. The Arabidopsis porB-1 porC-1 mutant displays a severe xantha (highly chlorophyll-deficient) phenotype characterized by smaller prolamellar bodies in etioplasts and decreased thylakoid stacking in chloroplasts. Here we have demonstrated the ability of an ectopic PORA overexpression construct to restore prolamellar body formation in the porB-1 porC-1 double mutant background. In response to illumination, light-dependent chlorophyll production, thylakoid stacking and photomorphogenesis are also restored in PORA-overexpressing porB-1 porC-1 seedlings and adult plants. An Arabidopsis porB-1 porC-1 double mutant can therefore be functionally rescued by the addition of ectopically expressed PORA, which suffices in the absence of either PORB or PORC to direct bulk chlorophyll synthesis and normal plant development.

  7. Click-chemistry approach to study mycoloylated proteins: Evidence for PorB and PorC porins mycoloylation in Corynebacterium glutamicum.

    PubMed

    Issa, Hanane; Huc-Claustre, Emilie; Reddad, Thamila; Bonadé Bottino, Nolwenn; Tropis, Maryelle; Houssin, Christine; Daffé, Mamadou; Bayan, Nicolas; Dautin, Nathalie

    2017-01-01

    Protein mycoloylation is a recently identified, new form of protein acylation. This post-translational modification consists in the covalent attachment of mycolic acids residues to serine. Mycolic acids are long chain, α-branched, β-hydroxylated fatty acids that are exclusively found in the cell envelope of Corynebacteriales, a bacterial order that includes important genera such as Mycobacterium, Nocardia or Corynebacterium. So far, only 3 mycoloylated proteins have been identified: PorA, PorH and ProtX from C. glutamicum. Whereas the identity and function of ProtX is unknown, PorH and PorA associate to form a membrane channel, the activity of which is dependent upon PorA mycoloylation. However, the exact role of mycoloylation and the generality of this phenomenon are still unknown. In particular, the identity of other mycoloylated proteins, if any, needs to be determined together with establishing whether such modification occurs in Corynebacteriales genera other than Corynebacterium. Here, we tested whether a metabolic labeling and click-chemistry approach could be used to detect mycoloylated proteins. Using a fatty acid alkyne analogue, we could indeed label PorA, PorH and ProtX and determine ProtX mycoloylation site. Importantly, we also show that two other porins from C. glutamicum, PorB and PorC are mycoloylated.

  8. Click-chemistry approach to study mycoloylated proteins: Evidence for PorB and PorC porins mycoloylation in Corynebacterium glutamicum

    PubMed Central

    Issa, Hanane; Huc-Claustre, Emilie; Reddad, Thamila; Bonadé Bottino, Nolwenn; Tropis, Maryelle; Houssin, Christine; Daffé, Mamadou; Bayan, Nicolas

    2017-01-01

    Protein mycoloylation is a recently identified, new form of protein acylation. This post-translational modification consists in the covalent attachment of mycolic acids residues to serine. Mycolic acids are long chain, α-branched, β-hydroxylated fatty acids that are exclusively found in the cell envelope of Corynebacteriales, a bacterial order that includes important genera such as Mycobacterium, Nocardia or Corynebacterium. So far, only 3 mycoloylated proteins have been identified: PorA, PorH and ProtX from C. glutamicum. Whereas the identity and function of ProtX is unknown, PorH and PorA associate to form a membrane channel, the activity of which is dependent upon PorA mycoloylation. However, the exact role of mycoloylation and the generality of this phenomenon are still unknown. In particular, the identity of other mycoloylated proteins, if any, needs to be determined together with establishing whether such modification occurs in Corynebacteriales genera other than Corynebacterium. Here, we tested whether a metabolic labeling and click-chemistry approach could be used to detect mycoloylated proteins. Using a fatty acid alkyne analogue, we could indeed label PorA, PorH and ProtX and determine ProtX mycoloylation site. Importantly, we also show that two other porins from C. glutamicum, PorB and PorC are mycoloylated. PMID:28199365

  9. Por La Vida intervention model for cancer prevention in Latinas.

    PubMed

    Navarro, A M; Senn, K L; Kaplan, R M; McNicholas, L; Campo, M C; Roppe, B

    1995-01-01

    Our goal was to describe the development and implementation of an intervention on cancer prevention for Latinas in San Diego, Calif. Thirty-six lay community workers ("consejeras") were recruited and trained to conduct educational group sessions. Each consejera recruited approximately 14 peers from the community to participate in the program (total number = 512). Half of the consejeras were randomly assigned to a control group, in which they participated in an equally engaging program entitled "Community Living Skills." Implementation of the intervention was assessed by qualitative and quantitative methods. Preintervention and postintervention self-report information was obtained from project participants on access to health care services, cancer knowledge, preventive measures, and previous cancer-screening examinations. Base-line data suggest that lack of knowledge, costs of cancer-screening tests, and the lack of a regular health care provider are the major obstacles against obtaining cancer-screening tests. Predisposing factors, such as fear and embarrassment, also constitute barriers to getting regular cervical cancer screening. Preliminary analysis indicates that the Por La Vida intervention increases use of cancer-screening tests in comparison to a community living skills control group. Universal access to health care would remove some of the major financial barriers to cancer screening. The Por La Vida program attempts to overcome the substantial barriers by reaching out to low-income Latinas and by providing information regarding the availability, acceptability, and preventive nature of cancer-screening tests.

  10. Prevalencia y factores de riesgo para infecciones del tracto urinario de inicio en la comunidad causadas por Escherichia coli productor de betalactamasas de espectro extendido en Colombia

    PubMed Central

    Blanco, Victor M.; Maya, Juan J.; Correa, Adriana; Perenguez, Marcela; Muñoz, Juan S.; Motoa, Gabriel; Pallares, Christian J.; Rosso, Fernando; Matta, Lorena; Celis, Yamile; Garzon, Martha; Villegas, y María V.

    2016-01-01

    RESUMEN Introducción Las infecciones del tracto urinario (ITU) son frecuentes en la comunidad. Sin embargo, la información de aislamientos resistentes en este contexto es limitada en Latinoamérica. Este estudio tiene como objetivo determinar la prevalencia y los factores de riesgo asociados con ITU de inicio en la comunidad (ITU-IC) causadas por Escherichia coli productor de betalactamasas de espectro extendido (BLEE) en Colombia. Materiales y métodos Entre agosto y diciembre de 2011 se realizó un estudio de casos y controles en 3 instituciones de salud de tercer nivel en Colombia. Se invitó a participar a todos los pacientes admitidos a urgencias con diagnóstico probable de ITU-IC, y se les pidió una muestra de orina. En los aislamien-tos de E. coli se realizaron pruebas confirmatorias para BLEE, susceptibilidad antibiótica, caracterización molecular (PCR en tiempo real para genes bla, repetitive element palindromic PCR [rep-PCR], multilocus sequence typing [MLST] y factores de virulencia por PCR). Se obtuvo información clínica y epidemiológica, y posteriormente se realizó el análisis estadístico. Resultados De los 2.124 pacientes seleccionados, 629 tuvieron un urocultivo positivo, en 431 de estos se aisló E. coli, 54 fueron positivos para BLEE y 29 correspondieron a CTX-M-15. La mayoría de los aislamientos de E. coli productor de BLEE fueron sensibles a ertapenem, fosfomicina y amikacina. La ITU complicada se asoció fuertemente con infecciones por E. coli productor de BLEE (OR = 3,89; IC 95%: 1,10–13,89; p = 0,03). E. coli productor de CTX-M-15 mostró 10 electroferotipos diferentes; de estos, el 65% correspondieron al ST131. La mayoría de estos aislamientos tuvieron 8 de los 9 factores de virulencia analizados. Discusión E. coli portador del gen blaCTX-M-15 asociado al ST131 sigue siendo frecuente en Colombia. La presencia de ITU-IC complicada aumenta el riesgo de tener E. coli productor de BLEE, lo cual debe tenerse en cuenta para ofrecer

  11. Acceptance test report for portable exhauster POR-007/Skid E

    SciTech Connect

    Kriskovich, J.R.

    1998-07-24

    This document describes Acceptance Testing performed on Portable Exhauster POR-007/Skid E. It includes measurements of bearing vibration levels, pressure decay testing, programmable logic controller interlocks, high vacuum, flow and pressure control functional testing. The purpose of Acceptance testing documented by this report was to demonstrate compliance of the exhausters with the performance criteria established within HNF-0490, Rev. 1 following a repair and upgrade effort at Hanford. In addition, data obtained during this testing is required for the resolution of outstanding Non-conformance Reports (NCR), and finally, to demonstrate the functionality of the associated software for the pressure control and high vacuum exhauster operating modes provided for by W-320. Additional testing not required by the ATP was also performed to assist in the disposition and close out of receiving inspection report and for application design information (system curve). Results of this testing are also captured within this document.

  12. Compton imaging with the PorGamRays spectrometer

    NASA Astrophysics Data System (ADS)

    Judson, D. S.; Boston, A. J.; Coleman-Smith, P. J.; Cullen, D. M.; Hardie, A.; Harkness, L. J.; Jones, L. L.; Jones, M.; Lazarus, I.; Nolan, P. J.; Pucknell, V.; Rigby, S. V.; Seller, P.; Scraggs, D. P.; Simpson, J.; Slee, M.; Sweeney, A.; PorGamRays Collaboration

    2011-10-01

    The PorGamRays project aims to develop a portable gamma-ray detection system with both spectroscopic and imaging capabilities. The system is designed around a stack of thin Cadmium Zinc Telluride (CZT) detectors. The imaging capability utilises the Compton camera principle. Each detector is segmented into 100 pixels which are read out through custom designed Application Specific Integrated Circuits (ASICs). This device has potential applications in the security, decommissioning and medical fields. This work focuses on the near-field imaging performance of a lab-based demonstrator consisting of two pixelated CZT detectors, each of which is bonded to a NUCAM II ASIC. Measurements have been made with point 133Ba and 57Co sources located ˜35 mm from the surface of the scattering detector. Position resolution of ˜20 mm FWHM in the x and y planes is demonstrated.

  13. Suppression of cytochrome P450 reductase (POR) expression in hepatoma cells replicates the hepatic lipidosis observed in hepatic POR-null mice.

    PubMed

    Porter, Todd D; Banerjee, Subhashis; Stolarczyk, Elzbieta I; Zou, Ling

    2011-06-01

    Cytochrome P450 reductase (POR) is a microsomal electron transport protein essential to cytochrome P450-mediated drug metabolism and sterol and bile acid synthesis. The conditional deletion of hepatic POR gene expression in mice results in a marked decrease in plasma cholesterol levels counterbalanced by the accumulation of triglycerides in lipid droplets in hepatocytes. To evaluate the role of cholesterol and bile acid synthesis in this hepatic lipidosis, as well as the possible role of lipid transport from peripheral tissues, we developed a stable, small interfering RNA (siRNA)-mediated cell culture model for the suppression of POR. POR mRNA and protein expression were decreased by greater than 50% in McArdle-RH7777 rat hepatoma cells 10 days after transfection with a POR-siRNA expression plasmid, and POR expression was nearly completely extinguished by day 20. Immunofluorescent analysis revealed a marked accumulation of lipid droplets in cells by day 15, accompanied by a nearly 2-fold increase in cellular triglyceride content, replicating the lipidosis seen in hepatic POR-null mouse liver. In contrast, suppression of CYP51A1 (lanosterol demethylase) did not result in lipid accumulation, indicating that loss of cholesterol synthesis is not the basis for this lipidosis. Indeed, addition of cholesterol to the medium appeared to augment the lipidosis in POR-suppressed cells, whereas removal of lipids from the medium reversed the lipidosis. Oxysterols did not accumulate in POR-suppressed cells, discounting a role for liver X receptor in stimulating triglyceride synthesis, but addition of chenodeoxycholate significantly repressed lipid accumulation, suggesting that the absence of bile acids and loss of farnesoid X receptor stimulation lead to excessive triglyceride synthesis.

  14. Suppression of Cytochrome P450 Reductase (POR) Expression in Hepatoma Cells Replicates the Hepatic Lipidosis Observed in Hepatic POR-Null Mice

    PubMed Central

    Banerjee, Subhashis; Stolarczyk, Elzbieta I.; Zou, Ling

    2011-01-01

    Cytochrome P450 reductase (POR) is a microsomal electron transport protein essential to cytochrome P450-mediated drug metabolism and sterol and bile acid synthesis. The conditional deletion of hepatic POR gene expression in mice results in a marked decrease in plasma cholesterol levels counterbalanced by the accumulation of triglycerides in lipid droplets in hepatocytes. To evaluate the role of cholesterol and bile acid synthesis in this hepatic lipidosis, as well as the possible role of lipid transport from peripheral tissues, we developed a stable, small interfering RNA (siRNA)-mediated cell culture model for the suppression of POR. POR mRNA and protein expression were decreased by greater than 50% in McArdle-RH7777 rat hepatoma cells 10 days after transfection with a POR-siRNA expression plasmid, and POR expression was nearly completely extinguished by day 20. Immunofluorescent analysis revealed a marked accumulation of lipid droplets in cells by day 15, accompanied by a nearly 2-fold increase in cellular triglyceride content, replicating the lipidosis seen in hepatic POR-null mouse liver. In contrast, suppression of CYP51A1 (lanosterol demethylase) did not result in lipid accumulation, indicating that loss of cholesterol synthesis is not the basis for this lipidosis. Indeed, addition of cholesterol to the medium appeared to augment the lipidosis in POR-suppressed cells, whereas removal of lipids from the medium reversed the lipidosis. Oxysterols did not accumulate in POR-suppressed cells, discounting a role for liver X receptor in stimulating triglyceride synthesis, but addition of chenodeoxycholate significantly repressed lipid accumulation, suggesting that the absence of bile acids and loss of farnesoid X receptor stimulation lead to excessive triglyceride synthesis. PMID:21368239

  15. Acceptance test report for portable exhauster POR-008/Skid F

    SciTech Connect

    Kriskovich, J.R.

    1998-07-24

    Portable Exhauster POR-008 was procured via HNF-0490, Specification for a Portable Exhausted System for Waste Tank Ventilation. Prior to taking ownership, acceptance testing was performed at the vendors. However at the conclusion of testing a number of issues remained that required resolution before the exhausters could be used by Project W-320. The purpose of acceptance testing documented by this report was to demonstrate compliance of the exhausters with the performance criteria established within HNF-O49O, Rev. 1 following a repair and upgrade effort at Hanford. In addition, data obtained during this testing is required for the resolution of outstanding Non-conformance Reports (NCR), and finally, to demonstrate the functionality of the associated software for the pressure control and high vacuum exhauster operating modes provided for by W-320. Additional testing not required by the ATP was also performed to assist in the disposition and close out of receiving inspection report and for application design information (system curve). Results of this testing are also captured within this document.

  16. Prenatal Diagnosis of Antley-Bixler Syndrome and POR Deficiency.

    PubMed

    Oldani, Elena; Garel, Catherine; Bucourt, Martine; Carbillon, Lionel

    2015-12-16

    Prenatal diagnosis of severe bone diseases is challenging and requires complete and precise analysis of fetal anomalies to guide genetic investigation and parental counselling. We report a rare case of Antley-Bixler syndrome prenatally diagnosed at 26 weeks' gestation by ultrasound and computed tomography in a 28-year-old woman with a history of early termination of pregnancy for "malposition of the inferior limbs". The prenatal ultrasound scan showed severe femoral bowing and frontal bossing. Taking into account the high probability of a recurrent severe skeletal disorder, a computed tomography (CT) scan was proposed. CT findings revealed bilateral femora deformation, craniosynostosis, severe midface hypoplasia, and radiohumeral synostosis. These anomalies strongly suggested Antley-Bixler syndrome. Sequencing of the POR gene in the fetus and the parents revealed compound heterozygous mutations in exon 9 and intron 7, both inherited from each parent, and this finding allowed genetic counseling. The first step in the proper prenatal diagnosis of fetal bone disorders is the precise analysis of ultrasonographic images. However, when a severe fetal inherited disorder is strongly suspected in late mid-trimester, CT may be discussed and usefully contribute to diagnosis and prognosis assessment.

  17. Lunar Phases and Earthly Events: Beliefs from Different Education Levels. (Spanish Title: Fases de la Luna y Acontecimientos Terrestres: Creencia de Distintos Niveles de Instrucción.) As Fases da Lua e os Acontecimentos Terrestres: a Crença de Diferentes Níveis de Instrução

    NASA Astrophysics Data System (ADS)

    Darroz, Luiz Marcelo; da Rosa, Cleci Teresinha Werner; Alves Vizzotto, Patrick; Becker da Rosa, Álvaro

    2013-12-01

    This article presents the result of a research carried out in the first semester of 2013 with a group of 80 subjects from different education levels. In this research, we sought to investigate the earthly events that this group of people attributes to the phenomenon of lunar phases. For data collection we used semi-structured interviews guided by questions that aimed to keep the focus on subjects of the investigation. Interviews were recorded and transcribed, and the results were compared to scientific studies in the area after being quantitatively and qualitatively analyzed. Research data showed that the Moon and the phenomenon of lunar phases still fascinate and raise the interest of people. However, the lack of knowledge to find correct explanations to the phenomena involving the moon ends up originating a series of beliefs about its influence on earthly events. En este artículo se presenta el resultado de una investigación realizada en el primer semestre de 2013 con a un grupo de 80 individuos de distintos niveles de instrucción. En esta investigación, tratamos de averiguar cuáles son los acontecimientos terrestres que este grupo de personas atribuye al fenómeno las fases lunares. Como instrumento de colecta de datos, se emplearon entrevistas semiestructuradas guiadas por preguntas que trataban de mantener la atención de los entrevistados en el objeto investigado. Las entrevistas fueron grabadas y transcriptas, y los resultados, después de ser analizados cuantitatativa y cualitativamente, fueron confrontados con estudios científicos del área. Los datos de la investigación demuestran que la Luna y de sus fases continúan fascinando y despertando el interés de la población. Sin embargo, la falta de conocimientos para encontrar explicaciones correctas relacionadas a los fenómenos que ocurren con el astro acaba originando una serie de creencias en la población sobre su influencia en los sucesos terrestres. Apresenta-se neste artigo o resultado de uma

  18. Purification of neuropathy target esterase from avian brain after prelabelling with [3H]diisopropyl phosphorofluoridate.

    PubMed

    Rüffer-Turner, M E; Read, D J; Johnson, M K

    1992-01-01

    Neuropathy target esterase from hen brains was radiolabelled at the active site with [3H]diisopropyl phosphorofluoridate. The labelled protein was purified by differential centrifugation and Nonidet P40 solubilization, detergent phase partitioning, anion exchange, and preparative sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The volatilizable counts assay and analytical SDS-PAGE were used to monitor the protein. The 150-kDa subunit polypeptide appears as a single band on analytical SDS-PAGE.

  19. P450 (Cytochrome) Oxidoreductase Gene (POR) Common Variant (POR*28) Significantly Alters CYP2C9 Activity in Swedish, But Not in Korean Healthy Subjects.

    PubMed

    Hatta, Fazleen H M; Aklillu, Eleni

    2015-12-01

    CYP2C9 enzyme contributes to the metabolism of several pharmaceuticals and xenobiotics and yet displays large person-to-person and interethnic variation. Understanding the mechanisms of CYP2C9 variation is thus of immense importance for personalized medicine and rational therapeutics. A genetic variant of P450 (cytochrome) oxidoreductase (POR), a CYP450 redox partner, is reported to influence CYP2C9 metabolic activity in vitro. We investigated the impact of a common variant, POR*28, on CYP2C9 metabolic activity in humans. 148 healthy Swedish and 146 healthy Korean volunteers were genotyped for known CYP2C9 defective variant alleles (CYP2C9*2, *3). The CYP2C9 phenotype was determined using a single oral dose of 50 mg losartan. Excluding oral contraceptive (OC) users and carriers of 2C9*2 and *3 alleles, 117 Korean and 65 Swedish were genotyped for POR*5, *13 and *28 using Taqman assays. The urinary losartan to its metabolite E-3174 metabolic ratio (MR) was used as an index of CYP2C9 metabolic activity. The allele frequency of the POR*28 variant allele in Swedes and Koreans was 29% and 44%, respectively. POR*5 and *13 were absent in both study populations. Considering the CYP2C9*1/*1 genotypes only, the CYP2C9 metabolic activity was 1.40-fold higher in carriers of POR*28 allele than non-carriers among Swedes (p = 0.02). By contrast, no influence of the POR*28 on CYP2C9 activity was found in Koreans (p = 0.68). The multivariate analysis showed that ethnicity, POR genotype, and smoking were strong predictors of CYP2C9 MR (p < 0.05). This is the first report to implicate the importance of POR*28 genetic variation for CYP2C9 metabolic activity in humans. These findings contribute to current efforts for global personalized medicine and using medicines by taking into account pharmacogenetic and phenotypic variations.

  20. Mitigation of Disagreement in Peer Review among L2 Learners and Native Speakers in a College Writing Class (Mitigación del Impacto de las Opiniones de Desacuerdo en el Proceso de Revisión por Pares entre Estudiantes de una Segunda Lengua y Hablantes Nativos en una Clase de Escritura a Nivel Universitario)

    ERIC Educational Resources Information Center

    Christoffersen, Katherine O'Donnell

    2015-01-01

    Peer review is now a commonplace practice in process-oriented writing instruction. A crucial aspect of peer review is assessing another classmate's work, which encompasses the act of disagreement. Given its prevalence in the classroom, it is necessary to analyze how L2 learners mitigate disagreement in the context of peer review with other L2…

  1. Gliding motility and Por secretion system genes are widespread among members of the phylum bacteroidetes.

    PubMed

    McBride, Mark J; Zhu, Yongtao

    2013-01-01

    The phylum Bacteroidetes is large and diverse, with rapid gliding motility and the ability to digest macromolecules associated with many genera and species. Recently, a novel protein secretion system, the Por secretion system (PorSS), was identified in two members of the phylum, the gliding bacterium Flavobacterium johnsoniae and the nonmotile oral pathogen Porphyromonas gingivalis. The components of the PorSS are not similar in sequence to those of other well-studied bacterial secretion systems. The F. johnsoniae PorSS genes are a subset of the gliding motility genes, suggesting a role for the secretion system in motility. The F. johnsoniae PorSS is needed for assembly of the gliding motility apparatus and for secretion of a chitinase, and the P. gingivalis PorSS is involved in secretion of gingipain protease virulence factors. Comparative analysis of 37 genomes of members of the phylum Bacteroidetes revealed the widespread occurrence of gliding motility genes and PorSS genes. Genes associated with other bacterial protein secretion systems were less common. The results suggest that gliding motility is more common than previously reported. Microscopic observations confirmed that organisms previously described as nonmotile, including Croceibacter atlanticus, "Gramella forsetii," Paludibacter propionicigenes, Riemerella anatipestifer, and Robiginitalea biformata, exhibit gliding motility. Three genes (gldA, gldF, and gldG) that encode an apparent ATP-binding cassette transporter required for F. johnsoniae gliding were absent from two related gliding bacteria, suggesting that the transporter may not be central to gliding motility.

  2. Impact of POR*28 on the pharmacokinetics of tacrolimus and cyclosporine A in renal transplant patients.

    PubMed

    Elens, Laure; Hesselink, Dennis A; Bouamar, Rachida; Budde, Klemens; de Fijter, Johannes W; De Meyer, Martine; Mourad, Michel; Kuypers, Dirk R J; Haufroid, Vincent; van Gelder, Teun; van Schaik, Ron H N

    2014-02-01

    The P450 oxidoreductase (POR)*28 variant allele has been associated with altered cytochrome P450 3A enzyme activities. Both CYP3A5 and CYP3A4 are involved in the metabolism of calcineurin inhibitors and recent data show that POR*28 may explain part of the variability observed in tacrolimus (Tac) pharmacokinetics. The aim of this study was to investigate the impact of the POR*28 allele on Tac and cyclosporine A (CsA) immunosuppressive therapies. Kidney transplant recipients receiving either Tac (n = 184) or CsA (n = 174), participating in a prospective multicenter trial, were genotyped for POR*28, CYP3A4*22, and CYP3A5*3. CYP3A5 expressers that were carriers of at least 1 POR*28 allele had a 16.9% decrease in dose-adjusted predose concentrations when compared CYP3A5 expressers that carried the POR*1/*1 genotype (P = 0.03), indicating an increased CYP3A5 activity for POR*28 carriers. In CYP3A5, nonexpressers carrying 2 POR*28 alleles, a 24.1% (confidence interval95% = -39.4% to -4.9%; P = 0.02) decrease in dose-adjusted predose concentrations was observed for Tac, suggesting higher CYP3A4 activity. For CsA, POR*28/*28 patients not expressing CYP3A5 and not carrying the CYP3A4*22 decrease-of-function allele showed 15% lower CsA dose-adjusted predose concentrations (P = 0.01), indicating also increased CYP3A4 activity. In both cohorts (ie, Tac and CsA), the POR*28 allele was neither associated with the incidence of delayed graft function nor with biopsy-proven acute rejection. These results were further confirmed in 2 independent cohorts. Our results show that the POR*28 allele is associated with increased in vivo CYP3A5 activity for Tac in CYP3A5 expressers, whereas POR*28 homozygosity was associated with a significant higher CYP3A4 activity in CYP3A5 nonexpressers for both Tac and CsA.

  3. Successful Mnemonics for "por"/"para" and Affirmative Commands with Pronouns.

    ERIC Educational Resources Information Center

    Mason, Keith

    1992-01-01

    Two mnemonic devices, "4A Rule" and "PERFECT," are described to simplify the learning of two grammar points: the placement of object pronouns with respect to commands and the distinction between "por" and "para." (five references) (LB)

  4. Crystallographic analysis of Neisseria meningitidis PorB extracellular loops potentially implicated in TLR2 recognition

    PubMed Central

    Kattner, Christof; Toussi, Deana; Zaucha, Jan; Wetzler, Lee M.; Rüppel, Nadine; Zachariae, Ulrich; Massari, Paola; Tanabe, Mikio

    2014-01-01

    Among all Neisseriae species, N. meningitidis and N. gonorrhoeae are the only human pathogens, causative agents of bacterial meningitis and gonorrhoea, respectively. PorB, a pan-Neisseriae trimeric porin that mediates diffusive transport of essential molecules across the bacterial outer membrane, is also known to activate host innate immunity via Toll-like receptor 2 (TLR2)-mediated signaling. The molecular mechanism of PorB binding to TLR2 is not known, but it has been hypothesized that electrostatic interactions contribute to ligand/receptor binding. Strain-specific sequence variability in the surface-exposed loops of PorB which are potentially implicated in TLR2 binding, may explain the difference in TLR2-mediated cell activation in vitro by PorB homologs from the commensal N. lactamica and the pathogen N. meningitidis. Here, we report a comparative structural analysis of PorB from N. meningitidis serogroup B strain 8765 (63% sequence homology with PorB from N. meningitidis serogroup W135) and a mutant in which amino acid substitutions in the extracellular loop 7 lead to significantly reduced TLR2-dependent activity in vitro. We observe that this mutation both alters the loop conformation and causes dramatic changes of electrostatic surface charge, both of which may affect TLR2 recognition and signalling. PMID:24361688

  5. An investigation of exploitation versus exploration in GBEA optimization of PORS 15 and 16 Problems

    SciTech Connect

    Koch, Kaelynn

    2012-01-01

    It was hypothesized that the variations in time to solution are driven by the competing mechanisms of exploration and exploitation.This thesis explores this hypothesis by examining two contrasting problems that embody the hypothesized tradeoff between exploration and exploitation. Plus one recall store (PORS) is an optimization problem based on the idea of a simple calculator with four buttons: plus, one, store, and recall. Integer addition and store are classified as operations, and one and memory recall are classified as terminals. The goal is to arrange a fixed number of keystrokes in a way that maximizes the numerical result. PORS 15 (15 keystrokes) represents the subset of difficult PORS problems and PORS 16 (16 keystrokes) represents the subset of PORS problems that are easiest to optimize. The goal of this work is to examine the tradeoff between exploitation and exploration in graph based evolutionary algorithm (GBEA) optimization. To do this, computational experiments are used to examine how solutions evolve in PORS 15 and 16 problems when solved using GBEAs. The experiment is comprised of three components; the graphs and the population, the evolutionary algorithm rule set, and the example problems. The complete, hypercube, and cycle graphs were used for this experiment. A fixed population size was used.

  6. Molecular characterisation of Porcine rubulavirus (PorPV) isolates from different outbreaks in Mexico.

    PubMed

    Cuevas-Romero, S; Rivera-Benítez, J F; Blomström, A-L; Ramliden, M; Hernández-Baumgarten, E; Hernández-Jáuregui, P; Ramírez-Mendoza, H; Berg, M

    2016-02-01

    Since the report of the initial outbreak of Porcine rubulavirus (PorPV) infection in pigs, only one full-length genome from 1984 (PorPV-LPMV/1984) has been characterised. To investigate the overall genetic variation, full-length gene nucleotide sequences of current PorPV isolates were obtained from different clinical cases of infected swine. Genome organisation and sequence analysis of the encoded proteins (NP, P, F, M, HN and L) revealed high sequence conservation of the NP protein and the expression of the P and V proteins in all PorPV isolates. The V protein of one isolate displayed a mutation that has been implicated to antagonise the antiviral immune responses of the host. The M protein indicated a variation in a short region that could affect the electrostatic charge and the interaction with the membrane. One PorPV isolate recovered from the lungs showed a mutation at the cleavage site (HRKKR) of the F protein that could represent an important factor to determine the tissue tropism and pathogenicity of this virus. The HN protein showed high sequence identity through the years (up to 2013). Additionally, a number of sequence motifs of very high amino acid conservation among the PorPV isolates important for polymerase activity of the L protein have been identified. In summary, genetic comparisons and phylogenetic analyses indicated that three different genetic variants of PorPV are currently spreading within the swine population, and a new generation of circulating virus with different characteristics has begun to emerge.

  7. Por Secretion System-Dependent Secretion and Glycosylation of Porphyromonas gingivalis Hemin-Binding Protein 35

    PubMed Central

    Shoji, Mikio; Sato, Keiko; Yukitake, Hideharu; Kondo, Yoshio; Narita, Yuka; Kadowaki, Tomoko; Naito, Mariko; Nakayama, Koji

    2011-01-01

    The anaerobic Gram-negative bacterium Porphyromonas gingivalis is a major pathogen in severe forms of periodontal disease and refractory periapical perodontitis. We have recently found that P. gingivalis has a novel secretion system named the Por secretion system (PorSS), which is responsible for secretion of major extracellular proteinases, Arg-gingipains (Rgps) and Lys-gingipain. These proteinases contain conserved C-terminal domains (CTDs) in their C-termini. Hemin-binding protein 35 (HBP35), which is one of the outer membrane proteins of P. gingivalis and contributes to its haem utilization, also contains a CTD, suggesting that HBP35 is translocated to the cell surface via the PorSS. In this study, immunoblot analysis of P. gingivalis mutants deficient in the PorSS or in the biosynthesis of anionic polysaccharide-lipopolysaccharide (A-LPS) revealed that HBP35 is translocated to the cell surface via the PorSS and is glycosylated with A-LPS. From deletion analysis with a GFP-CTD[HBP35] green fluorescent protein fusion, the C-terminal 22 amino acid residues of CTD[HBP35] were found to be required for cell surface translocation and glycosylation. The GFP-CTD fusion study also revealed that the CTDs of CPG70, peptidylarginine deiminase, P27 and RgpB play roles in PorSS-dependent translocation and glycosylation. However, CTD-region peptides were not found in samples of glycosylated HBP35 protein by peptide map fingerprinting analysis, and antibodies against CTD-regions peptides did not react with glycosylated HBP35 protein. These results suggest both that the CTD region functions as a recognition signal for the PorSS and that glycosylation of CTD proteins occurs after removal of the CTD region. Rabbits were used for making antisera against bacterial proteins in this study. PMID:21731719

  8. Impact of POR*28 on the clinical pharmacokinetics of CYP3A phenotyping probes midazolam and erythromycin.

    PubMed

    Elens, Laure; Nieuweboer, Annemieke J M; Clarke, Stephen J; Charles, Kellie A; de Graan, Anne-Joy M; Haufroid, Vincent; van Gelder, Teun; Mathijssen, Ron H J; van Schaik, Ron H N

    2013-03-01

    P450 oxidoreductase (POR) is essential for cytochrome P450 (CYP) activity in humans. The POR*28 allele (A503V) has been shown to impact on in-vitro CYP-mediated metabolism, including CYP3A isoenzymes. The aim of the present study was to determine the in vivo impact of the POR*28 allele on the pharmacokinetics of the classic CYP3A phenotyping probes midazolam and erythromycin. Whereas midazolam is metabolized by both CYP3A4 and CYP3A5, erythromycin is exclusively oxidized by CYP3A4. To assess CYP3A activity, 108 cancer patients received midazolam and 45 others underwent the erythromycin breath test. Patients were genotyped for POR*28, CYP3A4*22 and CYP3A5*3. In patients expressing CYP3A5, POR*28 carriers showed 45% lower midazolam metabolic ratios compared with POR*1/*1 patients (P<0.001). This is in line with a lower CYP3A5 activity toward midazolam for POR*28 carriers. In CYP3A5 nonexpressers, POR*28 had no influence on midazolam pharmacokinetics. For erythromycin, POR*28 carriership did not influence its metabolism. Our data show that the POR*28 allele is associated with a lower in vivo CYP3A5 activity, but has no effects on CYP3A4-mediated erythromycin and midazolam metabolism.

  9. PorA Represents the Major Cell Wall Channel of the Gram-Positive Bacterium Corynebacterium glutamicum

    PubMed Central

    Costa-Riu, Noelia; Burkovski, Andreas; Krämer, Reinhard; Benz, Roland

    2003-01-01

    The cell wall of the gram-positive bacterium Corynebacterium glutamicum contains a channel (porin) for the passage of hydrophilic solutes. The channel-forming polypeptide PorA is a 45-amino-acid acidic polypeptide with an excess of four negatively charged amino acids, which is encoded by the 138-bp gene porA. porA was deleted from the chromosome of C.glutamicum wild-type strain ATCC 13032 to obtain mutant ATCC 13032ΔporA. Southern blot analysis demonstrated that porA was deleted. Lipid bilayer experiments revealed that PorA was not present in the cell wall of the mutant strain. Searches within the known chromosome of C. glutamicum by using National Center for Biotechnology Information BLAST and reverse transcription-PCR showed that no other PorA-like protein is encoded on the chromosome or is expressed in the deletion strain. The porA deletion strain exhibited slower growth and longer growth times than the C. glutamicum wild-type strain. Experiments with different antibiotics revealed that the susceptibility of the mutant strain was much lower than that of the wild-type C. glutamicum strain. The results presented here suggest that PorA represents a major hydrophilic pathway through the cell wall and that C. glutamicum contains cell wall channels which are not related to PorA. PMID:12896997

  10. Hexacoordinate oxy-globin models Fe(Por)(NH3)(O2) react with NO to form only the nitrato analogs Fe(Por)(NH3)(η1-ONO2), even at ~100 K.

    PubMed

    Kurtikyan, Tigran S; Ford, Peter C

    2010-12-07

    The oxy-globin models Fe(Por)(NH(3))(O(2)), prepared by sequential reactions of O(2) ((18)O(2)) and NH(3) with thin porous layers of Fe(II)(Por), react with NO ((15)NO) at 80-100 K to form only the low-spin nitrato complexes Fe(Por)(NH(3))(η(1)-ONO(2)), thus implying that peroxynitrite intermediates, if formed, must undergo very facile isomerization to the nitrato analog.

  11. Oral administration of recombinant Neisseria meningitidis PorA genetically fused to H. pylori HpaA antigen increases antibody levels in mouse serum, suggesting that PorA behaves as a putative adjuvant

    PubMed Central

    Vasquez, Abel E; Manzo, Ricardo A; Soto, Daniel A; Barrientos, Magaly J; Maldonado, Aurora E; Mosqueira, Macarena; Avila, Anastasia; Touma, Jorge; Bruce, Elsa; Harris, Paul R; Venegas, Alejandro

    2015-01-01

    The Neisseria meningitidis outer membrane protein PorA from a Chilean strain was purified as a recombinant protein. PorA mixed with AbISCO induced bactericidal antibodies against N. meningitidis in mice. When PorA was fused to the Helicobacter pylori HpaA antigen gene, the specific response against H. pylori protein increased. Splenocytes from PorA-immunized mice were stimulated with PorA, and an increase in the secretion of IL-4 was observed compared with that of IFN-γ. Moreover, in an immunoglobulin sub-typing analysis, a substantially higher IgG1 level was found compared with IgG2a levels, suggesting a Th2-type immune response. This study revealed a peculiar behavior of the purified recombinant PorA protein per se in the absence of AbISCO as an adjuvant. Therefore, the resistance of PorA to proteolytic enzymes, such as those in the gastrointestinal tract, was analyzed, because this is an important feature for an oral protein adjuvant. Finally, we found that PorA fused to the H. pylori HpaA antigen, when expressed in Lactococcus lactis and administered orally, could enhance the antibody response against the HpaA antigen approximately 3 fold. These observations strongly suggest that PorA behaves as an effective oral adjuvant. PMID:25750999

  12. Oral administration of recombinant Neisseria meningitidis PorA genetically fused to H. pylori HpaA antigen increases antibody levels in mouse serum, suggesting that PorA behaves as a putative adjuvant.

    PubMed

    Vasquez, Abel E; Manzo, Ricardo A; Soto, Daniel A; Barrientos, Magaly J; Maldonado, Aurora E; Mosqueira, Macarena; Avila, Anastasia; Touma, Jorge; Bruce, Elsa; Harris, Paul R; Venegas, Alejandro

    2015-01-01

    The Neisseria meningitidis outer membrane protein PorA from a Chilean strain was purified as a recombinant protein. PorA mixed with AbISCO induced bactericidal antibodies against N. meningitidis in mice. When PorA was fused to the Helicobacter pylori HpaA antigen gene, the specific response against H. pylori protein increased. Splenocytes from PorA-immunized mice were stimulated with PorA, and an increase in the secretion of IL-4 was observed compared with that of IFN-γ. Moreover, in an immunoglobulin sub-typing analysis, a substantially higher IgG1 level was found compared with IgG2a levels, suggesting a Th2-type immune response. This study revealed a peculiar behavior of the purified recombinant PorA protein per se in the absence of AbISCO as an adjuvant. Therefore, the resistance of PorA to proteolytic enzymes, such as those in the gastrointestinal tract, was analyzed, because this is an important feature for an oral protein adjuvant. Finally, we found that PorA fused to the H. pylori HpaA antigen, when expressed in Lactococcus lactis and administered orally, could enhance the antibody response against the HpaA antigen approximately 3 fold. These observations strongly suggest that PorA behaves as an effective oral adjuvant.

  13. A Streamlined Western Blot Exercise: An Efficient and Greener Approach in the Laboratory Classroom

    ERIC Educational Resources Information Center

    Ness, Traci L.; Robinson, Rebekah L.; Mojadedi, Wais; Peavy, Lydia; Weiland, Mitch H.

    2015-01-01

    SDS-PAGE and western blotting are two commonly taught protein detection techniques in biochemistry and molecular biology laboratory classrooms. A pitfall associated with incorporating these techniques into the laboratory is the significant wait times that do not allow students to obtain timely results. The waiting associated with SDS-PAGE comes…

  14. A Streamlined Western Blot Exercise: An Efficient and Greener Approach in the Laboratory Classroom

    ERIC Educational Resources Information Center

    Ness, Traci L.; Robinson, Rebekah L.; Mojadedi, Wais; Peavy, Lydia; Weiland, Mitch H.

    2015-01-01

    SDS-PAGE and western blotting are two commonly taught protein detection techniques in biochemistry and molecular biology laboratory classrooms. A pitfall associated with incorporating these techniques into the laboratory is the significant wait times that do not allow students to obtain timely results. The waiting associated with SDS-PAGE comes…

  15. Variation in the Neisseria lactamica porin, and its relationship to meningococcal PorB.

    PubMed

    Bennett, Julia S; Callaghan, Martin J; Derrick, Jeremy P; Maiden, Martin C J

    2008-05-01

    One potential vaccine strategy in the fight against meningococcal disease involves the exploitation of outer-membrane components of Neisseria lactamica, a commensal bacterium closely related to the meningococcus, Neisseria meningitidis. Although N. lactamica shares many surface structures with the meningococcus, little is known about the antigenic diversity of this commensal bacterium or the antigenic relationships between N. lactamica and N. meningitidis. Here, the N. lactamica porin protein (Por) was examined and compared to the related PorB antigens of N. meningitidis, to investigate potential involvement in anti-meningococcal immunity. Relationships among porin sequences were determined using distance-based methods and F(ST), and maximum-likelihood analyses were used to compare the selection pressures acting on the encoded proteins. These analyses demonstrated that the N. lactamica porin was less diverse than meningococcal PorB and although it was subject to positive selection, this was not as strong as the positive selection pressures acting on the meningococcal porin. In addition, the N. lactamica porin gene sequences and the protein sequences of the loop regions predicted to be exposed to the human immune system were dissimilar to the corresponding sequences in the meningococcus. This suggests that N. lactamica Por, contrary to previous suggestions, may have limited involvement in the development of natural immunity to meningococcal disease and might not be effective as a meningococcal vaccine component.

  16. An Analysis of Interlanguage Development Over Time: Part 1, "por" and "para".

    ERIC Educational Resources Information Center

    Guntermann, Gail

    1992-01-01

    The first part of a larger planned investigation, this study examines the use of "por" and "para" by nine Peace Corps volunteers in oral interviews at the end of training and roughly one year later, to trace their acquisition over time, in two learning contexts. (24 references) (LB)

  17. The Acquisition of Lexical Meaning in a Study Abroad Context: The Spanish Prepositions "por" and "para."

    ERIC Educational Resources Information Center

    Lafford, Barbara A.; Ryan, John M.

    1995-01-01

    Examination of the development of form/function relations of the prepositions "por" and "para" at different levels of proficiency in the interlanguage of study-abroad students in Granada, Spain, revealed "noncanonical" as well as "canonical" uses of these prepositions. The most common noncanonical uses were…

  18. Japon: Nivel 3 (Japan: Level 3).

    ERIC Educational Resources Information Center

    Sengor, Graciela

    An elementary school Spanish-as-a-foreign-language immersion program in Social Science is described. Explanations for the teacher are offered, and activities are described in detail. Exercises and evaluations are also offered. Different aspects of the Japanese way of living are used to practice the Spanish language. Some of the aspects presented…

  19. Ciencia: Nivel A (Science: Level A).

    ERIC Educational Resources Information Center

    Duron, Dolores; And Others

    A teacher's manual was developed for an elementary level science course in Spanish as part of an immersion program for English speaking children. The Level A manual is designed for kindergarten and grade 1 pupils. The five units cover the basic concepts of the weather, colors, animals, plants, and the five senses. Each unit includes vocabulary,…

  20. Matematicas: Nivel F (Mathematics: Level F).

    ERIC Educational Resources Information Center

    Duron, Dolores, Ed.; And Others

    A teacher's manual was developed for an elementary level mathematics course in Spanish as part of an immersion program for English speaking children. The Level F manual is designed for grade 5 pupils. Teaching procedures, conceptual and language objectives, vocabulary, structures, and learning activities are included. Activities are designed to…

  1. Matematicas: Nivel E (Mathematics: Level E).

    ERIC Educational Resources Information Center

    Duron, Dolores, Ed.; And Others

    A teacher's manual was developed for an elementary level mathematics course in Spanish as part of an immersion program for English speaking children. The Level E manual is designed for grade 4 pupils. Teaching procedures, conceptual and language objectives, vocabulary, structures, and learning activities are included. Activities are designed to…

  2. Matematicas: Nivel A (Mathematics: Level A).

    ERIC Educational Resources Information Center

    Duron, Dolores; And Others

    A teacher's manual was developed for an elementary level mathematics course in Spanish as part of an immersion program for English speaking children. The Level A manual is designed for kindergarten and grade 1 pupils. Teaching procedures, conceptual objectives, vocabulary, and structures are included. Activities are designed to teach either…

  3. Matematicas: Nivel A (Mathematics: Level A).

    ERIC Educational Resources Information Center

    Duron, Dolores; And Others

    A teacher's manual was developed for an elementary level mathematics course in Spanish as part of an immersion program for English speaking children. The Level A manual is designed for kindergarten and grade 1 pupils. Teaching procedures, conceptual objectives, vocabulary, and structures are included. Activities are designed to teach either…

  4. Matematicas: Nivel F (Mathematics: Level F).

    ERIC Educational Resources Information Center

    Duron, Dolores, Ed.; And Others

    A teacher's manual was developed for an elementary level mathematics course in Spanish as part of an immersion program for English speaking children. The Level F manual is designed for grade 5 pupils. Teaching procedures, conceptual and language objectives, vocabulary, structures, and learning activities are included. Activities are designed to…

  5. Matematicas: Nivel E (Mathematics: Level E).

    ERIC Educational Resources Information Center

    Duron, Dolores, Ed.; And Others

    A teacher's manual was developed for an elementary level mathematics course in Spanish as part of an immersion program for English speaking children. The Level E manual is designed for grade 4 pupils. Teaching procedures, conceptual and language objectives, vocabulary, structures, and learning activities are included. Activities are designed to…

  6. Ciencia: Nivel A (Science: Level A).

    ERIC Educational Resources Information Center

    Duron, Dolores; And Others

    A teacher's manual was developed for an elementary level science course in Spanish as part of an immersion program for English speaking children. The Level A manual is designed for kindergarten and grade 1 pupils. The five units cover the basic concepts of the weather, colors, animals, plants, and the five senses. Each unit includes vocabulary,…

  7. PREJUICIO Y DISTANCIA SOCIAL HACIA PERSONAS HOMOSEXUALES POR PARTE DE JÓVENES UNIVERSITARIOS

    PubMed Central

    Fernández Rodríguez, María del C.; Squiabro, José Calderón

    2014-01-01

    Se realizó un estudio descriptivo transversal con el propósito de explorar actitudes de rechazo y distancia social hacia las personas gays y lesbianas (GL) en 565 universitarios. Se utilizó una escala para medir Prejuicio y otra escala para medir Distancia Social. Los participantes reflejaron niveles moderados de prejuicio y distancia social (DS) hacia las personas gays y lesbianas. Los varones (M=104.5, DT= 27.47) mostraron significativamente más prejuicio que las mujeres (M=98.8, DT= 23.41). Los hombres (M=22.7, DT= 7.00) mostraron significativamente mayor DS que las mujeres (M=21.1, DT= 5.41). Las personas que asisten con regularidad a la iglesia mostraron más prejuicio y DS que los que no asisten. Se analiza importancia de incluir el tema de la diversidad sexual a través del currículo para desmontar prejuicios hacia la comunidad homosexual. PMID:25606066

  8. Detección y estudio mediante Fluorescencia Inducida por Láser de radicales libres formados por Disociación Multifotónica Infrarroja

    NASA Astrophysics Data System (ADS)

    Santos, M.; Díaz, L.; Torresano, J. A.; Rubio, L.; Samoudi, B.

    Una de las principales aplicaciones actuales de los procesos de disociación multifotónica inducidos por radiación láser infrarroja (DMI) es la producción de radiales libres, con el fin de estudiar sus propiedades cinéticas y espectroscópicas. La disociación de moléculas poliatómicas en el IR con láseres de CO2 tiene lugar desde la superficie de energía molecular mas baja y conduce generalmente a la formación de fragmentos en el estado electrónico fundamental, con diversos grados de excitación vibracional. En el Grupo de Procesos Multifotónicos del Instituto de Estructura de la Materia del C.S.I.C. hemos puesto a punto la técnica de Fluorescencia Inducida por Láser (LIF) para la detección y análisis en tiempo real de los fragmentos producidos en la DMI inducida mediante uno o dos campos láseres de diferentes longitudes de onda. Objetivos de nuestro trabajo han sido el estudio de los canales de disociación mayoritarios y de las especies transitoria producidas, así como de la distribución de energía interna con que éstas son generadas. En particular hemos detectado mediante LIF las especies: C2, CF, CH, SiH2, CF2, CH2, SiHCl, y CF3 a partir de la disociación de, entre otras, las siguientes moléculas: C2H3Br, C3F6, C4H8Si, C2H5ClSi y CH5ClSi. En este trabajo presentamos algunos de los resultados obtenidos mediante el estudio por LIF de estos radicales: estudio temporal de la señal LIF obtenida con determinación de tiempos de vida, espectros de excitación y fluorescencia, temperaturas vibracionales de formación, variación de la intensidad LIF con el tiempo de retraso entre los láseres de disociación y prueba, etc.

  9. Methodology for obtaining stakeholder assessments of obesity policy options in the PorGrow project.

    PubMed

    Stirling, A; Lobstein, T; Millstone, E

    2007-05-01

    The Policy Options for Responding to the Growing Challenge of Obesity Research Project (PorGrow) study provided a unique opportunity to develop a large-scale application of a semi-quantitative technique for exploring interviewees' views on options to tackle obesity, using multi-criteria mapping. This 'heuristic' approach utilizes the advantages of a structured interviews framework by predefining a set of options for appraisal, while leaving interviewees free to select their own criteria for making their judgements. Additional information can be gleaned from the interview transcripts and related materials to set the appraisals in their policy context, and allowing interviewees to express their views on the options presented and their own appraisals. The PorGrow study team agreed a predefined set of 20 options for appraisal, and interviewed sets of stakeholders representing more than 20 aspects of policy development in each of the nine participating countries. The details of the methodology adopted are set out in this paper.

  10. ATP for the portable 500 CFM exhauster POR-004 skid B

    SciTech Connect

    Keller, C.M.

    1997-05-06

    This Acceptance Test Plan is for a 500 CFM Portable Exhauster POR-004 to be used for saltwell pumping. The Portable Exhauster System will be utilized to eliminate potential flammable gases that may exist within the dome space of the tank. This Acceptance Plan will test and verify that the exhauster meets the specified design criteria, safety requirements, operations requirements, and will provide a record of the functional test results.

  11. ATP for the portable 500 CFM exhauster POR-005 skid C

    SciTech Connect

    Keller, C.M.

    1997-06-27

    This Acceptance Test Plan is for a 500 CFM Portable Exhauster POR-005 to be used for saltwell pumping. The Portable Exhauster System will be utilized to eliminate potential flammable gases that may exist within the dome space of the tank. This Acceptance Plan will test and verify that the exhauster meets the specified design criteria, safety requirements, operations requirements, and will provide a record of the functional test results.

  12. Delayed diagnosis of disorder of sex development (DSD) due to P450 oxidoreductase (POR) deficiency.

    PubMed

    Koika, Vasiliki; Armeni, Anastasia K; Georgopoulos, Neoklis A

    2016-04-01

    A 36-year old man, operated on for cryptorchidism at the age of 8 years, was referred to the Outpatient Clinic of Reproductive Endocrinology for investigation of infertility. Clinical examination revealed ambiguous genitalia: penis 4-5 cm, testicular volume 2-3 ml, hypospadias, hypertrophic foreskin and scrotum bifida. Mild hypertension was confirmed. No skeletal malformations were detected. Hormonal and electrolytic determinations as well as semen analysis were conducted. PCR of the coding regions of 17-hydroxylase/17,20 lyase (P450c17) and of P450 oxidoreductase (POR) genes was also performed. Normal levels of electrolytes, low levels of androgens, high levels of gonadotropins and 17-hydroxyprogesterone as well as azoospermia were detected. Karyotype was shown to be 46,XY. Both hCG and ACTH stimulation significantly increased 17-hydroxyprogesterone with no increase in androgens. The diagnosis was congenital adrenal hyperplasia with apparent combined P450c17 and P450c21 deficiency due to mutations in the POR gene. Sequencing of the POR gene revealed: one deletion in exon 12 (Del 1696_1698delGTC >del531Valine) and one missense mutation in exon 7 (A259G) as well as two polymorphisms: rs1057868 (C/T A503V) and rs1057870 (G/A S572S) in exons 12 and 13, respectively. No nucleotide changes were detected in the 8 exons of P450c17. Molecular findings were consistent with the diagnosis of P450 oxidoreductase deficiency. Despite this severe deficiency, skeletal malformations simulating Antley-Bixler syndrome, which usually characterize the most severe forms, were not confirmed. This discrepancy could be attributed to the differential impact of a POR variant on each one of the P450 enzymes.

  13. ATP for the portable 500 CFM exhauster POR-006 skid D

    SciTech Connect

    Keller, C.M.

    1997-07-29

    This Acceptance Test Plan is for a 500 CFM Portable Exhauster POR-006 to be used for saltwell pumping. The Portable Exhauster System will be utilized to eliminate potential flammable gases that may exist within the dome space of the tank. This Acceptance Plan will test and verify that the exhauster meets the specified design criteria, safety requirements, operations requirements, and will provide a record of the functional test results.

  14. A Lack of Significant Effect of POR*28 Allelic Variant on Tacrolimus Exposure in Kidney Transplant Recipients.

    PubMed

    Jannot, Anne-Sophie; Vuillemin, Xavier; Etienne, Isabelle; Buchler, Mathias; Hurault de Ligny, Bruno; Choukroun, Gabriel; Colosio, Charlotte; Thierry, Antoine; Vigneau, Cécile; Moulin, Bruno; Rerolle, Jean-Philippe; Heng, Anne-Elizabeth; Subra, Jean-Francois; Legendre, Christophe; Beaune, Philippe; Loriot, Marie Anne; Thervet, Eric; Pallet, Nicolas

    2016-04-01

    POR*28 is a recently newly described allelic variant of the cytochrome P450 oxidoreductase (POR), which might be associated with an increased metabolic activity of P450 cytochromes (CYP) 3A5 and 3A4. Consequently, carriers of at least 1 allele of this polymorphism could require increased calcineurin inhibitors doses to reach the target residual concentrations (C0). The objective of this study was to test whether the allelic variant of POR, which is associated with an increased metabolic activity of CYP3A, impacts tacrolimus (Tac) pharmacokinetics. We tested this hypothesis in a population of 229 kidney transplant recipients (KTR) from a large, multicenter, prospective and randomized study. We have analyzed the association between POR*28 genotype and the proportion of individuals reaching the target Tac residual concentration (Tac C0) 10 days after transplantation. We have also measured the association between POR*28 and the Tac C0, and adjusted Tac C0 (Tac C0/Tac dose) over time using generalized mixed linear models. Ten days after transplantation, there was no difference of frequencies of KTR within the target range of Tac C0 (C0 10-15 ng/mL) according to the POR*28 genotype (P = 0.8). The mean Tac C0 at day 10 in the POR*1/*1 group was 15.3 ± 9.7 ng/mL compared with 15.7 ± 7.8 ng/mL in the POR*1/*28 group and 14.2 ± 6.8 ng/mL, in the POR*28/*28 group, P = 0.8. The adjusted Tac C0 was not associated with POR*28 genotype over time (random effects model, P = 0.9). When restricted to KTR expressing CYP3A5, POR*28 genotype did not impact the proportion of individuals within the Tac C0 target range neither the adjusted Tac C0 (random effects model, P = 0.1). POR*28 does not significantly influence Tac pharmacokinetic parameters in a large cohort of KTR. This study does not confirm recent findings indicating that POR*28 carriers require more Tac to reach target C0.

  15. P450 oxidoreductase *28 (POR*28) and tacrolimus disposition in pediatric kidney transplant recipients--a pilot study.

    PubMed

    Gijsen, Violette M G J; van Schaik, Ron H N; Soldin, Offie P; Soldin, Steven J; Nulman, Irena; Koren, Gideon; de Wildt, Saskia N

    2014-04-01

    Both age and CYP3A5 genotype are important determinants of tacrolimus disposition in pediatric kidney transplant recipients. In a recent study in adults, POR*28 was associated with increased dosing requirements early after transplant of CYP3A5-expressing kidney transplant recipients. The authors aimed to evaluate the additional contribution of POR*28 to early tacrolimus disposition in pediatric kidney transplant recipients. Retrospective data of 43 pediatric kidney transplant recipients up to 14 days posttransplant were evaluated on tacrolimus dose and tacrolimus predose blood concentrations. Recipient POR*28 and CYP3A5 genotype were determined. CYP3A5 expressers carrying at least 1 POR*28 allele had on average 18.3% lower tacrolimus predose concentrations and 20.2% lower concentration/dose ratios compared with CYP3A5 expressers with POR*1/*1 genotype (P = 0.002 and P = 0.001, respectively). In CYP3A5 nonexpressers, tacrolimus disposition did not significantly differ between POR genotypes. In this small cohort of pediatric kidney transplant recipients, POR*28 genotype seems to explain part of the variability found in tacrolimus disposition, in addition to age and CYP3A5 genotype. This finding should be validated in a larger population, and it would be worthwhile to evaluate the clinical impact of this genotype.

  16. Two NADPH: Protochlorophyllide Oxidoreductase (POR) Isoforms Play Distinct Roles in Environmental Adaptation in Rice.

    PubMed

    Kwon, Choon-Tak; Kim, Suk-Hwan; Song, Giha; Kim, Dami; Paek, Nam-Chon

    2017-12-01

    NADPH: protochlorophyllide oxidoreductase (POR) is an essential enzyme that catalyzes the photoreduction of protochlorophyllide to chlorophyllide, which is ultimately converted to chlorophyll in developing leaves. Rice has two POR isoforms, OsPORA and OsPORB. OsPORA is expressed in the dark during early leaf development; OsPORB is expressed throughout leaf development regardless of light conditions. The faded green leaf (fgl) is a loss-of-function osporB mutant that displays necrotic lesions and variegation in the leaves due to destabilized grana thylakoids, and has increased numbers of plastoglobules in the chloroplasts. To investigate whether the function of OsPORA can complement that of OsPORB, we constitutively overexpressed OsPORA in fgl mutant. In the 35S:OsPORA/fgl (termed OPAO) transgenic plants, the necrotic lesions of the mutant disappeared and the levels of photosynthetic pigments and proteins, as well as plastid structure, were recovered in developing leaves under natural long days in the paddy field and under short days in an artificially controlled growth room. Under constant light conditions, however, total chlorophyll and carotenoid levels in the developing leaves of OPAO plants were lower than those of wild type. Moreover, the OPAO plants exhibited mild defects in mature leaves beginning at the early reproductive stage in the paddy field. The physiological function of OsPORB in response to constant light or during reproductive growth cannot be completely replaced by constitutive activity of OsPORA, although the biochemical functions of OsPORA and OsPORB are redundant. Therefore, we suggest that the two OsPORs have differentiated over the course of evolution, playing distinct roles in the adaptation of rice to the environment.

  17. Genetic polymorphisms and haplotypes of por, encoding cytochrome p450 oxidoreductase, in a Japanese population.

    PubMed

    Saito, Yoshiro; Yamamoto, Noboru; Katori, Noriko; Maekawa, Keiko; Fukushima-Uesaka, Hiromi; Sugimoto, Daisuke; Kurose, Kouichi; Sai, Kimie; Kaniwa, Nahoko; Sawada, Jun-Ichi; Kunitoh, Hideo; Ohe, Yuichiro; Yoshida, Teruhiko; Matsumura, Yasuhiro; Saijo, Nagahiro; Okuda, Haruhiro; Tamura, Tomohide

    2011-01-01

    Cytochrome P450 oxidoreductase (POR) transfers electrons from NADPH to all microsomal cytochrome P450 (CYP) enzymes and is necessary for microsomal CYP activities. In this study, to find genetic variations and to elucidate the haplotype structures of POR, we comprehensively screened the genetic variations in the 5'-flanking region, all the exons and their flanking introns of POR for 235 Japanese subjects. Seventy-five genetic variations including 26 novel ones were found: 7 were in the 5'-flanking region, 2 in the 5'-untranslated region (5'-UTR, non-coding exon 1), 16 in the coding exons (10 nonsynonymous and 6 synonymous), 45 in the introns, 4 in the 3'-UTR and 1 in the 3'-flanking region. Of these, 4 novel nonsynonymous variations, 86C>T (T29M), 1648C>T (R550W), 1708C>T (R570C) and 1975G>A (A659T), were detected with allele frequencies of 0.002. We also detected known nonsynonymous SNPs 683C>T (P228L), 1237G>A (G413S), 1453G>A (A485T), 1508C>T (A503V), 1510G>A (G504R) and 1738G>C (E580Q) with frequencies of 0.002, 0.009, 0.002, 0.434, 0.002 and 0.002, respectively. Based on the linkage disequilibrium (LD) profiles, the analyzed region could be divided into two LD blocks. For Blocks 1 and 2, 14 and 46 haplotypes were inferred, respectively, and 2 and 6 common haplotypes found in more than 0.03 frequencies accounted for more than 81% of the inferred haplotypes. This study provides fundamental and useful information for the pharmacogenetic studies of drugs metabolized by CYPs in the Japanese population.

  18. Zinc finger nuclease knock-out of NADPH:cytochrome P450 oxidoreductase (POR) in human tumor cell lines demonstrates that hypoxia-activated prodrugs differ in POR dependence.

    PubMed

    Su, Jiechuang; Gu, Yongchuan; Pruijn, Frederik B; Smaill, Jeff B; Patterson, Adam V; Guise, Christopher P; Wilson, William R

    2013-12-27

    Hypoxia, a ubiquitous feature of tumors, can be exploited by hypoxia-activated prodrugs (HAP) that are substrates for one-electron reduction in the absence of oxygen. NADPH:cytochrome P450 oxidoreductase (POR) is considered one of the major enzymes responsible, based on studies using purified enzyme or forced overexpression in cell lines. To examine the role of POR in HAP activation at endogenous levels of expression, POR knock-outs were generated in HCT116 and SiHa cells by targeted mutation of exon 8 using zinc finger nucleases. Absolute quantitation by proteotypic peptide mass spectrometry of DNA sequence-confirmed multiallelic mutants demonstrated expression of proteins with residual one-electron reductase activity in some clones and identified two (Hko2 from HCT116 and S2ko1 from SiHa) that were functionally null by multiple criteria. Sensitivities of the clones to 11 HAP (six nitroaromatics, three benzotriazine N-oxides, and two quinones) were compared with wild-type and POR-overexpressing cells. All except the quinones were potentiated by POR overexpression. Knocking out POR had a marked effect on antiproliferative activity of the 5-nitroquinoline SN24349 in both genetic backgrounds after anoxic exposure but little or no effect on activity of most other HAP, including the clinical stage 2-nitroimidazole mustard TH-302, dinitrobenzamide mustard PR-104A, and benzotriazine N-oxide SN30000. Clonogenic cell killing and reductive metabolism of PR-104A and SN30000 under anoxia also showed little change in the POR knock-outs. Thus, although POR expression is a potential biomarker of sensitivity to some HAP, identification of other one-electron reductases responsible for HAP activation is needed for their rational clinical development.

  19. Zinc Finger Nuclease Knock-out of NADPH:Cytochrome P450 Oxidoreductase (POR) in Human Tumor Cell Lines Demonstrates That Hypoxia-activated Prodrugs Differ in POR Dependence*

    PubMed Central

    Su, Jiechuang; Gu, Yongchuan; Pruijn, Frederik B.; Smaill, Jeff B.; Patterson, Adam V.; Guise, Christopher P.; Wilson, William R.

    2013-01-01

    Hypoxia, a ubiquitous feature of tumors, can be exploited by hypoxia-activated prodrugs (HAP) that are substrates for one-electron reduction in the absence of oxygen. NADPH:cytochrome P450 oxidoreductase (POR) is considered one of the major enzymes responsible, based on studies using purified enzyme or forced overexpression in cell lines. To examine the role of POR in HAP activation at endogenous levels of expression, POR knock-outs were generated in HCT116 and SiHa cells by targeted mutation of exon 8 using zinc finger nucleases. Absolute quantitation by proteotypic peptide mass spectrometry of DNA sequence-confirmed multiallelic mutants demonstrated expression of proteins with residual one-electron reductase activity in some clones and identified two (Hko2 from HCT116 and S2ko1 from SiHa) that were functionally null by multiple criteria. Sensitivities of the clones to 11 HAP (six nitroaromatics, three benzotriazine N-oxides, and two quinones) were compared with wild-type and POR-overexpressing cells. All except the quinones were potentiated by POR overexpression. Knocking out POR had a marked effect on antiproliferative activity of the 5-nitroquinoline SN24349 in both genetic backgrounds after anoxic exposure but little or no effect on activity of most other HAP, including the clinical stage 2-nitroimidazole mustard TH-302, dinitrobenzamide mustard PR-104A, and benzotriazine N-oxide SN30000. Clonogenic cell killing and reductive metabolism of PR-104A and SN30000 under anoxia also showed little change in the POR knock-outs. Thus, although POR expression is a potential biomarker of sensitivity to some HAP, identification of other one-electron reductases responsible for HAP activation is needed for their rational clinical development. PMID:24196959

  20. Comparison of Serologic and Genetic porB-Based Typing of Neisseria gonorrhoeae: Consequences for Future Characterization

    PubMed Central

    Unemo, Magnus; Olcén, Per; Albert, Jan; Fredlund, Hans

    2003-01-01

    Due to temporal changes in the epidemiology of gonorrhea, a precise characterization of Neisseria gonorrhoeae is essential. In the present study genetic heterogeneity in the porB genes of N. gonorrhoeae was examined, and serovar determination was compared to porB gene sequencing. Among 108 N. gonorrhoeae isolates, phylogenetic analysis of the entire porB alleles (924 to 993 bp) identified 87 unique sequences. By analyzing only the four to six most heterogeneous porB gene regions (174 to 363 bp), 86 out of these 87 genetic variants were identified. Consequently, analysis of shorter highly variable regions of the porB gene generates high-level discriminatory ability as well as fast, objective, reproducible, and portable data for epidemiological characterization of N. gonorrhoeae. Regarding putative antigenic epitopes of PorB for Genetic Systems monoclonal antibodies (MAbs), some of the previous findings were confirmed, but new findings were also observed. For several of the MAbs, however, the precise amino acid residues of PorB critical for single-MAb reactivity were difficult to identify. In addition, repeated serovar determination of 108 N. gonorrhoeae isolates revealed discrepancies for 34 isolates, mostly due to nonreproducible reactivity with single MAbs. Thus, the prospects of a genetic typing system with congruent translation of the serovar determination seem to be limited. In conclusion, analysis of short highly variable regions of the porB gene could form the basis for a fast molecular epidemiological tool for the examination of emergence and transmission of N. gonorrhoeae strains within the community. PMID:12958238

  1. Improved purification of native meningococcal porin PorB and studies on its structure/function.

    PubMed

    Massari, Paola; King, Carol A; MacLeod, Heather; Wetzler, Lee M

    2005-12-01

    The outer membrane protein PorB of Neisseria meningitidis is a pore-forming protein which has various effects on eukaryotic cells. It has been shown to (1) up-regulate the surface expression of the co-stimulatory molecule CD86 and of MHC class II (which are TLR2/MyD88 dependent and related to the porin's immune-potentiating ability), (2) be involved in prevention of apoptosis by modulating the mitochondrial membrane potential, and (3) form pores in eukaryotic cells. As an outer membrane protein, its native trimeric form isolation is complicated by its insoluble nature, requiring the presence of detergent throughout the whole procedure, and by its tight association with other outer membrane components, such as neisserial LOS or lipoproteins. In this study, an improved chromatographic purification method to obtain an homogeneous product free of endotoxin and lipoprotein is described, without loss of any of the above-mentioned properties of the porin. Furthermore, we have investigated the requirement of the native trimeric structure for the porin's activity. Inactivation of functional PorB trimers into non-functional monomers was achieved by incubation on ice. Thus, routine long- and medium-term storage at low temperature may be a cause of porin inactivation.

  2. Diagnóstico diferencial en la encefalitis por anticuerpos contra el receptor NMDA

    PubMed Central

    González-Valcárcel, J.; Rosenfeld, M.R.; Dalmau, J.

    2011-01-01

    Resumen Introducción La encefalitis por anticuerpos contra el receptor de NMDA (NMDAR) suele desarrollarse como un síndrome característico de evolución multifásica y diagnóstico diferencial amplio. Pacientes Presentamos a 2 pacientes diagnosticadas de encefalitis por anticuerpos NMDAR con un cuadro clínico típico, pero que inicialmente señaló otras etiologías. Discusión La afectación frecuente de pacientes jóvenes con manifestaciones psiquiátricas prominentes indica frecuentemente otras consideraciones diagnósticas; las más frecuentes son las encefalitis virales, los procesos psiquiátricos y el síndrome neuroléptico maligno. Varios síndromes previamente definidos de manera parcial o descriptiva en adultos y pacientes pediátricos probablemente eran casos de encefalitis anti-NMDAR. Conclusiones La encefalitis anti-NMDAR debe considerarse en pacientes jóvenes con manifestaciones psiquiátricas subagudas, movimientos anormales y alteraciones autonómicas. La caracterización clínica e inmunológica de esta enfermedad ha llevado a la identificación de nuevos anticuerpos que afectan a procesos de memoria, aprendizaje, conducta y psicosis. PMID:20964986

  3. Effect of SPM-based cleaning POR on EUV mask performance

    NASA Astrophysics Data System (ADS)

    Choi, Jaehyuck; Lee, Han-shin; Yoon, Jinsang; Shimomura, Takeya; Friz, Alex; Montgomery, Cecilia; Ma, Andy; Goodwin, Frank; Kang, Daehyuk; Chung, Paul; Shin, Inkyun; Cho, H.

    2011-11-01

    EUV masks include many different layers of various materials rarely used in optical masks, and each layer of material has a particular role in enhancing the performance of EUV lithography. Therefore, it is crucial to understand how the mask quality and patterning performance can change during mask fabrication, EUV exposure, maintenance cleaning, shipping, or storage. The fact that a pellicle is not used to protect the mask surface in EUV lithography suggests that EUV masks may have to undergo more cleaning cycles during their lifetime. More frequent cleaning, combined with the adoption of new materials for EUV masks, necessitates that mask manufacturers closely examine the performance change of EUV masks during cleaning process. We have investigated EUV mask quality and patterning performance during 30 cycles of Samsung's EUV mask SPM-based cleaning and 20 cycles of SEMATECH ADT exposure. We have observed that the quality and patterning performance of EUV masks does not significantly change during these processes except mask pattern CD change. To resolve this issue, we have developed an acid-free cleaning POR and substantially improved EUV mask film loss compared to the SPM-based cleaning POR.

  4. Multiple active site residues are important for photochemical efficiency in the light-activated enzyme protochlorophyllide oxidoreductase (POR).

    PubMed

    Menon, Binuraj R K; Hardman, Samantha J O; Scrutton, Nigel S; Heyes, Derren J

    2016-08-01

    Protochlorophyllide oxidoreductase (POR) catalyzes the light-driven reduction of protochlorophyllide (Pchlide), an essential, regulatory step in chlorophyll biosynthesis. The unique requirement of the enzyme for light has provided the opportunity to investigate how light energy can be harnessed to power biological catalysis and enzyme dynamics. Excited state interactions between the Pchlide molecule and the protein are known to drive the subsequent reaction chemistry. However, the structural features of POR and active site residues that are important for photochemistry and catalysis are currently unknown, because there is no crystal structure for POR. Here, we have used static and time-resolved spectroscopic measurements of a number of active site variants to study the role of a number of residues, which are located in the proposed NADPH/Pchlide binding site based on previous homology models, in the reaction mechanism of POR. Our findings, which are interpreted in the context of a new improved structural model, have identified several residues that are predicted to interact with the coenzyme or substrate. Several of the POR variants have a profound effect on the photochemistry, suggesting that multiple residues are important in stabilizing the excited state required for catalysis. Our work offers insight into how the POR active site geometry is finely tuned by multiple active site residues to support enzyme-mediated photochemistry and reduction of Pchlide, both of which are crucial to the existence of life on Earth.

  5. Lecciones de Quechua. Primer Nivel, Segundo Nivel (Quechua Lessons. First Level, Second Level).

    ERIC Educational Resources Information Center

    Herrero, Joaquin; And Others

    The two volumes comprising this text represent the beginning and intermediate levels of a course in the Quechua language for speakers of Spanish. Based on the "Audio Lingual Materials" format, the ten lessons in the the first volume generally contain: (1) a short dialog and a supplementary vocabulary section or adapted dialog which…

  6. Estudio de la estructura logica utilizada en la ensenanza y el aprendizaje de los conceptos sobre el comportamiento de gases en el curso introductorio de quimica a nivel universitario

    NASA Astrophysics Data System (ADS)

    Costa Diaz, Agnes

    El estudio que se presenta es de caracter cualitativo, un estudio multicasos donde se estudia la estructura logica utilizada por cuatro (4) profesores universitarios que ensenan el curso introductorio de quimica, en la planificacion, presentacion y evaluacion del tema sobre el comportamiento de los gases. Se utilizaron varias fuentes de informacion como: cuestionarios de profesores y estudiantes, entrevistas, grabaciones videomagnetofonicas, materiales didacticos y una prueba conceptual, entre otros. La informacion recopilada fue analizada de acuerdo al orden logico del contenido presentado, el estilo de ensenanza del profesor, las tecnicas y estrategias utilizadas para el desarrollo de destrezas de pensamiento, el ambiente fisico en el salon de clase y los instrumentos de evaluacion y avaluo. El estudio demuestra que lo que los profesores piensan y planifican para hacer sus presentaciones no necesariamente es lo que ocurre en el salon de clases. El desarrollo de destrezas de pensamiento, que constituye una prioridad de los profesores, no se elaboran efectivamente. El uso de las estrategias de resolucion de problemas numericos predomino. La participacion del estudiante en el salon de clases fue limitada y no se logro demostrar el desarrollo de las destrezas de pensamiento deseadas. Aunque los profesores tienen su propio estilo de ensenanza, el orden logico del contenido presentado en clase fue el mismo o siguio muy de cerca el orden establecido por el libro de texto. Los profesores utilizaron preferentemente la tiza y la pizarra para sus presentaciones y la dinamica en el salon de clases fue esencialmente tradicional. Los profesores hicieron su presentacion y los estudiantes copiaron pasivamente la informacion. Las evaluaciones de los estudiantes fueron esencialmente, pruebas escritas de seleccion multiple de acuerdo con el estilo en que se les enseno. El avaluo fue casi inexistente. La prueba conceptual administrada revela un aprendizaje pobre en los conceptos mas

  7. Effects of heme precursors on CYP1A2 and POR expression in the baculovirus/Spodoptera frugiperda system.

    PubMed

    Lu, Huiyuan; Ma, Jun; Liu, Nian; Wang, Shoulin

    2010-05-01

    CYP1A2 and NADPH-CYP450 oxidoreductase (POR) were expressed in the baculovirus/Spodoptera frugiperda (sf9) system. The aim of this study was to investigate the effects of heme precursors on the expression of CYP1A2 and POR. The heme precursors [δ-Aminolaevulinic Acid (5-ALA), Fe(3+) and hemin] were introduced into the system to evaluate their effects on the expression of CYP1A2, POR and their co-expression. All the proteins were identified using immunoblotting, CO-difference spectroscopy, or cytochrome c assay. In the present study, functional CYP1A2 and POR were successfully expressed in the baculovirus/sf9 system, and both of them showed high activities. Co-addition of 5-ALA and Fe(3+) significantly improved expression of CYP1A2 by about 50% compared with the addition of 5-ALA, Fe(3+) or hemin alone. Either co-addition of 5-ALA and Fe(3+) or addition of 5-ALA or Fe(3+) alone improved the POR expression level 2 fold and its activity 7-10 fold compared with control (no addition). However, unlike CYP1A2, there was no difference between the co-addition and addition of these heme precursors alone. Different ratios of BvCYP1A2 to BvPOR also affected the co-expression of CYP1A2 and POR, with a 3:1 ratio of BvCYP1A2 / BvPOR significantly increasing their co-expression. Surprisingly, the addition of 0.1 mM 5-ALA or Fe(3+) alone, but not their co-addition, could significantly improve the CYP1A2 and POR co-expression (P < 0.05). 5-ALA and Fe(3+) increased the expression of CYP1A2 and POR in a baculovirus/sf9 system, but the pattern of their expression was different between their expression alone and co-expression.

  8. Reconstrução tridimensional de arcos magnéticos por tomografia

    NASA Astrophysics Data System (ADS)

    Simões, P. J. A.; Costa, J. E. R.

    2003-08-01

    Uma explosão solar é uma variação súbita do brilho que ocorre nas regiões ativas da atmosfera solar. Estas regiões são constituídas por um plasma magnetizado com intensa indução magnética e em cenários bem complexos como visto recentemente através de experimentos embarcados em satélites operando instrumentos em raios X moles e ultra-violeta distante. A energia magnética, que pode ser armazenada por um período de horas até dias em configurações magnéticas estressadas, é subitamente lançada na atmosfera solar e transferida para partículas como elétrons, prótons e núcleos pesados, que são acelerados e/ou aquecidos, produzindo radiação eletromagnética. A proposta final deste projeto é determinar as características espaciais de alta resolução da emissão e polarização girossincrotrônica de explosões solares em ambientes complexos de campos magnéticos. Os recentes resultados da emissão difusa em EUV apresentado pelos satélites TRACE e SOHO dos arcos magnéticos conectando as diferentes polaridades magnéticas sobre as regiões ativas possibilitam novas abordagens sobre o papel do campo magnético na emissão em rádio. Nesta etapa apresentamos os resultados da reconstrução da geometria tridimensional das linhas de força destes arcos utilizando técnicas tomográficas, a partir de imagens de alta resolução espacial obtidas pelo instrumento EIT (Extreme ultraviolet Imaging Telescope), além da modelagem das induções magnéticas por um campo dipolar e as densidades de partículas aceleradas. Utilizamos para a reconstrução geométrica, imagens tomadas em vários ângulos dos arcos devido à rotacão solar. Com estes resultados, daremos continuidade ao projeto, com os cálculos da transferência radiativa nos modos ordinário e extraordinário de propagação da radiação girossincrotrônica de explosões solares.

  9. Intermitência alfvênica gerada por caos na atmosfera solar e no vento solar

    NASA Astrophysics Data System (ADS)

    Rempel, E. L.; Chian, A. C.-L.; Macau, E. E. N.; Rosa, R. R.

    2003-08-01

    Dados medidos no vento solar rápido proveniente dos buracos coronais revelam que os plasmas no meio interplanetário são dominados por flutuações Alfvênicas, caracterizadas por uma alta correlação entre as variações do campo magnético e da velocidade do plasma. As flutuações exibem muitas características esperadas em turbulência magneto-hidrodinâmica totalmente desenvolvida, tais como intermitência e espectros contínuos. Contudo, os mecanismos responsáveis pela evolução de turbulência Alfvênica intermitente não são completamente compreendidos. Neste trabalho a teoria de caos é usada para explicar como sistemas Alfvênicos, modelados pela equação Schrödinger não-linear derivativa e pela equação Kuramoto-Sivashinsky, podem se tornar fortemente caóticos à medida em que parâmetros do plasma são variados. Pequenas perturbações no parâmetro de dissipação podem fazer com que o sistema mude bruscamente de um regime periódico, ou fracamente caótico, para um regime fortemente caótico. As séries temporais das flutuações do campo magnético nos regimes fortemente caóticos exibem comportamento intermitente, em que fases laminares ou fracamente caóticas são interrompidas por fortes estouros caóticos. É mostrado que o regime fortemente caótico é atingido quando as soluções periódicas ou fracamente caóticas globalmente estáveis interagem com soluções do sistema que são fortemente caóticas, mas globalmente instáveis. Estas soluções globalmente instáveis são conjuntos caóticos não-atrativos conhecidos como selas caóticas, e são responsáveis pelos fortes estouros nos regimes intermitentes. Selas caóticas têm sido detectadas experimentalmente em uma grande variedade de sistemas, sendo provável que elas desempenhem um papel importante na turbulência intermitente observada em plasmas espaciais.

  10. Geometry sensing through POR1 regulates Rac1 activity controlling early osteoblast differentiation in response to nanofiber diameter.

    PubMed

    Higgins, A M; Banik, B L; Brown, J L

    2015-02-01

    Bone grafting procedures in the United States rely heavily upon autografts and allografts, which are donor-dependent, cause donor site pain, and can transmit disease. Synthetic bone grafts can reduce these risks; however, synthetics lack the bone differentiating (osteoinductive) abilities of auto- and allografts. Achieving innate osteoinductive properties of synthetics through surface modifications is currently under investigation. This study focuses on nanofibers, with emphasis on how fiber diameter and the potential curvature sensor POR1 affect the activation of the signaling molecules Rac1 and Arf1, and leading to expression of alkaline phosphatase (ALP), an osteoinductive marker. Diameters of 0.1, 0.3, and 1.0 μm were compared against a flat control. The highest level of Rac1 activation was achieved on the smallest fibers (0.1 μm), a trend that was lost in POR1 knockdowns. This supports the hypothesis that on small nanofibers, POR1 favorably binds to highly curved cell membranes, which allows Rac1 to subsequently dissociate and activate. When the curvature is insufficient to bind POR1, POR1 binds to inactive Rac1 and competitively inhibits its activation. Arf1 activation followed an opposite trend, with the largest nanofibers exhibiting the highest activity. This trend reinforces the known interaction between Rac1 and Arf1 through the GIT-PIX complex, an Arf1 GAP and Rac1 GEF, respectively. Large, (1.0 μm), nanofibers demonstrated the highest ALP activity, indicating that ALP expression is inversely dependent on Rac1 activation. Knockdown of POR1 resulted in increased ALP activity across the substrates but without regard to the curvature sensing trend seen previously. Thus, POR1 senses curvature and increases Rac1 activity, which negatively regulates bone differentiation.

  11. Interaction of nitrogen bases with iron-porphyrin nitrito complexes Fe(Por)(ONO) in sublimed solids.

    PubMed

    Kurtikyan, Tigran S; Hovhannisyan, Astghik A; Gulyan, Gurgen M; Ford, Peter C

    2007-08-20

    The reactions of the nitrogen Lewis bases (B) 1-methylimidazole (1-MeIm), pyridine (Py), and NH3 as gases with sublimed layers containing the 5-coordinate nitrito iron(III)-porphyrinato complexes Fe(Por)(eta1-ONO) (1) are described (Por = meso-tetraphenyl-porphyrinato or meso-tetra-p-tolyl-porphyrinato dianions). In situ FTIR and optical spectra are used to characterize the formation of the 6-coordinate nitro complexes formed by the reaction of 1 with B = 1-MeIm, Py, or NH3. These represent the first examples of 6-coordinate amino-nitro complexes with sterically unprotected iron-porphyrins. The interaction of ammonia with Fe(Por)(ONO) at 140 K initially led to the nitrito species Fe(Por)(NH3)(eta1-ONO), and this species isomerized to the nitro complexes Fe(Por)(NH3)(eta1-NO2) upon warming to 180 K. When the latter were warmed to room temperature under intense pumping, the initial nitrito complexes Fe(Por)(eta1-ONO) were restored. Assignments of vibrational frequencies for the coordinated nitro group in 6-coordinate iron-porphyrin complexes are given and confirmed using 15N-labeled nitrogen dioxide to identify characteristic infrared bands. For M(Por)(B)(NO2) complexes (M = Fe or Co), an inverse correlation between the net charge transfer from the axial ligand B to the nitro group and the value of Deltanu = nua(NO2) - nus(NO2) is proposed. These observations are discussed in the context of growing interest in potential physiological roles of nitrite ion reactions with ferro- and ferri-heme proteins.

  12. Using the PORS Problems to Examine Evolutionary Optimization of Multiscale Systems

    SciTech Connect

    Reinhart, Zachary; Molian, Vaelan; Bryden, Kenneth

    2013-01-01

    Nearly all systems of practical interest are composed of parts assembled across multiple scales. For example, an agrodynamic system is composed of flora and fauna on one scale; soil types, slope, and water runoff on another scale; and management practice and yield on another scale. Or consider an advanced coal-fired power plant: combustion and pollutant formation occurs on one scale, the plant components on another scale, and the overall performance of the power system is measured on another. In spite of this, there are few practical tools for the optimization of multiscale systems. This paper examines multiscale optimization of systems composed of discrete elements using the plus-one-recall-store (PORS) problem as a test case or study problem for multiscale systems. From this study, it is found that by recognizing the constraints and patterns present in discrete multiscale systems, the solution time can be significantly reduced and much more complex problems can be optimized.

  13. Fabrication of por-Si/SnO{sub x} nanocomposite layers for gas microsensors and nanosensors

    SciTech Connect

    Bolotov, V. V. Korusenko, P. M.; Nesov, S. N.; Povoroznyuk, S. N.; Roslikov, V. E.; Kurdyukova, E. A.; Sten'kin, Yu. A.; Shelyagin, R. V.; Knyazev, E. V.; Kan, V. E.; Ponomareva, I. V.

    2011-05-15

    Two-phase nanocomposite layers based on porous silicon and nonstoichiometric tin oxide were fabricated by various methods. The structure, as well as elemental and phase composition, of the obtained nanocomposites were studied using transmission and scanning electron microscopy, Raman spectroscopy, Auger electron spectroscopy, and X-ray photoelectron spectroscopy. The results obtained confirm the formation of nanocomposite layers with a thickness as large as 2 {mu}m thick and SnO{sub x} stoichiometry coefficients x = 1.0-2.0. Significant tin diffusion into the porous silicon matrix with D{sub eff} Almost-Equal-To 10{sup -14} cm{sup 2} s{sup -1} was observed upon annealing at 770 K. Test sensor structures based on por-Si/SnO{sub x} nanocomposite layers grown by magnetron deposition showed fairly high stability of properties and sensitivity to NO{sub 2}.

  14. Impact of PPARA and POR polymorphisms on tacrolimus pharmacokinetics and new-onset diabetes in kidney transplant recipients.

    PubMed

    Kurzawski, Mateusz; Malinowski, Damian; Dziewanowski, Krzysztof; Droździk, Marek

    2014-08-01

    Recent efforts have been made to identify genetic markers of CYP3A4 enzymatic activity within genes encoding for regulatory elements. The aim of the current study was to investigate the impact of polymorphism of PPARA and POR genes on tacrolimus (TAC) dose-adjusted trough concentration and risk of new-onset diabetes after transplantation (NODAT). A total of 241 White kidney transplant patients were genotyped for three functional single nucleotide polymorphisms: rs1057868 (*28) in POR, rs4253728:G>A, and rs4823613:A>G in PPARA. No significant genotype-dependent differences in TAC dose-adjusted trough concentration were observed for either POR or PPARA variants. No significant differences in the incidence of NODAT were observed between patients stratified by PPARA and POR genotypes. The frequency of NODAT among PPARA rs4253728 AA homozygotes (42%) was higher compared with heterozygotes (22%) and GG homozygotes (19%), but the difference was not significant. Testing TAC-medicated renal transplant recipients for POR and PPARA variants seems to have limited clinical application.

  15. Host Cell Virus Entry Mediated by Australian Bat Lyssavirus Envelope G glycoprotein

    DTIC Science & Technology

    2013-10-24

    constructs .............................................. 104 Figure 22. SDS PAGE (A) and Native PAGE (B) analysis of purified ABLVs Gs. ......... 107...centrifugation and analyzed by 4-12% BT SDS PAGE. An equivalent of 1x10 6 cells per well were analyzed as described in the methods. nAchR was detected...10 min at 4°C and 20 µl of prepared lysate were loaded per well (an equivalent of 1x10 6 cells/well) and analyzed by 4-12% Bis-Tris SDS PAGE

  16. POR*28 SNP is associated with lipid response to atorvastatin in children and adolescents with familial hypercholesterolemia.

    PubMed

    Drogari, Euridiki; Ragia, Georgia; Mollaki, Vasiliki; Elens, Laure; Van Schaik, Ron H N; Manolopoulos, Vangelis G

    2014-12-01

    In children and adolescents with familial hypercholesterolemia (FH) pharmacotherapy with statins is the cornerstone in the current regimen to reduce low-density lipoprotein cholesterol (LDLc) and premature coronary heart disease risk. There is, however, a great interindividual variation in response to therapy, partially attributed to genetic factors. The polymorphic enzyme POR transfers electrons from NADPH to CYP450 enzymes including CYP3A, which metabolize atorvastatin. POR*28 polymorphism is associated with increased CYP3A enzyme activity. We analyzed the association of POR*28 allele with response to atorvastatin. One hundred and five FH children and adolescents treated with atorvastatin at doses 10-40 mg were included in the study. Total cholesterol (TChol) and LDLc were measured at baseline and after 6 months of treatment. POR*28 allele was analyzed with TaqMan assay. CYP3A4*22, CYP3A5*3 and SLCO1B1 521T>C and 388A>G genotypes were also determined with TaqMan or PCR-RFLP methods. POR*28 carriers had significantly lower percent mean reduction of TChol (33.1% in *1/*1, 29.8% in *1/*28 and 25.9% in *28/*28 individuals, p = 0.045) and of LDLc (43.9% in *1/*1, 40.9% in *1/*28 and 30.8% in *28/*28 individuals, p = 0.013). In multivariable linear regression adjusted for confounding factors, POR*28 genotypes, additionally to baseline cholesterol level, accounted for an estimated 8.3% and 7.3% of overall variability in % TChol and LDLc reduction (β: 4.05; 95% CI: 1.73-6.37; p = 0.001 and β: 5.08; 95% CI: 1.62-8.54; p = 0.004, respectively). CYP3A4*22, CYP3A5*3 and SLCO1B1 521T>C and 388A>G polymorphisms were not associated with lipid reductions and did not modify the effect of POR*28 on atorvastatin response. In children with FH, carriage of POR*28 allele is associated with reduced effect of atorvastatin on TChol and LDLc and therefore identifies FH children that may require higher atorvastatin doses to achieve full therapeutic benefits. Additional studies in

  17. Interactions between CYP3A5*3 and POR*28 polymorphisms and lipid lowering response with atorvastatin.

    PubMed

    Wei, Kan-kan; Zhang, Li-rong

    2015-09-01

    The polymorphic enzyme P450 oxidoreductase (POR) transfers electrons from nicotinamide adenine dinucleotide phosphate (NADPH) to cytochrome P450 (CYP) 3A enzyme s, which metabolize atorvastatin. This suggests that variations in the CYP3A5 and POR genes may influence the response to statins. We aimed to investigate the association and interactions between CYP3A5*3 and POR*28 polymorphisms and the lipid-lowering effects of atorvastatin in a Chinese population. Genotypes were determined by polymerase chain reaction (PCR) with restriction fragment length polymorphism analysis and by PCR with direct sequencing analysis for 179 hyperlipidaemic patients treated with atorvastatin 20 mg once daily for 4 weeks. Serum levels of triglycerides (TGs), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C) were determined before and after treatment. For the 179 patients (including 100 males), the variant allele frequencies of CYP3A5*3 and POR*28 were 73.75 and 41.62 %, respectively. Among all patients, no significant association was found between CYP3A5*3 polymorphisms and TG, TC, LDL-C and HDL-C levels before and after treatment with 20 mg of atorvastatin daily for 4 weeks. Homozygotes for the POR*28 T allele showed a significantly lower mean concentration of LDL-C than homozygotes for the POR*28 C allele (POR*28 TT vs POR*28 CC: 2.46 ± 0.37 vs 2.69 ± 0.49 mmol/L, P = 0.019) after treatment with atorvastatin 20 mg daily for 4 weeks. After adjustment for age, sex and body mass index, CYP3A5 non-expressors who were POR*28 wild-type homozygotes showed significantly higher mean TC and LDL-C levels than those who were POR*28 variant homozygotes, both at baseline (*3/CC vs *3/TT; TC: 7.30 ± 0.73 vs 6.94 ± 0.36 mmol/L, P = 0.026; LDL-C: 3.88 ± 0.70 vs 3.47 ± 0.46 mmol/L, P = 0.009) and after atorvastatin treatment (*3/CC vs *3/TT; TC: 6.03 ± 0.64 vs 5.69 ± 0.34 mmol/L, P = 0

  18. [Control of Aedes aegypti breeding sites with the program Recicla por tu bienestar in Merida, Mexico].

    PubMed

    Barrera-Pérez, Mario A; Pavía-Ruz, Norma; Mendoza-Mezquita, Jorge Eduardo; Torres-Arcila, Nerio; Hernández-Hernández, Ramón; Castro-Gamboa, Francisco; Geded-Moreno, Eduardo; Cohuo-Rodríguez, Azael; Medina-Barreiro, Anuar; Koyoc-Cardeña, Edgar; Gómez-Dantés, Héctor; Kroeger, Axel; Vázquez-Prokopec, Gonzalo; Manrique-Saide, Pablo

    2015-01-01

    To determine the importance of Ae. aegypti breeding-sites in Merida;to evaluate the impact of Recicla por tu bienestar (RxB, a recycling program) on the reduction of breeding sites and the perception of participants. The relative importance for pupae production of the different types of breeding-sites was determined. Pre-and post-RxB entomological surveys were performed in participant neighborhoods to evaluate the impact on total containers and positive breeding-sites. A survey on the perception of participating people about dengue prevention and control and RxB was applied. Buckets/pots and "small diverse items" were the most important breeding-sites. RxB had a significant impact in the reduction of total containers (IRR = 0.74), positive containers (IRR = 0.33) and the risk of a house being positive for Ae. aegypti (OR = 0.41). All the interviewed participants referred RxB as needed and most consider it useful. RxB should be considered as a good practice for the dengue vector control.

  19. Mujeres felices por ser saludables: a breast cancer risk reduction program for Latino women.

    PubMed

    Fitzgibbon, Marian L; Gapstur, Susan M; Knight, Sara J

    2003-05-01

    Breast cancer is the most commonly diagnosed cancer and the most common cause of cancer mortality among Latino women. Several behavioral factors such as early detection and dietary practices could help decrease morbidity and mortality associated with breast cancer in this population. Unfortunately, there are few data regarding the efficacy of health-related interventions for young Latino women. Mujeres Felices por ser Saludables is a randomized intervention project designed to assess breast cancer risk reduction behavior among Latino women ages 20-40 years. The primary objectives of the project were to determine whether an 8-month integrated dietary/breast health intervention could lead to a greater reduction in dietary fat, increase in dietary fiber, increase in the frequency and proficiency of breast self examination (BSE), and reduction in anxiety related to BSE compared to controls. Herein we describe the overall design of the project and present baseline characteristics of the 256 randomized women. Our results suggest that the average daily intake of dietary fat (percentage of total energy) was slightly below 30% (percentage of total energy) among the women randomized. While over half of these women reported that they practice BSE, and few reported anxiety related to BSE, less than 27% of women were proficient in the recommended BSE technique. There are few data on the dietary and breast health behaviors of young low-acculturated Latino women. This study documents the feasibility of recruiting, randomizing, and obtaining both baseline dietary and breast health data on this unique and underserved population.

  20. Unidas por la Vida (United for Life): implementing a culturally-tailored, community-based, family-oriented lifestyle intervention.

    PubMed

    Sorkin, Dara H; Biegler, Kelly A; Peyreda, Margarita; Kilgore, David; Dow, Emily; Ngo-Metzger, Quyen

    2013-01-01

    Unidas por la Vida, a behavioral weight-loss program, was developed for use among low-income, Mexican-American women with diabetes and their overweight/obese adult daughters. The program leverages community resources in a partnership between primary care and community-based organizations. This paper describes the program's implementation, lessons learned, and implications for sustainability.

  1. [Professor Frantisek Por MD and Professor Robert Klopstock MD, students at Budapest and Prague Faculties of Medicine].

    PubMed

    Mydlík, M; Derzsiová, K

    2010-11-01

    Professor Frantisek Por MD and Professor Robert Klopstock MD were contemporaries, both born in 1899, one in Zvolen, the other in Dombovar, at the time of Austro-Hungarian Monarchy. Prof. Por attended the Faculty of Medicine in Budapest from 1918 to 1920, and Prof. Klopstock studied at the same place between 1917 and 1919. From 1920 until graduation on 6th February 1926, Prof. Por continued his studies at the German Faculty of Medicine, Charles University in Prague. Prof. Klopstock had to interrupt his studies in Budapest due to pulmonary tuberculosis; he received treatment at Tatranske Matliare where he befriended Franz Kafka. Later, upon Kafka's encouragement, he changed institutions and continued his studies at the German Faculty of Medicine, Charles University in Prague, where he graduated the first great go. It is very likely that, during their studies in Budapest and Prague, both professors met repeatedly, even though their life paths later separated. Following his graduation, Prof. Por practiced as an internist in Prague, later in Slovakia, and from 1945 in Kosice. In 1961, he was awarded the title of university professor of internal medicine at the Faculty of Medicine, Pavol Jozef Safarik University in Kosice, where he practiced until his death in 1980. Prof. Klopstock continued his studies in Kiel and Berlin. After his graduation in 1933, he practiced in Berlin as a surgeon and in 1938 left for USA. In 1962, he was awarded the title of university professor of pulmonary surgery in NewYork, where he died in 1972.

  2. jPOR: An ImageJ macro to quantify total optical porosity from blue-stained thin sections

    NASA Astrophysics Data System (ADS)

    Grove, Clayton; Jerram, Dougal A.

    2011-11-01

    A fast and effective method has been developed to measure total optical porosity (TOP) of blue resin-impregnated thin sections. This utilises a macro file (jPOR.txt) for ImageJ, which can be used on digital photomicrographs of thin sections. The method requires no specialised scientific equipment and can be run entirely using free to download software. Digital images are acquired from blue resin-impregnated thin sections using a conventional film scanner in the present study, though the technique can be applied to any high resolution colour digital acquired by different means (e.g., flat bed scanning, digital capture). Images are preprocessed using a newly developed custom 8-bit palette and analysed for porosity in ImageJ using the simple to use jPOR macro. Our method rapidly calculates TOP for batches of images with or without the option of user adjustment. Results are compared with conventional methods (e.g., to point counting), and tested with several users to estimate any user variability. jPOR provided comparable results to more time-consuming point counting, but with significantly less "counting error" and less interoperator variability than published point counting studies. The jPOR macro has been integrated into a macro tool set that can be configured to be run on ImageJ start up.

  3. Functional POR A503V is associated with the risk of bladder cancer in a Chinese population

    PubMed Central

    Xiao, Xue; Ma, Gaoxiang; Li, Shushu; Wang, Meilin; Liu, Nian; Ma, Lan; Zhang, Zhan; Chu, Haiyan; Zhang, Zhengdong; Wang, Shou-Lin

    2015-01-01

    Human cytochrome P450 oxidoreductase (POR) plays important roles in the metabolism of exogenous carcinogens and endogenous sterol hormones. However, few studies have explored the association between POR variants and the risk of bladder cancer. In this study, we first sequenced all 16 POR exons among 50 randomly selected controls, and found three variants, rs1135612, rs1057868 (A503V) and rs2228104, which were then assessed the relation to risk of bladder cancer in a case-control study of 1,050 bladder cancer cases and 1,404 cancer-free controls in a Chinese population. People with A503V TT genotype have a decreased risk of bladder cancer in a recessive model (TT vs. CC/CT, OR = 0.73, 95% CI = 0.57–0.93), which was more pronounced among elderly male, non-smoking, subjects. Especially, A503V TT genotype showed a protective effect in the invasive tumor stage. Functional analysis revealed that A503V activity decreased in cytochrome c reduction (50.5 units/mg vs. 135.4 units/mg), mitomycin C clearance (38.3% vs. 96.8%), and mitomycin C-induced colony formation (78.0 vs 34.3 colonies per dish). The results suggested that POR A503V might decrease the risk of bladder cancer by reducing its metabolic activity, and should be a potential biomarker for predicting the susceptibility to human bladder cancer. PMID:26123203

  4. Global Microlending in Education Reform: Enseñá Por Argentina and the Neoliberalization of the Grassroots

    ERIC Educational Resources Information Center

    Friedrich, Daniel S.

    2010-01-01

    This article examines the workings and underlying assumptions behind Enseñá por Argentina (Teach for Argentina), one specific program that takes part in the larger and expanding network of Teach for All, by thinking about the ways in which a global push for redefining teaching and teacher education encounters local characteristics and histories,…

  5. Functional POR A503V is associated with the risk of bladder cancer in a Chinese population.

    PubMed

    Xiao, Xue; Ma, Gaoxiang; Li, Shushu; Wang, Meilin; Liu, Nian; Ma, Lan; Zhang, Zhan; Chu, Haiyan; Zhang, Zhengdong; Wang, Shou-Lin

    2015-06-30

    Human cytochrome P450 oxidoreductase (POR) plays important roles in the metabolism of exogenous carcinogens and endogenous sterol hormones. However, few studies have explored the association between POR variants and the risk of bladder cancer. In this study, we first sequenced all 16 POR exons among 50 randomly selected controls, and found three variants, rs1135612, rs1057868 (A503V) and rs2228104, which were then assessed the relation to risk of bladder cancer in a case-control study of 1,050 bladder cancer cases and 1,404 cancer-free controls in a Chinese population. People with A503V TT genotype have a decreased risk of bladder cancer in a recessive model (TT vs. CC/CT, OR = 0.73, 95% CI = 0.57-0.93), which was more pronounced among elderly male, non-smoking, subjects. Especially, A503V TT genotype showed a protective effect in the invasive tumor stage. Functional analysis revealed that A503V activity decreased in cytochrome c reduction (50.5 units/mg vs. 135.4 units/mg), mitomycin C clearance (38.3% vs. 96.8%), and mitomycin C-induced colony formation (78.0 vs 34.3 colonies per dish). The results suggested that POR A503V might decrease the risk of bladder cancer by reducing its metabolic activity, and should be a potential biomarker for predicting the susceptibility to human bladder cancer.

  6. Global Microlending in Education Reform: Enseñá Por Argentina and the Neoliberalization of the Grassroots

    ERIC Educational Resources Information Center

    Friedrich, Daniel S.

    2010-01-01

    This article examines the workings and underlying assumptions behind Enseñá por Argentina (Teach for Argentina), one specific program that takes part in the larger and expanding network of Teach for All, by thinking about the ways in which a global push for redefining teaching and teacher education encounters local characteristics and histories,…

  7. Rescue of cytochrome P450 oxidoreductase (Por) mouse mutants reveals functions in vasculogenesis, brain and limb patterning linked to retinoic acid homeostasis.

    PubMed

    Ribes, Vanessa; Otto, Diana M E; Dickmann, Leslie; Schmidt, Katy; Schuhbaur, Brigitte; Henderson, Colin; Blomhoff, Rune; Wolf, C Roland; Tickle, Cheryll; Dollé, Pascal

    2007-03-01

    Cytochrome P450 oxidoreductase (POR) acts as an electron donor for all cytochrome P450 enzymes. Knockout mouse Por(-/-) mutants, which are early embryonic (E9.5) lethal, have been found to have overall elevated retinoic acid (RA) levels, leading to the idea that POR early developmental function is mainly linked to the activity of the CYP26 RA-metabolizing enzymes (Otto et al., Mol. Cell. Biol. 23, 6103-6116). By crossing Por mutants with a RA-reporter lacZ transgene, we show that Por(-/-) embryos exhibit both elevated and ectopic RA signaling activity e.g. in cephalic and caudal tissues. Two strategies were used to functionally demonstrate that decreasing retinoid levels can reverse Por(-/-) phenotypic defects, (i) by culturing Por(-/-) embryos in defined serum-free medium, and (ii) by generating compound mutants defective in RA synthesis due to haploinsufficiency of the retinaldehyde dehydrogenase 2 (Raldh2) gene. Both approaches clearly improved the Por(-/-) early phenotype, the latter allowing mutants to be recovered up until E13.5. Abnormal brain patterning, with posteriorization of hindbrain cell fates and defective mid- and forebrain development and vascular defects were rescued in E9.5 Por(-/-) embryos. E13.5 Por(-/-); Raldh2(+/-) embryos exhibited abdominal/caudal and limb defects that strikingly phenocopy those of Cyp26a1(-/-) and Cyp26b1(-/-) mutants, respectively. Por(-/-); Raldh2(+/-) limb buds were truncated and proximalized and the anterior-posterior patterning system was not established. Thus, POR function is indispensable for the proper regulation of RA levels and tissue distribution not only during early embryonic development but also in later morphogenesis and molecular patterning of the brain, abdominal/caudal region and limbs.

  8. Determinacion del error sistematico del momentum de muones producidos por interacciones neutrino-nucleon en el detector MINER$\

    SciTech Connect

    Diaz Bautista, Gonzalo A.

    2015-11-29

    El Modelo Estandar describe todas las partculas observadas en el naturaleza hasta el momento as como las caractersticas que gobiernan a las interacciones fundamentales entre ellas. En especial es posible identicar a las interacciones electromagnetica y debil, las cuales bajo determinadas condiciones de temperatura y energa pueden ser descritas a traves de una sola teora que engloba a ambas. A esta teora se le denomina electrodebil y tiene como nalidad caracterizar las propiedades de la interaccion maniesta a partir de la mezcla de las interacciones electromagnetica y debil, la que tambien lleva como nombre interaccion electrodebil. Particularmente, los neutrinos son de especial interes ya que, por un lado, interactuan por medio de la interaccion debil muy raramente en comparacion con otras partculas y, por el otro, no son acertadamente descritos por el Modelo Estandar. Por medio de observaciones experimentales que demostraban que los neutrinos cambian de sabor al propagarse, fenomeno llamado oscilaciones de neutrinos, se pudo llegar a la conclusion de que la implicancia de este fenomeno da como consecuencia que los neutrinos efectivamente s tienen masa, algo que entra en contradiccion con la descripcion inicial del Modelo Estandar, el cual los describe como partculas sin masa. Es de esta manera que las oscilaciones de neutrinos han sido y siguen siendo en la actualidad objeto de interes en la Fsica de Altas Energas tanto teorica como experimental. A n de poder realizar mediciones precisas de oscilaciones de neutrinos, los experimentos encargados de estas mediciones deben tratar de reducir sus incertidumbres en lo posible. Una de estas proviene de la caracterizacion de las secciones de choque de los neutrinos cuando interactuan con la materia, particularmente los nucleones al interior de los nucleos atomicos. El experimento MINERA esta orientado, entre otras cosas, a hacer una correcta caracterizacion de secciones de choque neutrino-nucleon por medio del estudio de

  9. Inversor Resonante de Tres Elementos L-LC con Caracteristica Cortocircuitable para Aplicaciones de Calentamiento por Induccion

    NASA Astrophysics Data System (ADS)

    Espi Huerta, Jose Miguel

    Los generadores de calentamiento por induccion son puentes inversores con carga resonante, cuya mision es basicamente crear una corriente sinusoidal de gran amplitud sobre la "bobina de caldeo", que forma parte del tanque resonante. En el interior de esta bobina se introduce la pieza que se desea calentar. EI campo magnetico creado induce corrientes superficiales (corrientes de Foucault) sobre la pieza, que producen su calentamiento. Los tanques resonantes (tambien llamados osciladores) utilizados en la actualidad son el resonante serie y el resonante paralelo. Aunque ya desde hace algun tiempo se vienen construyendo generadores de alta potencia basados en estos dos osciladores, el exito nunca ha. sido completo en ninguno de los dos casos. Tal y como se explica en la introduccion de esta memoria, los puentes inversores utilizados deben operar sobre una carga inductiva (corriente retrasada) para evitar el fenomeno de la recuperacion inversa de sus diodos y la consiguiente ruptura de los transistores. De la restriccion topologica anterior se deduce que el generador paralelo debe conmutar a frecuencias inferiores a la resonancia, y el serie a frecuencias superiores. A esta restriccion topologica hay que unir otra que es exclusiva del calentamiento por induccion: La corriente por la bobina de caldeo debe ser sinusoidal. De no ser asi, resultaria imposible disponer toda la potencia de calentamiento sobre la pieza en el espesor requerido por la aplicacion. Como consecuencia, los inversores no pueden operar por debajo de la frecuencia de resonancia del oscilador, pues en ese caso se amplifican los armonicos de orden superior de la tension/corriente de entrada situados sobre la resonancia, con la consiguiente distorsion de la corriente de salida. La conjuncion de las dos restricciones anteriores obligan al inversor paralelo a funcionar a la frecuencia de resonancia del oscilador. Esto imposibilita un control por variacion de frecuencia, regulandose la potencia desde la

  10. The amino acid sequence of Neisseria lactamica PorB surface-exposed loops influences Toll-like receptor 2-dependent cell activation.

    PubMed

    Toussi, Deana N; Carraway, Margaretha; Wetzler, Lee M; Lewis, Lisa A; Liu, Xiuping; Massari, Paola

    2012-10-01

    Toll-like receptors (TLRs) play a major role in host mucosal and systemic defense mechanisms by recognizing a diverse array of conserved pathogen-associated molecular patterns (PAMPs). TLR2, with TLR1 and TLR6, recognizes structurally diverse bacterial products such as lipidated factors (lipoproteins and peptidoglycans) and nonlipidated proteins, i.e., bacterial porins. PorB is a pan-neisserial porin expressed regardless of organisms' pathogenicity. However, commensal Neisseria lactamica organisms and purified N. lactamica PorB (published elsewhere as Nlac PorB) induce TLR2-dependent proinflammatory responses of lower magnitude than N. meningitidis organisms and N. meningitidis PorB (published elsewhere as Nme PorB). Both PorB types bind to TLR2 in vitro but with different apparent specificities. The structural and molecular details of PorB-TLR2 interaction are only beginning to be unraveled and may be due to electrostatic attraction. PorB molecules have significant strain-specific sequence variability within surface-exposed regions (loops) putatively involved in TLR2 interaction. By constructing chimeric recombinant PorB loop mutants in which surface-exposed loop residues have been switched between N. lactamica PorB and N. meningitidis PorB, we identified residues in loop 5 and loop 7 that influence TLR2-dependent cell activation using HEK cells and BEAS-2B cells. These loops are not uniquely responsible for PorB interaction with TLR2, but NF-κB and MAP kinases signaling downstream of TLR2 recognition are likely influenced by a hypothetical "TLR2-binding signature" within the sequence of PorB surface-exposed loops. Consistent with the effect of purified PorB in vitro, a chimeric N. meningitidis strain expressing N. lactamica PorB induces lower levels of interleukin 8 (IL-8) secretion than wild-type N. meningitidis, suggesting a role for PorB in induction of host cell activation by whole bacteria.

  11. Thermal properties and phase composition of water for frozen rocks of Por-Bajin island (Republic of Tuva, Russia)

    NASA Astrophysics Data System (ADS)

    Aleksyutina, Daria; Motenko, Rimma; Buldovich, Sergey; Koshurnikov, Andrey; Prosuncov, Kirill

    2010-05-01

    We investigated current permafrost state, thermal properties and phase composition of water for frozen rocks of Por-Bajin Island (Tere-Hol Lake, south-east part of Tuva republic, Russia). Rocks of the island are represented by dispersing grounds in permafrost state. The architectural monument Por-Bajin fortress (the middle of the VIII century) located at the island can be destructed due to thawing of frozen rocks resulted by water level rising in the surrounding Por-Bajin lake. Seasonally thawed layer on the island varies from 1.1 to 1.8 meters based on drilling observations from 13 drill holes the. Measured in the field the thermal conductivity indicates the low values in seasonally thawed layer (from 0.14 to 0.19 Wm-1K-1). Twenty most typical for the studied area frozen handsamples were collected from several holes in the depth interval from 1.5 to 10.15 meters. The humidity of sampled specimens varies from 13 to 270 percents and density varies from 1.21 to 2.17 g/cm3. Investigations of grain-size composition, thermal properties and liquid phase composition were undertaken using laboratory methods. The grain-size composition of Por-Bajin grounds varies from clays to gritstones and gravelites with clay-silt matrix. Phase composition of water in the frozen grounds was studied by contact and cryoscopic methods. Thermal properties were investigated by the I-st type regular mode method (a-calorimeter). Based on obtained data computer model allowed to forecast the evolution of permafrost at the island and predict the destiny of Por-Bajin monument. This work was supported by grant from President of the Russian Federation NSh - 4078.2008.5

  12. Encefalitis por anticuerpos contra el receptor de NMDA: experiencia con seis pacientes pediátricos. Potencial eficacia del metotrexato

    PubMed Central

    Bravo-Oro, Antonio; Abud-Mendoza, Carlos; Quezada-Corona, Arturo; Dalmau, Josep; Campos-Guevara, Verónica

    2016-01-01

    Introducción La encefalitis por anticuerpos contra el receptor de N-metil-D-aspartato (NMDA) es una entidad cada vez más diagnosticada en edad pediátrica. A diferencia de los adultos, en muchos casos no se asocia a tumores y las manifestaciones iniciales en niños más frecuentes son crisis convulsivas y trastornos del movimiento, mientras que en los adultos predominan las alteraciones psiquiátricas. Casos clínicos Presentamos seis casos pediátricos confirmados con anticuerpos contra la subunidad NR1 del receptor de NMDA en suero y líquido cefalorraquídeo. Cinco de los casos comenzaron con crisis convulsivas como manifestación clínica inicial antes de desarrollar el cuadro clásico de esta entidad. En todos los casos se utilizaron esteroides como primera línea de tratamiento, con los que sólo se observó control de las manifestaciones en uno, por lo que el resto de los pacientes requirió inmunomoduladores de segunda línea. Todos los pacientes recibieron metotrexato como tratamiento inmunomodulador para evitar recaídas y la evolución fue a la mejoría en todos ellos. Conclusiones En nuestra serie de pacientes con encefalitis por anticuerpos contra el receptor de NMDA, ninguno se asoció a tumores. Todos los casos recibieron metotrexato por lo menos durante un año, no observamos eventos adversos clínicos ni por laboratorio, ni hubo secuelas neurológicas ni recaídas durante el tratamiento. Aunque es una serie pequeña y es deseable incrementar el número y tiempo de evolución, consideramos el metotrexato una excelente alternativa como tratamiento inmunomodulador para esta patología. PMID:24150952

  13. The effect of essential oil on heart rate and blood pressure among solus por aqua workers.

    PubMed

    Chuang, Kai-Jen; Chen, Hua-Wei; Liu, I-Jung; Chuang, Hsiao-Chi; Lin, Lian-Yu

    2014-07-01

    Aromatherapy is widely used around the world for stress relief. Whether exposure to essential oil increases the risk of cardiovascular events is still unknown. The purpose of this study was to investigate the effect of essential oil on heart rate and blood pressure among solus por aqua (spa) workers. We recruited 100 healthy workers from various spa centres in Taipei, Taiwan. Between July and August of 2010, three repeated measurements - resting heart rate (HR), systolic blood pressure (SBP), and diastolic blood pressure (DBP) - were taken of each spa worker in our study room. Participants were exposed to essential oil vapour generated from an ultrasonic atomizer in the study room for two consecutive hours. The total volatile organic compound (VOC) level in the study room was measured during the study period. We used a linear mixed-effect model to determine the association between the total VOC level and the participants' HR, SBP, and DBP. For the times from 15 to 60 min after start of exposure, we found that the VOC level was significantly associated with reduced 15-min mean BP and HR. After exposure for more than 1 hour, from 75 to 120 min after start of exposure, we found that the VOC levels were associated with increased 15-min mean BP and HR. Exposure to essential oil for 1 hour was found to be an effective method of relaxation, as indicated by decreases in the HR and BP. Prolonged exposure for longer than 1 hour to essential oils may be harmful to cardiovascular health among spa workers. © The European Society of Cardiology 2012.

  14. A Ruthenium(II) Complex Supported by Trithiacyclononane and Aromatic Diimine Ligand as Luminescent Switch-On Probe for Biomolecule Detection and Protein Staining

    PubMed Central

    Wong, Chun-Yuen; Chung, Lai-Hon; Lin, Sheng; Chan, Daniel Shiu-Hin; Ma, Dik-Lung

    2014-01-01

    A new ruthenium(II) complex has been developed for detection of biomolecules. This complex is highly selective for histidine over other amino acids and has been applied to protein staining in an SDS-PAGE gel. PMID:25409703

  15. The Sangre Por Salud Biobank: Facilitating Genetic Research in an Underrepresented Latino Community.

    PubMed

    Shaibi, Gabriel; Singh, Davinder; De Filippis, Eleanna; Hernandez, Valentina; Rosenfeld, Bill; Otu, Essen; Montes de Oca, Gregorio; Levey, Sharon; Radecki Breitkopf, Carmen; Sharp, Richard; Olson, Janet; Cerhan, James; Thibodeau, Stephen; Winkler, Erin; Mandarino, Lawrence

    2016-01-01

    The Sangre Por Salud (Blood for Health; SPS) Biobank was created for the purpose of expanding precision medicine research to include underrepresented Latino patients. It is the result of a unique collaboration between Mayo Clinic and Mountain Park Health Center, a federally qualified community health center in Phoenix, Arizona. This report describes the rationale, development, implementation, and characteristics of the SPS Biobank. Latino adults (ages 18-85 years) who were active patients within Mountain Park Health Center's internal medicine practice in Phoenix, Ariz., and had no history of diabetes were eligible. Participants provided a personal and family history of chronic disease, completed a sociodemographic, psychosocial, and behavioral questionnaire, underwent a comprehensive cardiometabolic risk assessment (anthropometrics, blood pressure and labs), and provided blood samples for banking. Laboratory results of cardiometabolic testing were returned to the participants and their providers through the electronic health record. During the first 2 years of recruitment into the SPS Biobank, 2,335 patients were approached and 1,432 (61.3%) consented to participate; 1,354 (94.5%) ultimately completed all requisite questionnaires and medical evaluations. The cohort is primarily Spanish-speaking (72.9%), female (73.3%), with a mean age of 41.3 ± 12.5 years. Most participants were born outside of the US (77.9%) and do not have health insurance (77.5%). The prevalence of overweight (35.5%) and obesity (45.0%) was high, as was previously unidentified prediabetes (55.9%), type 2 diabetes (7.4%), prehypertension (46.8%), and hypertension (16.2%). The majority of participants rated their health as good to excellent (72.1%) and, as a whole, described their overall quality of life as high (7.9/10). Collaborative efforts such as the SPS Biobank are critical for ensuring that underrepresented minority populations are included in precision medicine initiatives and biomedical

  16. Leishmania Skin Test

    DTIC Science & Technology

    2010-03-01

    Ninhydrin ), SDS-PAGE and non-viability testing . See Table 3 below: Table 3: Drug Substance Specifications Test Method Specification SDS-PAGE...AD_________________ Award Number: DAMD17-00-C-0030 TITLE: Leishmania Skin Test PRINCIPAL INVESTIGATOR: Nielsen, H.S., Jr...TYPE FINAL, PHASE II ADDENDUM 3. DATES COVERED (From - To) 1 APR 2009 - 28 FEB 2010 4. TITLE AND SUBTITLE Leishmania Skin Test 5a

  17. Biochemical Markers of Brain Injury: An Integrated Proteomics-Based Approach

    DTIC Science & Technology

    2007-02-01

    biomarkers in humans (Ringger et al., J Neurotrauma, 2004). Currently, there is no definitive diagnostic test for TBI to help physicians determine the...use of differential cyanine dyes and SDS-PAGE protein separation (Figure 1). Protein bands that appeared different in abundance between samples stood...TBI tissue. Figure 1. Differential protein analysis using cyanine dyes and SDS-PAGE. Tissue lysate from injured and naïve samples were

  18. Proteomic analysis of brain mitochondrial proteome and mitochondrial complexes.

    PubMed

    Lopez-Campistrous, Ana; Fernandez-Patron, Carlos

    2013-01-01

    We describe various complementary techniques to achieve multidimensional mitochondrial proteome fractionation and analysis. Previously described methods for 2D-DIGE/mass spectrometry and 1D-SDS-PAGE/Western techniques and protein complex analysis by BN-PAGE/Western and sucrose gradient ultracentrifugation/SDS-PAGE/mass spectrometry are optimized to characterize the brain mitochondrial proteome. This approach allows for a comprehensive identification of mitochondrial proteomic differences between health and disease conditions.

  19. Prevalencia y tamizaje del Trastorno por Déficit de Atención con Hiperactividad en Costa Rica

    PubMed Central

    Weiss, Nicholas T.; Schuler, Jovita; Monge, Silvia; McGough, James J.; Chavira, Denise; Bagnarello, Monica; Herrera, Luis Diego; Mathews, Carol A.

    2015-01-01

    Resumen La investigación tuvo como propósito estimar la prevalencia del Trastorno por Déficit de Atención con Hiperactividad (TDAH) en Costa Rica y determinar si la versión en español del cuestionario Swanson Nolan and Pelham Scale IV (SNAP-IV) es un instrumento de tamizaje útil en una población de niños y niñas escolares costarricenses. El instrumento fue entregado a padres y maestros de 425 niños entre 5 y 13 años de edad (promedio = 8.8). Todos fueron evaluados con el instrumento Swanson, Kotkin, Agler, M-Flynn and Pelham Scale (SKAMP). Su diagnóstico fue confirmado con una entrevista clínica. La sensibilidad y la especificidad del SNAP-IV fueron evaluadas como predictores de criterios de diagnóstico según el DSM-IV. La prevalencia puntual en la muestra del TDAH fue del 5%. El tamizaje más preciso lo hizo el SNAP-IV completado por el maestro en un corte de 20%, con una sensibilidad de 96% y una especificidad de un 82%. La sensibilidad de los instrumentos completados por los padres fue más baja que aquella de los maestros. El SNAP-IV completado por las maestras con un corte aislando el 20% de los mayores puntajes categorizó correctamente a un 87% de los sujetos. PMID:22432094

  20. Racism in digital era: Development and initial validation of the Perceived Online Racism Scale (PORS v1.0).

    PubMed

    Keum, Brian TaeHyuk; Miller, Matthew J

    2017-04-01

    The purpose of this study was to develop the Perceived Online Racism Scale (PORS) to assess perceived online racist interpersonal interactions and exposure to online racist content among people of color. Items were developed through a multistage process involving a comprehensive literature review, focus-groups, qualitative data collection, and survey of online racism experiences. Based on a sample of 1,023 racial minority participants, exploratory and confirmatory factor analyses provided support for a 30-item bifactor model accounted by the general factor and the following 3 specific factors: (a) personal experience of racial cyber-aggression, (b) vicarious exposure to racial cyber-aggression, and (c) online-mediated exposure to racist reality. The PORS demonstrated measurement invariance across racial/ethnic groups in our sample. Internal reliability estimates for the total and subscale scores of the PORS were above .88 and the 4-week test-retest reliability was adequate. Limitations and future directions for research are discussed. (PsycINFO Database Record (c) 2017 APA, all rights reserved).

  1. The PorB porin from commensal Neisseria lactamica induces Th1 and Th2 immune responses to ovalbumin in mice and is a potential immune adjuvant.

    PubMed

    Liu, Xiuping; Wetzler, Lee M; Massari, Paola

    2008-02-06

    Porins from pathogenic Neisseriae are among several bacterial products with immune adjuvant activity. Neisseria meningitidis (Nme) PorB, has been shown to induce immune cells activation in a TLR2-dependent manner and acts as a vaccine immune adjuvant. The PorB porin from Neisseria lactamica (Nlac), a common nasopharyngeal commensal, shares significant structural and functional similarities with Nme PorB. In this work we ask whether the immune adjuvant ability of porins from pathogenic Neisserial strains is a characteristic shared with porins from non-pathogenic Neisserial species or whether it is unique for bacterial products derived from microorganisms capable of inducing inflammation and disease. We evaluated the potential immune adjuvant effect of Nlac PorB in mice using ovalbumin (OVA) as a prototype antigen. Immunization with Nlac PorB/OVA induced high OVA-specific IgG and IgM titers compared to OVA alone, similar to other adjuvants such as Nme PorB and alum. High titers of IgG1 and IgG2b were detected as well as production of IL-4, IL-10, IL-12 and INF-gamma in response to Nlac PorB, consistent with induction of both a Th1-type and a Th2-type immune response. OVA-specific proliferation was also determined in splenocytes from Nlac PorB/OVA-immunized mice. In addition, B cell activation in vitro and cytokine production in response to Nlac PorB was found to be mediated by TLR2, in a similar manner to Nme PorB.

  2. Chlorophyll Synthesis in a Deetiolated (det340) Mutant of Arabidopsis without NADPH-Protochlorophyllide (PChlide) Oxidoreductase (POR) A and Photoactive PChlide-F655.

    PubMed Central

    Lebedev, N.; Van Cleve, B.; Armstrong, G.; Apel, K.

    1995-01-01

    Chlorophyll (Chl) synthesis in Arabidopsis is controlled by two light-dependent NADPH-protochlorophyllide (PChlide) oxidoreductases (PORs), one (POR A) that is active transiently in etiolated seedlings at the beginning of illumination and another (POR B) that also operates in green plants. The function of these two enzymes during the light-induced greening of dark-grown seedlings has been studied in the wild type and a deetiolated (det340) mutant of Arabidopsis. One of the consequences of the det mutation is that POR A is constitutively down-regulated, and therefore, synthesis of the POR A enzyme is shut off. When grown in the dark, the det340 mutant lacks POR A and the photoactive PChlide-F655 species but maintains the second PChlide reductase, POR B. Previously, photoactive PChlide-F655 has often been considered to be the only PChlide form that leads to Chl formation. Despite its deficiency in POR A and photoactive PChlide-F655, the det340 mutant is able to green when placed in the light. Chl accumulation, however, proceeds abnormally. At the beginning of illumination, seedlings of det340 mutants are extremely susceptible to photooxidative damage and accumulate Chl only at extremely low light intensities. They form core complexes of photosystems I and II but are almost completely devoid of light-harvesting structures. The results of this study demonstrate that in addition to the route of Chl synthesis that has been studied extensively in illuminated dark-grown wild-type plants, a second branch of Chl synthesis exists that is driven by POR B and does not require POR A. PMID:12242369

  3. CYP2C9*2 allele increases risk for hypoglycemia in POR*1/*1 type 2 diabetic patients treated with sulfonylureas.

    PubMed

    Ragia, G; Tavridou, A; Elens, L; Van Schaik, R H N; Manolopoulos, V G

    2014-01-01

    It is previously shown that carriers of the defective allele CYP2C9*3 that leads to impaired sulfonylurea metabolism are at increased sulfonylurea-induced hypoglycemia risk due to diminished drug metabolism, whereas no effect of CYP2C9*2 allele was found. Recently, a polymorphism in P450 oxidoreductase (POR) gene, assigned as POR*28 allele, was associated with increased CYP2C9 activity. The aim of this study was to assess i) the effect of POR*28 allele on sulfonylurea-induced hypoglycemia risk and ii) the association of CYP2C9*2 allele with hypoglycemia risk in non-carriers of POR*28 allele. The study group consisted of 176 patients with diagnosed type 2 diabetes mellitus (T2DM) treated with sulfonylureas, of whom 92 patients had experienced at least one drug-associated hypoglycemic event (cases), while 84 had never experienced a hypoglycemic event (controls). POR*28 allele was detected by use of real-time TaqMan PCR. POR*28 allele was not associated with sulfonyl-urea-induced hypoglycemia. In POR*1/*1 patients, CYP2C9*1/*2 genotype was more common in cases than in controls (32.7 vs. 14.3%, p=0.041). In a model adjusted for age, BMI, duration of T2DM and renal function, and POR*1/*1 entered as a selection variable, CYP2C9*2 allele increased the hypoglycemia risk in response to sulfonylurea (odds ratio: 3.218, p=0.031). In conclusion, our results suggest that POR*28 allele is masking the association of CYP2C9*2 allele with sulfonyl-urea-induced hypoglycemia. Therefore, POR*28 allele is an important source of CYP2C9 activity variability and combined with CYP2C9 gene poly-morphisms may explain individual variability in the effect of sulfonylureas.

  4. Microfluidic integration of Western blotting is enabled by electrotransfer-assisted sodium dodecyl sulfate dilution.

    PubMed

    Hou, Chenlu; Herr, Amy E

    2013-01-07

    We integrate sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) with subsequent antibody probing in a single, monolithic microdevice to realize microfluidic Western blotting. A hurdle to successful on-chip Western blotting lies in restoring antibody recognition of previously sized (denatured, reduced) proteins. To surmount this hurdle, we locally dilute free SDS from SDS-protein complexes using differential electromigration of the species during electrotransfer between SDS-PAGE and blotting regions of a microchamber. Local dilution of SDS minimizes re-association of SDS with proteins offering means to restore antibody binding affinity to proteins after SDS-PAGE. To achieve automated, programmable operation in a single instrument, we utilize a 1 × 2 mm(2) glass microchamber photopatterned with spatially distinct, contiguous polyacrylamide regions for SDS-PAGE, electrotransfer, and antibody blotting. Optimization of both the SDS-PAGE and electrotransfer conditions yields transfer distances of <1 mm (40 s). The Western blot is completed in 180 s, with fully automated assay operation using programmable voltage control. After SDS-PAGE and electrotransfer, we observe ~80% capture of protein band mass on the blotting region for a model protein, C-reactive protein. This novel microfluidic Western blot approach introduces fine transport control for in-transit protein handling to form the basis for an automated, rapid alternative to conventional slab-gel Western blotting.

  5. Flavobacterium johnsoniae PorV is required for secretion of a subset of proteins targeted to the type IX secretion system.

    PubMed

    Kharade, Sampada S; McBride, Mark J

    2015-01-01

    Flavobacterium johnsoniae exhibits gliding motility and digests many polysaccharides, including chitin. A novel protein secretion system, the type IX secretion system (T9SS), is required for gliding and chitin utilization. The T9SS secretes the cell surface motility adhesins SprB and RemA and the chitinase ChiA. Proteins involved in secretion by the T9SS include GldK, GldL, GldM, GldN, SprA, SprE, and SprT. Porphyromonas gingivalis has orthologs for each of these that are required for secretion of gingipain protease virulence factors by its T9SS. P. gingivalis porU and porV have also been linked to T9SS-mediated secretion, and F. johnsoniae has orthologs of these. Mutations in F. johnsoniae porU and porV were constructed to determine if they function in secretion. Cells of a porV deletion mutant were deficient in chitin utilization and failed to secrete ChiA. They were also deficient in secretion of the motility adhesin RemA but retained the ability to secrete SprB. SprB is involved in gliding motility and is needed for formation of spreading colonies on agar, and the porV mutant exhibited gliding motility and formed spreading colonies. However, the porV mutant was partially deficient in attachment to glass, apparently because of the absence of RemA and other adhesins on the cell surface. The porV mutant also appeared to be deficient in secretion of numerous other proteins that have carboxy-terminal domains associated with targeting to the T9SS. PorU was not required for secretion of ChiA, RemA, or SprB, indicating that it does not play an essential role in the F. johnsoniae T9SS.

  6. Polymorphisms of POR, SULT2A1 and HSD11B1 in children with premature adrenarche.

    PubMed

    Utriainen, Pauliina; Laakso, Saila; Jääskeläinen, Jarmo; Voutilainen, Raimo

    2012-09-01

    Premature adrenarche (PA) refers to an earlier than normal increase in adrenocortical androgen production. The pathogenesis of PA remains largely unknown. We hypothesized that common polymorphisms at P450 oxidoreductase (POR), steroid sulfotransferase (SULT2A1), or 11β-hydroxysteroid dehydrogenase type 1 (HSD11B1) genes could contribute to the polygenic pathogenesis of PA. We performed a case-control study on the polymorphisms rs1057868 at POR, rs182420 at SULT2A1, and rs12086634 at HSD11B1. The study cohort comprised 73 prepubertal children with PA (defined by clinical signs) and 97 age- and gender-matched healthy controls from a Finnish Caucasian population. Genotype distributions and clinical and metabolic phenotypes were determined. The genotype distributions of the polymorphisms were similar between the study groups. No variant was associated with alterations in serum adrenal steroid concentrations. The minor C variant at SULT2A1 was associated with higher serum sex hormone binding globulin (SHBG) concentrations (T/T, n=64 vs T/C&C/C, n=33; mean 94 vs 116 nmol/L; P=.001) and a trend for lower dehydroepiandrosterone sulfate/dehydroepiandrosterone ratios in the controls (P=.06), and with higher plasma total cholesterol concentrations in the PA subjects (T/T, n=42 vs T/C&C/C, n=31; 4.0 vs 4.6 mmol/L; P<.001). The minor G variant at HSD11B1 was associated with lower plasma triglyceride concentration in the controls (T/T, n=65 vs T/G&G/G, n=32; 0.61 vs 0.49 mmol/L; P=.013). Common polymorphisms at POR, SULT2A1 or HSD11B1 were not associated with PA in a Finnish Caucasian population. Copyright © 2012 Elsevier Inc. All rights reserved.

  7. Evaluation of immunological responses to recombinant Porin A protein (rPoA) from native strains of Neisseria meningitidis serogroups A and B using OMV as an adjuvant in BALB/c mice.

    PubMed

    Afrough, Parviz; Bouzari, Saeid; Mousavi, Seyed Fazlollah; Asadi Karam, Mohammad Reza; Vaziri, Farzam; Fateh, Abolfazl; Behrouzi, Ava; Malekan, Mohammadali; Siadat, Seyed Davar

    2017-09-20

    Neisseria meningitidis is one of the main causes of sepsis and meningitis, which are two serious life-threatening diseases in both children and adolescents. Porin A (porA) from both serogroup A and B were cloned into the pET28a plasmid and expressed in E. coli BL21 (DE3). The protein was expressed in Escherichia coli BL21 (DE3) and confirmed by SDS-PAGE and Western blot analysis. BALB/c mice were subcutaneously injected three times with 25 μg of the recombinant PorA. Specific total IgG antibodies and isotypes were evaluated using ELISA assay. Opsonophagocytic assay (OPA) and Serum Bactericidal assay (SBA) were performed. Results showed that vaccinated mice exhibited higher levels of anti-Porin A (p < 0.05) with a predominant IgG1 response compared to the control group. Results from in vitro experiments indicated that N. meningitidis was opsonized with immunized-mice sera, and compared to non-immunized mice, immunized mice displayed significantly increased phagocytic uptake and effective intracellular killing. In this study, serogroup B N. meningitidis OMV of strain CSBPI G-245 and complete and incomplete Freund's adjuvant were used. Results demonstrated that Porin A could be a valuable target for the development of immunotherapeutic strategies against N. meningitidis. Copyright © 2017. Published by Elsevier Ltd.

  8. The POR rs1057868–rs2868177 GC-GT diplotype is associated with high tacrolimus concentrations in early post-renal transplant recipients

    PubMed Central

    Liu, Shu; Chen, Rong-xin; Li, Jun; Zhang, Yu; Wang, Xue-ding; Fu, Qian; Chen, Ling-yan; Liu, Xiao-man; Huang, Hong-bing; Huang, Min; Wang, Chang-xi; Li, Jia-li

    2016-01-01

    Aim: Cytochrome P450 oxidoreductase (POR) is the only flavoprotein that donates electrons to all microsomal P450 enzymes (CYP), and several POR SNPs have been shown to be important contributors to altered CYP activity or CYP-mediated drug metabolism. In this study we examined the association between 6 POR SNPs and tacrolimus concentrations in Chinese renal transplant recipients. Methods: A total of 154 renal transplant recipients were enrolled. Genotyping of CYP3A5*3 and 6 POR SNPs was performed. All patients received a triple immunosuppressive regimen comprising tacrolimus, mycophenolate mofetil and prednisone. Dose-adjusted tacrolimus trough concentrations were obtained on d 7 (C0D7/D) after transplantation when steady-state concentration of tacrolimus was achieved (dosage had been unchanged for more than 3 d). Results: Tacrolimus C0D7/D in CYP3A5*3/*3/ POR rs1057868–rs2868177 GC-GT diplotype carriers was 1.62- and 2.72-fold higher than those in CYP3A5*3/*3/ POR rs1057868–rs2868177 GC-GT diplotype non-carriers and CYP3A5*1 carriers (220.17±48.09 vs 135.69±6.86 and 80.84±5.27 ng/mL/mg/kg, respectively, P<0.0001). Of CYP3A5*3/*3/ POR rs1057868-rs2868177GC-GT diplotype carriers, 85.71% exceeded the upper limit of the target range (8 ng/mL), which was also significantly higher compared with the latter two groups (14.29% and 0.00%, respectively, P<0.0001). The CYP3A5*3 and POR rs1057868–rs2868177 GC-GT diplotype explained 31.7% and 5.7%, respectively, of the inter-individual variability of tacrolimus C0D7/D, whereas the POR rs1057868–rs2868177 GC-GT diplotype could explain 10.9% of the inter-individual variability of tacrolimus C0D7/D in CYP3A5 non-expressers. Conclusion: The CYP3A5*3 and POR rs1057868–rs2868177 GC-GT diplotype accounted for the inter-individual variation of tacrolimus C0D7/D. Genotyping of POR rs1057868–rs2868177 diplotypes would help to differentiate initial tacrolimus dose requirements and to achieve early target C0 ranges in Chinese

  9. The POR rs1057868-rs2868177 GC-GT diplotype is associated with high tacrolimus concentrations in early post-renal transplant recipients.

    PubMed

    Liu, Shu; Chen, Rong-Xin; Li, Jun; Zhang, Yu; Wang, Xue-Ding; Fu, Qian; Chen, Ling-Yan; Liu, Xiao-Man; Huang, Hong-Bing; Huang, Min; Wang, Chang-Xi; Li, Jia-Li

    2016-09-01

    Cytochrome P450 oxidoreductase (POR) is the only flavoprotein that donates electrons to all microsomal P450 enzymes (CYP), and several POR SNPs have been shown to be important contributors to altered CYP activity or CYP-mediated drug metabolism. In this study we examined the association between 6 POR SNPs and tacrolimus concentrations in Chinese renal transplant recipients. A total of 154 renal transplant recipients were enrolled. Genotyping of CYP3A5*3 and 6 POR SNPs was performed. All patients received a triple immunosuppressive regimen comprising tacrolimus, mycophenolate mofetil and prednisone. Dose-adjusted tacrolimus trough concentrations were obtained on d 7 (C0D7/D) after transplantation when steady-state concentration of tacrolimus was achieved (dosage had been unchanged for more than 3 d). Tacrolimus C0D7/D in CYP3A5*3/*3/ POR rs1057868-rs2868177 GC-GT diplotype carriers was 1.62- and 2.72-fold higher than those in CYP3A5*3/*3/ POR rs1057868-rs2868177 GC-GT diplotype non-carriers and CYP3A5*1 carriers (220.17±48.09 vs 135.69±6.86 and 80.84±5.27 ng/mL/mg/kg, respectively, P<0.0001). Of CYP3A5*3/*3/ POR rs1057868-rs2868177GC-GT diplotype carriers, 85.71% exceeded the upper limit of the target range (8 ng/mL), which was also significantly higher compared with the latter two groups (14.29% and 0.00%, respectively, P<0.0001). The CYP3A5*3 and POR rs1057868-rs2868177 GC-GT diplotype explained 31.7% and 5.7%, respectively, of the inter-individual variability of tacrolimus C0D7/D, whereas the POR rs1057868-rs2868177 GC-GT diplotype could explain 10.9% of the inter-individual variability of tacrolimus C0D7/D in CYP3A5 non-expressers. The CYP3A5*3 and POR rs1057868-rs2868177 GC-GT diplotype accounted for the inter-individual variation of tacrolimus C0D7/D. Genotyping of POR rs1057868-rs2868177 diplotypes would help to differentiate initial tacrolimus dose requirements and to achieve early target C0 ranges in Chinese renal transplant recipients.

  10. Clinical Neisseria gonorrhoeae isolate with a N. meningitidis porA gene and no prolyliminopeptidase activity, Sweden, 2011: danger of false-negative genetic and culture diagnostic results.

    PubMed

    Golparian, D; Johansson, E; Unemo, M

    2012-03-01

    We describe a Neisseria gonorrhoeae strain, found in Sweden in 2011, that harbours a N. meningitidis porA gene causing false-negative results in PCRs targeting the gonococcal porA pseudogene. Furthermore, the strain had no prolyliminopeptidase (PIP) activity that many commercial biochemical kits for species verification in culture rely on. Enhanced awareness of the spread of such strains and screening for them can be crucial.

  11. Monitoring Shifts in the Conformation Equilibrium of the Membrane Protein Cytochrome P450 Reductase (POR) in Nanodiscs*

    PubMed Central

    Wadsäter, Maria; Laursen, Tomas; Singha, Aparajita; Hatzakis, Nikos S.; Stamou, Dimitrios; Barker, Robert; Mortensen, Kell; Feidenhans'l, Robert; Møller, Birger Lindberg; Cárdenas, Marité

    2012-01-01

    Nanodiscs are self-assembled ∼50-nm2 patches of lipid bilayers stabilized by amphipathic belt proteins. We demonstrate that a well ordered dense film of nanodiscs serves for non-destructive, label-free studies of isolated membrane proteins in a native like environment using neutron reflectometry (NR). This method exceeds studies of membrane proteins in vesicle or supported lipid bilayer because membrane proteins can be selectively adsorbed with controlled orientation. As a proof of concept, the mechanism of action of the membrane-anchored cytochrome P450 reductase (POR) is studied here. This enzyme is responsible for catalyzing the transfer of electrons from NADPH to cytochrome P450s and thus is a key enzyme in the biosynthesis of numerous primary and secondary metabolites in plants. Neutron reflectometry shows a coexistence of two different POR conformations, a compact and an extended form with a thickness of 44 and 79 Å, respectively. Upon complete reduction by NADPH, the conformational equilibrium shifts toward the compact form protecting the reduced FMN cofactor from engaging in unspecific electron transfer reaction. PMID:22891242

  12. Monitoring shifts in the conformation equilibrium of the membrane protein cytochrome P450 reductase (POR) in nanodiscs.

    PubMed

    Wadsäter, Maria; Laursen, Tomas; Singha, Aparajita; Hatzakis, Nikos S; Stamou, Dimitrios; Barker, Robert; Mortensen, Kell; Feidenhans'l, Robert; Møller, Birger Lindberg; Cárdenas, Marité

    2012-10-05

    Nanodiscs are self-assembled ∼50-nm(2) patches of lipid bilayers stabilized by amphipathic belt proteins. We demonstrate that a well ordered dense film of nanodiscs serves for non-destructive, label-free studies of isolated membrane proteins in a native like environment using neutron reflectometry (NR). This method exceeds studies of membrane proteins in vesicle or supported lipid bilayer because membrane proteins can be selectively adsorbed with controlled orientation. As a proof of concept, the mechanism of action of the membrane-anchored cytochrome P450 reductase (POR) is studied here. This enzyme is responsible for catalyzing the transfer of electrons from NADPH to cytochrome P450s and thus is a key enzyme in the biosynthesis of numerous primary and secondary metabolites in plants. Neutron reflectometry shows a coexistence of two different POR conformations, a compact and an extended form with a thickness of 44 and 79 Å, respectively. Upon complete reduction by NADPH, the conformational equilibrium shifts toward the compact form protecting the reduced FMN cofactor from engaging in unspecific electron transfer reaction.

  13. Structural and functional probing of PorZ, an essential bacterial surface component of the type-IX secretion system of human oral-microbiomic Porphyromonas gingivalis.

    PubMed Central

    Lasica, Anna M.; Goulas, Theodoros; Mizgalska, Danuta; Zhou, Xiaoyan; de Diego, Iñaki; Ksiazek, Mirosław; Madej, Mariusz; Guo, Yonghua; Guevara, Tibisay; Nowak, Magdalena; Potempa, Barbara; Goel, Apoorv; Sztukowska, Maryta; Prabhakar, Apurva T.; Bzowska, Monika; Widziolek, Magdalena; Thøgersen, Ida B.; Enghild, Jan J.; Simonian, Mary; Kulczyk, Arkadiusz W.; Nguyen, Ky-Anh; Potempa, Jan; Gomis-Rüth, F. Xavier

    2016-01-01

    Porphyromonas gingivalis is a member of the human oral microbiome abundant in dysbiosis and implicated in the pathogenesis of periodontal (gum) disease. It employs a newly described type-IX secretion system (T9SS) for secretion of virulence factors. Cargo proteins destined for secretion through T9SS carry a recognition signal in the conserved C-terminal domain (CTD), which is removed by sortase PorU during translocation. Here, we identified a novel component of T9SS, PorZ, which is essential for surface exposure of PorU and posttranslational modification of T9SS cargo proteins. These include maturation of enzyme precursors, CTD removal and attachment of anionic lipopolysaccharide for anchorage in the outer membrane. The crystal structure of PorZ revealed two β-propeller domains and a C-terminal β-sandwich domain, which conforms to the canonical CTD architecture. We further documented that PorZ is itself transported to the cell surface via T9SS as a full-length protein with its CTD intact, independently of the presence or activity of PorU. Taken together, our results shed light on the architecture and possible function of a novel component of the T9SS. Knowledge of how T9SS operates will contribute to our understanding of protein secretion as part of host-microbiome interactions by dysbiotic members of the human oral cavity. PMID:27883039

  14. Influence of various polymorphic variants of cytochrome P450 oxidoreductase (POR) on drug metabolic activity of CYP3A4 and CYP2B6.

    PubMed

    Chen, Xuan; Pan, Li Qiang; Naranmandura, Hua; Zeng, Su; Chen, Shu Qing

    2012-01-01

    Cytochrome P450 oxidoreductase (POR) is known as the sole electron donor in the metabolism of drugs by cytochrome P450 (CYP) enzymes in human. However, little is known about the effect of polymorphic variants of POR on drug metabolic activities of CYP3A4 and CYP2B6. In order to better understand the mechanism of the activity of CYPs affected by polymorphic variants of POR, six full-length mutants of POR (e.g., Y181D, A287P, K49N, A115V, S244C and G413S) were designed and then co-expressed with CYP3A4 and CYP2B6 in the baculovirus-Sf9 insect cells to determine their kinetic parameters. Surprisingly, both mutants, Y181D and A287P in POR completely inhibited the CYP3A4 activity with testosterone, while the catalytic activity of CYP2B6 with bupropion was reduced to approximately ~70% of wild-type activity by Y181D and A287P mutations. In addition, the mutant K49N of POR increased the CLint (Vmax/Km) of CYP3A4 up to more than 31% of wild-type, while it reduced the catalytic efficiency of CYP2B6 to 74% of wild-type. Moreover, CLint values of CYP3A4-POR (A115V, G413S) were increased up to 36% and 65% of wild-type respectively. However, there were no appreciable effects observed by the remaining two mutants of POR (i.e., A115V and G413S) on activities of CYP2B6. In conclusion, the extent to which the catalytic activities of CYP were altered did not only depend on the specific POR mutations but also on the isoforms of different CYP redox partners. Thereby, we proposed that the POR-mutant patients should be carefully monitored for the activity of CYP3A4 and CYP2B6 on the prescribed medication.

  15. Influence of Various Polymorphic Variants of Cytochrome P450 Oxidoreductase (POR) on Drug Metabolic Activity of CYP3A4 and CYP2B6

    PubMed Central

    Naranmandura, Hua; Zeng, Su; Chen, Shu Qing

    2012-01-01

    Cytochrome P450 oxidoreductase (POR) is known as the sole electron donor in the metabolism of drugs by cytochrome P450 (CYP) enzymes in human. However, little is known about the effect of polymorphic variants of POR on drug metabolic activities of CYP3A4 and CYP2B6. In order to better understand the mechanism of the activity of CYPs affected by polymorphic variants of POR, six full-length mutants of POR (e.g., Y181D, A287P, K49N, A115V, S244C and G413S) were designed and then co-expressed with CYP3A4 and CYP2B6 in the baculovirus-Sf9 insect cells to determine their kinetic parameters. Surprisingly, both mutants, Y181D and A287P in POR completely inhibited the CYP3A4 activity with testosterone, while the catalytic activity of CYP2B6 with bupropion was reduced to approximately ∼70% of wild-type activity by Y181D and A287P mutations. In addition, the mutant K49N of POR increased the CLint (Vmax/Km) of CYP3A4 up to more than 31% of wild-type, while it reduced the catalytic efficiency of CYP2B6 to 74% of wild-type. Moreover, CLint values of CYP3A4-POR (A115V, G413S) were increased up to 36% and 65% of wild-type respectively. However, there were no appreciable effects observed by the remaining two mutants of POR (i.e., A115V and G413S) on activities of CYP2B6. In conclusion, the extent to which the catalytic activities of CYP were altered did not only depend on the specific POR mutations but also on the isoforms of different CYP redox partners. Thereby, we proposed that the POR-mutant patients should be carefully monitored for the activity of CYP3A4 and CYP2B6 on the prescribed medication. PMID:22719896

  16. Diseno de puertas moleculares controladas a nivel nanoscopico

    NASA Astrophysics Data System (ADS)

    Casasus Lis, Rosa

    The present thesis has been developed between the frontiers of different disciplines such as Coordination and Supramolecular Chemistry and Material Science. The main objective has been the design and construction of nanosupramolecular gate-ensemble, which can be defined as a basis device that modulate the access to a certain site and whose state (opened or closed) can be controlled at will by certain external stimuli, for example ionically, electrochemically and photochemically. One of the most important ideas of this thesis is the development of molecular gates using organic-inorganic hybrid systems. We have been working with a mesoporous siliceous matrix MCM-41 type and UVM-7 that possesses preorganized cavities, in that sense the porous system are homogeneous in size even in shape and periodicity. Furthermore, it has been possible to obtain systems highly functionalized due to its high specific surfaces areas (internal and external). First of all, it has been studying the design of ionically-controlled nanoscopic molecular gates. The idealized open-closed mechanism would arise from simple interactions between amines (open-gate) and Coulombic repulsion between ammonium groups (closed-gate). When protonated the open-chain polyamines in the external surface would adopt a rigid-like conformation and would be pushed away towards the pore openings due to repulsion between ammonium groups charged positively. A fundamental aspect related to molecular gates was the demonstration of specific functions like "open-close" could be controlled wilfully by certain external stimuli. In this sense, we used two different approximations to prove how works the molecular machine: (a) detect the access (controlled by external stimuli) to the pores of certain species in solution and (b) study the release of some molecules entrapped from the pore voids into the bulk solution. The first approximation, the most difficult to control, has been carried out by using a coupled reaction that would give one observable and easy signal such as change of colour and even more the only way to occur was if the studied species came inside the nanometric porous. In second place it was reported a complete study of the behaviour of a pH-driven and anion-controlled nano-supramolecular gate-like ensemble obtained by anchoring suitable polyamines on the pore outlets of mesoporous materials of the type MCM-41. The release of an entrapped dye (Ru(bipy)3 2+) from the pore voids into the bulk solution allows us to study the gating effect. This study was carried out by monitoring the dye released from the pore voids of the solid at a certain pH in the presence of a range of anions with different structural dimensions and charges, including chloride, sulphate, phosphate, and ATP. The choice of a certain anionic guest results in a different gate-like ensemble behaviour, ranging from basically no action (chloride) to complete (ATP) or partial pore blockage, depending on the pH (sulphate and phosphate). Molecular dynamics simulations using force field methods have been carried out to explain the pH-driven open/close mechanism and selectivity patterns have been discussed in terms of kinetic rates of the liberation of the dye. Furthermore, it has been applied the potential use of molecular gatelike systems as a new strategy for the chromogenic signalling of the target anions in aqueous solutions. The idea involves molecular-recognition events coupled with the control of dye transport. It entails the use of solids with nanoscopic 3D organized surfaces (mesoporous solids) that have been functionalized at the outer surface with certain binding moieties (for example amines) and additionally the pores have been loaded with a suitable dye. In absence of any species to detect there is an opened gatelike system that is able to deliver the enclosed dye to the solution. The addition of a target anionic guest capable of forming a suitable complex with the binding site might "close the gate" which would lead to recognition, thus signalling the target anion by the inhibition of the mass-delivery process. In this work we have confirmed the ATP recognition and signalling by inhibiting dye release with nanoscopic supramolecular gatelike systems on mesoporous MCM-41 supports. Finally, a dual functional hybrid material was designed for the simultaneous chromo-fluorogenic detection and removal of Hg2+ in aqueous environment. The mesoporous solid is functionalized with thiol groups that have been further reacted with the squaraine dye, resulting in the formation of a 2,4-bis(4-dialkylaminophenyl)-3-hydroxy-4-alkylsulfanylcyclobut-2-enone (PAS) derivative being anchored to the inorganic silica matrix. When the species to detect, the Hg2+ cation, is present in the solution this reacts with the PAS fragment in the solid, releasing the squaraine dye to the solution that turned deep blue and highly fluorescent. This allows a straightforward "naked-eye" detection of Hg2+ employing an easy-to-use procedure.

  17. Senda: Lectura nivel 5 (Senda: Level Five Reader).

    ERIC Educational Resources Information Center

    Peniche Leger, Maria Elena, Ed.

    This textbook, the fifth in a series of six, is designed to give the advanced elementary school student continued practice in reading Spanish. It contains some 30 sections with passages of increasing degrees of difficulty. Illustrations are included. At this level the maintenance of a central plot is considered unnecessary. The central narrative…

  18. Modulo Navideno, Nivel Primario. (Christmas Module, Primary Level.)

    ERIC Educational Resources Information Center

    Espinoza, Delia; Lopez, Santiago, III

    Four units are combined to form this primary level unit on Navidad (Christmas). It discusses and compares 3 cultures: the Mexican, the Chicano, and the Anglo-Saxon. The unit consists of: (1) "La Muneca Mas Bella de Wildrose", a story by Amado Nervo which shows children's feelings of love and tenderness; (2) the Mexican tale "El…

  19. Senda: Lectura Nivel 2 (Senda: Level Two Reader).

    ERIC Educational Resources Information Center

    Peniche Leger, Maria Elena, Ed.

    This textbook, the second in a series of six, is designed to give the beginning student practice in reading Spanish. It contains some 30 sections with passages of increasing degrees of difficulty. Colored illustrations are included. Subjects include animals, children's games, a pirate's flag, and astronauts. (SK)

  20. Senda: Lectura nivel 1 (Senda: Level One Reader).

    ERIC Educational Resources Information Center

    Peniche Leger, Maria Elena, Ed.

    This textbook, the first in a series of six, is designed to give the beginning student practice in reading Spanish as adapted to Mexican linguistics. It makes use of comic strips with few words and paragraphs describing pictures. Subjects included in the 37 sections include the park, the zoo, a birthday celebration, dreams, and school. (Author/SK)

  1. Senda: Lectura Nivel 2 (Senda: Level Two Reader).

    ERIC Educational Resources Information Center

    Peniche Leger, Maria Elena, Ed.

    This textbook, the second in a series of six, is designed to give the beginning student practice in reading Spanish. It contains some 30 sections with passages of increasing degrees of difficulty. Colored illustrations are included. Subjects include animals, children's games, a pirate's flag, and astronauts. (SK)

  2. Senda: Lectura nivel 3 (Senda: Level Three Reader).

    ERIC Educational Resources Information Center

    Peniche Leger, Maria Elena, Ed.

    This textbook, the third in a series of six, is designed to give the intermediate elementary school student continued practice in reading Spanish. It contains some 30 sections with passages of increasing degrees of difficulty. Colored illustrations are included. The book introduces simple literary excerpts, short poems, and narratives from…

  3. Senda: Lectura nivel 5 (Senda: Level Five Reader).

    ERIC Educational Resources Information Center

    Peniche Leger, Maria Elena, Ed.

    This textbook, the fifth in a series of six, is designed to give the advanced elementary school student continued practice in reading Spanish. It contains some 30 sections with passages of increasing degrees of difficulty. Illustrations are included. At this level the maintenance of a central plot is considered unnecessary. The central narrative…

  4. Libro de Lectura. Nivel A. (Reading Book. Level A.).

    ERIC Educational Resources Information Center

    Yeats, Alid; And Others

    This is the first in a series of four reading books written in Spanish and designed for use in elementary bilingual education programs. The stories are divided into two main sections, Estudios Sociales (Social Studies) and La Naturaleza (Nature). The five stories in the first section deal with such topics as the home, school, and cleaning. The…

  5. Libro de Lectura. Nivel C. (Reading Book. Level C.).

    ERIC Educational Resources Information Center

    Yeats, Alid

    This is the third in a series of four reading books written in Spanish and designed for use in elementary bilingual education programs. The reader contains nine stories, most of which deal with some aspect of nature study, such as plants or insects. Each story is followed by a list of new vocabulary and enrichment exercises and activities in the…

  6. Libro de Lectura. Nivel B. (Reading Book. Level B.).

    ERIC Educational Resources Information Center

    Yeats, Alid; And Others

    This is the second in a series of four reading books written in Spanish and designed for use in elementary bilingual education programs. The stories are divided into two main sections, Estudios Sociales (Social Studies), and La Comunidad (The Community). The stories in the first section have to do with activities in the home, particularly chores…

  7. Análise dos Conceitos Astronômicos Apresentados por Professores de Algumas Escolas Estaduais Brasileiras

    NASA Astrophysics Data System (ADS)

    Voelzke, Marcos Rincon; Gonzaga, Edson Pereira

    2011-12-01

    A razão para o desenvolvimento deste trabalho baseia-se no fato de que muitos professores da Educação Básica (EB) não lidam com conceitos relacionados à astronomia, e quando o fazem eles simplesmente seguem livros didáticos que podem conter erros conceituais. Como é de conhecimento geral a astronomia é um dos conteúdos a serem ensinados na EB fazendo parte dos Parâmetros Curriculares Nacionais e das Propostas Curriculares do Estado de São Paulo, mas é um fato, que vários pesquisadores apontam, a existência de muitos problemas no ensino da astronomia. Com o propósito de minimizar algumas dessas deficiências foi realizado um trabalho de pesquisa com a utilização de questionários pré e pós pesquisa, para tanto foi desenvolvido um Curso de Extensão Universitária para professores da Diretoria de Ensino Regional (DE) que abrange Mauá, Ribeirão Pires e Rio Grande da Serra (no Estado de São Paulo) com os seguintes objetivos: levantar concepções alternativas; subsidiar os professores por meio de palestras, debates e workshops, e verificar o sucesso da aprendizagem após o curso, adotando-se como referência, para a análise dos resultados, os dicionários de Língua Portuguesa (FERREIRA, 2004) e Enciclopédico de Astronomia e Astronáutica (MOURĀO, 1995). Portanto, dezesseis questões foram aplicadas antes e após o curso, assim pode-se verificar após a pesquisa que 100,0% dos professores sabiam os nomes das fases da Lua, 97,0% entenderam que o Sistema Solar é composto por oito planetas, 78,1% foram capazes de explicar como ocorre um eclipse lunar, um eclipse solar e um solstício, 72,7% sabiam como explicar a ocorrência das estações do ano; 64,5% explicaram corretamente a ocorrência do equinócio, 89,7% foram capazes de definir adequadamente o termo cometa; 63,6% definiram asteróide, 54,5% meteoro, 58,1% galáxia, e 42,4% planeta. Os resultados obtidos indicam uma aprendizagem significativa por parte dos participantes.

  8. The genetic polymorphisms of POR*28 and CYP3A5*3 significantly influence the pharmacokinetics of tacrolimus in Chinese renal transplant recipients.

    PubMed

    Zhang, Jing-Jing; Liu, Shuai-Bing; Xue, Ling; Ding, Xiao-Liang; Zhang, Hua; Miao, Li-Yan

    2015-09-01

    The aims of this study were to assess the influence of the polymorphism of cytochrome P450 oxidoreductase (POR) as well as other relevant genes (CYP3A4, CYP3A5, ABCB1) on individual variability of tacrolimus pharmacokinetics and perform population pharmacokinetic analysis of tacrolimus in Chinese renal transplant recipients. Tacrolimus trough whole blood concentrations and clinical details were retrospectively collected from 83 renal recipients. CYP3A4*1G, CYP3A5*3, and ABCB1 C3435T were genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), POR*28 and CYP3A4*22 were genotyped by sequencing method. Population pharmacokinetic analysis was performed using NONMEM program. The significant influences of CYP3A5*3, CYP3A4*1G, and POR*28 polymorphisms on tacrolimus dose-adjusted trough concentrations (C0/D) were observed in 83 renal recipients. Subgroup analysis showed that POR*28 polymorphisms significantly decreased tacrolimus C0/D by 1.50 - 1.84-fold (p < 0.05) in patients who were CYP3A5 expressers (CYP3A5*1 carriers, n = 46), while similar results could not be obtained from CYP3A5 non-expressers (CYP3A5*3/*3 carriers, n = 37). Additionally, population pharmacokinetic analysis identified that the combined genotype of CYP3A5-POR was the only covariant for the apparent clearance of tacrolimus (CL/F). The study demonstrated that the POR*28 C>T mutation could decrease the C0/D of tacrolimus in renal recipients who were CYP3A5 expressers. The population pharmacokinetic model showed that the combined genotype of CYP3A5-POR was associated with the CL/F of tacrolimus which might provide references for personalized use of tacrolimus in clinic.

  9. Reactions of nitrogen oxides with the five-coordinate Fe(III)(porphyrin) nitrito intermediate Fe(Por)(ONO) in sublimed solids.

    PubMed

    Kurtikyan, Tigran S; Hovhannisyan, Astghik A; Hakobyan, Manya E; Patterson, James C; Iretskii, Alexei; Ford, Peter C

    2007-03-28

    Detailed experimental studies are described for reactions of several nitrogen oxides with iron porphyrin models for heme/NxOy systems. It is shown by FTIR and optical spectroscopy and by isotope labeling experiments that reaction of small increments of NO2 with sublimed thin layers of the iron(II) complex Fe(Por) (Por = meso-tetraphenylporphyrinato dianion, TPP, or meso-tetra-p-tolylporphyrinato dianion, TTP) leads to formation of the 5-coordinate nitrito complexes Fe(Por)(eta1-ONO) (1), which are fairly stable but very slowly decompose under vacuum giving mostly the corresponding nitrosyl complexes Fe(Por)(NO). Further reaction of 1 with new NO2 increments leads to formation of the nitrato complex Fe(Por)(eta2-O2NO) (2). The interaction of NO with 1 at low temperature involves ligand addition to give the nitrito-nitrosyl complexes Fe(Por)(eta1-ONO)(NO) (3); however, these isomerize to the nitro-nitrosyl analogs Fe(Por)(eta1-NO2)(NO) (4) upon warming. Experiments with labeled nitrogen oxides argue for an intramolecular isomerization ("flipping") mechanism rather than one involving dissociation and rebinding of NO2. The Fe(III) centers in the 6-coordinate species 3 and 4 are low spin in contrast to 1, which appears to be high-spin, although DFT computations of the porphinato models Fe(P)(nitrite) suggest that the doublet nitro species and the quartet and sextet nitrito complexes are all relatively close in energy. The nitro-nitrosyl complex 4 is stable under an NO atmosphere but decomposes under intense pumping to give a mixture of the ferrous nitrosyl complex Fe(Por)(NO) and the ferric nitrito complex Fe(Por)(eta1-ONO) indicating the competitive dissociation of NO and NO2. Hence, loss of NO from 4 is accompanied with nitro --> nitrito isomerization consistent with 1 being the more stable of the 5-coordinate NO2 complexes of iron porphyrins.

  10. Crystallization and preliminary X-ray analysis of the C-terminal fragment of PorM, a subunit of the Porphyromonas gingivalis type IX secretion system

    PubMed Central

    Stathopulos, Julien; Cambillau, Christian; Cascales, Eric; Roussel, Alain; Leone, Philippe

    2015-01-01

    PorM is a membrane protein involved in the assembly of the type IX secretion system (T9SS) from Porphyromonas gingivalis, a major bacterial pathogen responsible for periodontal disease in humans. The periplasmic domain of PorM was overexpressed in Escherichia coli and purified. A fragment of the purified protein was obtained by limited proteolysis. Crystals of this fragment belonged to the tetragonal space group P43212. Native and MAD data sets were recorded to 2.85 and 3.1 Å resolution, respectively, using synchrotron radiation. PMID:25615973

  11. Most but not all laboratories can detect the recently emerged Neisseria gonorrhoeae porA mutants - results from the QCMD 2013 N. gonorrhoeae external quality assessment programme.

    PubMed

    Luijt, D; Di Lorenzo, C; van Loon, A M; Unemo, M

    2014-02-27

    We describe the results of the Quality Control for Molecular Diagnostics 2013 Neisseria gonorrhoeae external quality assessment programme that included an N. gonorrhoeae strain harbouring an N. meningitidis porA gene which causes false-negative results in molecular diagnostic assays targeting the gonococcal porA pseudogene. Enhanced awareness of the international transmission of such gonococcal strains is needed to avoid false-negative results in both in-house and commercial molecular diagnostic assays used in laboratories worldwide, but particularly in Europe.

  12. "Estudio tribologico de aceros para moldes. Aplicacion al moldeo por inyeccion de polibutilentereftalato reforzado con fibra de vidrio"

    NASA Astrophysics Data System (ADS)

    Martinez Mateo, Isidoro Jose

    Mould materials for injection moulding of polymers and polymer-matrix composites represent a relevant industrial economic sector due to the large quantity of pieces and components processed. The material selection for mould manufacturing, its composition and heat treatment, the hardening procedures and machining and finishing processes determine the service performance and life of the mould. In the first part of the present study, the relationship between the hardness and microstructure and the wear resistance of mould steels from large blocks has been studied by pin-on-disc tests, studying the main wear mechanisms. In order to determine the surface damage on mould steels under real injection conditions, different commercial steels have been studied by measuring the variation of surface roughness with the number of injected pieces with different reinforcement percentages and different mould geometries, by using optical profilometry and scanning electron microscopy techniques. It was important to determine the variation of surface roughness of the moulded pieces with the number of injection operations. The materials used were polybutyleneterephthalate pure and reinforced with either 20% or 50% glass fibre. For the different mould designs, the evolution of the glass fibre orientation with injection flow has been determined by image analysis and related to roughness changes and surface damage, both of the composite parts and of the mould steel surface. Finally, the abrasion resistance of the composite parts has been studied by scratch tests as a function of the number of injected parts and of the scratch direction with respect to injection flow and glass fibre orientation. Los materiales para moldes de inyeccion de polimeros y materiales compuestos representan un sector economicamente muy relevante debido al gran aumento del numero de componentes fabricados a partir de materiales polimericos obtenidos mediante moldeo por inyeccion. La seleccion del material para la

  13. Rapid characterization of outer-membrane proteins in Neisseria lactamica by SELDI-TOF-MS (surface-enhanced laser desorption ionization-time-of-flight MS) for use in a meningococcal vaccine.

    PubMed

    Mukhopadhyay, Tarit Kumar; Halliwell, Denise; O'Dwyer, Cliona; Shamlou, Parviz Ayazi; Levy, Myriam Susana; Allison, Nigel; Gorringe, Andrew; Reddin, Karen M

    2005-04-01

    Immunological and epidemiological evidence suggests that the development of natural immunity to meningococcal disease results from colonization of the nasopharynx by commensal Neisseria species, particularly with Neisseria lactamica. We have reported previously that immunization with N. lactamica outer-membrane vesicles containing the major OMPs (outer-membrane proteins) protected mice against lethal challenge with meningococci of diverse serogroups and serotypes and has the potential to form the basis of a vaccine against meningococcal diseases [Oliver, Reddin, Bracegirdle et al. (2002) Infect. Immun. 70, 3621-3626]. In the present study, we have shown that biomass production and the profile of outer-membrane vesicle proteins may be affected by fermentation conditions and, in particular, media composition. Ciphergen SELDI-TOF Protein Chips were used as a rapid and sensitive new method in comparison with conventional SDS/PAGE. SELDI-TOF-MS (surface-enhanced laser-desorption ionization-time-of-flight MS) reproducibly identified three major OMPs (NspA, RmpM and PorB) and detected the changes in the protein profile when the growth medium was altered. The findings of this work indicate that SELDI-TOF-MS is a useful tool for the rapid optimization of OMP production in industrial fermentation processes and can be adapted as a Process Analytical Technology.

  14. Bioinformatic analysis of outer membrane proteome of Neisseria meningitidis and Neisseria lactamica.

    PubMed

    Abel, Ana; Sánchez, Sandra; Arenas, Jesús; Criado, María T; Ferreirós, Carlos M

    2007-03-01

    Two-dimensional electrophoresis (isoelectric focusing/SDS-PAGE) and Western-blotting techniques were used to analyze and compare common and/or specific outer-membrane proteins and antigens from Neisseria meningitidis and Neisseria lactamica. Bioinformatic image analyses of proteome and immunoproteome maps indicated the presence of numerous proteins and several antigens shared by N. meningitidis and N. lactamica, although the inter-strain variation in the maps was of similar magnitude to the inter-species variation, and digital comparison of the maps did not reveal proteins found to be identical by MALDI-TOF fingerprinting analysis. PorA and RmpM, two relevant outer-membrane antigens, manifested as various spots at several different positions. While some of these were common to all the strains analyzed, others were exclusive to N. meningitidis and their electrophoretic mobilities were different than expected. One such spot, with a molecular mass of 19 kDa, may be the C-terminal fragment of RmpM (RmpM-Cter). The results demonstrate that computer-driven analysis based exclusively on spot positions in the proteome or immunoproteome maps is not a reliable approach to predict the identity of proteins or antigens; rather, other identification techniques are necessary to obtain accurate comparisons.

  15. Expression of the hemagglutinin HA1 subunit of the equine influenza virus using a baculovirus expression system.

    PubMed

    Sguazza, Guillermo H; Fuentealba, Nadia A; Tizzano, Marco A; Galosi, Cecilia M; Pecoraro, Marcelo R

    2013-01-01

    Equine influenza virus is a leading cause of respiratory disease in horses worldwide. Disease prevention is by vaccination with inactivated whole virus vaccines. Most current influenza vaccines are generated in embryonated hens' eggs. Virions are harvested from allantoic fluid and chemically inactivated. Although this system has served well over the years, the use of eggs as the substrate for vaccine production has several well-recognized disadvantages (cost, egg supply, waste disposal and yield in eggs). The aim of this study was to evaluate a baculovirus system as a potential method for producing recombinant equine influenza hemagglutinin to be used as a vaccine. The hemagglutinin ectodomain (HA1 subunit) was cloned and expressed using a baculovirus expression vector. The expression was determined by SDS-PAGE and immunoblotting. A high yield, 20μg/ml of viral protein, was obtained from recombinant baculovirus-infected cells. The immune response in BALB/c mice was examined following rHA1 inoculation. Preliminary results show that recombinant hemagglutinin expressed from baculovirus elicits a strong antibody response in mice; therefore it could be used as an antigen for subunit vaccines and diagnostic tests. Copyright © 2013 Asociación Argentina de Microbiología. Publicado por Elsevier España. All rights reserved.

  16. Palivizumab outcomes registry data from Spain: Infección Respiratoria Infantil por Virus Respiratorio Sincitial (IRIS) Study Group.

    PubMed

    Carbonell-Estrany, Xavier

    2003-02-01

    Respiratory syncytial virus (RSV) is the leading cause of lower respiratory illness in children <2 years of age. Severe RSV infection requiring hospitalization is linked to gestational age, chronic cardiopulmonary conditions and immunosuppression. The Infección Respiratoria Infantil por Virus Respiratorio Sincitial (IRIS) Study group in Spain conducted two pivotal epidemiologic studies establishing that serious RSV illness among premature infants was responsible for high rehospitalization rates (approximately 13%). RSV lower respiratory tract illness also correlated with prolonged hospital stay and more intensive care unit admissions. In Europe recent availability of palivizumab, a humanized monoclonal antibody to RSV, is a major therapeutic advancement directed against prevention of lower respiratory tract infection secondary to this viral pathogen. To ensure proper and optimal usage of palivizumab, the IRIS group, in conjunction with the Spanish Neonatology Group, developed prophylaxis guidelines for neonates. Palivizumab prophylaxis is strongly recommended in premature infants < or =28 weeks gestation or those affected with chronic lung disease. Additionally, palivizumab is recommended for infants with a gestational age of 29 to 32 weeks, without evidence of chronic lung disease and who are <6 months old at the onset of the RSV season. It was thought that slightly older premature infants (33 to 35 weeks gestational age) should be assessed on an individual basis to determine whether prophylaxis is warranted. The IRIS Study Group is currently determining the effectiveness of these recommendations by measuring the incidence of RSV-related hospital admissions in infants born at < or =32 weeks gestational age who are receiving palivizumab prophylaxis.

  17. Community-based education in nutrition and cancer: the Por La Vida Cuidándome curriculum.

    PubMed

    Navarro, A M; Rock, C L; McNicholas, L J; Senn, K L; Moreno, C

    2000-01-01

    The Por La Vida (PLV) intervention model relies on community lay health advisors trained to conduct education sessions among members of their existing social networks. PLV Cuidándome was funded by the NCI to develop, implement, and evaluate the PLV model with respect to nutrition and cancer prevention, as well as early detection of breast and cervical cancers. The target population is the Latino community, for which substantial barriers to health care access exist. This article presents the curriculum that guides the sessions and describes its development, which was based on semi-structured interviews with Latina lay-health community workers to explore relevant attitudes and behaviors. Also key to the process was the work of the educational materials committee, whose members offered community representation as well as expertise in nutritional sciences, educational technologies, and community-based health promotion interventions and research. The 12-session curriculum's goal was to increase both the variety and the quality of fruits and/or vegetables consumed. It included information about consumption of fiber and fat in the importance of balance between energy intake and physical activity. The program has been well received. An ongoing study examines how it enhances nutrition and cancer prevention.

  18. Avidity maturation following vaccination with a meningococcal recombinant hexavalent PorA OMV vaccine in UK infants.

    PubMed

    Longworth, Emma; Borrow, Ray; Goldblatt, David; Balmer, Paul; Dawson, Maureen; Andrews, Nick; Miller, Elizabeth; Cartwright, Keith

    2002-06-07

    To date, there are no data assessing the utility of avidity indices as a surrogate marker for the induction of immunological memory following meningococcal serogroup B outer membrane vesicle (OMV) vaccination. We studied infants who had been immunized with three doses of a recombinant hexavalent PorA OMV vaccine at ages 2-4 months, together with a fourth dose at age 12-18 months. A control group had received a single dose of the same vaccine at age 12-18 months. As previously reported, serum bactericidal antibody (SBA) titres increased after each of the first three doses, with a significant increase observed from 6 months post third dose to 1 month post fourth dose. The geometric mean avidity indices (GMAI), against strain H44/76 OMVs, increased from 1 month post first dose to 1 month post third dose. Significant increases in GMAI were observed at 6 months post third dose and again following the fourth dose. At 32-42 months of age, though the SBA titres had returned to post first dose levels, the GMAI remained elevated. No increase in avidity was observed in the control group. Antibody avidity indices are useful laboratory markers for the priming of immunological memory following vaccination with meningococcal serogroup B OMV vaccines.

  19. Spectroscopic and kinetic characterization of the light-dependent enzyme protochlorophyllide oxidoreductase (POR) using monovinyl and divinyl substrates.

    PubMed

    Heyes, Derren J; Kruk, Jerzy; Hunter, C Neil

    2006-02-15

    The enzyme POR [Pchlide (protochlorophyllide) oxidoreductase] catalyses the reduction of Pchlide to chlorophyllide, which is a key step in the chlorophyll biosynthesis pathway. This light-dependent reaction has previously been studied in great detail but recent reports suggest that a mixture of MV (monovinyl) and DV (divinyl) Pchlides may have influenced some of these properties of the reaction. Low-temperature absorbance and fluorescence spectroscopy have revealed several spectral differences between MV and DV Pchlides, which were purified from a Rhodobacter capsulatus strain that was shown to contain a mixture of the two pigments. A thorough steady-state kinetic characterization using both Pchlide forms demonstrates that neither pigment appears to affect the kinetic properties of the enzyme. The reaction has also been monitored following illumination at low temperatures and was shown to consist of an initial photochemical step followed by four 'dark' steps for both pigments. However, minor differences were observed in the spectral properties of some of the intermediates, although the temperature dependency of each step was nearly identical for the two pigments. This work provides the first detailed kinetic and spectroscopic study of this unique enzyme using biologically important MV and DV substrate analogues. It also has significant implications for the DV reductase enzyme, which is responsible for converting DV pigments into their MV counterparts, and its position in the sequence of reactions that comprise the chlorophyll biosynthesis pathway.

  20. Examination of P-OR bridging bond orders in phosphate monoesters using (18)O isotope shifts in 31P NMR.

    PubMed

    Sorensen-Stowell, Kerensa; Hengge, Alvan C

    2005-06-10

    Evidence indicates that phosphate monoesters undergo hydrolysis by a loose transition state with extensive bond fission to the leaving group. It has been proposed that part of the high dependence of the rate on the leaving group pKa (betalg = -1.23) arises from weaker ester bonds in the reactants as the pKa of the leaving group decreases, on the basis of X-ray structures and calculations. In contrast, IR and Raman studies suggest that the leaving group has little effect on the length of the P-OR bridging bond in solution. To gather additional data on this issue, we have used (18)O isotopic shifts in 31P NMR to monitor the bond order of P-O bonds in a range of phosphate esters with different leaving groups. Using this technique, we have been able to evaluate whether significant changes are observed in the P-O bond orders for the bridging and nonbridging positions of methyl, ethyl, phenethyl, propargyl, phenyl, and p-nitrophenyl phosphate using [(16)O(18)O] labeled species in deuterium oxide. The results indicate that the bridging and nonbridging bond orders to phosphorus in phosphate monoesters are not significantly altered by differences in the pKa of the leaving group or by the counterion of the phosphate ester dianion.

  1. P(O)R2-directed Pd-catalyzed C-H functionalization of biaryl derivatives to synthesize chiral phosphorous ligands.

    PubMed

    Hu, Rong-Bin; Wang, Hong-Li; Zhang, Hong-Yu; Zhang, Heng; Ma, Yan-Na; Yang, Shang-Dong

    2014-01-01

    Chiral phosphorus ligands have been widely used in transition metal-catalyzed asymmetric reactions. Herein, we report a new synthesis approach of chiral biaryls containing a phosphorus moiety using P(O)R2-directed Pd-catalyzed C-H activation; the functionalized products are produced with good enantioselectivity.

  2. Interaction between variants in the CYP2C9 and POR genes and the risk of sulfonylurea-induced hypoglycaemia: A GoDARTS Study.

    PubMed

    Dujic, Tanja; Zhou, Kaixin; Donnelly, Louise A; Leese, Graham; Palmer, Colin N A; Pearson, Ewan R

    2017-06-28

    Data on the association of CYP2C9 genetic polymorphisms with sulfonylurea (SU)-induced hypoglycaemia (SH) are inconsistent. Recent studies showed that variants in the P450 oxidoreductase (POR) gene could affect CYP2C9 activity. In this study, we explored the effects of POR*28 and combined CYP2C9*2 and CYP2C9*3 genotypes on SH and the efficacy of SU treatment in type 2 diabetes. A total of 1770 patients were included in the analysis of SU efficacy, assessed as the combined outcome of the HbA1c reduction and the prescribed SU daily dose. Sixty-nine patients with severe SH were compared with 311 control patients. The number of CYP2C9 deficient alleles was associated with nearly three-fold higher odds of hypoglycaemia (OR, 2.81; 95% CI, 1.30-6.09; P = .009) and better response to SU treatment (β, -0.218; SE, 0.074; P = .003) only in patients carrying the POR*1/*1 genotype. Our results indicate that interaction between CYP2C9 and POR genes may be an important determinant of efficacy and severe adverse effects of SU treatment. © 2017 The Authors. Diabetes, Obesity and Metabolism published by John Wiley & Sons Ltd.

  3. Adult Student Retention and Achievement with Language-Based Modular Materials. POR FIN: Program Organizing Related Family Instruction in the Neighborhood.

    ERIC Educational Resources Information Center

    Bexar County School Board, San Antonio, TX.

    The goal of the POR FIN research design was to develop a language-based curriculum emphasizing the audiolingual approach and integrating academic and social-functioning subject matter. The modular curriculum is designed so that each lesson is independent and complete in itself, and provides a high degree of motivation, retention, and achievement…

  4. El proceso hacia la integracion de la equidad por genero al curriculo.(The Process of the Integration of Gender Equity in the Curriculum.)

    ERIC Educational Resources Information Center

    Rivera-Bermudez, Carmen D.

    "El Proyecto Colaborativo de Equidad por Genero en la Educacion," or the Collaborative Project for Gender Equity in Education, was undertaken in Puerto Rico between 1990 and 1992 to study how to facilitate the integration of gender equity themes in the curriculum through the direct action of participating teachers. A study examined the…

  5. Recombinant PorA, the major outer membrane protein of Campylobacter jejuni, provides heterologous protection in an adult mouse intestinal colonization model.

    PubMed

    Islam, Anjum; Raghupathy, Raj; Albert, M John

    2010-11-01

    Immunity against Campylobacter jejuni, a major food-borne pathogen causing diarrhea, is largely serotype specific. The major outer membrane protein (MOMP) of C. jejuni, PorA, is a common antigen with the potential to provide broad protection. Adult BALB/c mice were orally immunized with a recombinant glutathione S-transferase (GST) fused to PorA prepared from Campylobacter jejuni C31 (O:6,7) (GST-PorA) combined with a modified heat-labile enterotoxin of Escherichia coli as an adjuvant and later orally challenged with C31 strain or three heterologous strains: 48 (O:19), 75 (O:3), and 111 (O:1,44). Protection from colonization with the challenge organism was studied by fecal screening daily for 9 days. Serum and intestinal lavage fluid antibodies against the vaccine and Sarkosyl-purified MOMP from C31 were measured by using an enzyme-linked immunosorbent assay. The vaccine produced robust antibody responses against both antigens in serum and secretion. Since strain C31 was a poor colonizer, homologous protection could not be studied. The protective efficacies of heterologous strains were 43% (for strain 48, P < 0.001), 29% (for strain 75, P < 0.005), and 42% (for strain 111, P < 0.001) for the 9-day period compared to control mice given phosphate-buffered saline. Thus, PorA provided appreciable protection against colonization with heterologous serotypes.

  6. Cell Growth Defect Factor1/CHAPERONE-LIKE PROTEIN OF POR1 Plays a Role in Stabilization of Light-Dependent Protochlorophyllide Oxidoreductase in Nicotiana benthamiana and Arabidopsis[C][W

    PubMed Central

    Lee, Jae-Yong; Lee, Ho-Seok; Song, Ji-Young; Jung, Young Jun; Reinbothe, Steffen; Park, Youn-Il; Lee, Sang Yeol; Pai, Hyun-Sook

    2013-01-01

    Angiosperms require light for chlorophyll biosynthesis because one reaction in the pathway, the reduction of protochlorophyllide (Pchlide) to chlorophyllide, is catalyzed by the light-dependent protochlorophyllide oxidoreductase (POR). Here, we report that Cell growth defect factor1 (Cdf1), renamed here as CHAPERONE-LIKE PROTEIN OF POR1 (CPP1), an essential protein for chloroplast development, plays a role in the regulation of POR stability and function. Cdf1/CPP1 contains a J-like domain and three transmembrane domains, is localized in the thylakoid and envelope membranes, and interacts with POR isoforms in chloroplasts. CPP1 can stabilize POR proteins with its holdase chaperone activity. CPP1 deficiency results in diminished POR protein accumulation and defective chlorophyll synthesis, leading to photobleaching and growth inhibition of plants under light conditions. CPP1 depletion also causes reduced POR accumulation in etioplasts of dark-grown plants and as a result impairs the formation of prolamellar bodies, which subsequently affects chloroplast biogenesis upon illumination. Furthermore, in cyanobacteria, the CPP1 homolog critically regulates POR accumulation and chlorophyll synthesis under high-light conditions, in which the dark-operative Pchlide oxidoreductase is repressed by its oxygen sensitivity. These findings and the ubiquitous presence of CPP1 in oxygenic photosynthetic organisms suggest the conserved nature of CPP1 function in the regulation of POR. PMID:24151298

  7. The influence of CYP3A, PPARA, and POR genetic variants on the pharmacokinetics of tacrolimus and cyclosporine in renal transplant recipients.

    PubMed

    Lunde, Ingrid; Bremer, Sara; Midtvedt, Karsten; Mohebi, Beata; Dahl, Miriam; Bergan, Stein; Åsberg, Anders; Christensen, Hege

    2014-06-01

    Tacrolimus (Tac) and cyclosporine (CsA) are mainly metabolized by CYP3A4 and CYP3A5. Several studies have demonstrated an association between the CYP3A5 genotype and Tac dose requirements. Recently, CYP3A4, PPARA, and POR gene variants have been shown to influence CYP3A metabolism. The present study investigated potential associations between CYP3A5*3, CYP3A4*22, PPARA c.209- 1003G>A and c.208+3819A>G, and POR*28 alleles and dose-adjusted concentrations (C/D) of Tac and CsA in 177 renal transplant patients early post-transplant. All patients (n=177) were genotyped for CYP3A4*22, CYP3A5*3, POR*28, PPARA c.209-1003G>A, and PPARA c.208+3819A>G using real-time polymerase chain reaction (PCR) and melting curve analysis with allelespecific hybridization probes or PCR restriction fragment length polymorphisms (RFLP) methods. Drug concentrations and administered doses were retrospectively collected from patient charts at Oslo University Hospital, Rikshospitalet, Norway. One steady-state concentration was collected for each patient. We confirmed a significant impact of the CYP3A5*3 allele on Tac exposure. Patients with POR*28 and PPARA variant alleles demonstrated 15 % lower (P=0.04) and 19 % higher (P=0.01) Tac C0/D respectively. CsA C2/D was 53 % higher among CYP3A4*22 carriers (P=0.03). The results support the use of pre-transplant CYP3A5 genotyping to improve initial dosing of Tac, and suggest that Tac dosing may be further individualized by additional POR and PPARA genotyping. Furthermore, initial CsA dosing may be improved by pre-transplant CYP3A4*22 determination.

  8. Compound heterozygosity of a paternal submicroscopic deletion and a maternal missense mutation in POR gene: Antley-bixler syndrome phenotype in three sibling fetuses.

    PubMed

    Tzetis, Maria; Konstantinidou, Anastasia; Sofocleous, Christalena; Kosma, Konstantina; Mitrakos, Anastasios; Tzannatos, Christina; Kitsiou-Tzeli, Sofia

    2016-07-01

    Antley-Bixler syndrome (ABS) is an exceptionally rare craniosynostosis syndrome that can be accompanied by disordered steroidogenesis, and is mainly caused by mutations in the POR gene, inherited in an autosomal recessive manner. Here we report the prenatal and postmortem findings of three sibling fetuses with ABS as a result of compound heterozygosity of a paternal submicroscopic deletion and a maternal missense mutation in the POR gene. Prenatal ultrasound and postmortem examination were performed in three sibling fetuses with termination of pregnancy at 22, 23, and 17 weeks of gestation, respectively. Molecular analysis of fetus 2 and 3 included (a) bidirectional sequencing of exon 8 of the POR gene after amplification of the specific locus by polymerase chain reaction, to detect single nucleotide variants (SNVs) and (b) high resolution comparative genomic hybridization (CGH) positive single nucleotide polymorphism array CGH (aCGH) analysis to detect copy number variants (CNVs), copy neutral areas of loss of heterozygosity and uniparental disomy. The diagnosis of ABS was suggested by the postmortem examination findings. The combination of the POR gene molecular analysis and aCGH revealed a compound heterozygous genotype of a maternal SNV (p.A287P) and a paternal CNV (NC_000007.13:g.(?_75608488)_(75615534_?)del). To the best of our knowledge, these sibling fetuses add to the few reported cases of ABS, caused by a combination of a SNV and a CNV in the POR gene. The detailed description of the pathologic and radiographic findings of second trimester fetuses affected with ABS adds novel knowledge concerning the early ABS phenotype, in lack of previous relevant reports. Birth Defects Research (Part A) 106:536-541, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  9. Acrylamide concentration determines the direction and magnitude of helical membrane protein gel shifts

    PubMed Central

    Rath, Arianna; Cunningham, Fiona; Deber, Charles M.

    2013-01-01

    SDS/PAGE is universally used in biochemistry, cell biology, and immunology to resolve minute protein amounts readily from tissue and cell extracts. Although molecular weights of water-soluble proteins are reliably determined from their SDS/PAGE mobility, most helical membrane proteins, which comprise 20–30% of the human genome and the majority of drug targets, migrate to positions that have for decades been unpredictably slower or faster than their actual formula weight, often confounding their identification. Using de novo designed transmembrane-mimetic polypeptides that match the composition of helical membrane-spanning sequences, we quantitate anomalous SDS/PAGE fractionation of helical membrane proteins by comparing the relative mobilities of these polypeptides with typical water-soluble reference proteins on Laemmli gels. We find that both the net charge and effective molecular size of the migrating particles of transmembrane-mimetic species exceed those of the corresponding reference proteins and that gel acrylamide concentration dictates the impact of these two factors on the direction and magnitude of anomalous migration. Algorithms we derived from these data compensate for this differential effect of acrylamide concentration on the SDS/PAGE mobility of a variety of natural membrane proteins. Our results provide a unique means to predict anomalous migration of membrane proteins, thereby facilitating straightforward determination of their molecular weights via SDS/PAGE. PMID:24019476

  10. High-precision quantitation of a tuberculosis vaccine antigen with capillary-gel electrophoresis using an injection standard.

    PubMed

    Shala-Lawrence, Agnesa; Beheshti, Samaneh; Newman, Elena; Tang, Mei; Krylova, Svetlana M; Leach, Michael; Carpick, Bruce; Krylov, Sergey N

    2017-12-01

    Analysis of proteinogenic vaccine antigens in a quality control environment requires an accurate, precise, and reliable method for protein separation and quantitation. While having multiple advantages over the classical SDS-PAGE, capillary gel electrophoresis (CGE) has not yet become a standard tool in vaccine antigen analysis. Here we report on development of a CGE-based method for quantitative analysis of a tuberculosis vaccine fusion antigen protein, H4, currently in clinical trials. We demonstrate that our method can monitor antigen purity and relative quantity with greater precision and accuracy versus SDS-PAGE. In addition, due to use of direct light-absorbance detection, the CGE method is suitable for absolute quantitation, an application for which SDS-PAGE is limited due to the need for staining and limited dynamic range of detection. To further improve the performance of our quantitation method, we introduced Bovine Serum Albumin (BSA) as an injection standard to correct for signal variance associated with the injected sample volume. We found that, for our specific application, BSA was more appropriate as an injection standard versus one provided in a commercial kit, in terms of precision and accuracy for quantitation of H4. In addition to providing better method performance versus SDS-PAGE, CGE is also faster and less resource-intensive. We conclude that CGE should be considered as a replacement for traditional SDS-PAGE methods for vaccine antigen quantitation in a quality-control environment. Copyright © 2017 Elsevier B.V. All rights reserved.

  11. Estimaciones de Prevalencia del VIH por Género y Grupo de Riesgo en Tijuana, México: 2006

    PubMed Central

    Iñiguez-Stevens, Esmeralda; Brouwer, Kimberly C.; Hogg, Robert S.; Patterson, Thomas L.; Lozada, Remedios; Magis-Rodriguez, Carlos; Elder, John P.; Viani, Rolando M.; Strathdee, Steffanie A.

    2010-01-01

    OBJETIVO Estimar la prevalencia del VIH en adultos de 15-49 años de edad en Tijuana, México - en la población general y en subgrupos de riesgo en el 2006. METODOS Se obtuvieron datos demográficos del censo Mexicano del 2005, y la prevalencia del VIH se obtuvo de la literatura. Se construyó un modelo de prevalencia del VIH para la población general y de acuerdo al género. El análisis de sensibilidad consistió en estimar errores estándar del promedio-ponderado de la prevalencia del VIH y tomar derivados parciales con respecto a cada parámetro. RESULTADOS La prevalencia del VIH es 0.54%(N = 4,347) (Rango: 0.22%–0.86%, (N = 1,750–6,944)). Esto sugiere que 0.85%(Rango: 0.39%–1.31%) de los hombres y 0.22%(Rango: 0.04%–0.40%) de las mujeres podrían ser VIH-positivos. Los hombres que tienen sexo con hombres (HSH), las trabajadoras sexuales usuarias de drogas inyectables (MTS-UDI), MTS-noUDI, mujeres UDI, y los hombres UDI contribuyeron las proporciones más elevadas de personas infectadas por el VIH. CONCLUSIONES El número de adultos VIH-positivos entre subgrupos de riesgo en la población de Tijuana es considerable, marcando la necesidad de enforcar las intervenciones de prevención en sus necesidades específicas. El presente modelo estima que hasta 1 en cada 116 adultos podrían ser VIH-positivos. PMID:19685824

  12. Results of Mujeres Felices por ser Saludables: a dietary/breast health randomized clinical trial for Latino women.

    PubMed

    Fitzgibbon, Marian L; Gapstur, Susan M; Knight, Sara J

    2004-10-01

    Data are limited on the efficacy of health-focused interventions for young, low-acculturated Latino women. Because breast cancer is the most commonly diagnosed cancer and the most common cause of cancer mortality in this population, combined interventions that address both early detection and dietary patterns could help reduce both morbidity and mortality associated with breast cancer in this underserved population. Mujeres Felices por ser Saludables was randomized intervention study designed to assess the efficacy of an 8-month combined dietary and breast health intervention to reduce fat and increase fiber intake as well as to increase the frequency and proficiency of breast self-examination (BSE) and reduce anxiety related to BSE among Latinas. Blocked randomization in blocks of 6 was used to randomize 256 20- to 40-year-old Latinas to the intervention (n = 127) or control group (n = 129). The intervention group attended an 8-month multicomponent education program designed specifically for low-acculturated Latinas. The control group received mailed health education material on a schedule comparable to the intervention. A total of 195 women (76.2%) completed both the baseline and 8-month follow-up interviews. The intervention and control groups were similar on baseline sociodemographic characteristics. At the 8-month follow up, the intervention group reported lower dietary fat (P < .001) and higher fiber intake (p = .06); a higher proportion reported practicing BSE at the recommended interval (p < .001) and showed improved BSE proficiency (p < .001) compared to the control group. BSE-related anxiety was low for both groups at baseline, and no difference in reduction was observed. This project provides a successful model for achieving dietary change and improving breast health behavior in young, low-acculturated Latinas.

  13. Study of the interaction mechanisms between absorbed NO{sub 2} and por-Si/SnO{sub x} nanocomposite layers

    SciTech Connect

    Bolotov, V. V.; Kan, V. E. Makushenko, R. K.; Biryukov, M. Yu.; Ivlev, K. E.; Roslikov, V. E.

    2013-10-15

    The interaction mechanisms between NO{sub 2} molecules and the surface of por-Si/SnO{sub x} nanocomposites obtained by magnetron deposition and chemical vapor deposition (CVD) are studied by infrared absorption spectroscopy and electron paramagnetic resonance methods. The observed increase in the free carrier concentration in the por-Si/SnO{sub x} nanocomposite layers is explained by a change in the charge state of P{sub b} centers due to the formation of neutral 'surface defect-adsorbed NO{sub 2} molecule' complexes with free carrier generation in the crystallite bulk. In the nanocomposite layers grown by the CVD method, the increase in the free hole concentration during NO{sub 2} adsorption is much less pronounced in comparison with the composite grown by magnetron deposition, which is caused by the competing interaction channel of NO{sub 2} molecules with electrically neutral P{sub b} centers.

  14. Adjuvant effects elicited by novel oligosaccharide variants of detoxified meningococcal lipopolysaccharides on Neisseria meningitidis recombinant PorA protein: a comparison in mice.

    PubMed

    Mehta, Ojas H; Norheim, Gunnstein; Hoe, J Claire; Rollier, Christine S; Nagaputra, Jerry C; Makepeace, Katherine; Saleem, Muhammad; Chan, Hannah; Ferguson, David J P; Jones, Claire; Sadarangani, Manish; Hood, Derek W; Feavers, Ian; Derrick, Jeremy P; Pollard, Andrew J; Moxon, E Richard

    2014-01-01

    Neisseria meningitidis lipopolysaccharide (LPS) has adjuvant properties that can be exploited to assist vaccine immunogenicity. The modified penta-acylated LPS retains the adjuvant properties of hexa-acylated LPS but has a reduced toxicity profile. In this study we investigated whether two modified glycoform structures (LgtE and IcsB) of detoxified penta-acylated LPS exhibited differential adjuvant properties when formulated as native outer membrane vesicles (nOMVs) as compared to the previously described LgtB variant. Detoxified penta-acylated LPS was obtained by disruption of the lpxL1 gene (LpxL1 LPS), and three different glycoforms were obtained by disruption of the lgtB, lgtE or icsB genes respectively. Mice (mus musculus) were immunized with a recombinant PorA P1.7-2,4 (rPorA) protein co-administered with different nOMVs (containing a different PorA serosubtype P1.7,16), each of which expressed one of the three penta-acylated LPS glycoforms. All nOMVs induced IgG responses against the rPorA, but the nOMVs containing the penta-acylated LgtB-LpxL1 LPS glycoform induced significantly greater bactericidal activity compared to the other nOMVs or when the adjuvant was Alhydrogel. Compared to LgtE or IcsB LPS glycoforms, these data support the use of nOMVs containing detoxified, modified LgtB-LpxL1 LPS as a potential adjuvant for future meningococcal protein vaccines.

  15. IgG antibody subclass responses determined by immunoblot in infants' sera following vaccination with a meningococcal recombinant hexavalent PorA OMV vaccine.

    PubMed

    Martin, S; Sadler, F; Borrow, R; Dawson, M; Fox, A; Cartwright, K

    2001-08-14

    The introduction of meningococcal serogroup C conjugate vaccines into the UK immunisation schedule has led to the decline of serogroup C disease in those vaccinated but there is no imminent vaccine solution for serogroup B disease. The PorA outer membrane protein (OMP) is a potential serogroup B vaccine candidate and an outer membrane vesicle (OMV) vaccine containing six different PorA OMPs (each representing a different serosubtype) has been evaluated in phase II trials with encouraging results. Little is known about the IgG subclass response to the various antigens contained within this vaccine. These responses are important due to the different half-lives and complement fixing abilities of these antibodies. In this study, immunoblotting was undertaken with infants' sera following either three or four doses of vaccine, and OMVs from six isogenic meningococcal strains differing only in their PorA serosubtype. Following either three or four doses of the vaccine, IgG(3) and IgG(1) subclass antibodies were induced to all six of the isogenic strains, although sera collected after four doses of vaccine showed stronger antibody levels. IgG(3) was found in more sera than IgG(1). For both sets of sera, the two isogenic strains expressing P1.5,2 and P1.5(c),10 induced stronger IgG subclass antibody responses than the other four meningococcal strains. The recombinant hexavalent PorA OMV vaccine stimulates both IgG(1) and IgG(3) subclass antibodies, the subclasses that are most effective in activating the complement system.

  16. Corynebacterium jeikeium jk0268 Constitutes for the 40 Amino Acid Long PorACj, Which Forms a Homooligomeric and Anion-Selective Cell Wall Channel

    PubMed Central

    Norouzy, Amir; Schulz, Robert; Nau, Werner M.; Kleinekathöfer, Ulrich; Tauch, Andreas; Benz, Roland

    2013-01-01

    Corynebacterium jeikeium, a resident of human skin, is often associated with multidrug resistant nosocomial infections in immunodepressed patients. C. jeikeium K411 belongs to mycolic acid-containing actinomycetes, the mycolata and contains a channel-forming protein as judged from reconstitution experiments with artificial lipid bilayer experiments. The channel-forming protein was present in detergent treated cell walls and in extracts of whole cells using organic solvents. A gene coding for a 40 amino acid long polypeptide possibly responsible for the pore-forming activity was identified in the known genome of C. jeikeium by its similar chromosomal localization to known porH and porA genes of other Corynebacterium strains. The gene jk0268 was expressed in a porin deficient Corynebacterium glutamicum strain. For purification temporarily histidine-tailed or with a GST-tag at the N-terminus, the homogeneous protein caused channel-forming activity with an average conductance of 1.25 nS in 1M KCl identical to the channels formed by the detergent extracts. Zero-current membrane potential measurements of the voltage dependent channel implied selectivity for anions. This preference is according to single-channel analysis caused by some excess of cationic charges located in the channel lumen formed by oligomeric alpha-helical wheels. The channel has a suggested diameter of 1.4 nm as judged from the permeability of different sized hydrated anions using the Renkin correction factor. Surprisingly, the genome of C. jeikeium contained only one gene coding for a cell wall channel of the PorA/PorH type found in other Corynebacterium species. The possible evolutionary relationship between the heterooligomeric channels formed by certain Corynebacterium strains and the homooligomeric pore of C. jeikeium is discussed. PMID:24116064

  17. Astronomy in the Classroom: Why? (Spanish Title: Astronomía en la Clase: ¿Por Qué?) Astronomia na Sala de Aula: Por Quê?

    NASA Astrophysics Data System (ADS)

    Daros Gama, Leandro; Bagdonas Henrique, Alexandre

    2010-07-01

    There are many discussions about the relevance of the topics covered in classes. One subject in particular is the focus of this essay: astronomy. In what sense and to what extent it would be worth to teach it in science or other kind of classes? In this paper we discuss some aspects of the advantages of dealing with this area of knowledge in schools, taking into account the epistemological and axiological dimensions of astronomy, in light of the vision of science as an intelligent dialogue with the world (Bachelard), in addition to the "problematization" knowledge of Paulo Freire. We propose that in fact the Astronomy does not need to be seen as just a new set of contents to be taught, but appears as a set of motivational contents for historical-philosophical discussions, and permit the discussion of concepts of other disciplines. Numerosas discusiones se están llevando a cabo acerca de la pertinencia de los temas tradicionalmente tratados en las clases. Uno de los temas, en particular, es el foco de este ensayo: la astronomía. ¿En qué sentido y en qué medida sería conveniente tratarla en clase, ya sea en clases de ciencias naturales, específicamente en las de astronomía o asignaturas afines? Elaboramos en este artículo algunos aspectos de las ventajas de tratar esta área del conocimiento en las escuelas, teniendo en cuenta las dimensiones epistemológica y axiológica de la astronomía, a la luz de la visión de la ciencia como un diálogo inteligente con el mundo (Bachelard), además de la propuesta del conocimiento "problematizador" de Paulo Freire. Proponemos que en realidad la astronomía no tiene por qué ser vista sólo como un nuevo conjunto de contenidos que se enseñan, sino que aparece como un conjunto de temas de motivación para el debate histórico-filosófico y para permitir la discusión de los conceptos típicos de otras disciplinas. Muitas discussões vêm acontecendo sobre a relevância dos temas abordados em sala de aula. Um tema, em

  18. [Optimization of tri-expression of human CYP3A4 with POR and cyt b5 in Sf 9 cells].

    PubMed

    Xie, Zhangming; Liu, Wenhui; Xu, Yingchun; Chen, Shuqing

    2013-01-01

    To investigate the optimal conditions of tri-expression of CYP3A4, POR and cyt b5 in Sf 9 cells. The Sf 9 cells expressing CYP3A4, POR and cyt b5 were cultured in shaker flasks. The optimized conditions, including the temperature and rotation speed, the culture volume, the amount of surfactant and the culture time were studied. The expressed products in microsomes were used to metabolize the testosterone and their metabolic activity was determined. When the temperature and rotation speed of the shaker were 27 degree and 90 r/min, the cell density and culture volume were 5X105 cells/ml and 80-120 ml per 250 ml shaker flasks, respectively. When Pluronic F-68 was 0.1% and the culture time was 72 h, the condition was most suitable for culture of Sf 9 cells and expression of targeted proteins. When the ratio of the volume of three added viruses was 1:1:1, the expression condition was optimal, under which the Km, Vmax, and CLint for testosterone metabolism were 119.6 μmol/L,0.52 μmol/(min*g protein) and 4.34 ml/(min*g protein), respectively. The conditions of tri-expressing of CYP3A4, POR and cyt b5 have been optimized in the study and the product CYP3A4 is obtained with higher metabolic activity.

  19. Obtención de la curva de luz en la ocultación de 35 Sgr por Júpiter el 6 de marzo de 1996

    NASA Astrophysics Data System (ADS)

    Paolantonio, S.; Duffard, R.; Carranza, G.

    La ocultación de la estrella de quinta magnitud 35 Sgr por Júpiter, se produjo el 6 de Marzo de 1996 a las 13 hs. TU. El objetivo era medir el cambio del flujo de la estrella en el ingreso y egreso por el limbo del planeta. Con estos datos se pueden determinar parámetros físicos del planeta (radio, eccentricidad) y de su atmósfera (escala de altura, temperatura, densidad, presión) Para lograr ésto se programó la cámara CCD TH 7896 1024 x 1025 instalada en el telescopio de 1.54 m de Bosque Alegre con el objetivo de lograr 2 imágenes por segundo. De esta forma se obtuvieron 2100 imágenes de la inmersión y otras tantas de la emersión. Hubo que tener grandes precauciones para evitar la saturación del CCD ya que la observación se realizó de día. En este momento las imágenes se encuentran en el Department of Planetary Sciences, Lunar and Planetary Laboratory, University of Arizona, para su reducción.

  20. [Determination and analysis of protein profile of different transfer factors].

    PubMed

    Guidos-Fogelbach, Guillermo Arturo; Paredes-Aguilar, Jorge Antonio; Colín-Martínez, Nayeli Montserrat; Rojo-Gutiérrez, María Isabel; López-Hidalgo, Marisol; Reyes-López, César Augusto Sandino

    2016-01-01

    Introducción: El factor de transferencia (FT) es el extracto dializable de leucocitos con propiedades de transferencia de inmunidad celular. Su uso se ha extendido en el tratamiento de una amplia gama de padecimientos inmunológicos, infecciosos y como coadyuvante de padecimientos oncológicos. A pesar de ello, no se conocen completamente aspectos importantes de su perfil proteico, concentraciones de componentes y mecanismos de acción. Objetivos: Analizar los perfiles proteicos de diferentes factores de transferencia comercializados en México. Métodos: Se obtuvieron y analizaron 6 FT comercializados en México. Se realizó la cuantificación de proteínas por el método de Bradford, cromatografía líquida de alta resolución (HPLC) y electroforesis en geles de poliacrilamida (SDS-PAGE). Todas las muestras fueron analizadas por duplicado. Resultados: Las concentraciones de proteínas totales de todos los FT analizados fueron menores de 0.2 mg/mL. Los perfiles cromatográficos mostraron diferencias en algunos FT. La concentración de proteínas resultó de 6 hasta casi mil veces más baja en comparación con lo informado por algunos fabricantes. Conclusión: Casi la totalidad de los factores de transferencia comercializados en México carecen de un etiquetado y registro sanitario que cumpla con las normas oficiales vigentes.

  1. Expression patterns of chitinase and chitosanase produced from Bacillus cereus in suppression of phytopathogen.

    PubMed

    Seo, Dong-Jun; Lee, Ji-Hae; Song, Yong-Su; Park, Ro-Dong; Jung, Woo-Jin

    2014-08-01

    Bacillus cereus MP-310 was incubated on various culture media substrates as LB, colloidal chitin, chitosan powder, and chitosan beads to investigate the concurrent expression patterns of chitinase and chitosanase isozymes by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Chitinase activity increased rapidly with a maximum level after 6 days of incubation in CM-chitin medium. Major bands of chitinase isozymes were strongly expressed on SDS-PAGE in LB medium (four bands) and in colloidal chitin medium (five bands) after 6 days after incubation, and in chitosan powder medium (one band) and in chitosan beads medium (five bands) after 12 days after incubation. A major band of chitosanase isozymes was strongly expressed on SDS-PAGE in chitosan powder medium (one band) and in chitosan beads medium (one band) after 12 days of incubation. Copyright © 2014 Elsevier Ltd. All rights reserved.

  2. Enhanced protein electrophoresis technique for separating human skeletal muscle myosin heavy chain isoforms

    NASA Technical Reports Server (NTRS)

    Bamman, M. M.; Clarke, M. S.; Talmadge, R. J.; Feeback, D. L.

    1999-01-01

    Talmadge and Roy (J. Appl. Physiol. 1993, 75, 2337-2340) previously established a sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE) protocol for separating all four rat skeletal muscle myosin heavy chain (MHC) isoforms (MHC I, IIa, IIx, IIb); however, when applied to human muscle, the type II MHC isoforms (Ila, IIx) are not clearly distinguished. In this brief paper we describe a modification of the SDS-PAGE protocol which yields distinct and consistent separation of all three adult human MHC isoforms (MHC I, IIa, IIx) in a minigel system. MHC specificity of each band was confirmed by Western blot using three monoclonal IgG antibodies (mAbs) immunoreactive against MHCI (mAb MHCs, Novacastra Laboratories), MHCI+IIa (mAb BF-35), and MHCIIa+IIx (mAb SC-71). Results provide a valuable SDS-PAGE minigel technique for separating MHC isoforms in human muscle without the difficult task of casting gradient gels.

  3. Pincharse sin infectarse: estrategias para prevenir la infección por el VIH y el VHC entre usuarios de drogas inyectables

    PubMed Central

    MATEU-GELABERT, P.; FRIEDMAN, S.; SANDOVAL, M.

    2011-01-01

    Resumen Objetivo Desde principios de los noventa, en la ciudad de Nueva York se han implementado con éxito programas para reducir la incidencia del virus de la inmunodeficiencia humana (VIH) y, en menor medida, del virus de la hepatitis C (VHC). A pesar de ello, aproximadamente el 70% de los usuario de drogas inyectables (UDI) están infectados por el VHC. Queremos investigar cómo el 30% restante se las ha arreglado para no infectarse. El Staying safe (nombre original del estudio) explora los comportamientos y mecanismos que ayudan a evitar la infección por el VHC y el VIH a largo plazo. Material y métodos Hemos utilizado el concepto de «desviación positiva» aplicado en otros campos de salud pública. Estudiamos las estrategias, prácticas y tácticas de prevención de aquellos UDI que, viviendo en contextos de alta prevalencia, se mantienen sin infectar por VIH y el VHC, a pesar de haberse inyectado heroína durante años. Los resultados preliminares presentados en este artículo incluyen el análisis de las entrevistas realizadas a 25 UDI (17 doble negativos, 3 doble positivos y 5 con infección por el VHC y sin infección por el VIH). Se usaron entrevistas semiestructuradas que exploraban con detalle la historia de vida de los sujetos, incluyendo su consumo de drogas, redes sociales, contacto con instituciones, relaciones sexuales y estrategias de protección y vigilancia. Resultados La intencionalidad es importante para no infectarse, especialmente durante períodos de involución (períodos donde hay un deterioro económico y/o social que llevan al que se inyecta a situaciones de mayor riesgo). Presentamos tres dimensiones independientes de intencionalidad que conllevan comportamientos que pueden ayudar a prevenir la infección: a) evitar «el mono» (síntomas de abstención) asegurando el acceso a la droga; b) «llevarlo bien» para no convertirse en un junkie y así evitar la «muerte social» y la falta de acceso a los recursos, y c) seguir sin

  4. Comparison and validation of methods to quantify Cry1Ab toxin from Bacillus thuringiensis for standardization of insect bioassays.

    PubMed

    Crespo, André L B; Spencer, Terence A; Nekl, Emily; Pusztai-Carey, Marianne; Moar, William J; Siegfried, Blair D

    2008-01-01

    Standardization of toxin preparations derived from Bacillus thuringiensis (Berliner) used in laboratory bioassays is critical for accurately assessing possible changes in the susceptibility of field populations of target pests. Different methods were evaluated to quantify Cry1Ab, the toxin expressed by 80% of the commercially available transgenic maize that targets the European corn borer, Ostrinia nubilalis (Hübner). We compared three methods of quantification on three different toxin preparations from independent sources: enzyme-linked immunosorbent assay (ELISA), sodium dodecyl sulfate-polyacrylamide gel electrophoresis and densitometry (SDS-PAGE/densitometry), and the Bradford assay for total protein. The results were compared to those obtained by immunoblot analysis and with the results of toxin bioassays against susceptible laboratory colonies of O. nubilalis. The Bradford method resulted in statistically higher estimates than either ELISA or SDS-PAGE/densitometry but also provided the lowest coefficients of variation (CVs) for estimates of the Cry1Ab concentration (from 2.4 to 5.4%). The CV of estimates obtained by ELISA ranged from 12.8 to 26.5%, whereas the CV of estimates obtained by SDS-PAGE/densitometry ranged from 0.2 to 15.4%. We standardized toxin concentration by using SDS-PAGE/densitometry, which is the only method specific for the 65-kDa Cry1Ab protein and is not confounded by impurities detected by ELISA and Bradford assay for total protein. Bioassays with standardized Cry1Ab preparations based on SDS-PAGE/densitometry showed no significant differences in LC(50) values, although there were significant differences in growth inhibition for two of the three Cry1Ab preparations. However, the variation in larval weight caused by toxin source was only 4% of the total variation, and we conclude that standardization of Cry1Ab production and quantification by SDS-PAGE/densitometry may improve data consistency in monitoring efforts to identify changes in

  5. In Situ Evaluation of the Role of the Small GTPase Rac3 in Breast Cancer

    DTIC Science & Technology

    2005-07-01

    sample buffer. Proteins from the total cell lysate, as well as the bead pellet, were separated by 10% SDS-PAGE gel , transferred to a nitrocellulose...buffer, of EGF is in the range used to activate EGFR and elicit ef- equally loaded, and separated on 10% SDS-PAGE gels . fects on the actin cytoskeleton...Burns J, Yokota T, Ashihara H, Lean ME, and Crozier A (2002). Plant foods and herbal sources of resveratrol. J Agric Food Chem 50,ERI3(+) metastatic

  6. Assays for determination of protein concentration.

    PubMed

    Olson, Bradley J S C; Markwell, John

    2007-05-01

    Biochemical analysis of proteins relies on accurate quantitation of protein concentration. This unit describes how to perform commonly used protein assays, e.g., Lowry, Bradford, BCA, and UV spectroscopic protein assays. The primary focus of the unit is assay selection, emphasizing sample and buffer compatibility. Protein assay standard curves and data processing fundamentals are discussed in detail. This unit also details high-throughput adaptations of the commonly used protein assays, and also contains a protocol for BCA assay of total protein in SDS-PAGE sample buffer that is used for equal loading of SDS-PAGE gels, which is reliable, inexpensive, and quick.

  7. Assays for determination of protein concentration.

    PubMed

    Olson, Bradley J S C; Markwell, John

    2007-09-01

    Biochemical analysis of proteins relies on accurate quantitation of protein concentration. This appendix describes how to perform commonly used protein assays, e.g., Lowry, Bradford, BCA, and UV spectroscopic protein assays. The primary focus of the appendix is assay selection, emphasizing sample and buffer compatibility. Protein assay standard curves and data processing fundamentals are discussed in detail. This appendix also details high-throughput adaptations of the commonly used protein assays, and also contains a protocol for BCA assay of total protein in SDS-PAGE sample buffer that is used for equal loading of SDS-PAGE gels, which is reliable, inexpensive, and quick.

  8. Acid and alkaline phosphatases of Capnocytophaga species. II. Isolation, purification, and characterization of the enzymes from Capnocytophaga ochracea.

    PubMed

    Poirier, T P; Holt, S C

    1983-10-01

    Capnocytophaga ochracea acid (AcP; EC 3.1.3.2) and alkaline (AlP; EC 3.1.3.1) phosphatase was isolated by Ribi cell disruption and purified by sodium dodecyl sulphate - polyacrylamide gel electrophoresis (SDS-PAGE.) Both phosphatases eluted from Sephadex G-150 consistent with molecular weights (migration) of 140 000 and 110 000. SDS-PAGE demonstrated a 72 000 and 55 000 subunit molecular migration for AcP and AlP, respectively. The kinetics of activity of purified AcP and AlP on p-nitrophenol phosphate and phosphoseryl residues of the phosphoproteins are presented.

  9. Purification of elastase-like chymotrypsin from cardamom shoot and Capsule borer [corrected].

    PubMed

    Josephrajkumar, A; Chakrabarty, R; Thomas, G

    2007-11-01

    An elastase-like chymotrypsin was purified by aprotinin-agarose affinity chromatography from the midgut extract of cardamom shoot and capsule borer, Conogethes punctiferalis. The purified enzyme had a Vmax of 687.6 +/- 22.1 nmole pNA released/min/mg protein, Km of 0.168 +/- 0.012 mM with SAAPLpNA as substrate and gave a single band on SDS-PAGE with a molecular mass of 72.1 kDa. Casein zymogram revealed one clear zone of proteolytic activity, which corresponded to the band obtained with SDS-PAGE indicating that this could be a single-polypeptide enzyme.

  10. A Fusion-Inhibiting Peptide against Rift Valley Fever Virus Inhibits Multiple, Diverse Viruses

    DTIC Science & Technology

    2013-09-12

    crystals which are solubilized by detergent . Absorbance was read at 570 nm with a 96-well plate spectrophotometer (Promega/Turner Biosystems, Madison, WI...beads were washed with TBS (or low pH medium for the pH treated beads) to remove unbound virus, and SDS -PAGE loading buffer (Life Technologies, Grand...Island, NY) was added to the beads. After a 5 min incubation at 70uC, samples were resolved on a SDS -PAGE gel. The resolved proteins were transferred to a

  11. Challenges of glycoprotein analysis by microchip capillary gel electrophoresis.

    PubMed

    Engel, Nicole; Weiss, Victor U; Wenz, Christian; Rüfer, Andreas; Kratzmeier, Martin; Glück, Susanne; Marchetti-Deschmann, Martina; Allmaier, Günter

    2015-08-01

    Glycosylations severely influence a protein's biological and physicochemical properties. Five exemplary proteins with varying glycan moieties were chosen to establish molecular weight (MW) determination (sizing), quantitation, and sensitivity of detection for microchip capillary gel electrophoresis (MCGE). Although sizing showed increasing deviations from literature values (SDS-PAGE or MALDI-MS) with a concomitant higher degree of analyte glycosylation, the reproducibility of MW determination and accuracy of quantitation with high sensitivity and reliability were demonstrated. Additionally, speed of analysis together with the low level of analyte consumption render MCGE attractive as an alternative to conventional SDS-PAGE.

  12. CYP3A5*3 and POR*28 genetic variants influence the required dose of tacrolimus in heart transplant recipients.

    PubMed

    Lesche, Dorothea; Sigurdardottir, Vilborg; Setoud, Raschid; Oberhänsli, Markus; Carrel, Thierry; Fiedler, Georg M; Largiadèr, Carlo R; Mohacsi, Paul; Sistonen, Johanna

    2014-12-01

    After heart transplantation (HTx), the interindividual pharmacokinetic variability of immunosuppressive drugs represents a major therapeutic challenge due to the narrow therapeutic window between over-immunosuppression causing toxicity and under-immunosuppression leading to graft rejection. Although genetic polymorphisms have been shown to influence pharmacokinetics of immunosuppressants, data in the context of HTx are scarce. We thus assessed the role of genetic variation in CYP3A4, CYP3A5, POR, NR1I2, and ABCB1 acting jointly in immunosuppressive drug pathways in tacrolimus (TAC) and ciclosporin (CSA) dose requirement in HTx recipients. Associations between 7 functional genetic variants and blood dose-adjusted trough (C0) concentrations of TAC and CSA at 1, 3, 6, and 12 months after HTx were evaluated in cohorts of 52 and 45 patients, respectively. Compared with CYP3A5 nonexpressors (*3/*3 genotype), CYP3A5 expressors (*1/*3 or *1/*1 genotype) required around 2.2- to 2.6-fold higher daily TAC doses to reach the targeted C0 concentration at all studied time points (P ≤ 0.003). Additionally, the POR*28 variant carriers showed higher dose-adjusted TAC-C0 concentrations at all time points resulting in significant differences at 3 (P = 0.025) and 6 months (P = 0.047) after HTx. No significant associations were observed between the genetic variants and the CSA dose requirement. The CYP3A5*3 variant has a major influence on the required TAC dose in HTx recipients, whereas the POR*28 may additionally contribute to the observed variability. These results support the importance of genetic markers in TAC dose optimization after HTx.

  13. Spatial and stimulus-type tuning in the LEC, MEC, POR, PrC, CA1, and CA3 during spontaneous item recognition memory.

    PubMed

    Beer, Zachery; Chwiesko, Caroline; Kitsukawa, Takashi; Sauvage, Magdalena M

    2013-12-01

    According to the "two streams" hypothesis, the lateral entorhinal (LEC) and the perirhinal (PrC) cortices process information related to items (a "what" stream), the postrhinal (POR) and the medial entorhinal cortices (MEC) process spatial information (a "where" stream), and both types of information are integrated in the hippocampus (HIP). However, within the framework of memory function, only the HIP is reliably shown to preferentially process spatial information, and the PrC items' features. In contrast, the role of the LEC and MEC in memory is virtually unexplored, and conflicting results emerge for the POR. Moreover, the specific contribution of the hippocampal subfields CA1 and CA3 to spatial and non-spatial memory is not thoroughly understood. To investigate which of these areas is specifically tuned to spatial demands or stimulus identity (odor or object), we assessed the pattern of activation of these areas during recognition memory by detecting the immediate-early gene Arc, commonly used as a marker of neuronal activation. We report that all MTL areas were recruited during the spatial and the non-spatial tasks. However, the LEC, MEC, POR, and CA1 were activated to a comparable level in spatial and non-spatial tasks, while the PrC was tuned to stimulus-type, not spatial demands, and CA3 to spatial demands but not stimulus-type. Results are discussed within the frame of a recent model suggesting that the MTL could be segregated in terms of memory processes, such as recollection and familiarity, rather than information content. Copyright © 2013 Wiley Periodicals, Inc.

  14. Cytochrome P450 oxidoreductase deficiency caused by R457H mutation in POR gene in Chinese: case report and literature review.

    PubMed

    Bai, Yang; Li, Jinhui; Wang, Xiaoli

    2017-03-14

    Cytochrome P450 oxidoreductase deficiency (PORD) is a rare disease exhibiting a variety of clinical manifestations. It can be difficult to differentiate with other diseases such as 21-hydroxylase deficiency (21-OHD), polycystic ovary syndrome (PCOS) and Antley-Bixler syndrome (ABS). Nearly 100 cases of PORD have been reported worldwide. However, the genetic characters and clinical management are still unclear, especially in China. In this study, we report a 27-year-old female Chinese patient who first presented with amenorrhea and recurrence of large ovary cyst. She was misdiagnosed with PCOS and non-classical 21-OHD due to ovary cysts and elevated 17-hydroxy-progesterone. The patient's complaining of a mild difficulty of bending the metacarpophalangeal joints reminded us to consider PORD, which usually presents with skeletal deformities and sexual dysfunction. The diagnosis of PORD was confirmed by genetic analyses, which showed the patient harboring a homozygous missense mutation in the POR gene (R457H) and her parents carrying the heterozygous mutation. The patient was treated with low-dose corticosteroids and estrogen/progesterone sequential therapy, and her ovarian cyst gradually reduced with regular menstruation in the follow-up. Moreover, the clinical and genetic characteristics of 104 previously reported PORD cases were also summarized and analyzed. PORD is a very rare disease which can be easily misdiagnosed in mild cases. Clinicians should keep in mind of this disease in patients with sexual dysfunction, especially combined with special skeletal deformities. Our data could provide a consciously understanding of this disease for clinic practicers. Low-dose corticosteroids combined with estrogen/progesterone sequential therapy will be effective in PORD patients with recurrence of large ovary cyst. The fact that the reported PORD patients in China carrying an identical variant R457H in POR gene also give us a viewpoint that R457H mutation in POR gene maybe

  15. Preclinical safety and immunogenicity evaluation of a nonavalent PorA native outer membrane vesicle vaccine against serogroup B meningococcal disease.

    PubMed

    Kaaijk, Patricia; van Straaten, Ineke; van de Waterbeemd, Bas; Boot, Elmieke P J; Levels, Lonneke M A R; van Dijken, Harry H; van den Dobbelsteen, Germie P J M

    2013-02-04

    An improved nonavalent PorA native outer membrane vesicle vaccine was developed with intrinsic adjuvating activity due to presence of less-toxic (lpxL1) LPS. In the present study, the safety and immunogenicity of this next-generation NonaMen vaccine were evaluated following repeated vaccination in rabbits and mice. A repeated-dose toxicology study was performed in rabbits. Immunogenicity of next-generation NonaMen was evaluated by determining the serum bactericidal antibody (SBA) titers against meningococcal serogroup B strains containing several PorA subtypes. Release of the pro-inflammatory cytokine, interleukin-6 (IL-6), by the human monocytic cell line (MM6) was measured to estimate pyrogenic activity. No toxicologically relevant findings were noted in vaccinated rabbits receiving plain next-generation NonaMen. In agreement, next-generation NonaMen induced reduced amounts of the pro-inflammatory cytokine, IL-6, released by human monocyte cell line. In both rabbits and mice, next-generation NonaMen induced high SBA titers against all tested MenB strains regardless of whether or not aluminium phosphate adjuvant is used. The data suggest that next-generation NonaMen is a safe vaccine with the potential to develop a broadly protective immune response and encourage the start of the first clinical studies. Copyright © 2012 Elsevier Ltd. All rights reserved.

  16. A survey of homopteran species (Auchenorrhyncha) from coffee shrubs and poró and laurel trees in shaded coffee plantations, in Turrialba, Costa Rica.

    PubMed

    Rojas, L; Godoy, C; Hanson, P; Hilje, L

    2001-01-01

    A survey of homopteran species (Auchenorryncha) was conducted in coffee plantations with no shade (C), and in those with shade of either poró (Erythrina poeppigiana) (CP) or poró plus laurel (Cordia alliodora) (CPL), in Turrialba, Costa Rica. A total of 130 species in ten families were collected, dominated by Cicadellidae (82 species). Species richness was highest in the CP system (88), followed by CPL (74) and C systems (60). Five most common species for all systems were Fusigonalia lativittata, Hebralebra nicaraguensis, Neocoelidia sp., Oliarus sp. and Clastoptera sp. Diversification of the coffee agroecosystem favors some species while limiting others, and have no effect on the majority of species. Thus, only F. lativittata, Neocoelidia sp. and Scaphytopius ca. latidens were well represented in all systems, but were more abundant in coffee shrubs. Additionally, the following were the dominant species in each system: Graphocephala sp. 1 (C), F. lativittata (CP) and H. nicaraguensis (CPL). Four species abundant on laurel trees, including H. nicaraguensis, appeared almost exclusively on these tree species. Species similarity was highest on the CP and CPL systems (51% of the species in common), followed by the C and CP (39%) and the C and CPL systems (38%). These findings show that even disturbed systems can harbor many insect species, so that they deserve attention from conservation advocates and biologists.

  17. A Fast Real-Time Polymerase Chain Reaction Method for Sensitive and Specific Detection of the Neisseria gonorrhoeae porA Pseudogene

    PubMed Central

    Hjelmevoll, Stig Ove; Olsen, Merethe Elise; Sollid, Johanna U. Ericson; Haaheim, Håkon; Unemo, Magnus; Skogen, Vegard

    2006-01-01

    Ever since the advent of molecular methods, the diagnostics of Neisseria gonorrhoeae has been troubled by false negative and false positive results compared with culture. Commensal Neisseria species and Neisseria meningitidis are closely related to N. gonorrhoeae and may cross-react when using molecular tests comprising too-low specificity. We have devised a real-time polymerase chain reaction (PCR), including an internal amplification control, that targets the N. gonorrhoeae porA pseudogene. DNA was automatically isolated on a BioRobot M48. Our subsequent PCR method amplified all of the different N. gonorrhoeae international reference strains (n = 34) and N. gonorrhoeae clinical isolates (n = 176) but not isolates of the 13 different nongonococcal Neisseria species (n = 68) that we tested. Furthermore, a panel of gram-negative bacterial (n = 18), gram-positive bacterial (n = 23), fungal (n = 1), and viral (n = 4) as well as human DNA did not amplify. The limit of detection was determined to be less than 7.5 genome equivalents/PCR reaction. In conclusion, the N. gonorrhoeae porA pseudogene real-time PCR developed in the present study is highly sensitive, specific, robust, rapid and reproducible, making it suitable for diagnosis of N. gonorrhoeae infection. PMID:17065426

  18. Effect of CYP3A4*22, POR*28, and PPARA rs4253728 on sirolimus in vitro metabolism and trough concentrations in kidney transplant recipients.

    PubMed

    Woillard, Jean-Baptiste; Kamar, Nassim; Coste, Sandra; Rostaing, Lionel; Marquet, Pierre; Picard, Nicolas

    2013-12-01

    Recent studies have identified new candidate polymorphisms in the genes related to CYP3A activity or calcineurin inhibitor dose requirements in kidney transplant recipients. These genes and polymorphisms are CYP3A4 (cytochrome P450, family 3, subfamily A, polypeptide 4) (rs35599367-C>T; *22); POR [P450 (cytochrome) oxidoreductase] (rs1057868-C>T; *28); and PPARA (peroxisome proliferator-activated receptor alpha) (rs4253728-G>A). We investigated the impact of these polymorphisms on sirolimus (SRL) in vitro hepatic metabolism, SRL trough concentrations (C0), and SRL adverse events in kidney transplant recipients. The clinical study included 113 stable kidney transplant patients switched from a calcineurin inhibitor to SRL (SRL C0 measured at 1, 3, and 6 months thereafter). We investigated SRL metabolism in vitro using human liver microsomes derived from individual donors (n = 31). Microsomes and patients were genotyped by use of Taqman® allelic discrimination assays. The effects of polymorphisms and covariates were studied using multilinear regression imbedded in linear mixed-effect models or logistic regressions. In vitro, the CYP3A4*22 allele resulted in approximately 20% lower metabolic rates of SRL (P = 0.0411). No significant association was found between CYP3A4, CYP3A5, or PPARA genotypes and SRL dose, C0, or C0/dose in kidney transplant patients. POR*28 was associated with a minor but significant decrease in SRL log-transformed C0 [CT/TT vs CC, β = -0.15 (0.05); P = 0.0197] but this did not have any impact on the dose administered, which limited the relevance of the finding. After adjustment for nongenetic covariates and correction for false discovery finding, none of the single-nucleotide polymorphisms tested showed significant association with SRL adverse events. These recently described polymorphisms do not seem to substantially influence the pharmacokinetics of SRL or the occurrence of SRL adverse events in kidney transplant recipients.

  19. Variation in P450 oxidoreductase (POR) A503V and flavin-containing monooxygenase (FMO)-3 E158K is associated with minor alterations in nicotine metabolism, but does not alter cigarette consumption.

    PubMed

    Chenoweth, Meghan J; Zhu, Andy Z X; Sanderson Cox, Lisa; Ahluwalia, Jasjit S; Benowitz, Neal L; Tyndale, Rachel F

    2014-03-01

    The rates of nicotine metabolism differ widely, even after controlling for genetic variation in the major nicotine-metabolizing enzyme, CYP2A6. Genetic variants in an additional nicotine-metabolizing enzyme, flavin-containing monooxygenase (FMO)-3, and an obligate microsomal CYP-supportive enzyme, cytochrome P450 oxidoreductase (POR), were investigated. We examined the impact of FMO3 E158K and POR A503V before and after stratifying by CYP2A6 metabolism group. In 130 nonsmokers of African descent who received 4 mg oral nicotine, FMO3 158K trended toward slower nicotine metabolism in reduced CYP2A6 metabolizers (P=0.07) only, whereas POR 503V was associated with faster CYP2A6 activity (nicotine metabolite ratio) in normal (P=0.03), but not reduced, CYP2A6 metabolizers. Neither FMO3 158K nor POR 503V significantly altered the nicotine metabolic ratio (N=659), cigarette consumption (N=667), or urine total nicotine equivalents (N=418) in smokers of African descent. Thus, FMO3 E158K and POR A503V are minor sources of nicotine metabolism variation, insufficient to appreciably alter smoking.

  20. Effect of CYP3A4*1G, CYP3A5*3, POR*28, and ABCB1 C3435T on the pharmacokinetics of nifedipine in healthy Chinese volunteers.

    PubMed

    Wang, Xiao-Fei; Yan, Liang; Cao, Hui-Min; Wei, Lu-Man; Yang, Wei-Hong; Zhang, Sheng-Jun; Zhang, Li-Rong

    2015-09-01

    Nifedipine is a calcium channel blocker that is widely used in the treatment of cardiovascular disease. However, significant individual variances in the disposition of nifedipine have been reported, and genetic factors are considered to play an important role. The aim of the present study was to investigate the effect of CYP3A4*1G, CYP3A5*3, ABCB1-C3435T, and POR*28 genetic polymorphisms on nifedipine pharmacokinetics in healthy Chinese volunteers. 45 healthy Chinese volunteers enrolled in this study received a single oral dose of 90 mg nifedipine after providing written informed consent. Volunteers were genotyped for CYP3A4*1G, CYP3A5*3, POR*28, and ABCB1-C3435T. The blood concentrations of nifedipine were determined by high performance liquid chromatography tandem mass spectrometry (LC-MS/MS) method. There were significant differences of AUC00-∞ and AUC0-48h in the different CYP3A5*3 genotype groups (p = 0.043 and p = 0.048, respectively). The CYP3A5*3 GG group and POR*28 CT/TT group were found to have lower AUC00-∞ and Cmax compared with the POR*28 CC group (p = 0.046 and p = 0.002, respectively). In addition, the POR*28 CT/TT group was found to have longer t1/2 but lower Cmax than the CYP3A4*1G GG group (p = 0.032 and p = 0.002, respectively) as well as the CYP3A4*1G GG and the CYP3A5*3 GG group (p = 0.038 and p = 0.036, respectively) compared with the POR*28 CC group. No significant associations were found between CYP3A4*1G/ABCB1-C3435T polymorphism and pharmacokinetics of nifedipine. Both CYP3A5*3 and POR*28 polymorphisms are found to be associated with the difference in disposition of nifedipine; POR*28 is considered to have an impact on CYP3A4 activity.

  1. Unique combined penA/mtrR/porB mutations and NG-MAST strain types associated with ceftriaxone and cefixime MIC increases in a 'susceptible' Neisseria gonorrhoeae population.

    PubMed

    Thakur, S D; Starnino, S; Horsman, G B; Levett, P N; Dillon, J R

    2014-06-01

    To determine which mutations in penA, mtrR and porB are implicated in increasing minimum MICs of ceftriaxone and cefixime in a susceptible gonococcal population and to ascertain associations with gonococcal strain types (STs). One hundred and forty-six Neisseria gonorrhoeae isolates formed two extended-spectrum cephalosporin susceptibility groups: group 1 isolates with cefixime and ceftriaxone MICs of 0.0005-0.016 mg/L; and group 2 isolates with cefixime MICs of 0.03-0.125 mg/L (n = 24) and ceftriaxone MICs of 0.03-0.06 mg/L (n = 23). Mutation patterns in penicillin-binding protein 2 (PBP2; penA), multiple transfer resistance repressor (MtrR; mtrR) and porin B (PorB; porB) were ascertained by DNA sequence and bioinformatic analysis. STs were determined using N. gonorrhoeae multiantigen sequence typing (NG-MAST). Most isolates carried PBP2 mutation pattern IX (D345a, F504L, A510V, A516G and P551L; 50/146, 34.2%), a G45D substitution in MtrR (37.7%) and a wild-type (WT) sequence for PorB (43.2%). Group 2 gonococcal isolates were significantly associated with: penA pattern IX; dual mutations in the promoter (A-) and DNA dimerization domain (H105Y) of MtrR; and G120K;A121D substitutions in PorB. There were 50 combined penA/mtrR/porB mutation patterns, with corresponding patterns I/WT/WT and IX/G45D/G120K;A121D predominating. Gonococci susceptible to ceftriaxone and cefixime were significantly associated with NG-MAST ST 25 (33/36; 92%) and the combined penA/mtrR/porB mutation pattern I/WT/WT. No combined mutation pattern or specific ST was associated with elevated ceftriaxone MICs. NG-MAST ST 3654 was significantly associated with the pattern IX/G45D/G120K;A121D and cefixime group 2 isolates. Specific single or combined mutation patterns in penA, mtrR and porB and specific STs were associated with differences in susceptibility to ceftriaxone and cefixime. © The Author 2014. Published by Oxford University Press on behalf of the British Society for Antimicrobial

  2. Expression of recombinant AccMRJP1 protein from royal jelly of Chinese honeybee in Pichia pastoris and its proliferation activity in an insect cell line

    USDA-ARS?s Scientific Manuscript database

    Main royal jelly protein 1 (MRJP1) is the most abundant member of the main royal jelly protein (MRJP) family among honeybees. Mature MRJP1 cDNA of the Chinese honeybee (Apis cerana cerana MRJP1, or AccMRJP1) was expressed in Pichia pastoris. SDS-PAGE showed that recombinant AccMRJP1 was identical in...

  3. Localization of thioredoxin in Fasciola hepatica

    USDA-ARS?s Scientific Manuscript database

    Thioredoxin is a member of a group of small redox proteins that may protect parasites from the host immune response. Experiments were designed to localize thioredoxin anatomically within the adult and immature stages of Fasciola hepatica. SDS-PAGE and Western blotting were performed to determine t...

  4. Chromosomal integration of recombinant alpha-amylase and glucoamylase genes in saccharomyces cerevisiae for starch conversion

    USDA-ARS?s Scientific Manuscript database

    Recombinant constructs of barley '-amylase and Lentinula edodes glucoamylase genes were integrated into the chromosomes of Saccharomyces cerevisiae. The insertion was confirmed by PCR amplification of the gene sequence in the chromosomes. The expression was analyzed by SDS-PAGE of the enzymes puri...

  5. Lygus hesperus polygalacturonase Characterization and Role in Plant Damage

    USDA-ARS?s Scientific Manuscript database

    The amino terminus, of a Lygus hesperus salivary gland protein revealing polygalacturonase (PG) activity in an SDS-PAGE activity gel assay, has been sequenced via Edman degradation. The N-terminal amino acid sequence shares homology with the predicted amino acid sequence for putative L. lineolaris P...

  6. Beta-Carotene chemical stability in nanoemulsions was improved by stabilized with Beta-Lactoglobulin-Catechin conjugates through free radical method

    USDA-ARS?s Scientific Manuscript database

    Beta-lactoglobulin (BLG)-catechin conjugates were prepared by a free radical method and investigated with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), electrospray ionization-mass spectrometry (ESI-MS), and far-UV circular dichroism (CD). Covalent binding between BLG and cat...

  7. Guaiacol Peroxidase Zymography for the Undergraduate Laboratory

    ERIC Educational Resources Information Center

    Wilkesman, Jeff; Castro, Diana; Contreras, Lellys M.; Kurz, Liliana

    2014-01-01

    This laboratory exercise presents a novel way to introduce undergraduate students to the specific detection of enzymatic activity by electrophoresis. First, students prepare a crude peroxidase extract and then analyze the homogenate via electrophoresis. Zymography, that is, a SDS-PAGE method to detect enzyme activity, is used to specifically…

  8. Comparison of in-gel protein separation techniques commonly used for fractionation in mass spectrometry-based proteomic profiling

    PubMed Central

    Jafari, Mohieddin; Primo, Vincent; Smejkal, Gary B.; Moskovets, Eugene V.; Kuo, Winston P.; Ivanov, Alexander R.

    2014-01-01

    Fractionation of complex samples at the cellular, subcellular, protein or peptide level is an indispensable strategy to improve the sensitivity in mass spectrometry-based proteomic profiling. This study revisits, evaluates, and compares the most common gel-based protein separation techniques i.e., 1-D SDS PAGE, preparative 1-D SDS PAGE, isoelectric focusing in immobilized pH gradients (IEF-IPG), and 2-D PAGE in their performance as fractionation approaches in nanoLC-ESI-MS/MS analysis of a mixture of protein standards and mitochondrial extracts isolated from rat liver. This work demonstrates that all the above techniques provide complementary protein identification results, but 1-D SDS PAGE and IEF-IPG had the highest number of identifications. The IEF-IPG technique resulted in the highest average number of detected peptides per protein. The 2-D PAGE was evaluated as a protein fractionation approach. This work shows that the recovery of proteins and resulting proteolytic digests is highly dependent on the total volume of the gel matrix. The performed comparison of the fractionation techniques demonstrates the potential of a combination of orthogonal 1-D SDS PAGE and IEF-IPG for the improved sensitivity of profiling without significant decrease in throughput. PMID:22899259

  9. PURIFICATION AND PARTIAL CHARACTERIZATION OF AN ACID PHOSPHATASE FROM SPIRODELA OLIGORRHIZA AND ITS AFFINITY FOR SELECTED ORGANOPHOSPHATE PESTICIDES

    EPA Science Inventory

    An acid phosphatase from the aquatic plant Spirodela oligorrhiza (duckweed) was isolated by fast protein liquid chromatography (FPLC) and partially characterized. The enzyme was purified 1871-fold with a total yield of 40%. SDS-PAGE electrophoresis of the pure acid phosphatase ...

  10. Changes in the antigenic and molecular structure of. gamma. -irradiated bacterial lipopolysaccharide (LPS)

    SciTech Connect

    Csako, G.; Suba, E.A.; Tsai, C.M.; Elin, R.J.

    1986-03-01

    Ionization radiation is known to alter the biological properties of LPS. The author treated a highly purified LPS from E. coli in aqueous medium with /sup 60/Co-radiation. The changes in the antigenic and molecular structure of LPS were studied in double immunodiffusion/immunoelectrophoresis (rabbit antiserum) and sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE), respectively. Untreated LPS revealed two major antigenic components and, due to varying lengths of the O-polysaccharide side chain, a series of homopolymers (SDS-PAGE). High doses of ..gamma..-radiation destroyed all antigenic reactivities and all stainable bands on SDS-PAGE. However, lower doses of radiation were selective. Disappearance of the more radiation-sensitive, electrophoretically fast-migrating antigenic component paralleled elimination of the long O-side chain containing molecules. The relatively radiation-resistant, less anodic second antigenic component cross-reacted with LPS of another E. coli strain and corresponded to LPS molecules composed of R-core and lipid A (SDS-PAGE). These findings explain the in vivo loss of antibody protection from shock before non-specific resistance with ..gamma..-irradiation of LPS.

  11. Effects of Fe deficiency on the protein profile of Brassica napus phloem sap

    USDA-ARS?s Scientific Manuscript database

    The aim of this work was to study the effect of Fe deficiency on the protein profile of phloem sap exudates from Brassica napus using 2-DE (IEF-SDS PAGE). The experiment was repeated thrice and two technical replicates per treatment were done. Two hundred sixty-three spots were consistently detected...

  12. Characteristics of an Extensive Mycobacterium avium subspecies paratuberculosis Recombinant Protein Set

    USDA-ARS?s Scientific Manuscript database

    In the first step of a comprehensive large-scale antigen discovery project, 651 Mycobacterium avium subspecies paratuberculosis proteins were produced in Escherichia coli. All of these were purified by affinity chromatography, dialyzed in phosphate buffered saline, and analyzed on SDS-PAGE gels. C...

  13. Confirmation of the genome position and mass of the viral protien, VP3, of Solenopsis Invicta Virus 1 (Picornavirales: Dicistroviridae) infecting the Red Imported Fire Ant (Hymenoptera: Formicidae)

    USDA-ARS?s Scientific Manuscript database

    A predicted peptide sequence corresponding to the capsid protein VP3 of SINV-1 was used to prepare a polyclonal antibody preparation and probe SDS-PAGE separated proteins from SINV-1 particles and S. invicta ants. The SINV-1 VP3 antibody preparation recognized a 24 kDa protein from denatured SINV-1...

  14. Identification and distribution of Bacillus species in doenjang by whole-cell protein patterns and 16S rRNA gene sequence analysis.

    PubMed

    Kim, Tae-Woon; Kim, Young-Hoon; Kim, Sung-Eon; Lee, Jun-Hwa; Park, Cheon-Seok; Kim, Hae-Yeong

    2010-08-01

    Many bacteria are involved in fermentation of doenjang and Bacillus species are known to perform significant roles. Although the SDS-PAGE technique has been frequently used for classification and identification of bacteria in various samples, there has been no investigation of the microbial diversity in doenjang. This study aims to investigate the identification and distribution of dominant Bacillus species in doenjang using SDS-PAGE profiles of whole cell proteins and 16S rDNA sequencing. SDS-PAGE of whole cell proteins of the reference Bacillus strains yielded differential banding patterns that could be considered to be highly specific fingerprints. Bacterial strains isolated from doenjang samples were grouped using whole cell protein patterns, which were confirmed by the analysis of 16S rDNA sequencing. B. subtilis was found to be the most dominant strain in most of the samples, and B. licheniformis and B. amyloliquefaciens were less frequently detected. The results obtained in this study showed that a combined identification method, SDS-PAGE patterns of whole cell proteins and subsequent 16S rDNA sequence analysis, could successfully identify Bacillus species isolated from doenjang.

  15. PURIFICATION AND PARTIAL CHARACTERIZATION OF AN ACID PHOSPHATASE FROM SPIRODELA OLIGORRHIZA AND ITS AFFINITY FOR SELECTED ORGANOPHOSPHATE PESTICIDES

    EPA Science Inventory

    An acid phosphatase from the aquatic plant Spirodela oligorrhiza (duckweed) was isolated by fast protein liquid chromatography (FPLC) and partially characterized. The enzyme was purified 1871-fold with a total yield of 40%. SDS-PAGE electrophoresis of the pure acid phosphatase ...

  16. Erythrocyte membrane protein analysis by sodium dodecyl sulphate-capillary gel electrophoresis in the diagnosis of hereditary spherocytosis.

    PubMed

    Debaugnies, France; Cotton, Frédéric; Boutique, Charles; Gulbis, Béatrice

    2011-03-01

    Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) is currently the reference method for detecting protein deficiencies related to hereditary spherocytosis. The aim of the study was to evaluate an automated capillary gel electrophoresis system, the Experion instrument from BioRad, for its ability to separate and quantify the erythrocyte membrane proteins. The major erythrocyte membrane proteins (actin, protein 4.2, protein 4.1, band 3, ankyrin, α- and β-spectrin) were extracted and purified from membrane ghosts by centrifugation, immunoprecipitation and electroelution. Analyses were performed using SDS-PAGE and sodium dodecyl sulphate capillary gel electrophoresis (SDS-CGE) to establish a separation profile of the total ghosts. Then, the samples from patients received for investigations of erythrocyte membrane defects were analysed. Five of the seven expected erythrocyte membrane proteins were finally separated and identified. In the 20 studied cases, taking into account the screening test results and the clinical and family histories, the SDS-CGE method allowed us to achieve the same conclusion as with SDS-PAGE, except for the patient with elliptocytosis. The new SDS-CGE method presents interesting features that could make this instrument a powerful diagnostic tool for detection of erythrocyte membrane protein abnormalities, and can be proposed as an automated alternative method to the labour intensive SDS-PAGE analysis.

  17. Oncoprotein protein kinase

    DOEpatents

    Karin, Michael; Hibi, Masahiko; Linn, Anning

    1996-01-01

    An isolated polypeptide (JNK) characterized by having a molecular weight of 46kD as determined by reducing SDS-PAGE, having serine and threonine kinase activity, phosphorylating the c-Jun N-terminal activation domain and polynucleotide sequences and method of detection of JNK.

  18. Effect of Pulsed Ultraviolet Light and High Hydrostatic Pressure on the Antigenicity of Almond Protein Extracts.

    USDA-ARS?s Scientific Manuscript database

    The efficacy of pulsed ultraviolet light (PUV) and high hydrostatic pressure (HHP) on reducing the IgE binding to the almond extracts, was studied using SDS-PAGE, Western Blot, and ELISA probed with human plasma containing IgE antibodies to almond allergens, and a polyclonal antibody against almond ...

  19. [Isolation of plant insulin from Momordica charantia seeds by gel filtration and RP-HPLC].

    PubMed

    Sheng, Qingkai; Yao, Huiyuan; Xu, Huajun; Ling, Xiaoyun; He, Ting

    2004-06-01

    Hypoglycemic polypeptide (PA) was extracted from Momordica charantia seeds with organic acid and ethanol and purified with Sephadex G-50 gel filtration and RP-HPLC. PA was judged as plant insulin on the base of the analysis of its SDS-PAGE electrophoresis and amino acid composition.

  20. Identification of c-di-AMP-Binding Proteins Using Magnetic Beads.

    PubMed

    Kampf, Jan; Gundlach, Jan; Herzberg, Christina; Treffon, Katrin; Stülke, Jörg

    2017-01-01

    To identify cytosolic proteins that bind to cyclic di-AMP, a biotinylated analog of the nucleotide is used for protein pull-down experiments. In this approach, biotinylated c-di-AMP is coupled to Streptactin-covered beads. After protein separation using standard SDS-PAGE, the protein(s) of interest are identified by mass spectrometric analyses.

  1. Differentiation of Staphylococci from Iberian dry fermented sausages by protein fingerprinting.

    PubMed

    Benito, María J; Serradilla, Manuel J; Martín, Alberto; Aranda, Emilio; Hernández, Alejandro; Córdoba, María G

    2008-08-01

    The Staphylococci populations in different types of Iberian dry fermented sausages from central-west Spain were identified. A simple electrophoretic method of whole-cell proteins and extracellular protein profiling was evaluated for speed of identification. This study was correlated with a 16S rRNA gene sequence analysis and biochemical identification by API Staph. A total of 81 isolates were identified by SDS-PAGE of the whole-cell proteins. These showed stable profiles in the range 99-14kDa that were clearly different for the different species, and were grouped into clusters together with the profiles of the eight reference strains. SDS-PAGE of the extracellular protein extracts provided additional characteristic banding patterns for the characterization of the Staphylococcus species present. The whole-cell SDS-PAGE showed that the predominant species was Staphylococcus saprophyticus (61.7%) followed by Staphylococcus aureus (19.7%). The identifications were confirmed by the 16S rRNA gene sequencing and by a BLAST search of the GenBank database. However, the API Staph biochemical identifications were frequently erroneous at the species level. In sum, SDS-PAGE analysis showed itself to be rapid and accurate in identifying the most commonly encountered Staphylococcus isolates in dry fermented sausages.

  2. Two-dimensional gel electrophoresis: vertical isoelectric focusing.

    PubMed

    Dorri, Yaser

    2012-01-01

    Two-dimensional gel electrophoresis (2-DE) is one of the most powerful tools for separating proteins based on their size and charge. 2-DE is very useful to separate two proteins with identical molecular weights but different charges, which cannot be achieved with just sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Here, a simpler and easier version of 2-DE is presented which is also faster than all the currently available techniques. In this modified version of 2-DE, isoelectric focusing is carried out in the first dimension using a vertical SDS-PAGE apparatus. Following the first-dimensional IEF, each individual lane is excised from the IEF gel and, after a 90° rotation, is inserted into a second-dimensional SDS-PAGE, which can be stained with Coomassie Brilliant Blue for protein analysis or immunoblotted for further analysis. This version of IEF can be run in less than 2 h compared to the overnight run required by O'Farrell's method. Difficult tube gel casting and gel extrusion as well as tube gel distortion are eliminated in our method. This method is simpler, faster, and inexpensive. Both dimensions can be done on the same SDS-PAGE apparatus, and up to ten samples can be run simultaneously using one gel.

  3. Improved adsorption-desorption extraction applied to the partial characterization of the antilisterial bacteriocin produced by Carnobacterium maltaromaticum C2.

    PubMed

    Tulini, F L; De Martinis, E C P

    2010-04-01

    Bacteriocins are ribosomally produced peptides useful for food biopreservation. An improved adsorption-desorption process is proposed for the partial purification of the bacteriocin produced by the fish isolate Carnobacterium maltaromaticum C2. Analyzis of extract by SDS-PAGE indicated this method may offer an alternative to improve the yield of purification of bacteriocins.

  4. Compatibility of imported fire ant whole body extract with cat, ragweed, Dermatophagoides pteronyssinus, and timothy grass allergens.

    PubMed

    Rans, Tonya S; Hrabak, Todd M; Whisman, Bonnie A; Grier, Thomas J; LeFevre, Dawn M; Kwon, Paul O; Tankersley, Michael S

    2009-01-01

    Recommendations regarding the administration of imported fire ant whole body extract (IFA WBE) combined with aeroallergens or environmental allergens in a single immunotherapy injection are lacking. To evaluate the degradative effect of IFA WBE on cat, ragweed, Dermatophagoides pteronyssinus, and timothy grass allergens. Imported fire ant whole body extract was combined with extracts of cat, ragweed, D pteronyssinus, and timothy grass. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed on each sample after storage for 0, 1, 3, and 6 months at 4 degrees C. In addition, cat and ragweed combinations were evaluated by radial immunodiffusion (RID); D pteronyssinus by enzyme-linked immunosorbent assay (ELISA) inhibition; and timothy grass by ELISA inhibition and Western blot. Imported fire ant whole body extract combined with timothy grass demonstrated degradation of timothy grass allergens by SDS-PAGE, ELISA inhibition, and Western blot results. Cat and ragweed allergens were stable after mixing with IFA WBE, based on SDS-PAGE and RID analyses. Stability of D pteronyssinus allergens with IFA WBE was evident from SDS-PAGE and ELISA inhibition data. Imported fire ant whole body extract combined with timothy grass resulted in significant and rapid timothy protein degradation. Imported fire ant whole body extract mixed with cat, ragweed, or D pteronyssinus revealed aeroallergen stability, yielding the possibility of combining these extracts in a single immunotherapy injection. Compatibilities of IFA WBE with other common aeroallergens remain undetermined and thus are not recommended for single-injection immunotherapy formulations.

  5. Guaiacol Peroxidase Zymography for the Undergraduate Laboratory

    ERIC Educational Resources Information Center

    Wilkesman, Jeff; Castro, Diana; Contreras, Lellys M.; Kurz, Liliana

    2014-01-01

    This laboratory exercise presents a novel way to introduce undergraduate students to the specific detection of enzymatic activity by electrophoresis. First, students prepare a crude peroxidase extract and then analyze the homogenate via electrophoresis. Zymography, that is, a SDS-PAGE method to detect enzyme activity, is used to specifically…

  6. Proteomic Profiling of Rat Thyroarytenoid Muscle

    ERIC Educational Resources Information Center

    Welham, Nathan V.; Marriott, Gerard; Bless, Diane M.

    2006-01-01

    Purpose: Proteomic methodologies offer promise in elucidating the systemwide cellular and molecular processes that characterize normal and diseased thyroarytenoid (TA) muscle. This study examined methodological issues central to the application of 2-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (2D SDS-PAGE) to the study of…

  7. Staining proteins in gels with coomassie blue.

    PubMed

    Simpson, Richard J

    2007-04-01

    INTRODUCTIONThe most commonly used dye for visualizing proteins in SDS-PAGE gels is Coomassie Brilliant Blue R250 (CBR-250) because of its relatively high sensitivity. This protocol describes the standard CBR-250 staining method, along with a simple method for preparing stained gels for long-term storage.

  8. Myrmecia pilosula (Jack Jumper) ant venom: validation of a procedure to standardise an allergy vaccine.

    PubMed

    Wiese, Michael D; Milne, Robert W; Davies, Noel W; Chataway, Tim K; Brown, Simon G A; Heddle, Robert J

    2008-01-07

    Ant sting allergy is relatively common within south-eastern Australia and is predominantly due to Myrmecia pilosula (Jack Jumper Ant, JJA). Venom immunotherapy has been shown to be effective in preventing anaphylaxis to the sting of the JJA, but analytical techniques to standardise the venom have not been validated. The purpose of this study was to develop assays to analyse JJA venom and apply these to the standardisation of venom prior to new batches being used for the diagnosis and treatment of JJA sting allergy. Venom was analysed by protein content, HPLC-UV, enzyme-linked immunosorbent assay (ELISA) inhibition, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and SDS-PAGE immunoblot. The protein content in JJA venom was adjusted so that all batches were equivalent. A HPLC-UV assay was used to quantify the relative amount of the major allergen Myr p 2 and two minor allergens Myr p 1 and Myr p 3 and allergenic potency was determined by ELISA inhibition. SDS-PAGE and SDS-PAGE immunoblot were used as qualitative tools to determine the protein profile and presence or absence of additional high molecular weight allergens not quantifiable by HPLC-UV. A standardisation procedure has been developed that complies with the requirements described in the European Pharmacopoeia. Techniques used to determine the content of some of the other minor allergens could be developed, which would further improve the standardisation methodology.

  9. Pig heart calpastatin: identification of repetitive domain structures and anomalous behavior in polyacrylamide gel electrophoresis.

    PubMed

    Takano, E; Maki, M; Mori, H; Hatanaka, M; Marti, T; Titani, K; Kannagi, R; Ooi, T; Murachi, T

    1988-03-22

    Isolation and nucleotide sequencing of the complementary DNA for pig heart calpastatin have been completed. The amino acid sequence of 713 residues predicted from the nucleotide sequence contains five domains, each composed of approximately 140 amino acid residues. A unique N-terminal domain is followed by four mutually homologous domains. The best fit alignment of these four domains gives residue identities between any two domains of 22.5-36.0%. The analysis of the sequence similarities by several methods also suggests the existence of additional shorter repeats at intervals of 60-80 residues. The calculated molecular weight of pig calpastatin of 713 amino acid residues (Mr 77,122) is significantly lower than the value of purified pig heart calpastatin (Mr 107,000) estimated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE). The expression of the calpastatin genes in Escherichia coli and the detection of the translation products of 713, 366, and 140 amino acid residues by the specific anti-calpastatin antibody indicate that the products always migrate considerably slow on SDS-PAGE, giving an average of 1.53 for the ratio of the molecular weight estimated by SDS-PAGE to the value calculated from the amino acid sequences. It is most likely that the discrepancy in the molecular weight is caused by an anomalous behavior of calpastatin in SDS-PAGE.

  10. A cysteine endopeptidase ("dionain") is involved in the digestive fluid of Dionaea muscipula (Venus's fly-trap).

    PubMed

    Takahashi, Kenji; Suzuki, Takehiro; Nishii, Wataru; Kubota, Keiko; Shibata, Chiaki; Isobe, Toshiaki; Dohmae, Naoshi

    2011-01-01

    The carnivorous plant Dionaea muscipula (Venus's flytrap) secretes proteinases into the digestive fluid to digest prey proteins. In this study, we obtained evidence that the digestive fluid contains a cysteine endopeptidase, presumably belonging to the papain family, through inhibitor studies and partial amino acid sequencing of the major SDS-PAGE band protein. The name "dionain" is proposed for the enzyme.

  11. Confirmation of a blocked amino terminus of sulfhydryl oxidase

    SciTech Connect

    Janolino, V.G.; Morrison-Rowe, S.J.; Swaisgood, H.E. )

    1990-09-01

    The isolation of sulfhydryl oxidase from bovine milk in a suitably pure form for sequencing was carried out by transient covalent affinity chromatography of diafiltered whey using cysteinylsuccinamidopropyl-glass as matrix. The glutathione-eluted proteins were separated by SDS-PAGE. By radiolabeling the affinity chromatography-purified enzyme with ({sup 14}C)iodoacetate before subjecting to SDS-PAGE, the sulfhydryl oxidase band was identified, because sulfhydryl oxidase is known to be inactivated by alkylation of one sulfhydryl group per mole. The results confirmed that sulfhydryl oxidase corresponds to the 85 ({plus minus} 5)-kDa band observed on SDS-PAGE. The protein band corresponding to radiolabeled sulfhydryl oxidase was recovered from SDS-PAGE gels by electrophoretic elution and by electroblotting on polyvinylidene difluoride membrane and subjected to gas phase sequencing. Precautions were taken during electrophoretic elution to prevent reactions that result in N-terminal blocking. Both methods of protein recovery yielded negative results when subjected to sequence analysis indicating that the N-terminus of sulfhydryl oxidase is blocked.

  12. Glycation inhibits trichloroacetic acid (TCA)-induced whey protein precipitation

    USDA-ARS?s Scientific Manuscript database

    Four different WPI saccharide conjugates were successfully prepared to test whether glycation could inhibit WPI precipitation induced by trichloroacetic acid (TCA). Conjugates molecular weights after glycation were analyzed with SDS-PAGE. No significant secondary structure change due to glycation wa...

  13. Proteomic Profiling of Rat Thyroarytenoid Muscle

    ERIC Educational Resources Information Center

    Welham, Nathan V.; Marriott, Gerard; Bless, Diane M.

    2006-01-01

    Purpose: Proteomic methodologies offer promise in elucidating the systemwide cellular and molecular processes that characterize normal and diseased thyroarytenoid (TA) muscle. This study examined methodological issues central to the application of 2-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (2D SDS-PAGE) to the study of…

  14. Effect of multiple extrusion passes on zein

    USDA-ARS?s Scientific Manuscript database

    Zein was repeatedly processed up to seven times using a single screw extruder at a temperature of 145 °C and at approximately 15 grams per minute to determine the extent of degradation that occurs with multiple extrusion passes. SDS-PAGE shows that with the second pass, and each additional pass, the...

  15. Envelope protein complexes of Mycobacterium avium subsp. paratuberculosis and their antigenicity

    USDA-ARS?s Scientific Manuscript database

    Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of Johne’s disease, a chronic enteric disease of ruminant animals. In the present study, blue native PAGE electrophoresis and 2D SDS-PAGE were used to separate MAP envelope protein complexes, followed by mass spectrometry (MS) ...

  16. Characterization of carotenoid-protein complexes and gene expression analysis associated with carotenoid sequestration in pigmented cassava (Manihot esculenta Crantz) storage root

    USDA-ARS?s Scientific Manuscript database

    Carotenoid-protein complex separation by size exclusion chromatography, protein fractionation by SDS-PAGE, and shotgun PROTEOMICS technology were used to identify and characterize carotenoid associated proteins (CAPs) of chromoplast-enriched suspensions from cassava intense yellow storage root. A no...

  17. Agarose isoelectric focusing can improve resolution of membrane proteins in the two-dimensional electrophoresis of bacterial proteins.

    PubMed

    Altenhofer, Pia; Schierhorn, Angelika; Fricke, Beate

    2006-10-01

    2-D separation of bacterial membrane proteins is still difficult despite using high-resolution IPG-IEF/SDS-PAGE. We were searching for alternative methods to avoid typical problems such as precipitation, low solubility, and aggregation of membrane proteins in the 1-D separation with IPG-IEF. Blue native electrophoresis (BNE) and agarose IEF (A-IEF) were tested for their separation capacity and their capability of replacing IPG-IEF in the first dimension. SDS-PAGE was chosen for the second dimension on account of its outstanding resolution. We could confirm that only A-IEF was a useful replacement for the IPG-IEF in the first dimension resulting in 2-D protein distributions with additional membrane protein spots not being found after IPG-IEF/SDS-PAGE. A second interesting result was that the agarose IEF mediates the possibility of separation of membrane proteins in a partially native state in the first dimension. This native A-IEF resulted in drastically changed spot patterns with an acidic shift of nearly all spots and divergent distribution of proteins compared to non-native A-IEF and IPG-IEF. We found out that native and non-native A-IEF are powerful tools to supplement IPG-IEF/SDS-PAGE.

  18. External Factors, Produced by Growing Nerves, Trigger a Regenerative Response in a Non-Regenerative Central Nervous System: Purification and Mode of Action

    DTIC Science & Technology

    1988-01-01

    against mune EHS sarcoma lamlinin (diluted 1:1000, gift of Dr. H. Kleinman, NIH) or PBS containing 0. 2rq/ml BSA were then applied for 2 h at roan...polypeptide, the 220 kDa, may be fibronectin. Figure 5: Analysis of carp plasma lipoproteins by SDS-PAGE. The lipo - proteins having the densities

  19. A sensitive and rapid method for identification and characterization of low abundance receptors

    SciTech Connect

    Sunahara, R.K.; Murphy, B.J.; Tuana, B.S. )

    1990-02-15

    An improved method for detection of low intensity radioligand-receptor complexes resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) is described. (3H)Azidopine-labeled 1,4-dihydropyridine (DHP) receptor from skeletal muscle resolved by SDS-PAGE was transferred to nitrocellulose and cut into strips and individual slices were analyzed for radioincorporation by liquid scintillation counting. (3H)Azidopine-labeled DHP binding subunit migrated as a single entity with a mass of 170 kDa and was confirmed using conventional methods. Results were obtained within 4 h after resolution by SDS-PAGE compared to 3-40 days using conventional methods. In addition, detection of extremely low signals (less than 50 cpm/lane), otherwise overwhelmed by background noise using conventional methods, was possible due to removal of free ligand during electro-transfer to nitrocellulose. This technique offers a rapid sensitive, cost effective alternative to fluorography or other conventional gel slice analysis methods for detecting low intensity radiolabeled complexes resolved by SDS-PAGE.

  20. Personal Docente del Nivel Primario. Series Estadisticas Basicas, Nivel Educativo: Cordoba (Teaching Personnel in Primary Schools. Basic Statistics Series , Level of Education: Cordoba).

    ERIC Educational Resources Information Center

    Ministerio de Educacion Nacional, Bogota (Colombia). Instituto Colombiano de Pedagogia.

    This document provides statistical data on the distribution and education of teaching personnel working the elementary schools of Cordoba, Colombia, between 1958 and 1967. The statistics cover the number of men and women, public and private schools, urban and rural location, and the amount of education of the teachers. For overall statistics in…

  1. Personal Docente del Nivel Primario. Series Estadisticas Basicas, Nivel Educativo: Cauca (Teaching Personnel in Primary Schools. Basic Statistics Series, Level of Education: Cauca).

    ERIC Educational Resources Information Center

    Ministerio de Educacion Nacional, Bogota (Colombia). Instituto Colombiano de Pedagogia.

    This document provides statistical data on the distribution and education of teaching personnel working in the elementary schools of Cauca, Colombia, between 1958 and 1967. The statistics cover the number of men and women, public and private schools, urban and rural location, and the amount of education of the teachers. For overall statistics in…

  2. Personal Docente del Nivel Primario. Series Estadisticas Basicas, Nivel Educativo: Huila (Teaching Personnel in Primary Schools. Basic Statistics Series, Level of Education: Huila).

    ERIC Educational Resources Information Center

    Ministerio de Educacion Nacional, Bogota (Colombia). Instituto Colombiano de Pedagogia.

    This document provides statistical data on the distribution and education of teaching personnel working in the elementary schools of Huila, Colombia, between 1958 and 1967. The statistics cover the number of men and women, public and private schools, urban and rural location, and the amount of education of the teachers. For overall statistics in…

  3. Personal Docente des Nivel Primario. Series Estadisticas Basicas, Nivel Educativo: Narino (Teaching Personnel in Primary Schools. Basic Statistics Series, Level of Education: Narino).

    ERIC Educational Resources Information Center

    Ministerio de Educacion Nacional, Bogota (Colombia). Instituto Colombiano de Pedagogia.

    This document provides statistical data on the distribution and education of teaching personnel working in the elementary schools of Narino, Colombia, between 1958 and 1967. The statistics cover the number of men and women, public and private schools, urban and rural location, and the amount of education of the teachers. For overall statistics in…

  4. Personal Docente del Nivel Primario. Series Estadisticas Basicas, Nivel Educativo: Boyaca (Teaching Personnel in Primary Schools. Basic Statistics Series, Level of Education: Boyaca).

    ERIC Educational Resources Information Center

    Ministerio de Educacion Nacional, Bogota (Colombia). Instituto Colombiano de Pedagogia.

    This document provides statistical data on the distribution and education of teaching personnel working in the elementary schools of Boyaca, Colombia, between 1958 and 1967. The statistics cover the number of men and women, public and private schools, urban and rural location, and the amount of education of the teachers. For overall statistics in…

  5. Personal Docente del Nivel Primario. Series Estadisticas Basicas, Nivel Educativo: Caldas (Teaching Personnel in Primary Schools. Basic Statistics Series, Level of Education: Caldas).

    ERIC Educational Resources Information Center

    Ministerio de Educacion Nacional, Bogota (Colombia). Instituto Colombiano de Pedagogia.

    This document provides statistical data on the distribution and education of teaching personnel working in the elementary schools of Caldas, Colombia, between 1958 and 1967. The statistics cover the number of men and women, public and private schools, urban and rural location, and the amount of education of the teachers. For overall statistics in…

  6. Characterization of the key antigenic components and pre-clinical immune responses to a meningococcal disease vaccine based on Neisseria lactamica outer membrane vesicles.

    PubMed

    Finney, Michelle; Vaughan, Thomas; Taylor, Stephen; Hudson, Michael J; Pratt, Catherine; Wheeler, Jun X; Vipond, Caroline; Feavers, Ian; Jones, Christopher; Findlow, Jamie; Borrow, Ray; Gorringe, Andrew

    2008-01-01

    Serogroup B strains are now responsible for over 80% of meningococcal disease in the UK and no suitable vaccine is available that confers universal protection against all serogroup B strains. Neisseria lactamica shares many antigens with the meningococcus, except capsule and the surface protein PorA. Many of these antigens are thought to be responsible for providing cross-protective immunity to meningococcal disease. We have developed an N. lactamica vaccine using methods developed for meningococcal outer membrane vesicle (OMV) vaccines. The major antigenic components were identified by excision of 11 major protein bands from an SDS-PAGE gel, followed by mass spectrometric identification. These bands contained at least 22 proteins identified from an unassembled N. lactamica genome, 15 of which having orthologues in published pathogenic Neisseria genomes. Western blotting revealed that most of these bands were immunogenic, and antibodies to these proteins generally cross-reacted with N. meningitidis proteins. Sera from mice and rabbits immunized with either N. lactamica or N. meningitidis OMVs produced comparable cross-reactive ELISA titres against OMVs prepared from a panel of diverse meningococcal strains. Mice immunized with either N. meningitidis or N. lactamica OMVs showed no detectable serum bactericidal activity against the panel of target strains except N. meningitidis OMV sera against the homologous strain. Similarly, rabbit antisera to N. lactamica OMVs elicited little or no bactericidal antibodies against the panel of serogroup B meningococcal strains. However, such antisera did mediate opsonophagocytosis, suggestingthat this may did mediate opsonophagocytosis, suggesting that this may be a mechanism by which this vaccine protects in a mouse model of meningococcal bacteraemia.

  7. Bifurcación de las soluciones de vientos impulsados por radiación en estrellas Be: formación de líneas

    NASA Astrophysics Data System (ADS)

    Curé, M.; Rial, D.; Cidale, L.; Venero, R.

    Se ha estudiado la topología de la ecuación hidrodinámica no-lineal que describe el perfil de velocidades de vientos impulsados por radiación en estrellas tempranas. Al aplicar este modelo a estrellas Be se encuentra que existen dos tipos De soluciones: la estándar, que describe el viento polar, y una nueva, que describe un viento más denso y lento y que explicaría el disco que se encuentra alrededor de estos objetos. Existe una región de transición en donde ambas soluciones coexisten (bifurcación}). Ambas soluciones satisfacen en esta región las mismas condiciones de borde. Para estas dos soluciones se han obtenido los perfiles de líneas de hidrógeno del visible y del IR, resolviendo el transporte de radiación en el ``comoving frame". Para la solución estándar, se obtienen perfiles con componentes en emisión, mientras que para la nueva solución se obtienen perfiles en absorción. Se comparan cualitativamente los resultados con las observaciones.

  8. Weight of ABCB1 and POR genes on oral tacrolimus exposure in CYP3A5 nonexpressor pediatric patients with stable kidney transplant.

    PubMed

    Almeida-Paulo, G N; Dapía García, I; Lubomirov, R; Borobia, A M; Alonso-Sánchez, N L; Espinosa, L; Carcas-Sansuán, A J

    2017-01-17

    Tacrolimus (TAC) is highly effective for the prevention of acute organ rejection. However, its clinical use may be challenging due to its large interindividual pharmacokinetic variability, which can be partially explained by genetic variations in TAC-metabolizing enzymes and transporters. The aim of this study was to evaluate the influence of genetic and clinical factors on TAC pharmacokinetic variability in 21 stable pediatric renal transplant patients. This study was nested in a previous Prograf to Advagraf conversion clinical trial. CYP3A5, ABCB1 and two POR genotypes were assessed by real-time PCR. The impact on TAC pharmacokinetics of individual genetic variants on CYP3A5 nonexpressors was evaluated by genetic score. Explicative models for TAC AUC0-24h, Cmax and Cmin after Advagraf were developed by linear regression. The built genetic scores explain 13.7 and 26.5% of the total AUC0-24h and Cmin total variability, respectively. Patients genetic information should be considered to monitorizate and predict TAC exposure.The Pharmacogenomics Journal advance online publication, 17 January 2017; doi:10.1038/tpj.2016.93.

  9. Relación masa-radio para estrellas enanas blancas y la interpretación de recientes mediciones hechas por Hipparcos

    NASA Astrophysics Data System (ADS)

    Panei, J. A.; Althaus, L. G.; Benvenuto, O. G.

    Recientes mediciones de la masa y el radio hechas por Hipparcos de las estrellas enanas blancas 40 Eri B y Procyon B (Shipman, H. & Provencal, J. - ApJ. 1998, 494, 759), sugieren un núcleo compuesto de hierro para dichas estrellas, en lugar de carbono y oxígeno como predice la teoría standard de evolución estelar. Para interpretar estas observaciones, presentamos aquí, relaciones masa-radio para configuraciones degeneradas a temperatura finita para distintas composiciones químicas centrales. Para tal fin hemos calculado secuencias evolutivas de enanas blancas utilizando el código de evolución estelar, desarrollado en el Observatorio de La Plata. Dicho código resuelve las ecuaciones de estructura y evolución estelar mediante la técnica de relajación de Henyey, y esta basado en una descripción física muy detallada y actualizada.

  10. Population pharmacokinetic approach to evaluate the effect of CYP2D6, CYP3A, ABCB1, POR and NR1I2 genotypes on donepezil clearance.

    PubMed

    Noetzli, Muriel; Guidi, Monia; Ebbing, Karsten; Eyer, Stephan; Wilhelm, Laurence; Michon, Agnès; Thomazic, Valérie; Stancu, Ioana; Alnawaqil, Abdel-Messieh; Bula, Christophe; Zumbach, Serge; Gaillard, Michel; Giannakopoulos, Panteleimon; von Gunten, Armin; Csajka, Chantal; Eap, Chin B

    2014-07-01

    A large interindividual variability in plasma concentrations has been reported in patients treated with donepezil, the most frequently prescribed antidementia drug. We aimed to evaluate clinical and genetic factors influencing donepezil disposition in a patient population recruited from a naturalistic setting. A population pharmacokinetic study was performed including data from 129 older patients treated with donepezil. The patients were genotyped for common polymorphisms in the metabolic enzymes CYP2D6 and CYP3A, in the electron transferring protein POR and the nuclear factor NR1I2 involved in CYP activity and expression, and in the drug transporter ABCB1. The average donepezil clearance was 7.3 l h(-1) with a 30% interindividual variability. Gender markedly influenced donepezil clearance (P < 0.01). Functional alleles of CYP2D6 were identified as unique significant genetic covariate for donepezil clearance (P < 0.01), with poor metabolizers and ultrarapid metabolizers demonstrating, respectively, a 32% slower and a 67% faster donepezil elimination compared with extensive metabolizers. The pharmacokinetic parameters of donepezil were well described by the developed population model. Functional alleles of CYP2D6 significantly contributed to the variability in donepezil disposition in the patient population and should be further investigated in the context of individual dose optimization to improve clinical outcome and tolerability of the treatment. © 2014 The British Pharmacological Society.

  11. IgE Reactivity of Blue Swimmer Crab (Portunus pelagicus) Tropomyosin, Por p 1, and Other Allergens; Cross-Reactivity with Black Tiger Prawn and Effects of Heating

    PubMed Central

    Varese, Nirupama; Zubrinich, Celia; Lopata, Andreas L.; O'Hehir, Robyn E.; Rolland, Jennifer M.

    2013-01-01

    Shellfish allergy is a major cause of food-induced anaphylaxis, but the allergens are not well characterized. This study examined the effects of heating on blue swimmer crab (Portunus pelagicus) allergens in comparison with those of black tiger prawn (Penaeus monodon) by testing reactivity with shellfish-allergic subjects' serum IgE. Cooked extracts of both species showed markedly increased IgE reactivity by ELISA and immunoblotting, and clinical relevance of IgE reactivity was confirmed by basophil activation tests. Inhibition IgE ELISA and immunoblotting demonstrated cross-reactivity between the crab and prawn extracts, predominantly due to tropomyosin, but crab-specific IgE-reactivity was also observed. The major blue swimmer crab allergen tropomyosin, Por p 1, was cloned and sequenced, showing strong homology with tropomyosin of other crustacean species but also sequence variation within known and predicted linear IgE epitopes. These findings will advance more reliable diagnosis and management of potentially severe food allergy due to crustaceans. PMID:23840718

  12. Genetic linkage between protein and DNA polymorphisms and antioxidant capacity of Cuminum cyminum L. accessions.

    PubMed

    Abdelhaliem, E; Al-Huqail, A A

    2016-10-06

    This study aimed to link the genetic variation observed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and random amplified polymorphic DNA (RAPD) analysis among 11 Cuminum cyminum L. accessions, collected from diverse ecogeographical areas in Saudi Arabia, with their antioxidant capacity to better identify potential genotypes for breeding programs for this medicinal spice. SDS-PAGE analysis revealed genetic variation among cumin germplasms and distinct polymorphisms (100%). Protein polymorphisms were identified based on the number of polypeptide bands (288) with molecular weights ranging from 13.85 to 350 kDa, band intensity, the appearance of new bands, and the absence of other bands. RAPD analysis revealed 363 amplified DNA products with a high polymorphism value (98.88%) based on DNA band type (unique, non-unique, and monomorphic), DNA 90 to 1085-bp long, and band intensity. The unweighted pair group method with arithmetic mean clustering based on SDS-PAGE or RAPD and Jaccard's similarity coefficient divided cumin accessions into similar but distinct clusters with respect to their location of collection. The antioxidant potential of cumin accessions based on 1, 1-diphenyl-2-picrylhydrazyl radical scavenging activity, the β-carotene-linoleate model system, and total phenolic and flavonoid contents revealed distinct variability. These data indicate that cumin is a valuable genetic resource with high antioxidant activity. Additionally, clustering based on antioxidant activity was not identical to that based on SDS-PAGE and RAPD. Data and clustering of SDS-PAGE and RAPD, combined with the high antioxidant capacity of cumin accessions, are important for the efficient use of genetic resources of cumin in breeding strategies and genetic improvement programs.

  13. Phenotypic and Genotypic Analyses of Neisseria gonorrhoeae Isolates That Express Frequently Recovered PorB PIA Variable Region Types Suggest that Certain P1a Porin Sequences Confer a Selective Advantage for Urogenital Tract Infection▿

    PubMed Central

    Garvin, Lotisha E.; Bash, Margaret C.; Keys, Christine; Warner, Douglas M.; Ram, Sanjay; Shafer, William M.; Jerse, Ann E.

    2008-01-01

    Typing of the porB variable region (VR) is an epidemiological tool that classifies gonococcal strains based on sequence differences in regions of the porB gene that encode surface-exposed loops. The frequent isolation of certain porB VR types suggests that some porin sequences confer a selective advantage during infection and/or transmission. Alternatively, certain porin types may be markers of strains that are successful due to factors unrelated to porin. In support of the first hypothesis, here we show urogenital tract isolates representing the most common PIA VR types identified in an urban clinic in Baltimore, MD, over a 10-year period belonged to several different clonal types, as determined by pulsed-field gel electrophoresis (PFGE). Serum resistance, which was confirmed by factor H and C4b-binding protein binding studies, was more often associated with gonococcal the most common VR types. In contrast, three porin-independent phenotypes, namely, lactoferrin utilization, β-lactamase production, and multiple transferable resistance (Mtr), were segregated with the PFGE cluster and not with the VR type. Data combined with another PIA strain collection showed a strong correlation between serum resistance and the most common VR types. A comparison of VR typing hybridization patterns and nucleotide sequences of 12 porB1a genes suggests that certain porin loop 1, 3, 6, and/or 7 sequences may play a role in the serum resistance phenotype. We conclude that some PorB PIA sequences confer a survival or transmission advantage in the urogenital tract, perhaps via increased resistance to complement-mediated killing. The capacity of some porin types to evade a porin-specific adaptive immune response must also be considered. PMID:18541655

  14. Fuentes de variabilidad en el diagnóstico de gastritis atrófica multifocal asociada con la infección por Helicobacter pylori1

    PubMed Central

    Bravo, Luis Eduardo; Bravo, Juan Carlos; Realpe, José Luis; Zarama, Guillermo; Piazuelo, MarÍa Blanca; Correa, Pelayo

    2014-01-01

    RESUMEN Introducción El mapeo de las diferentes regiones del estómago y el número de fragmentos de mucosa gástrica disponibles para evaluación histopatológica son fuentes importantes de variación en el momento de clasificar y hacer la gradación de la gastritis crónica. Objetivos Estimar la sensibilidad del número de fragmentos de mucosa gástrica necesarios para establecer los diagnósticos de gastritis atrófica con metaplasia intestinal (MI), displasia y estado de infección por Helicobacter pylori. Además evaluar la variabilidad intra-observador en la clasificación de estas lesiones precursoras del cáncer gástrico. Materiales y métodos En una cohorte de 6 años de seguimiento se evaluaron 1,958 procedimientos de endoscopia realizados por dos gastroenterólogos. En cada procedimiento y de cada participante se obtuvieron 5 biopsias de mucosa gástrica que representaban antro, incisura angularis y cuerpo. Un único patólogo hizo la interpretación histológica de las 5 biopsias y proporcionó un diagnóstico definitivo global que se utilizó como patrón de referencia. Cada fragmento de mucosa gástrica examinado condujo a un diagnóstico individual para cada biopsia que se comparó con el patrón de referencia. La variabilidad intra-observador se evaluó en 127 personas que corresponden a una muestra aleatoria de 20% del total de endoscopias hechas a los 72 meses de seguimiento. Resultados La sensibilidad del diagnóstico de MI y displasia gástrica aumentó de manera significativa con el número de fragmentos de mucosa gástrica evaluados El sitio anatómico de mayor sensibilidad para el diagnóstico de MI y displasia fue la incisura angularis. Para descubrir H. pylori se logró alta sensibilidad con el estudio de un solo fragmento de mucosa gástrica (95.9%) y fue independiente del sitio de obtención de la biopsia. El acuerdo intra-observador para el diagnóstico de gastritis crónica fue 86.1% con valor kappa de 0.79 IC 95% (0.76-0.85). Las

  15. A UV-vis study of the effects of alcohols on formation and stability of Mn(por)(O)(OAc) complexes.

    PubMed

    Mohajer, Daryoush; Jahanbani, Maryam

    2012-06-01

    Interactions of three different (acetato) (tetraarylporphyrinato) manganese (III) Mn(III)(por) with tetra-n-butylammonium hydrogen monopersulfate (n-Bu(4)NHSO(5)), in the presence of excess tetra-n-butylammonium acetate (n-Bu(4)NOAc) and in the absence or presence of various alcohols (alcohols=CH(3)OH, C(2)H(5)OH, i-C(3)H(7)OH, t-C(4)H(9)OH) in CH(2)Cl(2), were monitored by their UV-vis spectral changes, under identical conditions, at room temperature. (Acetato) (tetrakispentafluorophenylporphyrinato) manganese (III) Mn(III)(tpfpp)(OAc) and (acetato) (tetramesitylporphyrinato) manganese (III) Mn(III)(tmp)(OAc) produced their corresponding high valent Mn(tpfpp)(O)(OAc) and Mn(tmp)(O)(OAc) both in the absence or presence of alcohols. Whereas, (acetato) (tetraphenylporphyrinato) manganese (III) Mn(III)(tpp)(OAc) only generated Mn(tpp)(O)(OAc) in the presence of less bulky alcohols. In the absence of alcohols or in the presence of t-C(4)H(9)OH, the UV-vis spectra displayed a very weak sign of formation of Mn(tpp)(O)(OAc) complex. It was observed that alcohols generally increased the rate of formation of Mn-oxo species in accordance with their acidity or hydrogen bonding strength, and enhanced the stability of Mn-oxo complexes, as their size increases. Attempts are made to explain these effects. A mechanistic scheme is also suggested for the decomposition of HSO(5)(-) to O(2) and HSO(4)(-), through the formation and dimerization of Mn-oxo species.

  16. A UV-vis study of the effects of alcohols on formation and stability of Mn(por)(O)(OAc) complexes

    NASA Astrophysics Data System (ADS)

    Mohajer, Daryoush; Jahanbani, Maryam

    2012-06-01

    Interactions of three different (acetato) (tetraarylporphyrinato) manganese (III) MnIII(por) with tetra-n-butylammonium hydrogen monopersulfate (n-Bu4NHSO5), in the presence of excess tetra-n-butylammonium acetate (n-Bu4NOAc) and in the absence or presence of various alcohols (alcohols = CH3OH, C2H5OH, i-C3H7OH, t-C4H9OH) in CH2Cl2, were monitored by their UV-vis spectral changes, under identical conditions, at room temperature. (Acetato) (tetrakispentafluorophenylporphyrinato) manganese (III) MnIII(tpfpp)(OAc) and (acetato) (tetramesitylporphyrinato) manganese (III) MnIII(tmp)(OAc) produced their corresponding high valent Mn(tpfpp)(O)(OAc) and Mn(tmp)(O)(OAc) both in the absence or presence of alcohols. Whereas, (acetato) (tetraphenylporphyrinato) manganese (III) MnIII(tpp)(OAc) only generated Mn(tpp)(O)(OAc) in the presence of less bulky alcohols. In the absence of alcohols or in the presence of t-C4H9OH, the UV-vis spectra displayed a very weak sign of formation of Mn(tpp)(O)(OAc) complex. It was observed that alcohols generally increased the rate of formation of Mn-oxo species in accordance with their acidity or hydrogen bonding strength, and enhanced the stability of Mn-oxo complexes, as their size increases. Attempts are made to explain these effects. A mechanistic scheme is also suggested for the decomposition of HSO5- to O2 and HSO4-, through the formation and dimerization of Mn-oxo species.

  17. Impact of the CYP3A5, CYP3A4, COMT, IL-10 and POR genetic polymorphisms on tacrolimus metabolism in Chinese renal transplant recipients.

    PubMed

    Li, Chuan-Jiang; Li, Liang; Lin, Li; Jiang, Hai-Xia; Zhong, Ze-Yan; Li, Wei-Mo; Zhang, Yan-Jun; Zheng, Ping; Tan, Xu-Hui; Zhou, Lin

    2014-01-01

    Tacrolimus is a widely used immunosuppressive drug for preventing the rejection of solid organ transplants. The efficacy of tacrolimus shows considerable variability, which might be related to genetic variation among recipients. We conducted a retrospective study of 240 Chinese renal transplant recipients receiving tacrolimus as immunosuppressive drug. The retrospective data of all patients were collected for 40 days after transplantation. Seventeen SNPs of CYP3A5, CYP3A4, COMT, IL-10 and POR were identified by the SNaPshot assay. Tacrolimus blood concentrations were obtained on days 1-3, days 6-8 and days 12-14 after transplantation, as well as during the period of the predefined therapeutic concentration range. Kruskal-Wallis test was used to examine the effect of genetic variation on the tacrolimus concentration/dose ratio (C 0/D) at different time points. Chi-square test was used to compare the proportions of patients who achieved the target C 0 range in the different genotypic groups at weeks 1, 2, 3 and 4 after transplantation. After correction for multiple testing, there was a significant association of C 0/D with CYP3A5*3, CYP3A4*1G and CYP3A4 rs4646437 T>C at different time points after transplantation. The proportion of patients in the IL-10 rs1800871-TT group who achieved the target C 0 range was greater (p = 0.004) compared to the IL-10 rs1800871-CT and IL-10 rs1800871-CC groups at week 3 after transplantation. CYP3A5*3, CYP3A4 *1G, CYP3A4 rs4646437 T>C and IL-10 rs1800871 C>T might be potential polymorphisms affecting the interindividual variability in tacrolimus metabolism among Chinese renal transplant recipients.

  18. Legal Knowledge as a Tool for Social Change: La Mesa por la Vida y la Salud de las Mujeres as an Expert on Colombian Abortion Law.

    PubMed

    González Vélez, Ana Cristina; Jaramillo, Isabel Cristina

    2017-06-01

    In May 2006, Colombia's Constitutional Court liberalized abortion, introducing three circumstances under which the procedure would not be considered a crime: (1) rape or incest; (2) a risk to the woman's health or life; and (3) fetal malformations incompatible with life. Immediately following the court's ruling, known as Sentence C-355, members of La Mesa por la Vida y Salud de las Mujeres (hereinafter La Mesa) began to mobilize to ensure the decision's implementation, bearing in mind the limited impact that the legal framework endorsed by the court has had in other countries in the region. We argue that La Mesa's strategy is an innovative one in the field of legal mobilization insofar as it presumes that law can be shaped not just by public officials and universities but also by social actors engaged in the creation and diffusion of legal knowledge. In this regard, La Mesa has become a legal expert on abortion by accumulating knowledge about the multiple legal rules affecting the practice of abortion and about the situations in which these rules are to be applied. In addition, by becoming a legal expert, La Mesa has been able to persuade health providers that they will not risk criminal prosecution or being fired if they perform abortions. We call this effect of legal mobilization a "pedagogical effect" insofar as it involves the production of expertise and appropriation of knowledge by health professionals. We conclude by discussing La Mesa's choice to become a legal expert on abortion as opposed to recruiting academics to do this work or encouraging women to produce and disseminate this knowledge.

  19. Estudio teórico de la desorción de Na y K de SiO2 estimulada por la acción de fotones o electrones

    NASA Astrophysics Data System (ADS)

    Domínguez Ariza, D.; López, N.; Illas, F.; Pacchioni, G.; Madey, T. E.

    Se ha estudiado el mecanismo de generación de sodio y potasio atómico a partir de muestras de SiO2 utilizando cálculos basados tanto en la teoría del funcional de la densidad como en métodos post-Hartree Fock, así como en el método de cluster para modelar el sólido. Como consecuencia del estudio se han propuesto distintos caminos posibles para la desorción, estimulada por la acción de fotones o electrones, de sodio y potasio desde el óxido de silicio, proporcionando por lo tanto una explicación a la atmósfera tenue de sodio y potasio de La Luna.

  20. The P450 oxidoreductase (POR) rs2868177 and cytochrome P450 (CYP) 2B6*6 polymorphisms contribute to the interindividual variability in human CYP2B6 activity.

    PubMed

    Gao, Li-Chen; Liu, Fang-Qun; Yang, Li; Cheng, Lin; Dai, Hai-Ying; Tao, Ran; Cao, Shi-Peng; Wang, Di; Tang, Jie

    2016-10-01

    To investigate whether single-nucleotide polymorphisms (SNPs) in the P450 oxidoreductase (POR) gene were correlated with interindividual variations in cytochrome P450 (CYP) 2B6 activity. Thirty-six healthy volunteers who tested CYP2B6 and POR polymorphisms were enrolled in the study. CYP2B6 activity was measured by bupropion hydroxylation with LC/MS/MS. The ratio of hydroxybupropion versus bupropion (AUC_hyd/AUC_bup) in terms of area under the time-concentration curve (AUC) was used to represent the CYP2B6 activity. The volunteers carrying CYP2B6*1/*1 showed a significantly higher mean AUC_hyd/ AUC_bup than those CYP2B6*1/*6 and CYP2B6*6/*6 variants (15.66 ± 1.65 vs. 9.25 ± 1.92, P = 0.008 and 15.66 ± 1.65 vs. 8.21 ± 1.74, P = 0.006, respectively). POR rs2868177 (6593 A > G) AA homozygotes showed a significantly lower mean AUC_hyd/ AUC_bup than that of POR rs2868177 AG heterozygotes or GG homozygotes (8.13 ± 1.37 vs. 12.15 ± 2.97, P = 0.005 and 8.13 ± 1.37 vs. 17.59 ± 3.25, P = 0.001, respectively). Moreover, POR rs2868177 AG heterozygotes and GG homozygotes showed a significantly increased mean AUC_hyd/AUC_bup than AA homozygotes in the CYP2B6*1/*1 and CYP2B6*6 carriers (16.40 ± 2.01 vs. 12.40 ± 1.45, P = 0.006 and 10.65 ± 1.47 vs. 6.54 ± 1.25, P = 0.004, respectively). Meanwhile, a strong correlation between the genetic variations (POR rs2868177 and CYP2B6*6) and AUC_hyd/ AUC_bup was found (P = 0.009 and P = 0.001, respectively). There was no significant difference in the mean AUC_hyd/AUC_bup among different POR *28 genotypes (P > 0.05). POR rs2868177 and CYP2B6*6 variants contribute to the interindividual variability in human CYP2B6 activity, which may affect the disposition and interaction of other CYP2B6 substrate drugs.

  1. Long-term RNA persistence of porcine rubulavirus (PorPV-LPMV) after an outbreak of a natural infection: the detection of viral mRNA in sentinel pigs suggests viral transmission.

    PubMed

    Cuevas-Romero, S; Hernández-Baumgarten, E; Kennedy, S; Hernández-Jáuregui, P; Berg, M; Moreno-López, J

    2014-08-08

    The persistence of porcine rubulavirus (PorPV-LPMV) in five pigs that had survived an outbreak of a natural infection was determined. After the resolution of the outbreak, each animal was housed in an isolation pen together with one sentinel pig. Approximately every 2 months thereafter one group of animals was euthanized and tissue samples taken for virological and serological analysis. Infectious virus was not isolated from any samples; antibodies to PorPV-LPMV were detected in convalescent pigs by virus neutralisation test and blocking ELISA but not in sentinel pigs. PorPV-LPMV mRNA of the nucleoprotein (NP) and phosphoprotein (P) genes was detected by a nested polymerase chain reaction (nPCR) in samples of trigeminal and optic nerves, cervical spinal cord, tonsils, salivary gland, lung and pancreas from convalescent pigs. mRNA was also detected in the midbrain, corpus callosum, or olfactory bulb in four out of five pigs by nRT-PCR, this result was confirmed by the sequencing of a 260bp PCR product of P gene region. The highest average viral copies/μg of total RNA occurred in the olfactory bulb and pancreas tissues of convalescent pigs and midbrain, tonsil and pancreas of sentinel pigs housed with the convalescent pigs. Satellitosis and gliosis of the midbrain, olfactory bulb, corpus callosum, medulla oblongata or choroid plexus were microscopically observed in four convalescent pigs. The control pig remained negative in all tests. The results indicate that PorPV-LPMV mRNA persists and induces a durable humoral immune response in pigs that have recovered from a natural infection. After a possible reactivation of the virus, it was transmitted to sentinel pigs in contact with the convalescent pigs.

  2. Combined effects of CYP3A5*1, POR*28, and CYP3A4*22 single nucleotide polymorphisms on early concentration-controlled tacrolimus exposure in de-novo renal recipients.

    PubMed

    Kuypers, Dirk R J; de Loor, Henriette; Naesens, Maarten; Coopmans, Tamara; de Jonge, Hylke

    2014-12-01

    In a cohort of 298 de-novo renal recipients treated with a standard tacrolimus loading dose of 0.2 mg/kg, the combined effects of the CYP3A5*1, POR*28, and CYP3A4*22 genotypes on early tacrolimus exposure (C0), dose requirements, and achievement of the therapeutic target, C0, were examined. The incidence of clinical events (e.g. acute rejection, diabetes mellitus) was compared between genotypes. Fast metabolizers (CYP3A5*1/POR*28T carriers) had two-fold to three-fold higher tacrolimus dose requirements compared with slow metabolizers (CYP3A5*3/*3/CYP3A4*22 carriers) and needed significantly more time to achieve the target tacrolimus C0 of a minimum 10 ng/ml (3.3±1.7 vs. 1.34±0.75 days; P<0.0001). No differences in acute rejection incidence and time to first rejection were observed. Slow metabolizers more frequently had tacrolimus C0 above the target range early after transplantation (70 vs. 13% on day 3); however, this did not translate into a higher incidence of post-transplantation diabetes mellitus or graft dysfunction. Multivariate analyses identified the CYP3A5*1/POR*28/CYP3A4*22 genotype combination as the single strongest determinant of tacrolimus dose requirements throughout the first year, explaining between 24-40% of its variability, whereas recipient age, hematocrit, and delayed graft function were additional nongenetic determinants of tacrolimus dose. Combining the CYP3A5*1, POR*28 and CYP3A4*22 genotypes allows partial differentiation of early tacrolimus dose requirements and the time to reach therapeutic target concentrations after transplantation, but without obvious clinical implications. Larger prospective studies need to address the clinical relevance of early combined genotype-based tacrolimus dosing in de-novo renal recipients.

  3. Characterisation by multilocus sequence and porA and flaA typing of Campylobacter jejuni isolated from samples of dog faeces collected in one city in New Zealand.

    PubMed

    Mohan, V; Stevenson, M A; Marshall, J C; French, N P

    2017-07-01

    To investigate the prevalence of Campylobacter spp. and C. jejuni in dog faecal material collected from dog walkways in the city of Palmerston North, New Zealand, and to characterise the C. jejuni isolates by multilocus sequence typing (MLST) and porA and flaA antigen gene typing. A total of 355 fresh samples of dogs faeces were collected from bins provided for the disposal of dog faeces in 10 walkways in Palmerston North, New Zealand, between August 2008-July 2009. Presumptive Campylobacter colonies, cultured on modified charcoal cefoperazone deoxycholate plates, were screened for genus Campylobacter and C. jejuni by PCR. The C. jejuni isolates were subsequently characterised by MLST and porA and flaA typing, and C. jejuni sequence types (ST) were assigned. Of the 355 samples collected, 72 (20 (95% CI=16-25)%) were positive for Campylobacter spp. and 22 (6 (95% CI=4-9)%) were positive for C. jejuni. Of the 22 C. jejuni isolates, 19 were fully typed by MLST. Ten isolates were assigned to the clonal complex ST-45 and three to ST-52. The allelic combinations of ST-45/flaA 21/porA 44 (n=3), ST-45/flaA 22/porA 53 (n=3) and ST-52/ flaA 57/porA 905 (n=3) were most frequent. The successful isolation of C. jejuni from canine faecal samples collected from faecal bins provides evidence that Campylobacter spp. may survive outside the host for at least several hours despite requiring fastidious growth conditions in culture. The results show that dogs carry C. jejuni genotypes (ST-45, ST-50, ST-52 and ST-696) that have been reported in human clinical cases. Although these results do not provide any evidence either for the direction of infection or for dogs being a potential risk factor for human campylobacteriosis, dog owners are advised to practice good hygiene with respect to their pets to reduce potential exposure to infection.

  4. La Noche de las Brujas Module. Nivel Primario. [The Night of the Witches Module. Primary Level.

    ERIC Educational Resources Information Center

    Espinoza, Delia

    La Noche de las Brujas (Halloween) is the topic of this primary level unit. The objectives are to enable the child to: (1) draw scenery, using his imagination, about witches, castles, and devils; (2) write compositions on witches, devils, and Halloween; (3) explain the story "La Noche de las Brujas"; (4) tell about any adventures or…

  5. The Danger of Poison. Level 1 = El Veneno es Peligroso. [Nivel 1.

    ERIC Educational Resources Information Center

    Pennsylvania State Dept. of Education, Harrisburg.

    Developed especially for migrant children, this field-tested curriculum teaches the benefits and hazards of pesticides to early primary children. Materials are prepared in Spanish and English versions and can be used as a separate science/health unit on pesticides or integrated into the regular math and reading curriculum. Topics include benefits…

  6. The Danger of Poison. Level 4 = El Veneno es Peligroso. [Nivel 4.

    ERIC Educational Resources Information Center

    Pennsylvania State Dept. of Education, Harrisburg.

    Developed especially for migrant children, this field-tested curriculum teaches the benefits and hazards of pesticides to intermediate grade students. Materials, prepared in Spanish and English, can be used as a separate science/health unit on pesticides or integrated into the regular math and reading curriculum. Topics include benefits of…

  7. The Danger of Poison. Level 2 = El Veneno es Peligroso. [Nivel 2.

    ERIC Educational Resources Information Center

    Pennsylvania State Dept. of Education, Harrisburg.

    Developed especially for migrant children, this field-tested curriculum teaches the benefits and hazards of pesticides to primary students. Materials are prepared in Spanish and English versions and can be used as a separate science/health unit on pesticides or integrated into the regular math and reading curriculum. Topics include benefits of…

  8. Las Matematicas: Lenguaje Universal. Nivel 3: La Medida (Mathematics: A Universal Language. Level 3: Measurement).

    ERIC Educational Resources Information Center

    Dissemination and Assessment Center for Bilingual Education, Austin, TX.

    This is one of a series of student booklets designed for use in a bilingual mathematics program in grades 6-8. The general format is to present each page in both Spanish and English. The mathematical topics in this booklet include liquid, dry, linear, weight, and time measures. (MK)

  9. Linea abierta -- Comunicacion en espanol. Nivel Uno (Open Line -- Communication in Spanish. Level One).

    ERIC Educational Resources Information Center

    Dissemination and Assessment Center for Bilingual Education, Austin, TX.

    This text is the first of a series of four units concerning language skills for Spanish speakers of limited English-speaking ability in grades 6-9. The text includes a variety of learning experiences designed to develop competence in the student's cognitive and affective skills derived from the following basic areas: listening, speaking, reading,…

  10. Dia de Dar Gracias. Modulo Nivel Primario. (Day to Give Thanks. Module Primary Level.)

    ERIC Educational Resources Information Center

    Espinoza, Delia; Lopez, Santiago, III

    Dia de Dar Gracias (Thanksgiving) is the subject of this primary level unit. The unit objectives are to: (1) know about El Dia de Dar Gracias as it is celebrated in the United States; (2) know how the Mayas celebrated it; (3) understand the context of the stories in the unit; (4) know about the main food used, the turkey; (5) distinguish other…

  11. Las Matematicas: Lenguaje Universal. Nivel 3: La Medida (Mathematics: A Universal Language. Level 3: Measurement).

    ERIC Educational Resources Information Center

    Dissemination and Assessment Center for Bilingual Education, Austin, TX.

    This is one of a series of student booklets designed for use in a bilingual mathematics program in grades 6-8. The general format is to present each page in both Spanish and English. The mathematical topics in this booklet include liquid, dry, linear, weight, and time measures. (MK)

  12. Mira y lee: Libro de lectura 1, nivel 1 (Look and Read: Reader 1, Level 1).

    ERIC Educational Resources Information Center

    Martinez, Emiliano; And Others

    This reading textbook contains a series of stories designed to relate to the natural interest of the school child. Students learn about a Puerto Rican family living in New York. New vocabulary is introduced gradually for the student to memorize. Included are exercises in oral expression and comprehension, auditory discrimination, intonation,…

  13. La ciudad: Libro de lectura 1, nivel 2 (The City: Reader 1, Level 2).

    ERIC Educational Resources Information Center

    Martinez, Emiliano; And Others

    This reading textbook was designed for the elementary school Spanish-speaking student. It presents short selections pertaining to city life, with special emphasis on streets, transportation, public services, personal relations, and the urban environment. The presentation includes color illustrations. See FL 004 070 for the accompanying workbook.…

  14. Lee y trabaja: Libro de lectura 2, nivel 1 (Read and Work: Reader 2, Level 1).

    ERIC Educational Resources Information Center

    Martinez, Emiliano; and Others

    This reading textbook, the second of a series, is an anthology of stories designed to relate to the natural interest of the elementary school child. On this level the number of words to memorize is increased (on the average, four per unit) while at the same time, the study of word variants is introduced to begin analysis exercises based on the…

  15. Trabaja y aprende: Libro de lectura 3, nivel 1 (Work and Learn: Reader 3, Level 1).

    ERIC Educational Resources Information Center

    Martinez, Emiliano; And Others

    This reading textbook, the third of a series, is an anthology of stories designed to relate to the natural interest of the elementary school child. In the book, students learn about a Puerto Rican family living in New York. Attention is paid to visual and auditory discrimination of consonant groups and normal endings. Included are exercises which…

  16. The Danger of Poison. Level 3 = El Veneno es Peligroso. [Nivel 3.

    ERIC Educational Resources Information Center

    Pennsylvania State Dept. of Education, Harrisburg.

    Developed especially for migrant children, this field-tested curriculum teaches the benefits and hazards of pesticides to early intermediate grade children. Materials are prepared in Spanish and English versions and can be used as a separate science/health unit on pesticides or integrated into the regular math and reading curriculum. Topics…

  17. Lee y trabaja: Libro de lectura 2, nivel 1 (Read and Work: Reader 2, Level 1).

    ERIC Educational Resources Information Center

    Martinez, Emiliano; and Others

    This reading textbook, the second of a series, is an anthology of stories designed to relate to the natural interest of the elementary school child. On this level the number of words to memorize is increased (on the average, four per unit) while at the same time, the study of word variants is introduced to begin analysis exercises based on the…

  18. Trabaja y aprende: Libro de lectura 3, nivel 1 (Work and Learn: Reader 3, Level 1).

    ERIC Educational Resources Information Center

    Martinez, Emiliano; And Others

    This reading textbook, the third of a series, is an anthology of stories designed to relate to the natural interest of the elementary school child. In the book, students learn about a Puerto Rican family living in New York. Attention is paid to visual and auditory discrimination of consonant groups and normal endings. Included are exercises which…

  19. Mira y lee: Libro de lectura 1, nivel 1 (Look and Read: Reader 1, Level 1).

    ERIC Educational Resources Information Center

    Martinez, Emiliano; And Others

    This reading textbook contains a series of stories designed to relate to the natural interest of the school child. Students learn about a Puerto Rican family living in New York. New vocabulary is introduced gradually for the student to memorize. Included are exercises in oral expression and comprehension, auditory discrimination, intonation,…

  20. La ciudad: Libro de lectura 1, nivel 2 (The City: Reader 1, Level 2).

    ERIC Educational Resources Information Center

    Martinez, Emiliano; And Others

    This reading textbook was designed for the elementary school Spanish-speaking student. It presents short selections pertaining to city life, with special emphasis on streets, transportation, public services, personal relations, and the urban environment. The presentation includes color illustrations. See FL 004 070 for the accompanying workbook.…

  1. Plan de estudios de nivel secundario para adultos (Study Plan for Adult Secondary Education).

    ERIC Educational Resources Information Center

    Boletin del Centro Nacional de Documentacion e Informacion Educativa, Parte II: Informaciones, 1970

    1970-01-01

    This document describes an experimental, multinational plan for adult secondary education sponsored through the Organization of American States and the Argentine Ministry of Culture and Education. General and specific goals of the program are listed here along with details of the proposed curriculum and areas of study, entrance requirements,…

  2. Bases para el Curriculum de las Escuelas de Nivel Elemental (Bases for the Elementary School Curriculum).

    ERIC Educational Resources Information Center

    Ministerio de Cultura y Educacion, Buenos Aires (Argentina). Centro National de Documentacion e Informacion Educativa.

    This document proposes a detailed foundation for curriculum planning in grades 1, 2, and 3 in the Argentine elementary schools. The book covers such topics as curriculum objectives, contents and activities, personalization and individualization, socialization and regionalization, quality, organization, and suggestions for subject matter and…

  3. The Danger of Poison. Level R = El Veneno es Peligroso. [Nivel R.

    ERIC Educational Resources Information Center

    Pennsylvania State Dept. of Education, Harrisburg.

    Designed by Project TEACH (Teaching Environmental Awareness to the Children of Harvest), the skill-sequenced curriculum unit was developed to teach 3- to 5-year-old migrant children about the benefits and possible hazards of pesticides. Prepared in both Spanish and English, the field-tested unit can be used as a separate teaching unit or…

  4. Comparación de resultados del método de clasificación de órbitas por análisis de frecuencias con el método de exponentes de Lyapunov

    NASA Astrophysics Data System (ADS)

    Carpintero, D. D.; Muzzio, J. C.; Wachlin, F. C.

    Hemos realizado extensas comparaciones del método de análisis de frecuencias con el de exponentes de Lyapunov. El primero resulta claramente superior por las siguientes razones: 1) permite distinguir distintos tipos de órbitas y no sólo si son regulares o caóticas 2) es mucho más veloz requiriendo mucho menos tiempo de cómputo. La concordancia de resultados es, en general, buena y se discuten algunas discrepancias.

  5. Providing Meaningful Learning for Students of the Sixth Grade of Middle School: a Study on the Moon Phases. (Breton Title: Propiciando Aprendizagem Significativa Para Alunos do Sexto Ano do Ensino Fundamental: um Estudo sobre as Fases da Lua.) Propiciando el Aprendizaje Significativo Para Alumnos del Sexto Nivel de la Educación General Básica: un Estudio sobre Las Fases de la Luna

    NASA Astrophysics Data System (ADS)

    Darroz, Luiz Marcelo; Samudio Pérez, Carlos Ariel; da Rosa, Cleci Werner; Heineck, Renato

    2012-07-01

    ía del Aprendizaje Significativo de David Ausubel, se buscó desenvolver la propuesta a partir de un modelo didáctico que simula las Fases de la Luna, usando como base las concepciones previas de los estudiantes. Los indicios del aprendizaje fueron verificados a través de registros de memorias de la actividad. Por los resultados obtenidos creemos que la propuesta alcanzó sus objetivos, una vez que los estudiantes consiguieron identificar y transferir el fenómeno de las fases de la Luna para nuevos contextos. Así, se concluye que una metodología con enfoque en un contenido significativo para el estudiante es fundamental para la construcción y comprensión genuina de lo que está siendo aprendido.

  6. Aquisição fonológica do português brasileiro por crianças ouvintes bilíngues bimodais e surdas usuárias de implante coclear

    PubMed Central

    Cruz, Carina Rebello; Finger, Ingrid

    2014-01-01

    Resumo O presente estudo investiga a aquisição fonológica do Português Brasileiro (PB) por 24 crianças ouvintes bilíngues bimodais, com acesso irrestrito à Língua Brasileira de Sinais (Libras), e por 6 crianças surdas que utilizam implante coclear (IC), com acesso restrito ou irrestrito à Libras. Para a avaliação do sistema fonológico das crianças em PB, foi utilizada a Parte A, Prova de Nomeação, do ABFW – Teste de Linguagem Infantil (ANDRADE et al. 2004). Os resultados revelaram que as crianças ouvintes bilíngues bimodais e a criança surda usuária de IC com acesso irrestrito à Libras apresentaram processo de aquisição fonológica esperada (normal) para a sua faixa etária. Considera-se que a aquisição precoce e o acesso irrestrito à Libras podem ter sido determinantes para o desempenho dessas crianças no teste oral utilizado. PMID:25506105

  7. La inserción en el mercado laboral de los inmigrantes latinos en España y en los Estados Unidos: Diferencias por país de origen y estatus legal

    PubMed Central

    Connor, Phillip; Massey, Douglas

    2013-01-01

    Resumen Este artículo compara los resultados económicos entre los inmigrantes latinoamericanos en España y Estados Unidos. Detectamos un efecto de selección por el que la mayoría de los inmigrantes latinoamericanos en España proceden de Sudamérica de un entorno de clases medias, mientras la mayoría de los inmigrantes que van a los Estados Unidos son centroamericanos de clase baja. Este efecto de selección explica las diferencias transnacionales en la probabilidad de empleo, logro ocupacional y salarios obtenidos. A pesar de las diferencias en los orígenes y las características de los latinoamericanos en ambos países, los factores demográficos, humanos y de capital social parecen operar de forma similar en ambos países; y cuando los modelos se estiman separadamente por estatus legal, descubrimos que los efectos se acentúan más entre los inmigrantes irregulares cuando se los compara con los regulares, especialmente en Estados Unidos. PMID:24532857

  8. The Understanding of Astronomy Concepts by Students from Basic Education of a Public School. (Spanish Title: El Entendimiento de Conceptos de Aastronmía Por Los Alumnos de Educación Básica en Una Escuela Pública.) O Entendimento de Conceitos de Astronomia Por Alunos da Educação Básica: O Caso de Uma Escola Pública Brasileira

    NASA Astrophysics Data System (ADS)

    Iria Machado, Daniel; dos Santos, Carlos

    2011-07-01

    We present the results obtained in a research on the comprehension of basic astronomical concepts, in which 561 students from fifth grade middle school to third grade high school of a public school of the city of Foz do Iguaçu (Brazil) took part. A test with 20 multiple-choice questions was applied to indentify the most common conceptions expressed by the students. This test was elaborated based on the literature about misconceptions and covered the following topics: the day-night cycle; the time zones; the seasons of the year; the phases of the Moon; the movement of the Moon; the apparent movement of the Sun in the celestial sphere; the eclipses; the dimensions and distances in the Universe; the brightness of the stars and its observation from Earth. Though a small progress was verified in the proportion of scientifically acceptable answers when comparing the eighth grade of middle school to the fifth, and the third grade of high school to the first, there was an overall predominance of alternative conceptions regarding most of the explored subjects, which persisted up to the last year of secondary school. The comparison to data found in this research made in other socio-cultural contexts revealed, in many aspects, similar notions and difficulties revealed by the students. Se presentan los resultados de una investigación sobre la comprensión de conceptos astronómicos básicos, en la cual participaron 561 estudiantes que cursaban entre el quinto grado de la enseñanza primaria y el tercer año de la enseñanza secundaria de una escuela pública de la ciudad de Foz do Iguaçu (Brasil). Se utilizó un test de 20 preguntas de opción múltiple para identificar las concepciones más comunes expresadas por los estudiantes. Este instrumento de recolección de datos se desarrolló en base a la literatura sobre las concepciones alternativas y trató los siguientes temas: el ciclo día-noche, los husos horarios, las estaciones del año, las fases de la Luna, el

  9. Antihemorrhagin in the blood serum of king cobra (Ophiophagus hannah): purification and characterization.

    PubMed

    Chanhome, Lawan; Khow, Orawan; Omori-Satoh, Tamotsu; Sitprija, Visith

    2003-06-01

    King cobra (Ophiophagus hannah) serum was found to possess antihemorrhagic activity against king cobra hemorrhagin. The activity was stronger than that in commercial king cobra antivenom. An antihemorrhagin has been purified by ion exchange chromatography, affinity chromatography and gel filtration with a 22-fold purification and an overall yield of 12% of the total antihemorrhagic activity contained in crude serum. The purified antihemorrhagin was homogeneous in disc-PAGE and SDS-PAGE. Its apparent molecular weight determined by SDS-PAGE was 120 kDa. The antihemorrhagin was also active against other hemorrhagic snake venoms obtained in Thailand and Japan such as Calloselasma rhodostoma, Trimeresurus albolabris, Trimeresurus macrops and Trimeresurus flavoviridis (Japanese Habu). It inhibited the proteolytic activity of king cobra venom. It is an acid- and thermolabile protein and does not form precipitin lines against king cobra venom.

  10. Analysis of amyloid fibrils in the cheetah (Acinonyx jubatus).

    PubMed

    Bergström, Joakim; Ueda, Mitsuharu; Une, Yumi; Sun, Xuguo; Misumi, Shogo; Shoji, Shozo; Ando, Yukio

    2006-06-01

    Recently, a high prevalence of amyloid A (AA) amyloidosis has been documented among captive cheetahs worldwide. Biochemical analysis of amyloid fibrils extracted from the liver of a Japanese captive cheetah unequivocally showed that protein AA was the main fibril constituent. Further characterization of the AA fibril components by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis revealed three main protein AA bands with approximate molecular weights of 8, 10 and 12 kDa. Mass spectrometry analysis of the 12-kDa component observed in SDS-PAGE and Western blotting confirmed the molecular weight of a 12,381-Da peak. Our finding of a 12-kDa protein AA component provides evidence that the cheetah SAA sequence is longer than the previously reported 90 amino acid residues (approximately 10 kDa), and hence SAA is part of the amyloid fibril.

  11. Characterization of human red cell Rh (rhesus-)specific polypeptides by limited proteolysis.

    PubMed

    Krahmer, M; Prohaska, R

    1987-12-21

    Human red cells of various Rh phenotypes were surface-labelled with 125I and the Rh-specific labelled polypeptides were isolated by preparative SDS-PAGE. The polypeptides were subjected to limited proteolysis and the resulting fragments were analysed by SDS-PAGE and autoradiography. Chymotryptic peptide maps of proteins obtained from Rh(D)-positive and -negative types appeared completely identical, whereas tryptic peptide maps revealed a difference: a fragment of Mr 17,500 was associated with the Rh(D) antigen, and one of Mr 19,000 with the Rh(C/c,E/e) antigens. Treatment of Rh polypeptides with carboxypeptidase Y prior to tryptic digestion resulted in a shift of nearly all tryptic fragments, including a fragment of Mr 8,000, indicating that the surface label was incorporated into the C-terminal part of the molecule.

  12. Gel electrophoretic methods for the analysis of biosimilar pharmaceuticals using the example of recombinant erythropoietin.

    PubMed

    Reichel, Christian; Thevis, Mario

    2013-03-01

    Due to their versatility and cost-effectiveness, gel electrophoretic methods provide an important set of tools for the analysis of therapeutic proteins. As an increasing number of biosimilar pharmaceuticals are entering the market, techniques are required that allow reliable demonstration of comparability of these products with the reference products. Isoelectric focusing, SDS-PAGE, native PAGE and 2D electrophoresis (2D-PAGE) have been frequently applied for this purpose. Supplementary techniques are fluorophore-assisted carbohydrate electrophoresis and sarcosyl-PAGE. Of additional importance is the comparison of recombinant with endogenously synthesized glycoproteins. Reagent array analysis combined with SDS-PAGE and western blotting proved especially useful for this purpose. As an example for the application of these methods, the analysis of recombinant originator erythropoietins and some of their biosimilar counterparts is described.

  13. Lipids and proteins in the Rathke's gland secretions of the North American mud turtle (Kinosternon subrubrum)

    USGS Publications Warehouse

    Seifert, W.E.; Gotte, S.W.; Leto, T.L.; Weldon, P.J.

    1994-01-01

    Lipids and proteins in the Rathke's gland secretions of the North American mud turtle (Kinosternon subrubrum, Kinosternidae) were analyzed by gas chromatography-mass spectrometry (GC-MS) and SDS-polyacrylamide gel electrophoresis (SDS-PAGE), respectively. Analysis by GC-MS indicates 2,3-dihydroxypropanal and C3–C24 free or esterified fatty acids. Analysis by SDS-PAGE indicates a major protein component with an approximate molecular mass of 60 kDa and minor components ranging from ca. 23 to 34 kDa. The major component of K. subrubrum glandular secretions exhibits a mobility that matches that of the Kemp's ridley sea turtle (Lepidochelys kempi, Cheloniidae), suggesting that these proteins are evolutionarily conserved.

  14. Purification and some properties of acidocin 8912, a novel bacteriocin produced by Lactobacillus acidophilus TK8912.

    PubMed

    Tahara, T; Kanatani, K; Yoshida, K; Miura, H; Sakamoto, M; Oshimura, M

    1992-08-01

    Acidocin 8912, a bacteriocin produced by Lactobacillus acidophilus TK8912, was purified by ammonium sulfate fractionation and successive chromatographies on CM-cellulose, Sephadex G-50, Sephadex G-25, and reversed-phase HPLC on Aquapore RP-300. The purified acidocin 8912 migrated as a single band on SDS-PAGE. The molecular weight was estimated to be 5200 by SDS-PAGE, and 5400 by HPLC gel filtration on TSKgel G3000PWXL. Both the amino acid composition and the N-terminal amino acid sequence analysis indicated that acidocin 8912 was a peptide composed of presumably 50 amino acids containing a Lys residue at the N-terminus. The purified acidocin 8912 showed a bactericidal effect on sensitive cells but not a bacteriolytic effect.

  15. Quantitation of yeast total proteins in sodium dodecyl sulfate-polyacrylamide gel electrophoresis sample buffer for uniform loading.

    PubMed

    Sheen, Hyukho

    2016-04-01

    Proteins in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer are difficult to quantitate due to SDS and reducing agents being in the buffer. Although acetone precipitation has long been used to clean up proteins from detergents and salts, previous studies showed that protein recovery from acetone precipitation varies from 50 to 100% depending on the samples tested. Here, this article shows that acetone precipitates proteins highly efficiently from SDS-PAGE sample buffer and that quantitative recovery is achieved in 5 min at room temperature. Moreover, precipitated proteins are resolubilized with urea/guanidine, rather than with SDS. Thus, the resolubilized samples are readily quantifiable with Bradford reagent without using SDS-compatible assays. Copyright © 2016 Elsevier Inc. All rights reserved.

  16. Three-dimensional electrophoresis for quantitative profiling of complex proteomes.

    PubMed

    Mauro, Sergio; Colignon, Bertrand; Dieu, Marc; Delaive, Edouard; Raes, Martine

    2015-01-01

    Quantitative 2D-gel-dependent proteomics became feasible with 2D fluorescence difference gel electrophoresis (2D-DIGE), and this technique has gained wide acceptance because it has eliminated the gel to gel variations and greatly facilitated the quantitative comparisons across gels for many different experimental conditions. However, the co-migration of several proteins in the same spot is still a major limitation which detracts from the accuracy of comparative quantification and prevents unambiguous post-translational modifications (PTMs) detection.A protocol based on traditional polyacrylamide gel IEF sample fractionation, and followed by two consecutive SDS-PAGE electrophoreses alleviates co-migration limitations. The use of two different buffer systems for SDS-PAGE is central to the proposed approach.

  17. Reducing sample complexity in proteomics by chromatofocusing with simple buffer mixtures.

    PubMed

    Shen, Hong; Li, Xiang; Bieberich, Charles J; Frey, Douglas D

    2008-01-01

    Chromatofocusing has many potential applications in the field of proteomics, such as for the isolation and removal of major sample components to facilitate the analysis of low-abundance components, and for sample prefractionation prior to a subsequent separation using SDS-PAGE, narrow-pI-range 2D-PAGE, or additional chromatography steps. However, the chromatofocusing techniques that are most commonly used employ propriety polyampholyte elution buffers and highly specialized column packings, both of which limit the use of chromatofocusing in practice. To expand the range of application for this technique, this chapter considers chromatofocusing methods which employ common ion-exchange column packings and elution buffers which are simple mixtures of readily available buffering species. Of particular interest is the use of chromatofocusing with a multistep pH gradient for the fractionation of protein mixtures into narrow-pI-range fractions. The cross-contamination characteristics of these fractions using SDS-PAGE are also assessed.

  18. Purification of saliva agglutinin of Streptococcus intermedius and its association with bacterial aggregation and adherence.

    PubMed

    Yamaguchi, Taihei

    2004-02-01

    Streptococcus intermedius strain 1208-1 cells were aggregated in the presence of saliva. The saliva agglutinin was purified by centrifugation, filtration, and gel filtration. SDS-PAGE analyses indicated that the purified agglutinin consisted of two high-molecular-mass proteins. Aggregation was dependent on calcium over pH 5.5, with 1 mM being the most effective concentration. Boiling inactivated purified agglutinin. S. intermedius strain 3 and Streptococcus mutans strain 1 were aggregated in the purified agglutinin. After adsorption with strain 1208-1 cells, the saliva sample did not exhibit any aggregation activity, and the agglutinin bands were no longer visible by SDS-PAGE. Adherence analyses demonstrated that the purified agglutinin immobilized on the surfaces of polystyrene wells, actinomyces cells, and apatite beads accounted for the binding of streptococcus cells. Agglutinin also effectively inhibited adherence to apatite beads coated with native saliva.

  19. Non-denaturing gel electrophoresis system for the purification of membrane bound proteins

    SciTech Connect

    Cavinato, A.G.; Macleod, R.M.; Ahmed, M.S.

    1988-01-01

    A new method is described for the purification of a membrane bound glycoprotein, the kappa opioid receptor from human placental tissue. The method uses preparative slab-gel electrophoresis in the presence of the non-denaturing detergent CHAPS. A linear relationship between log molecular weight and SDS PAGE electrophoretic mobility of known molecular weight markers, in the presence of CHAPS, is observed. Using this method, we were able partially to purify an /sup 3/H-etorphine binding glycoprotein, from placental villus tissue, with an apparent molecular weight range of 60-70,000. The iodinated glycoprotein migrates in SDS PAGE with an apparent molecular weight of 63,000. This method may be useful for the isolation of membrane bound proteins, especially when an affinity ligand is not available.

  20. Salivary protein changes in response to acute stress in medical residents performing advanced clinical simulations: a pilot proteomics study.

    PubMed

    Marvin, Rachel K; Saepoo, Muncharie B; Ye, Simiao; White, Donald B; Liu, Rong; Hensley, Kenneth; Rega, Paul; Kazan, Viviane; Giovannucci, David R; Isailovic, Dragan

    Quantitative changes of salivary proteins due to acute stress were detected. To explore protein markers of stress in saliva of eight medical residents who performed emergency medicine simulations. Saliva was collected before the simulations, after the simulations, and following morning upon waking. Proteins were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), identified by mass spectrometry (MS), and relatively quantified by densitometry. Salivary alpha-amylase and S-type cystatins significantly increased, while the ∼26 kDa and low-molecular weight (MW) (<10 kDa) SDS-PAGE bands exhibited changes after stress. Alpha-amylase and cystatins are potential salivary markers of acute stress, but further validation should be performed using larger sample populations.

  1. Molecular and biochemical evaluation of genetic effect of Calotropis procera (Ait.) latex on Aspergillus terreus (Thom).

    PubMed

    Qari, Sameer H

    2008-10-01

    The present study was conducted to observe the genotoxic effect of latex of Calotropis procera. Dense conidial suspensions of Aspergillus terreus Thorn were treated with different concentrations of Calotropis latex and assayed for percentage survival, auxotrophic mutants, DNA and total protein contents and further subjected to RAPD and SDS-PAGE tests. It was found that latex of Calotropis procera had potent lethal and mutagenic activities. Percentage survival decreased as concentration or time of exposure increased. Frequency of auxotrophic mutants increased with increase in concentration or exposure time. Most auxotrophic mutants were amino acid requiring mutants. DNA and total protein contents of each mutant was significantly lower than wild type of Aspergillus terreus. RAPD demonstrated polymorphic genetic bands which were the electrophoretic products of PCR for all mutants compared with the wild type strain. SDS-PAGE results expressed a polymorphism of protein bands as well. All these results indicated the mutagenicity of latex of Calotropis procera.

  2. Genetic variability of the low-molecular-weight glutenin subunits in spelt wheat (Triticum aestivum ssp. spelta L. em Thell.).

    PubMed

    Caballero, L; Martín, L M; Alvarez, J B

    2004-03-01

    The low-molecular-weight glutenin subunit composition of a collection of 403 accessions of spelt wheat ( Triticum aestivum ssp. spelta L. em. Thell) was analyzed by SDS-PAGE. Extensive variation was found, including 46 different patterns for zone B and 16 for zone C. Patterns within zone B exhibited from two to six bands and patterns in zone C had between four and six bands in SDS-PAGE gels. A higher number of bands was observed when urea was added to the gels. Zone B exhibited between six and 11 bands, and we identified 14 new patterns in this zone. For zone C, up to ten new patterns that comprised between five and nine bands were detected. For both zones, 86 patterns were found. The variability detected in this material is greater than that detected in other hulled wheats.

  3. Lipids and proteins in the Rathke's gland secretions of the North American mud turtle (Kinosternon subrubrum)

    USGS Publications Warehouse

    Seifert, W.E.; Gotte, S.W.; Leto, T.L.; Weldon, P.J.

    1994-01-01

    Lipids and proteins in the Rathke's gland secretions of the North American mud turtle (Kinosternon subrubrum, Kinosternidae) were analyzed by gas chromatography-mass spectrometry (GC-MS) and SDS-polyacrylamide gel electrophoresis (SDS-PAGE), respectively. Analysis by GC-MS indicates 2,3-dihydroxypropanal and C3?C24 free or esterified fatty acids. Analysis by SDS-PAGE indicates a major protein component with an approximate molecular mass of 60 kDa and minor components ranging from ca. 23 to 34 kDa. The major component of K. subrubrum glandular secretions exhibits a mobility that matches that of the Kemp's ridley sea turtle (Lepidochelys kempi, Cheloniidae), suggesting that these proteins are evolutionarily conserved.

  4. The antibacterial mechanism of berberine against Actinobacillus pleuropneumoniae.

    PubMed

    Kang, Shuai; Li, Zhengwen; Yin, Zhongqiong; Jia, Renyong; Song, Xu; Li, Li; Chen, Zhenzhen; Peng, Lianci; Qu, Jing; Hu, Zhiqiang; Lai, Xin; Wang, Guangxi; Liang, Xiaoxia; He, Changliang; Yin, Lizi

    2015-01-01

    This study demonstrated berberine to be a potential natural compound against Actinobacillus pleuropneumoniae. Liquid doubling dilution, transmission electron microscopy (TEM), SDS-PAGE and 4',6-diamidino-2-phenylindole (DAPI) staining were employed to elucidate the antibacterial activity and mechanism of berberine. The minimal inhibitory concentration of berberine was 0.3125 mg/mL, and time-kill curves showed concentration and time dependence. The TEM micrographs displayed damaged cell wall, concentrated cytoplasm, cytoplasmic content leakage and cell death. SDS-PAGE and DAPI assays revealed that berberine can restrain DNA and protein syntheses. Berberine inhibited the synthesis of proteins associated with the growth and cleavage of bacteria and then blocked the division and development of bacteria. The compound ultimately induced cytoplasm pyknosis and bacterial death.

  5. Is nephrocalcin related to the urinary derivative (bikunin) of inter-alpha-trypsin inhibitor?

    PubMed

    Tang, Y; Grover, P K; Moritz, R L; Simpson, R J; Ryall, R L

    1995-10-01

    To isolate, purify, sequence and characterize nephrocalcin (NC), a urinary protein that may be an important determinant of calcium oxalate (CaOx) kidney-stone disease. Proteins were isolated from human urine using cellulose and resin columns and were sequenced using Edman degradation and SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Inhibition of CaOx crystal growth by the isolated proteins was assessed by measuring the deposition of 14C-labelled CaOx. A protein assumed to be NC on the basis of SDS-PAGE, inhibitory and gel filtration properties was isolated from healthy human urine. Its molecular weight and the amino acid sequences of two of its peptides suggested it was identical to fragment HI-14 of the light chain (bikunin) of inter-alpha-trypsin inhibitor (ITI). NC represents a portion of the light chain of ITI, although this conclusion must remain tentative until confirmed using authentic NC.

  6. High molecular weight glutenin subunits and the classification used in Brazilian wheat industry.

    PubMed

    Chapla, P I; Vieira, E S N; Franco, F A; Linde, G A; Silva, G J; Colauto, N B; Marchioro, V S; Schuster, I

    2017-09-21

    Industrial wheat quality flour is related to gluten amount in grain. This study aimed to evaluate the relationship between high molecular weight (HMW) glutenins obtained by SDS-PAGE and gluten strength (W) obtained by the alveograph test and cropping environmental effect on wheat flour quality for Brazilian industry. Fifty-one cultivars/breeding lines were evaluated in three environments. The W value and HMW glutenin score were evaluated by SDS-PAGE. The environment effects on wheat flour were also evaluated. There was a relationship between the W value used in wheat flour industrial classification and score 10 of HMW glutenins, but there was no relation with scores 9 or lower. Cultivars/breeding lines with score 10 of HMW glutenin are less susceptible to environmental effects and produce breeding type wheat flour (W value ≥300) of interest for industry. The cultivars/breeding lines with score 10 for HMW glutenins is the main choice for a wheat breeding program.

  7. [Irritant stability of raphides and tubers from Pinellia ternate].

    PubMed

    Yu, Hong-Li; Wu, Hao; Zhang, Xing-De; Liu, Fu-Yan

    2010-06-01

    To investigate the irritation stability of raphides from Pinellia ternata and the contribution of raphides proteins on irritation. The irritation of raphides and tubers from P. ternata treated with different solvents or protease digestion were evaluated by the Draize test. The shape and appearance of raphides treated with immersion in different solvents were showed by scanning electron microscopy, and protein bands from raphides before and after protease digestion were showed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The raphides gradually lost the irritation when immersed in methanol and ethanol, while scanning electron micrograph showed the fragility of the methanol and ethanol treated raphides. The crude tubers of P. ternata immersed in 75% solution of ethanol also lost the acridity. When treated with protease digestion, raphides lost the irritation as well as the many protein bands on the SDS-PAGE gel gradually disappeared. Protein of the raphides could be involved in the raphides irritation.

  8. Assays for Determination of Protein Concentration.

    PubMed

    Olson, Bradley J S C

    2016-06-01

    Biochemical analysis of proteins relies on accurate quantification of protein concentration. Detailed in this appendix are some commonly used methods for protein analysis, e.g., Lowry, Bradford, bicinchoninic acid (BCA), UV spectroscopic, and 3-(4-carboxybenzoyl)quinoline-2-carboxaldehyde (CBQCA) assays. The primary focus of this report is assay selection, emphasizing sample and buffer compatibility. The fundamentals of generating protein assay standard curves and of data processing are considered, as are high-throughput adaptations of the more commonly used protein assays. Also included is a rapid, inexpensive, and reliable BCA assay of total protein in SDS-PAGE sample buffer that is used for equal loading of SDS-PAGE gels. © 2016 by John Wiley & Sons, Inc. Copyright © 2016 John Wiley & Sons, Inc.

  9. A neonate with Coombs-negative hemolytic jaundice with spherocytes but normal erythrocyte indices: a rare case of autosomal-recessive hereditary spherocytosis due to alpha-spectrin deficiency.

    PubMed

    Yaish, H M; Christensen, R D; Agarwal, A

    2013-05-01

    The diagnosis of hereditary spherocytosis (HS) in a newborn infant is generally made on the basis of a positive family history, spherocytes on blood film and Coombs-negative hemolytic jaundice of variable severity with an elevated mean corpuscular hemoglobin concentration (MCHC) and a low mean corpuscular volume (MCV). In general, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) quantification of erythrocyte membrane proteins is not needed to make the clinical diagnosis of HS. However, we observed that a neonate with no family history of HS, but with abundant spherocytosis on repeated blood films, Coombs-negative hemolytic jaundice and normal MCHC and MCV measurements, where SDS-PAGE revealed alpha-spectrin deficiency, a rare autosomal-recessive variety of HS that generally has a severe clinical phenotype.

  10. Purification and characterisation of α-amylase produced by mutant strain of Aspergillus oryzae EMS-18.

    PubMed

    Abdullah, Roheena; Ikram-ul-Haq

    2015-01-01

    α-Amylase produced by a mutant strain of Aspergillus oryzae EMS-18 has been purified to homogeneity as judged by sodium dodecyle sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme was purified by using 70% ammonium sulphate precipitation followed by anion exchange chromatography on DEAE-Sephadex column and gel filtration on Sephadex G-100. An enzyme purification factor of 9.5-fold was achieved with a final specific activity of 1987.7 U/mg protein and overall yield of 23.8%. The molecular weight of purified α-amylase was estimated to be 48 kDa by SDS-PAGE. The purified enzyme revealed an optimum assay temperature and pH 40°C and 5.0, respectively. Except Ca(++) all other metal ions such as Mg, Mn, Na, Zn, Ni, Fe, Cu, Co and Ba were found to be inhibitory to enzyme activity.

  11. Characterization of a highly pathogenic Bacillus thuringiensis strain isolated from common cockchafer, Melolontha melolontha.

    PubMed

    Kati, H; Sezen, K; Demirbağ, Z

    2007-01-01

    A bacterial isolate (Mm2) of Melolontha melolontha was identified and characterized. Based on various morphological, physiological, biochemical and molecular characteristics, it was identified as Bacillus thuringiensis subsp. tenebrionis. This isolate was compared to the reference strains by electron microscopy, SDS-PAGE analysis, plasmid pattern, cry gene content and insecticidal activity. Cells of the isolate harbored flat square inclusions containing a protein component of approximately equal to65 kDa. After trypsin digestion of solubilized crystals, SDS-PAGE resolved a unique proteinase-resistant peptide of approximately equal to 50 kDa. Plasmid pattern showed similar bands to those of the reference strain, PCR analysis showed that the isolate has cry3 gene. Toxicity tests (against 5 coleopteran species) showed 80 % insecticidal activity against the larvae of M. melolontha. The isolate Mm2 may be valuable as biological control agent for M. melolontha and other coleopteran insects.

  12. Fluorescent staining of glycoproteins in sodium dodecyl sulfate polyacrylamide gels by 4H-[1]-benzopyrano[4,3-b]thiophene-2-carboxylic acid hydrazide.

    PubMed

    Zhu, Zhongxin; Zhou, Xuan; Wang, Yang; Chi, Lisha; Ruan, Dandan; Xuan, Yuanhu; Cong, Weitao; Jin, Litai

    2014-06-07

    A fluorescent detection method for glycoproteins in SDS-PAGE by using 4H-[1]-benzopyrano[4,3-b]thiophene-2-carboxylic acid hydrazide (BH) was developed in this study. As low as 4-8 ng glycoproteins (transferrin, α1-acid glycoprotein) could be specifically detected by the BH staining method, which is twofold more sensitive than that of the most commonly used Pro-Q Emerald 488 glycoprotein stain. Furthermore, the specificity of the newly developed stain for glycoproteins was demonstrated by 1-D and 2-D SDS-PAGE, deglycosylation, glycoprotein affinity enrichment and LC-MS/MS, respectively. According to the results, it is concluded that BH stain may provide new choices for convenient, sensitive, specific and economic visualization of gel-separated glycoproteins.

  13. The effects of electroendosmosis in agarose electrophoresis.

    PubMed

    Guo, Y; Li, X; Fang, Y

    1998-06-01

    The effects of agarose gel strips without and with 0.03 m(r) electroendosmosis (EEO) on isoelectric focusing (IEF) were studied. It is shown that only agarose without EEO can be used for IEF. The effects of electrode buffer strips using agarose with different EEO of 0, 0.03, 0.08, 0.20 m(r) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and native PAGE were also studied. It apparently did not affect SDS-PAGE, but affected native PAGE to a certain extent. The higher the EEO value was, the more water was present on the agarose gel strip during a separation. Agarose gel strips with an EEO of 0.2 m(r) are not suitable as electrode buffer strips for native PAGE.

  14. Use of principal component analysis for differentiation of gelatine sources based on polypeptide molecular weights.

    PubMed

    Nur Azira, T; Che Man, Y B; Raja Mohd Hafidz, R N; Aina, M A; Amin, I

    2014-05-15

    The study was aimed to differentiate between porcine and bovine gelatines in adulterated samples by utilising sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) combined with principal component analysis (PCA). The distinct polypeptide patterns of 6 porcine type A and 6 bovine type B gelatines at molecular weight ranged from 50 to 220 kDa were studied. Experimental samples of raw gelatine were prepared by adding porcine gelatine in a proportion ranging from 5% to 50% (v/v) to bovine gelatine and vice versa. The method used was able to detect 5% porcine gelatine added to the bovine gelatine. There were no differences in the electrophoretic profiles of the jelly samples when the proteins were extracted with an acetone precipitation method. The simple approach employing SDS-PAGE and PCA reported in this paper may provide a useful tool for food authenticity issues concerning gelatine.

  15. The proteins of the grape (Vitis vinifera L.) seed endosperm: fractionation and identification of the major components.

    PubMed

    Gazzola, Diana; Vincenzi, Simone; Gastaldon, Luca; Tolin, Serena; Pasini, Gabriella; Curioni, Andrea

    2014-07-15

    In the present study, grape (Vitis vinifera L.) seed endosperm proteins were characterized after sequential fractionation, according to a modified Osborne procedure. The salt-soluble fraction (albumins and globulins) comprised the majority (58.4%) of the total extracted protein. The protein fractions analysed by SDS-PAGE showed similar bands, indicating different solubility of the same protein components. SDS-PAGE in non-reducing and reducing conditions revealed the polypeptide composition of the protein bands. The main polypeptides, which were similar in all the grape varieties analysed, were identified by LC-MS/MS as homologous to the 11S globulin-like seed storage proteins of other plant species, while a monomeric 43 kDa protein presented high homology with the 7S globulins of legume seeds. The results provide new insights about the identity, structure and polypeptide composition of the grape seed storage proteins. Copyright © 2014 Elsevier Ltd. All rights reserved.

  16. Insights into the interactions between enzyme and co-solvents: stability and activity of stem bromelain.

    PubMed

    Rani, Anjeeta; Venkatesu, Pannuru

    2015-02-01

    In present study, an attempt is made to elucidate the effects of various naturally occurring osmolytes and denaturants on BM at pH 7.0. The effects of the varying concentrations of glycerol, sorbitol, sucrose, trehalose, urea and guanidinium chloride (GdnHCl) on structure, stability and activity of BM are explored by fluorescence spectroscopy, circular dichroism (CD), UV-vis spectroscopy and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Our experimental observations reveal that glycerol and sorbitol are acting as stabilizers at all concentrations while sucrose and trehalose are found to be destabilizers at lower concentrations, however, acted as stabilizers at higher concentrations. On the other hand, urea and GdnHCl are denaturants except at lower concentrations. There is a direct relationship between activity and conformational stability as the activity data are found to be in accordance with conformational stability parameters (ΔGu, Tm, ΔCp) and BM profile on SDS-PAGE.

  17. Oxidized low-density lipoprotein (Ox-LDL) impacts on erythrocyte viscoelasticity and its molecular mechanism.

    PubMed

    Wang, Xiang; Yang, Li; Liu, Yao; Gao, Wei; Peng, Weiyan; Sung, K-L Paul; Sung, Lanping Amy

    2009-10-16

    The oxidized low-density lipoprotein (Ox-LDL) plays an important role in atherosclerosis, yet it remains unclear if it damages circulating erythrocytes. In this study, erythrocyte deformability and its membrane proteins after Ox-LDL incubations are investigated by micropipette aspiration, thiol radical measurement, and sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Results show that Ox-LDL incubation reduces the erythrocyte deformability, decreases free thiol radical contents in erythrocytes, and induces the cross-linking among membrane proteins. SDS-PAGE analysis reveals a high molecular weight (HMW) complex as well as new bands between spectrins and band 3 and reduced ratios between band 3 and other major membrane skeletal proteins. Analyses indicate that Ox-LDL makes erythrocytes harder to deform through a molecular mechanism by which the oxidation of free thiol radicals forms disulfide bonds among membrane skeletal proteins.

  18. One-step purification and porin transport activity of the major outer membrane proteins P2 from Haemophilus influenzae, FomA from Fusobacterium nucleatum and PorB from Neisseria meningitidis.

    PubMed

    Kattner, Christof; Pfennig, Sabrina; Massari, Paola; Tanabe, Mikio

    2015-03-01

    Bacterial porins are major outer membrane proteins that function as essential solute transporters between the bacteria and the extracellular environment. Structural features of porins are also recognized by eukaryotic cell receptors involved in innate and adaptive immunity. To better investigate the function of porins, proper refolding is necessary following purification from inclusion bodies [1, 2]. Using a single-step size exclusion chromatographic method, we have purified three major porins from pathogenic bacteria, the OmpP2 (P2) from Haemophilus influenzae, FomA from Fusobacterium nucleatum and PorB from Neisseria meningitidis, at high yield and report their unique solute transport activity with size exclusion limit. Furthermore, we have optimized their purification method and achieved improvement of their thermostability for facilitating functional and structural analyses.

  19. Development of activity-based probes for trypsin-family serine proteases.

    PubMed

    Pan, Zhengying; Jeffery, Douglas A; Chehade, Kareem; Beltman, Jerlyn; Clark, James M; Grothaus, Paul; Bogyo, Matthew; Baruch, Amos

    2006-06-01

    A series of diphenylphosphonate-based probes were developed for the trypsin-like serine proteases. These probes selectively target serine proteases rather than general serine hydrolases that are targets for fluorophosphonate-based probes. This increased selectivity allows detection of low abundance serine proteases in complex proteomes using simple SDS-PAGE methods. We present here the application of multiple probes in enzyme activity profiling of intact mast cells, a type of inflammatory cell implicated in allergy and autoimmune diseases.

  20. Oncoprotein protein kinase

    DOEpatents

    Karin, M.; Hibi, M.; Lin, A.

    1997-02-25

    An isolated polypeptide (JNK) characterized by having a molecular weight of 46 kD as determined by reducing SDS-PAGE is disclosed. The polypeptide has serine and threonine kinase activity, phosphorylating the c-Jun N-terminal activation domain and polynucleotide sequences. The method of detection of JNK is also provided. JNK phosphorylates c-Jun N-terminal activation domain which affects gene expression from AP-1 sites. 44 figs.

  1. Development of Purification Protocol Specific for Bacteriocin 105B

    DTIC Science & Technology

    2017-02-09

    Chromatography. Representative PAGE gel with samples collected from column chromatography using DEAE Sephadex. Lane 1: Novex Sharp Pre- Stained Protein...8  Figure 4. Confirmation of bacteriocin 105b in the flow-through of ion exchange column chromatography. A.) SDS-PAGE gel stained ...with Coomassie Blue. Lanes 1 and 2: flow- through collected after applying 30k retentate to the DEAE Sephadex column. Lane 3: Novex Sharp Pre- Stained

  2. A New Cell-Free System to Study BRCA1 Function

    DTIC Science & Technology

    2016-06-01

    insect cells was analysed via SDS page alongside a MW ladder and stained with Coomassie blue. Figure 5: Modified forms of MCM7 are ubiquitylated...photobleaching, we sampled fresh fields of view every !18 s after extract addition and obtained FRET efficiency histo- grams at each time point, which are...model on super-resolution images of fixed cells in which a fraction of immunostained XRCC4, XLF, and LIG4 foci co-localized with Ku or TUNEL staining

  3. Imaging Prostate Cancer Microenvironment by Collagen Hybridization

    DTIC Science & Technology

    2015-10-01

    undergoing normal (e.g. skeleton) and pathological (arthritis) remodeling. 2. Demonstration of the use of CMP as collagen staining agent in SDS-PAGE gel...goal of the proposed project; however musculoskeletal injuries are the number one medical issue for the DOD when it comes to encounters and...CMP can be used to detect mechanical damage to collagen in tendon which could be used for diagnostic and therapeutics of musculoskeletal injury which

  4. Oncoprotein protein kinase antibody kit

    DOEpatents

    Karin, Michael; Hibi, Masahiko; Lin, Anning

    2008-12-23

    An isolated polypeptide (JNK) characterized by having a molecular weight of 46 kD as determined by reducing SDS-PAGE, having serine and threonine kinase activity, phosphorylating the c-Jun N-terminal activation domain and polynucleotide sequences and method of detection of JNK are provided herein. JNK phosphorylates c-Jun N-terminal activation domain which affects gene expression from AP-1 sites.

  5. Purification and characterization of Fe(III)-EDTA reductase from Bacillus sp. B-3.

    PubMed

    Shinagawa, Emiko

    2011-01-01

    Fe(III)-EDTA reductase was purified from Bacillus sp. B-3 isolated as a Fe(III)-EDTA-degrading bacterium. The purified enzyme showed a single protein band corresponding to a molecular mass of 19 kDa on SDS-PAGE, and had FMN as cofactor. It was alkali-thermostable. Its N-terminal amino acid sequence was identical with that of NADPH azoreductase from several species of Bacillus.

  6. Protein bodies from the cotyledons of Cytisus scoparius L. (Link). Ultrastructure, isolation, and subunit composition of albumin, legumin and vicilin.

    PubMed

    Citharel, L; Citharel, J

    1985-09-01

    The structure of protein bodies differs in the upper and lower parts of the cotyledons of mature seeds of Cytisus scoparius L. The palisade-mesophyll cells contain essentially homogeneous protein bodies, without globoids, but the protein bodies of the spongy-mesophyll cells are heterogeneous, with numerous globoids. Albumins, legumins and vicilins were selectively extracted from isolated protein bodies and their subunits separated by SDS-PAGE, under non-reducing and reducing conditions.

  7. Review of DoD Malaria Research Programs,

    DTIC Science & Technology

    1992-05-01

    pGEMEX were subjected to preparative SDS-PAGE, transferred to nitrocellulose paper, and stained with 0.01% amido black. Bands corresponding to the...Commander. COL George Lewis said the Pentagon is concerned about a lack of prioritization in the programs. U CDR Richard Oberst stressed the need for...Sadoff also stressed the need for open and immediate sharing of information between the Army or Navy. Recommendations from Advisors I All three

  8. Differential Phosphoprotein Profiling of Tamoxifen Response

    DTIC Science & Technology

    2008-08-01

    cataloging global phosphorylation events in response to tamoxifen treatment in tamoxifen sensitive and resistant cells we will provide better... global phosphoprotein profiles. Our methodology involves stable isotope labeling 2, a phosphoprotein affinity step, 1-D SDS-PAGE and LC-MS/MS 3. I...with Xpress ratio less than 0.6 and about 28 proteins with ratio larger than 1.66. Manual analysis is underway to confirm the protein abundance

  9. Early Life Stress and Sleep Restriction as Risk Factors in PTSD: An Integrative Pre-Clinical Approach

    DTIC Science & Technology

    2014-04-01

    of each sample was loaded for 12 % SDS-polyacrylamide gel for electrophoresis (SDS-PAGE). After semi-dry transfer (nitrocellulose membrane ) and...As a highly organized structure, the DG receives inputs from the entorhinal cortex into the outer two-thirds of its molecular layer. The granule cell...mm – -4.16 mm from Bregma; Paxinos & Watson, 1998) and thaw mounted on poly-L-lysine coated RNase-free membrane slides (Leica Microsystems, Wetzlar

  10. Metaproteome analysis of the microbial communities in agricultural biogas plants.

    PubMed

    Heyer, R; Kohrs, F; Benndorf, D; Rapp, E; Kausmann, R; Heiermann, M; Klocke, M; Reichl, U

    2013-09-25

    In biogas plants agricultural waste and energy crops are converted by complex microbial communities to methane for the production of renewable energy. In Germany, this process is widely applied namely in context of agricultural production systems. However, process disturbances, are one of the major causes for economic losses. In addition, the conversion of biomass, in particular of cellulose, is in most cases incomplete and, hence, insufficient. Besides technical aspects, a more profound characterization concerning the functionality of the microbial communities involved would strongly support the improvement of yield and stability in biogas production. To monitor these communities on the functional level, metaproteome analysis was applied in this study to full-scale agricultural biogas plants. Proteins were extracted directly from sludge for separation by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and subsequent identification with mass spectrometry. Protein profiles obtained with SDS-PAGE were specific for different biogas plants and often stable for several months. Differences of protein profiles were visualized by clustering, which allowed not only the discrimination between mesophilic and thermophilic operated biogas plants but also the detection of process disturbances such as acidification. In particular, acidification of a biogas plant was detected in advance by disappearance of major bands in SDS-PAGE. Identification of proteins from SDS-PAGE gels revealed that methyl CoM reductase, which is responsible for the release of methane during methanogenesis, from the order Methanosarcinales was significantly decreased. Hence, it is assumed that this enzyme might be a promising candidate to serve as a predictive biomarker for acidification.

  11. Signal Transduction Pathway in Maspin-induced Tumor Suppression of Prostate Cancer

    DTIC Science & Technology

    2002-03-01

    min, resulting in the heavy membrane SDS-PAGE and Western Blot Analysis-These procedures were car- fraction in the pellet containing mitochondria ...prostasin antibody, heparin, and somes and mitochondria , and in lipid particles (33). Prostasin serine protease inhibitors. In a membrane overlay...mechanism. This idea is also potentially applicable to diseases of the respiratory system since there is recent evidence that prostasin may be involved

  12. Identification of New Substrates for Breast Tumor-Specific LMW Cyclin E/CDk2 Kinase

    DTIC Science & Technology

    2011-09-01

    induced breast cancer and that CDK2 inhibitor (such as roscovitine ) also delays mammary tumor formation (Akli et al., 2011). The hypothesis is that...in the presence of 32P-γ-ATP and with or without roscovitine . The samples were separated by SDS-PAGE and exposed to x-ray films. (E) Schematic of...phosphorylate Hbo1 at relatively similar levels, and addition of roscovitine efficiently inhibited the Hbo1 phosphorylation signal (Figure 2C). Similar

  13. Affinity crosslinking of /sup 125/I-human beta-endorphin to cell lines possessing either mu or delta type opioid binding sites

    SciTech Connect

    Keren, O.; Gioannini, T.L.; Hiller, J.M.; Simon, E.J.

    1986-01-01

    Affinity crosslinking of human /sup 125/I-beta-Endorphin to cell lines possessing either mu or delta binding sites was carried out. Autoradiography of SDS-PAGE gels from these crosslinked cell lines revealed that these two sites contain major peptide subunits that differ in molecular size. This confirms our earlier finding in mammalian brain which demonstrated separate and distinct subunits for mu and delta opioid receptors.

  14. Oncoprotein protein kinase

    DOEpatents

    Karin, Michael; Hibi, Masahiko; Lin, Anning

    2004-03-16

    An isolated polypeptide (JNK) characterized by having a molecular weight of 46 kD as determined by reducing SDS-PAGE, having serine and threonine kinase activity, phosphorylating the c-Jun N-terminal activation domain and polynucleotide sequences and method of detection of JNK are provided herein. JNK phosphorylates c-Jun N-terminal activation domain which affects gene expression from AP-1 sites.

  15. Alterations in membrane protein-profile during cold treatment of alfalfa

    SciTech Connect

    Mohapatra, S.S.; Poole, R.J.; Dhindsa, R.S. )

    1988-04-01

    Changes in pattern of membrane proteins during cold acclimation of alfalfa have been examined. Cold acclimation for 2 to 3 days increases membrane protein content. Labeling of membrane proteins in vivo with ({sup 35}S)methionine indicates increases in the rate of incorporation as acclimation progresses. Cold acclimation induces the synthesis of about 10 new polypeptides as shown by SDS-PAGE and fluorography of membrane proteins labeled in vivo.

  16. Rapid scanning thermal lens/laser transmission densitometer.

    PubMed

    Peck, K; Demana, T; Morris, M D

    1988-01-01

    An automated densitometer based on the thermal lens principle is described. The apparatus also operates as a conventional laser transmission densitometer. Comparison of the performance in both modes shows that thermal lens densitometry provides lower detection limits, but that transmission densitometry is more satisfactory at high optical densities. The instrument is characterized with proteins separated by SDS-PAGE and stained with Coomassie Brilliant Blue G250.

  17. Oncoprotein protein kinase

    DOEpatents

    Karin, Michael; Lin, Anning

    1999-11-30

    An isolated polypeptide (JNK) characterized by having a molecular weight of 46 kD or 55 kD as determined by reducing SDS-PAGE, having serine and theonine kinase activity, phosphorylating the c-Jun N-terminal activation domain and polynucleotide sequences and method of detection of JNK are provided herein. JNK phosphorylates c-Jun N-terminal activation domain which affects gene expression from AP-1 sites.

  18. HbzF catalyzes direct hydrolysis of maleylpyruvate in the gentisate pathway of Pseudomonas alcaligenes NCIMB 9867.

    PubMed

    Liu, Kun; Liu, Ting-Ting; Zhou, Ning-Yi

    2013-02-01

    HbzF from Pseudomonas alcaligenes NCIMB 9867 was purified to homogeneity as a His-tagged protein and likely a dimer by SDS-PAGE and gel filtration. This protein was demonstrated to be a novel maleylpyruvate hydrolase, catalyzing direct hydrolysis of maleylpyruvate to maleate and pyruvate, and belongs to the fumarylacetoacetate hydrolase superfamily. This study reveals the genetic determinate for the direct maleylpyruvate hydrolysis in the gentisate pathway, complementary to the well-studied maleylpyruvate isomerization route.

  19. Oncoprotein kinase

    DOEpatents

    Karin, Michael; Hibi, Masahiko; Lin, Anning

    2001-02-27

    An isolated polypeptide (JNK) characterized by having a molecular weight of 46 kD or 55 kD as determined by reducing SDS-PAGE, having serine and theonine kinase activity, phosphorylating the c-Jun N-terminal activation domain and polynucleotide sequences and method of detection of JNK are provided herein. JNK phosphorylates c-Jun N-terminal activation domain which affects gene expression from AP-1 sites.

  20. Oncoprotein protein kinase

    DOEpatents

    Karin, Michael; Hibi, Masahiko; Lin, Anning

    1998-01-01

    An isolated polypeptide (JNK) characterized by having a molecular weight of 46 kD as determined by reducing SDS-PAGE, having serine and threonine kinase activity, phosphorylating the c-Jun N-terminal activation domain and polynucleotide sequences and method of detection of JNK are provided herein. JNK phosphorylates c-Jun N-terminal activation domain which affects gene expression from AP-1 sites.

  1. Oncoprotein protein kinase

    DOEpatents

    Davis, Roger; Derijard, Benoit; Karin, Michael; Hibi, Masahiko; Lin, Anning

    2005-01-25

    An isolated polypeptide (JNK) characterized by having a molecular weight of 46 kD as determined by reducing SDS-PAGE, having serine and threonine kinase activity, phosphorylating the c-Jun N-terminal activation domain and polynucleotide sequences and method of detection of JNK are provided herein. JNK phosphorylates c-Jun N-terminal activation domain which affects gene expression from AP-1 sites.

  2. Oncoprotein protein kinase

    DOEpatents

    Karin, Michael; Hibi, Masahiko; Lin, Anning; Davis, Roger; Derijard, Benoit

    2003-02-04

    An isolated polypeptide (JNK) characterized by having a molecular weight of 46kD as determined by reducing SDS-PAGE, having serine and threonine kinase activity, phosphorylating the c-Jun N-terminal activation domain and polynucleotide sequences and method of detection of JNK are provided herein. JNK phosphorylates c-Jun N-terminal activation domain which affects gene expression from AP-1 sites.

  3. Oncoprotein protein kinase

    DOEpatents

    Karin, Michael; Hibi, Masahiko; Lin, Anning

    1997-01-01

    An isolated polypeptide (JNK) characterized by having a molecular weight of 46kD as determined by reducing SDS-PAGE, having serine and threonine kinase activity, phosphorylating the c-Jun N-terminal activation domain and polynucleotide sequences and method of detection of JNK are provided herein. JNK phosphorylates c-Jun N-terminal activation domain which affects gene expression from AP-1 sites.

  4. Oncoprotein protein kinase

    DOEpatents

    Karin, Michael; Hibi, Masahiko; Lin, Anning; Davis, Roger; Derijard, Benoit

    2005-03-08

    An isolated polypeptide (JNK) characterized by having a molecular weight of 46 kD as determined by reducing SDS-PAGE, having serine and threonine kinase activity, phosphorylating the c-Jun N-terminal activation domain and polynucleotide sequences and method of detection of JNK are provided herein. JNK phosphorylates c-Jun N-terminal activation domain which affects gene expression from AP-1 sites.

  5. Oncoprotein protein kinase

    DOEpatents

    Karin, Michael; Hibi, Masahiko; Lin, Anning

    1999-01-01

    An isolated polypeptide (JNK) characterized by having a molecular weight of 46 kD or 55 kD as determined by reducing SDS-PAGE, having serine and theonine kinase activity, phosphorylating the c-Jun N-terminal activation domain and polynucleotide sequences and method of detection of JNK are provided herein. JNK phosphorylates c-Jun N-terminal activation domain which affects gene expression from AP-1 sites.

  6. Oncoprotein protein kinase

    DOEpatents

    Karin, Michael; Hibi, Masahiko; Lin, Anning

    1997-01-01

    An isolated polypeptide (JNK) characterized by having a molecular weight of 46 kD as determined by reducing SDS-PAGE, having serine and threonine kinase activity, phosphorylating the c-Jun N-terminal activation domain and polynucleotide sequences and method of detection of JNK are provided herein. JNK phosphorylates c-Jun N-terminal activation domain which affects gene expression from AP-1 sites.

  7. A fluorescence-based Coomassie Blue protocol for two-dimensional gel-based proteomics.

    PubMed

    Yang, Yongxin; Wang, Jiaqi; Bu, Dengpan; Zhang, Leying; Li, Shanshan; Zhou, Lingyun; Wei, Hongyang

    2011-01-01

    A sensitive and convenient "visible SYPRO" staining protocol was developed for visualizing proteins after SDS-PAGE. Gels were sensitized with SYPRO Ruby and then stained with the Coomassie Brilliant Blue G-250 protocol (Blue Silver). This combined protocol had similar or better linearity than staining with only SYPRO Ruby or Blue Silver, respectively. In addition, this method was more sensitive than that of Blue Silver, simpler than that of SYPRO Ruby, and compatible with subsequent mass spectrometry analysis.

  8. SIRT3 is a Mitochondrial Tumor Suppressor and Genetic Loss Results in a Murine Model for ER/PR-Positive Mammary Tumors Connecting Metabolism and Carcinogenesis Mitochondrial Tumor Suppressor. Revision

    DTIC Science & Technology

    2013-11-01

    address this question a series of in vitro deacetylation assays were preformed. Affinity purified ATP synthase was obtained from bovine mitochondria...resolved by SDS-PAGE and immunoblotted with an anti-acetyl-lysine antibody (Fig. 1, left panel lane 1) or stained with Coomassie (lane 2). These...from lane 2, middle panel were mixed with recombinant SIRT3 without and with NAD for an in vitro deacetylation assay , similar to the experiments above

  9. Tromantadine inhibits HSV-1 induced syncytia formation and viral glycoprotein processing

    SciTech Connect

    Ickes, D.E.

    1989-01-01

    Tromantadine inhibits a late event in Herpes Simplex Virus Type 1 (HSV-1) replication, visualized by the inhibition of both the size and number of syncytia. Tromantadine can be added at any time between 1 and 9 h post infection with complete inhibition of syncytia formation. Glycan synthesis of the viral glycoproteins, important for syncytia formation, is incomplete due to tromantadine treatment. Tromantadine does not inhibit the initiation of glycosylation, since viral glycoproteins, gX{sub t}, synthesized in the presence of tromantadine still incorporate {sup 3}H-glucosamine. Tromantadine does not inhibit the transport of t e viral glycoproteins to the cell surface, since glycoproteins B, C, and D are expressed, as demonstrated by immunofluorescence. Tromantadine inhibition of HSV-1 glycoprotein processing is demonstrated by an increase in mobility of the radioimmunoprecipitated gX{sub t}, on SDS-PAGE. The gX{sub t} of KOS, a non-syncytial strain of HSV-1, had a similar increase in mobility, suggesting that the block in glycoprotein processing is a general effect of tromantadine treatment. Fucose, which is incorporated into oligosaccharides in the medial Golgi, is incorporated into gX{sub t}, indicating that the tromantadine block in glycoprotein processing occurs after this step. Lectin binding studies and SDS-PAGE analysis of gC processed in the presence of tromantadine, gC{sub t}, indicates that it has terminal galactose residues in both N- and O-linked glycans (binds Peanut and Ricin Agglutinins, respectively). The inhibition of sialylation of N-linked glycans by tromantadine was indicated by the extent of the increase in SDS-PAGE mobility of the G protein from Vesicular Stomatitis Virus. O-glycanase digestion and SDS-PAGE analysis of gC{sub t} indicate that the O-linked disaccharide NAcGal-Galactose is present.

  10. Protein changes in leaf-sheath pulvini of barley (hordeum) induced by gravistimulation

    NASA Technical Reports Server (NTRS)

    Kaufman, P. B.; Song, I.

    1984-01-01

    Sodium dodecyl sulfate polyacrylamade gel electrophoresis (SDS-PAGE) pattern of salt soluble proteins elicite by gravistimulation were shown in the top and the bottom halves of the gravistimulated pulvini as follows: at least five proteins were increased in the tissues derived from the bottom halves of the pulvini in the approximate molecular weight range of 91, 57, 50, 22, 17 kilodatons. SDS densitometric scans indicated that the two of them are probably cellulase and invertase.

  11. Quantification of Protein Signatures in Archived Human Prostate Tissues Using Shotgun Proteomic Methods

    DTIC Science & Technology

    2012-05-01

    Fig. 2). The detergent-solubilized proteins were subsequently incubated with wheat - germ agglutinin (WGA) and concanavalin-A (ConA) beads, washed...optimizing protocols to extract and profile proteins in matched normal and diseased tissue samples using directed mass spectrometry methods. The ultimate...able to extract up to 100 micrograms of total protein using whole- mount FFPE RP tissue block samples. This Figure 2. Silver-stained SDS-PAGE gel

  12. Role of the Neddylation Enzyme Uba3, a New Estrogen Receptor Corepressor, in Breast Cancer

    DTIC Science & Technology

    2003-09-01

    malignant transformation of prostate cancer was recently reported (13). Because our results show an intact NEDD8 pathway is essential for ERcc...Proteomic analysis of normal and malignant prostate tissue to identify novel proteins lost in cancer . Prostate 50:54-63 APPENDICES Reprint: Fan M...transfection efficiency and SDS-PAGE of prostate cancer was recently reported (60). Be- loading. cause our results show an intact NEDD8 pathway is Stable

  13. [Glutathione S-transferase of alpha class from pike liver].

    PubMed

    Borvinskaia, E V; Smirnov, L P; Nemova, N N

    2013-01-01

    In this study, glutathione S-transferase (GST) was isolated from the liver of pike Esox lucius, which was homogenous according to SDS-PAGE and isoelectrofocusing. It is a homodimer with subunits mass 25235.36 Da (according to HPLC-MS/MS) and pI about 6.4. Substrate specificity, thermostability, some kinetic characteristics and optimum pH were determined. The enzyme was identified as Alpha class GST.

  14. Neurogenin1 Expression and Function in the Developing Mouse Cerebellum

    DTIC Science & Technology

    2011-12-14

    natural Cre recombinase from Bacteriophage P1 at the predicted 38.5 kDa size by SDS-PAGE Western blot (manufacturer’s technical information). (7) 1...L-glutamine, taurine , putrescine, L-alanine, and carnosine. The antibody has weak cross reactivity to β-alanine and ε-aminocaproic acid (all...cell lineages. Many studies of bHLH factors have highlighted the combinatorial nature of these proteins in their specification of neuronal subtypes

  15. Targeting MED1 LxxLL Motifs for Tissue-Selective Treatment of Human Breast Cancer

    DTIC Science & Technology

    2013-09-01

    surface markers using this approach insufficient, because the extraction of these cell surface proteins requires detergent that tends to diminish the...phenol chloroform/ chloroform extractions to separate the bound aptamers from the protein. After ethanol/NaAC precipitations, the RNA was reverse...beads through centrifugation, and subjected to western blot analyses after separation by SDS /PAGE gel. Negative controls in this experiment consisted

  16. [Evolution of tocopherols in relation of unsaturated fatty acids during maturation of seeds of rapeseed (Brassica napus L.)].

    PubMed

    Sebei, Khaled; Boukhchina, Sadok; Kallel, Habib

    2007-01-01

    The oil content increases during the maturation of seeds (rise of 30%), but decreases at the end of seed maturation. Differences between SDS-PAGE total protein profiles were shown. Polyunsaturated fatty acids contents increase during middle-maturation. Contents of alpha and gamma tocopherols increase with time. This increase is explained by the fact that tocopherols participate actively in the protection of membranes whose phospholipids consist of polyunsaturated fatty acids (PUFAs).

  17. Protein changes in leaf-sheath pulvini of barley (hordeum) induced by gravistimulation

    NASA Technical Reports Server (NTRS)

    Kaufman, P. B.; Song, I.

    1984-01-01

    Sodium dodecyl sulfate polyacrylamade gel electrophoresis (SDS-PAGE) pattern of salt soluble proteins elicite by gravistimulation were shown in the top and the bottom halves of the gravistimulated pulvini as follows: at least five proteins were increased in the tissues derived from the bottom halves of the pulvini in the approximate molecular weight range of 91, 57, 50, 22, 17 kilodatons. SDS densitometric scans indicated that the two of them are probably cellulase and invertase.

  18. Activity staining on polyacrylamide gels of trypsin inhibitors from leaves of sweet potato (Ipomoea batatas L. Lam) varieties.

    PubMed

    Hou, W C; Lin, Y H

    1998-02-01

    The failure of activity staining of trypsin inhibitors in crude leaf extracts of sweet potato varieties including Tainong 27, Tainong 34, and Tainong 57 on a 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel was prevented by dipping the gels in solutions containing 10-40 mM hydrogen peroxide, 10 mM Tris buffer (pH 7.9) for 30 min before staining.

  19. Butyrate-Induced Apoptosis in Prostate Cancer Cell Lines

    DTIC Science & Technology

    2001-09-01

    butyrate-induced apoptosis was independent of cell cycle phase. 14. SUBJECT TERMS 15. NUMBER OF PAGES prostate cancer, histone deacetylase inhibitors, bone...of cells plated) HDI histone deacetylase inhibitor SBHA suberoylbishydroxamate PKC protein kinase C activator SDS-PAGE SDS polyacrylamide gel...cancer cell lines 1. Summary of goals and findings Histone deacetylase inhibitors (HDI) such as butyrate and suberoylbishydroxamate (SBHA) have

  20. Individual venom profiling of Crotalus durissus terrificus specimens from a geographically limited region: crotamine assessment and captivity evaluation on the biological activities.

    PubMed

    Lourenço, Airton; Zorzella Creste, Camila Fernanda; de Barros, Luciana Curtolo; Delazari dos Santos, Lucilene; Pimenta, Daniel C; Barraviera, Benedito; Ferreira, Rui Seabra

    2013-07-01

    Crotalus durissus terrificus (Cdt) venom major components comprise crotoxin, crotamine, gyroxin and convulxin. Crotamine exerts a myotoxic action, among others, but its expression varies even amid snakes from the same region. Biochemical, enzymatic and pharmacological variations of venoms may be associated with the geography, climate, gender, age, and diet, as well as captivity time and venom extraction intervals. The present study aimed to characterize the Cdt venom from the Botucatu region, (SP, Brazil), by assessing its biochemical, pharmacological and enzymatic properties. Venoms from newly captured snakes and already-captured animals were characterized comparatively to verify the sexual, environmental (length of captivity) and ontogenetic variations that could influence the venom composition. Protein concentration, SDS-PAGE and RP-HPLC were performed and the coagulant, toxic (LD50) and crotamine activities were assayed. Individual SDS-PAGE analyses (315 samples) were performed and the biological activities of the venom of 60 adults (captive and newly captured males and females) and 18 newborns were compared with the Brazilian Reference Venom. Crotamine was found in 39.7% (125/315) of the samples, as determined by SDS-PAGE and RP-HPLC. Protein concentration differed significantly between adults (75%) and newborns (60%). RP-HPLC and SDS-PAGE analyses showed highly variable protein concentration and copious crotoxin isoforms; however, the LD50 values decreased during the captivity time. Cdt venom biological activities were similar among adult groups, but diminished during the captivity period. The current findings demonstrate that venoms vary significantly in terms activity and protein concentration, despite originating from the same specie and region. Copyright © 2013 Elsevier Ltd. All rights reserved.

  1. Antigenic and physical diversity of Neisseria gonorrhoeae lipooligosaccharides.

    PubMed Central

    Mandrell, R; Schneider, H; Apicella, M; Zollinger, W; Rice, P A; Griffiss, J M

    1986-01-01

    We used mouse monoclonal antibodies (MAbs) to characterize Neisseria gonorrhoeae lipooligosaccharide (LOS). LOSs that bound two or more MAbs in a solid-phase radioimmunoassay usually bound them to different LOS components, as separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE); strains with multiple LOS components on SDS-PAGE usually bound more than one MAb. However, the LOS of some strains bound the same MAb to two LOS components with different relative molecular weights, and some individual LOS components bound more than one MAb. LOSs from different strains bound different amounts of the same MAb at saturation, reflecting differences in the quantitative expression of individual LOS components. Not all components recognized by MAbs were stained by silver after periodate oxidation. Treatment with NaOH variously affected epitopes defined by different MAbs. MAb 3F11 completely inhibited and MAb 2-1-L8 partially inhibited the binding of 125I-labeled 06B4 MAb to WR220 LOS and WR220 outer membranes in competitive binding studies. Other MAbs did not compete with the binding of 125I-labeled 06B4 to either antigen. We conclude that a strain of N. gonorrhoeae elaborates multiple LOSs that can be separated by SDS-PAGE and that are antigenically distinct. Epitope expression within these glycolipids is complex. Images PMID:2428752

  2. Partial purification and characterization of protease enzyme from Bacillus subtilis and Bacillus cereus.

    PubMed

    Orhan, Elif; Omay, Didem; Güvenilir, Yüksel

    2005-01-01

    The aim of this experimental study was to isolate and partially purify protease enzyme from Bacillus cereus and Bacillus subtilis. Protease enzyme is obtained by inducing spore genesis of bacteria from Bacillus species in suitable nutrient plates. The partial purification was realized by applying, respectively, ammonium sulfate precipitation, dialysis, and DEAE-cellulose ion-exchange chromatography to the supernatant that was produced later. Optimum pH, optimum temperature, pH stability, and temperature stability were determined, as well as the effects of pH, temperature, substrate concentration, reaction time, and inhibitors and activators on enzyme activity. In addition, the molecular mass of the obtained enzyme was investigated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The specific activity of partially purified enzyme from B. subtilis was determined to be 84 U/mg. The final enzyme preparation was eight-fold more pure than the crude homogenate. The molecular mass of the partially purified enzyme was found to be 45 kDa by using SDS-PAGE. The protease enzyme that was partially purified from B. cereus was purified 1.2-fold after ammonium sulfate precipitation. The molecular mass of the partially purified enzyme was determined to be 37 kDa by using SDS-PAGE.

  3. The molecular heterogeneity of nonspecific cross-reacting antigen synthesized by tumor cells and granulocytes.

    PubMed

    Kuroki, M; Kuroki, M; Moore, G E; Ichiki, S; Matsuoka, Y

    1988-01-01

    The molecular heterogeneity of nonspecific cross-reacting antigen (NCA) was examined. Metabolically-labeled glycoproteins were precipitated from cell lysates of human tumor cell lines and of normal peripheral granulocytes with antibodies specific for NCA, and analyzed by SDS-PAGE. NCA components synthesized by three tumor cell lines, QGP-1 (pancreas), HLC-1 (lung) and CAOV-2 (ovary) showed slightly different migration patterns on SDS-PAGE, but the molecular weights of their unglycosylated peptides synthesized in the presence of tunicamycin were all found to be 35K. On the other hand, two molecular species of NCA were identified in normal granulocytes: an 80K mature form derived from a 69K precursor peptide and a 58K mature form from a 41K precursor peptide. Upon SDS-PAGE, the migration pattern of the unglycosylated NCA peptides from tumor cells was affected by the presence of 2-mercaptoethanol, while that of the peptides of granulocytes was not. All the NCAs identified in this study possessed antigenic determinants common to carcinoembryonic antigen as well as those unique to NCA. These results suggest that the molecular heterogeneity of NCA observed thus far resulted from diverse glycosylation of the three fundamental molecular forms of unglycosylated peptides: one with a molecular weight of 35K produced by tumor cells and two with molecular weights of 69K and 41K produced by granulocytes.

  4. Structural properties of lipopolysaccharides from Rickettsia typhi and Rickettsia prowazekii and their chemical similarity to the lipopolysaccharide from Proteus vulgaris OX19 used in the Weil-Felix test.

    PubMed

    Amano, K I; Williams, J C; Dasch, G A

    1998-03-01

    The lipopolysaccharides (LPSs) isolated from typhus group (TG) rickettsiae Rickettsia typhi and Rickettsia prowazekii were characterized by chemical analysis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by silver staining. LPSs from two species of TG rickettsiae contained glucose, 3-deoxy-D-manno-octulosonic acid, glucosamine, quinovosamine, phosphate, and fatty acids (beta-hydroxylmyristic acid and heneicosanoic acid) but not heptose. The O-polysaccharides of these LPSs were composed of glucose, glucosamine, quinovosamine, and phosphorylated hexosamine. Resolution of these LPSs by their apparent molecular masses by SDS-PAGE showed that they have a common ladder-like pattern. Based on the results of chemical composition and SDS-PAGE pattern, we suggest that these LPSs act as group-specific antigens. Furthermore, glucosamine, quinovosamine, and phosphorylated hexosamine were also found in the O-polysaccharide of the LPS from Proteus vulgaris OX19 used in the Weil-Felix test, suggesting that they may represent the antigens common to LPSs from TG rickettsiae and P. vulgaris OX19.

  5. A protein complex from Choristoneura fumiferana gut-juice involved in the precipitation of delta-endotoxin from Bacillus thuringiensis subsp. sotto.

    PubMed

    Milne, R E; Pang, A S; Kaplan, H

    1995-12-01

    A 75 kDa protein from spruce budworm (Choristoneura fumiferana) gut-juice has been isolated and shown to cause a specific precipitation of the delta-endotoxin from Bacillus thuringiensis subsp. sotto. This 75 kDa protein, separated by either column chromatography or SDS-PAGE, caused precipitation of the sotto toxin in both agarose diffusion gels and the PAGE gels. The precipitation event leads to limited proteolysis of the toxin and loss of larval toxicity. SDS-PAGE analysis of the precipitated toxin indicates that proteolysis of the toxin is not a prerequisite for precipitation. The protein responsible for precipitation, exhibits elastase-like activity and appears to be a complex which partially dissociates during boiling in SDS-PAGE sample buffer. Gut-juice from gypsy moth, forest tent caterpillar and white mark tussock moth also precipitated delta-endotoxin, but silkworm gut-juice gave a much weaker response. These results provide further evidence that, in the larval gut, differential processing of delta-endotoxin may play a role in the expression of activity towards various insect larvae.

  6. Chip electrophoresis as a novel approach to measure the polyphenols reactivity toward human saliva.

    PubMed

    Rinaldi, Alessandra; Iturmendi, Néréa; Gambuti, Angelita; Jourdes, Michael; Teissedre, Pierre-Louis; Moio, Luigi

    2014-06-01

    Saliva is a biological fluid with a multifunctional role that makes it interesting in terms of research and diagnostic possibilities. In food research, human saliva represented a useful tool by which we measure the tactile sensation elicited by polyphenol-rich beverages called astringency. A method based on SDS-PAGE analysis of saliva before and after the binding reaction with wine polyphenols has been successfully used in previous studies for measuring wine astringency by means of the saliva precipitation index. In this work, chip electrophoresis was used alternatively to SDS-PAGE and results were compared. Chip electrophoresis provides a very good reproducibility for wine and grape astringency. Moreover, this approach is much faster than the conventional SDS-PAGE method requiring several hours for an analysis. Another advantage over traditional gel is lower sample and reagent volume requirements, as well as the lower and less toxic wastes, contributing benefits to health and environment. The application of this novel method allowed, using the principal component analysis, to distinguish grapes and wines according to the saliva precipitation index and structural characteristics determined by the phoroglucinolysis analysis.

  7. Characterization of seeds of selected wild species of rice (Oryza) stored under high temperature and humidity conditions.

    PubMed

    Das, Smruti; Nayak, Monalisa; Patra, B C; Ramakrishnan, B; Krishnan, P

    2010-06-01

    Wild progenitors of rice (Oryza) are an invaluable resource for restoring genetic diversity and incorporating useful traits back into cultivars. Studies were conducted to characterize the biochemical changes, including SDS-PAGE banding pattern of storage proteins in seeds of six wild species (Oryza alta, O. grandiglumis, O. meridionalis, O. nivara, O. officinalis and O. rhizomatis) of rice stored under high temperature (45 degrees C) and humidity (approixmately 100%) for 15 days, which facilitated accelerated deterioration. Under the treated conditions, seeds of different wild rice species showed decrease in per cent germination and concentrations of protein and starch, but increase in conductivity of leachate and content of sugar. The SDS-PAGE analysis of seed proteins showed that not only the total number of bands, but also their intensity in terms of thickness differed for each species under storage. The total number of bands ranged from 11 to 22, but none of the species showed all the bands. Similarity index for protein bands between the control and treated seeds was observed to be least in O. rhizomatis and O. alta, while the indices were 0.7 and 0.625 for O. officinalis and O. nivara, respectively. This study clearly showed that seed deterioration led to distinctive biochemical changes, including the presence or absence as well as altered levels of intensity of proteins. Hence, SDS-PAGE protein banding pattern can be used effectively to characterize deterioration of seeds of different wild species of rice.

  8. Studies on bioprospecting potential of a gastropod mollusc Cantharus tranquebaricus (Gmelin, 1791)

    PubMed Central

    Sarumathi, G; Arumugam, M; Kumaresan, S; Balasubramanian, T

    2012-01-01

    Objective To study the biological activities of the tissue extract of Cantharus tranquebaricus (C. tranquebaricus). Methods Crude extract of gastropod was tested for inhibition of bacterial growth. Antibacterial assay was carried out by disc diffusion method and the activity was measured accordingly based on the inhibition zone around the disc impregnated with gastropod extract. Molecular weight of the extract was determined by using SDS-PAGE. Plasma coagulation, Fibrin plate assay and substrate SDS-PAGE were used to determine the effect of sample on plasma coagulation, fibrin (ogen) olytic and proteolytic activity. Results The maximum inhibition zone (10 mm) was observed against Vibrio cholera (V. cholera) and minimum inhibition zone (2 mm) was noticed against Proteus mirablis (P. mirablis). The molecular weight was determined as 47-106 kDa. The tissue extract shows proteolytic activity above 48 kDa. SDS-PAGE analysis of fibrinogen after incubation with the tissue extract showed fibrinogenolytic activity. In plasma coagulation assay C. tranquebaricus tissue extract showed procoagulant property and it coagulated chicken plasma within 150 s, while control took 5 min to clot. The 9 HU hemolytic units were found against chicken blood and also exhibit high level of brine shrimp lethality. Conclusions This study suggests that C. tranquebaricus could be used as potential source for isolating bioactive compounds, since it is explored first time and found with promising results. PMID:23569843

  9. Accounting for adjuvant-induced artifacts in the characterization of vaccine formulations by polyacrylamide gel electrophoresis.

    PubMed

    Jakob, Virginie; Brunner, Livia; Barnier-Quer, Christophe; Blust, Molly; Collin, Nicolas; Carter, Lauren; Carter, Darrick; Rausch, Kelly M; Fox, Christopher B

    2017-04-01

    Several vaccine adjuvants comprise complex nano- or micro-particle formulations, such as oil-in-water emulsions. In order to characterize interactions and compatibility of oil-in-water emulsion adjuvants with protein antigens in vaccines, effective protein characterization methods that can accommodate potential interference from high concentrations of lipid-based particles are needed. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is a standard protein characterization technique which is affected by the presence of adjuvants such as oil-in-water emulsions. In this article, we investigate variations in SDS-PAGE methods that result in a reduction of adjuvant-induced staining artifacts. We have investigated whether the SDS method or the adjuvant composition were the reason for these artifacts and succeeded in reducing the artifacts with a modified sample preparation and different staining procedures. The best results were obtained by using gold staining or silver staining instead of a Coomassie Blue staining procedure. Moreover, the replacement of the dilution buffer (20% SDS to disrupt emulsion) by alternative detergents such as Tween® 80 and Triton® X-100 removed adjuvant-induced streaking artifacts at the top of the gel. These methods may be useful for improving characterization approaches of antigen-adjuvant mixtures by SDS-PAGE.

  10. Polar Residues in Transmembrane Helices can Decrease Electrophoretic Mobility in Polyacrylamide Gels Without Causing Helix Dimerization

    PubMed Central

    Walkenhorst, William F.; Merzlyakov, Mikhail; Hristova, Kalina; Wimley, William C.

    2010-01-01

    There are only a few available methods to study lateral interactions and self assembly of transmembrane helices. One of the most frequently used methods is sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) which can report on strong interactions between peptides in SDS solution. Here we offer a cautionary tale about studying the folding and assembly of membrane proteins using peptides and SDS-PAGE experiments as membrane mimetic systems. At least for the specific peptide and detergent systems studied here, we show that a polar asparagine residue in the 12th position of an otherwise hydrophobic helical segment of 20 amino acids causes a peptide to migrate on SDS-PAGE gels with an apparent molecular weight that is twice its true molecular weight, suggesting dimerization. However when examined carefully in SDS solutions and in situ in the polyacrylamide gel itself using Forster resonance energy transfer no interaction can be detected. Instead we show evidence suggesting that differential interactions between peptide and detergent drive the differences in electrophoretic mobility without any interaction between peptides. These results emphasize the need to apply multiple independent techniques to the study of membrane protein folding, and they highlight the usefulness of studying folding and structure of membrane proteins in lipid membranes rather than in detergents. PMID:19265670

  11. A streamlined Western blot exercise: An efficient and greener approach in the laboratory classroom.

    PubMed

    Ness, Traci L; Robinson, Rebekah L; Mojadedi, Wais; Peavy, Lydia; Weiland, Mitch H

    2015-01-01

    SDS-PAGE and western blotting are two commonly taught protein detection techniques in biochemistry and molecular biology laboratory classrooms. A pitfall associated with incorporating these techniques into the laboratory is the significant wait times that do not allow students to obtain timely results. The waiting associated with SDS-PAGE comes from staining and destaining, whereas with western blotting it is the times required for antibody incubations and the numerous wash steps. This laboratory exercise incorporates 2,2,2-trichloroethanol (TCE) into the SDS-PAGE gel allowing for visualization of migrated proteins in a matter of minutes, saving both the time and chemical waste associated with traditional Coomassie staining. Additionally, TCE staining does not affect protein transfer eliminating the requirement for duplicated gels for total protein and western analyses. Protein transfer can be confirmed immediately without the use of Ponceau S staining. Lastly, this western blot procedure has been further shortened by using an HRP-conjugated primary antibody, which eliminates the secondary antibody incubation and washes, and uses a colorimetric detection to allow for visualization by students without the need for specialized equipment. © 2015 The International Union of Biochemistry and Molecular Biology.

  12. Intermonomer hydrogen bonds enhance GxxxG-driven dimerization of the BNIP3 transmembrane domain: roles for sequence context in helix-helix association in membranes

    PubMed Central

    Lawrie, Charles M.; Sulistijo, Endah S.; MacKenzie, Kevin R.

    2009-01-01

    We determined the sequence dependence of human BNIP3 transmembrane domain dimerization using the biological assay TOXCAT. Mutants in which intermonomer hydrogen bonds between Ser 172 and His 173 are abolished show moderate interaction, indicating that side chain hydrogen bonds contribute to dimer stability but are not essential to dimerization. Mutants in which a GxxxG motif composed of Gly 180 and Gly 184 has been abolished show little or no interaction, demonstrating the critical nature of the GxxxG motif to BNIP3 dimerization. These findings show that side chain hydrogen bonds can enhance the intrinsic dimerization of a GxxxG motif and that sequence context can control how hydrogen bonds influence helix-helix interactions in membranes. The dimer interface mapped by TOXCAT mutagenesis agrees closely with the interfaces observed in the NMR structure and inferred from mutational analysis of dimerization on SDS-PAGE, showing that the native dimer structure is retained in detergents. We show that TOXCAT and SDS-PAGE give complementary and consistent information about BNIP3 TMD dimerization: TOXCAT is insensitive to mutations that have modest effects on self-association in detergents but readily discriminates among mutations that completely disrupt detergent-resistant dimerization. The close agreement between conclusions reached from TOXCAT and SDS-PAGE data for BNIP3 suggests that accurate estimates of the relative effects of mutations on native state protein-protein interactions can be obtained even when the detergent environment is strongly disruptive. PMID:20026130

  13. Purification and characterization of C-Phycocyanin from cyanobacterial species of marine and freshwater habitat.

    PubMed

    Patel, Anamika; Mishra, Sandhya; Pawar, Richa; Ghosh, P K

    2005-04-01

    The present paper describes an efficient single step chromatographic method for purification of C-Phycocyanin from three cyanobacterial species, i.e., Spirulina sp. (freshwater), Phormidium sp. (marine water) and Lyngbya sp. (marine water). C-Phycocyanin from these cyanobacterial species was purified to homogeneity and some of their properties were investigated. The purification involves a multistep treatment of the crude extract by fractional precipitation with ammonium sulfate, followed by ion-exchange chromatography on DEAE-Sepharose CL-6B column. Pure C-Phycocyanin was finally obtained from Spirulina, Phormidium, and Lyngbya spp. with purity ratio (A620/A280) 4.42, 4.43, and 4.59, respectively, further the purity and homogeneity were confirmed by native and SDS-PAGE. The estimated molecular weights of purified C-PC from Spirulina, Phormidium, and Lyngbya spp. were 112, 131, and 81 kDa, respectively. SDS-PAGE of pure C-Phycocyanin yielded two bands corresponding to alpha and beta subunits. The results of SDS-PAGE demonstrate the same molecular weight of beta subunits (24.4 kDa) for all the three cyanobacterial species, whereas the molecular weight of the alpha subunit is different for all (17 kDa Spirulina sp., 19.1 kDa Phormidium sp., 15.2 kDa Lyngbya sp.). Thus, the C-Phycocyanin was characterized as (alphabeta)3 for Spirulina and Phormidium spp., while as (alphabeta)2 for Lyngbya sp.

  14. [An easy way to purify the inclusion body protein with high purity from prokaryotic expression cells].

    PubMed

    Liu, Rong; Zhong, Qin-Ping; Jiang, Ming-Sen; Dong, Hui-Fen

    2011-10-01

    To clone partial ORF of SjBMP and to construct the recombinant SjBMP-pET-28a(+) plasmids, and then to transform them into the competent cells E. coli BL21 (DE3), finally a positive clone was used to be induced by IPTG. The bacterial aggregates with target protein expressed as inclusion bodies were purified by the methods of Ni(2+)-NTA affinity purification under denaturation condition and SDS-PAGE gel extraction. The purified protein was used to immune rabbits and make antiserum against the SjBMP, and the antiserum were then used to identify the rSjBMP by Western blotting. The target protein obtained by Ni(2+)-NTA Agarose affinity purification was not pure with unspecific proteins, but the protein further purified by SDS-PAGE gel extraction and the dialysis bag horizontal electrophoresis was quite pure, and the recovery rate was more than 11.0%. Meanwhile, Western blotting was used to identify the recombinant SjBMP protein by antiserum, only a specific single strip appeared, which suggested the protein purified by this method kept its antigenicity, and could be used for common immunological studies. Therefore, the SDS-PAGE gel extraction combining with electroosmosis and dialysis recycling are good and easy to purify the inclusion body proteins.

  15. Electroelution without gel sectioning of proteins from sodium dodecyl sulfate-polyacrylamide gel electrophoresis: fluorescent detection, recovery, isoelectric focusing and matrix assisted laser desorption/ionization-time of flight of the electroeluate.

    PubMed

    Radko, Sergey P; Chang, Huan-Tsung; Zakharov, Sergey F; Bezrukov, Ludmila; Yergey, Alfred L; Vieira, Nancy E; Chrambach, Andreas; Chen, Huan-Tsung

    2002-04-01

    A method of direct electroelution of intact proteins, without gel sectioning and orthogonal to the orientation of electrophoretic migration, was developed in application to Novex gels, using a simple home-made experimental setup. Six model proteins covering the molecular mass range of 14-120 kDa were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), stained with an aqueous solution of the fluorescent dye, SYPRO-red, and electroeluted from the intact gel. The sensitivity of visual detection was 0.1-0.2 microg upon illumination by a green laser and 0.5-1 microg of protein on side-ways UV-illumination. Duration (for each protein) and field strength were optimized to render protein electroelution from the gel near-quantitative (above 80%) and relatively fast (1-12 min at 1 kV). At a given field strength, the optimal duration was found to be inversely proportional to the mobility of proteins in SDS-PAGE. Sequential ultrafiltration was evaluated as a simple approach to concentrate electroeluted proteins and deplete SDS to a level compatible with mass spectrometry of proteins: protein yields in the electroeluate were 25-33% (depending on the protein used) after three steps of ultrafiltration with water. The analysis of the electroeluate by isoelectric focusing in an immobilized pH gradient, to reveal protein heterogeneity under a single SDS-PAGE band (prior, e.g., to mass spectrometric analysis), was demonstrated.

  16. Purification of a secreted lectin from Andrias davidianus skin and its antibacterial activity.

    PubMed

    Qu, Min; Tong, Changqing; Kong, Liang; Yan, Xin; Chernikov, Oleg V; Lukyanov, Pavel A; Jin, Qiao; Li, Wei

    2015-01-01

    A lectin secreted from Andrias davidianus skin (ADL) was purified by affinity chromatography on porcine stomach mucin (type III) (PSM)-crosslinked albumin, followed by gel filtration on Sephadex G-100 and HPLC on TSK gel G3000PWXL. The purified lectin was found to be a dimeric protein, as revealed by SDS-PAGE and MALDI-TOF analysis. SDS-PAGE showed that the ADL protein had a molecular mass of 17 kDa. ADL produced an 8.5 kDa band when examined using SDS-PAGE under reducing conditions. ADL agglutinated native and trypsinized human B erythrocytes. The hemagglutination activity was inhibited by glycoproteins, such as PSM and asialo-PSM, but not by any of the monosaccharides tested. The activity was stable between 4 °C and 50 °C. Significant ADL activity was observed between pH 4–5. The lectin reaction did not depend on the presence of the divalent cation Ca2+ or Mg2+. The N-terminal ADL sequence was determined to be VGYTVGATPM. The lectin exhibited antibacterial activity, involving growth and respiration inhibition in Escherichia coli, Enterobacter aerogenes, Staphylococcus aureus, Bacillus subtilis and Shewanella sp. Furthermore, ADL showed inhibition activity against the yeast Saccharomyces cerevisiae. These findings suggest that ADL plays an important role in the innate immunity of A. davidianus on the body surface.

  17. Isolation and characterisation of a cysteine protease (phytolacain G), from Phytolacca americana roots.

    PubMed

    Sussner, Ursula; Abel, Gudrun; Schulte, Ralf; Kreis, Wolfgang

    2004-10-01

    Protein extracts obtained from dried and fresh roots of Phytolacca americana L. (Phytolaccaceae) were examined in order to identify and characterise individual proteins. The extracts were compared with a commercial pokeweed mitogen standard using SDS polyacrylamide gel electrophoresis (SDS-PAGE). A dominant protein, present in both the extracts and the pokeweed mitogen standard, was isolated by subsequent ammonium sulphate fractionation, anion exchange chromatography, gel filtration and SDS-PAGE. In this way it was purified 140-fold with about 20 % yield and 70-fold with about 13 % yield from dried and fresh roots, respectively. Its molecular mass as determined by gel filtration and SDS-PAGE was estimated to be about 25 kDa. Subsequent isoelectric focussing revealed one single protein band at pH 6.0. LysC digestion of the 25 kDa protein yielded several peptides which were subjected to micro-sequencing. Comparison with published sequences revealed that the protein isolated was phytolacain G, a cysteine protease previously isolated from unripe fruits of P. americana L. The enzyme showed a high affinity towards the oxidised insulin B-chain and was completely inhibited by trans-epoxysuccinyl- L-leucylamido(4-guanidino)-butane (E64). The purified phytolacain G showed "lectin-like" activities such as haemagglutination and mitogenic effects towards human lymphocytes.

  18. Detection and identification of Vibrio species using whole-cell protein pattern analysis.

    PubMed

    Lee, Chae-Yoon; Hong, Yeun; Ryu, Jio; Kim, Young-Rok; Oh, Sang-Suk; Lee, Soon-Ho; Hwang, In-Gyun; Kim, Hae-Yeong

    2012-08-01

    Outbreaks of foodborne diseases associated with Vibrio species such as V. parahaemolyticus, V. vulnificus, and V. cholerae frequently occur in countries having a dietary habit of raw seafood consumption. For rapid identification of different Vibrio species involved in foodborne diseases, whole-cell protein pattern analysis for 13 type strains of 12 Vibrio species was performed using SDS-PAGE analysis. Pathogenic Vibrio species such as V. parahaemolyticus, V. vulnificus, V. cholerae, V. alginolyticus, V. fluvialis, and V. mimicus were included in the 12 Vibrio species used in this study. Each of the 12 Vibrio species showed clearly specific band patterns of its own. Two different strains of V. parahaemolyticus showed two different SDS-PAGE wholecell protein patterns, giving the possibility of categorizing isolated strains in the same V. parahaemolyticus species into two subgroups. The 36 Vibrio isolates collected from sushi restaurants in Busan were all identified as V. parahaemolyticus by comparing their protein patterns with those of Vibrio type strains. The identified isolates were categorized into two different subgroups of V. parahaemolyticus. The whole-cell protein pattern analysis by SDS-PAGE can be used as a specific, rapid, and simple identification method for Vibrio spp. involved in foodborne diseases at the subspecies level.

  19. Enzymatic properties of chitinase-producing antagonistic bacterium Paenibacillus chitinolyticus with various substrates.

    PubMed

    Song, Yong-Su; Seo, Dong-Jun; Ju, Wan-Taek; Lee, Yong-Seong; Jung, Woo-Jin

    2015-12-01

    Various chitin substrates were used to investigate the properties of enzymes produced from the chitinase-producing bacterium Paenibacillus chitinolyticus MP-306 against phytopathogens. The MP-306 bacterium was incubated in nine culture media [crab shell powder chitin (CRS), chitin-protein complex powder (CPC), carboxymethyl-chitin powder (CMC), yeast extract only (YE), LB (Trypton, NaCl, and yeast extract), GT (Trypton, NaCl, and glucose), crab shell colloidal chitin (CSC), squid pen powder chitin (SPC), and cicada slough powder chitin (CSP)] at 30 °C for 3 days. Chitinase isozymes in CPC medium were expressed strongly as CN1, CN2, CN3, CN4, CN5, and CN6 bands on native-PAGE gels. Chitinase isozymes in CPC and CMC medium were expressed as 13 bands (CS1-CS13) on SDS-PAGE gels. Chitinase isozymes were expressed strongly on SDS-PAGE gels as two bands (CS6 and CS8) on YE and LB medium and 13 bands (CS1-CS13) on SPC medium. In crude enzyme, chitinase isozymes at pH 7 and pH 9 in chitin media appeared strongly on SDS-PAGE gels. Partial purified enzyme indicated high stability of enzyme activity at various temperatures and pHs in chitin medium, while these enzymes indicated low activity staining of enzyme on electrophoresis gels at various temperatures and pHs condition of chitin medium. Copyright © 2015 Elsevier Ltd. All rights reserved.

  20. Accounting for adjuvant-induced artifacts in the characterization of vaccine formulations by polyacrylamide gel electrophoresis

    PubMed Central

    Jakob, Virginie; Brunner, Livia; Barnier-Quer, Christophe; Blust, Molly; Collin, Nicolas; Carter, Lauren; Carter, Darrick; Rausch, Kelly M.; Fox, Christopher B.

    2017-01-01

    Objectives: Several vaccine adjuvants comprise complex nano- or micro-particle formulations, such as oil-in-water emulsions. In order to characterize interactions and compatibility of oil-in-water emulsion adjuvants with protein antigens in vaccines, effective protein characterization methods that can accommodate potential interference from high concentrations of lipid-based particles are needed. Methods: Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is a standard protein characterization technique which is affected by the presence of adjuvants such as oil-in-water emulsions. In this article, we investigate variations in SDS-PAGE methods that result in a reduction of adjuvant-induced staining artifacts. We have investigated whether the SDS method or the adjuvant composition were the reason for these artifacts and succeeded in reducing the artifacts with a modified sample preparation and different staining procedures. Results: The best results were obtained by using gold staining or silver staining instead of a Coomassie Blue staining procedure. Moreover, the replacement of the dilution buffer (20% SDS to disrupt emulsion) by alternative detergents such as Tween® 80 and Triton® X-100 removed adjuvant-induced streaking artifacts at the top of the gel. Conclusions: These methods may be useful for improving characterization approaches of antigen–adjuvant mixtures by SDS-PAGE. PMID:28515938

  1. Two-dimensional blue native polyacrylamide gel electrophoresis.

    PubMed

    Schamel, Wolfgang W A

    2008-03-01

    Multiprotein complexes play crucial roles in nearly all cell biological processes. Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE) is a powerful method to study these complexes. It is a native protein separation method that relies on the dye Coomassie blue to confer negative charge for separation. It has a higher resolution than gel filtration or sucrose density ultracentrifugation and can be used for protein complexes from 10 kDa to 10 MDa. If a second-dimension SDS-PAGE is applied (two-dimensional BN/SDS-PAGE), the size, subunit composition, and relative abundance of the different multiprotein complexes can be studied. In recent years, there has been a large increase in the number of publications where BN-PAGE was used to study protein-protein interactions. Here, we give detailed protocols for the separation of multiprotein complexes by two-dimensional BN/SDS-PAGE and for a related technique to determine the stoichiometry of these complexes. (c) 2008 by John Wiley & Sons, Inc.

  2. Analysis of proteins stained by Alexa dyes.

    PubMed

    Huang, Shijun; Wang, Houyi; Carroll, Christopher A; Hayes, Shirley J; Weintraub, Susan T; Serwer, Philip

    2004-03-01

    Alexa dye staining of proteins is used for the fluorescence microscopy of single particles that are sometimes multimolecular protein complexes. To characterize the staining, post-staining determination must be made of which protein(s) in a complex have been Alexa-stained. The present communication describes the use of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for performing this determination. The Alexa-stained proteins are observed directly in gels by illumination with an ultraviolet transilluminator. The test multimolecular particle is bacteriophage T7. The protein capsid of T7 is a multimolecular complex that has both external and internal proteins. SDS-PAGE of Alexa-stained bacteriophage T7 produces fluorescent capsid proteins each of which usually comigrates with an unstained protein. However, one Alexa-induced modification of protein migration was observed by SDS-PAGE. Mass spectrometry shows that the protein with modified migration is the major protein of the outer shell of the T7 capsid. The procedures used are generally applicable. The distribution of Alexa staining among T7 capsid proteins depends on the size of the dye molecule used. The larger the dye molecule is, the greater the preference for external proteins.

  3. Sodium dodecyl sulfate-polyacrylamide gel protein electrophoresis of freshwater photosynthetic sulfur bacteria.

    PubMed

    Osuna, M Begoña; Casamayor, Emilio O

    2011-01-01

    Sodium dodecyl sulfate-polyacrylamide gel protein electrophoresis (SDS-PAGE) was carried out using different bacterial strains of the photosynthetic sulfur bacteria Chlorobium, Thiocapsa, Thiocystis, and Chromatium cultured in the laboratory, and the natural blooms in two karstic lakes (Lake Cisó and Lake Vilar, NE Spain) where planktonic photosynthetic bacteria (purple and green sulfur bacteria) massively developed accounting for most of the microbial biomass. Several extraction, solubilization, and electrophoresis methods were tested to develop an optimal protocol for the best resolution of the SDS-PAGE. Protein composition from different water depths and at different times of the year was visualized within a molecular mass range between 100 and 15 kDa yielding up to 20 different protein bands. Protein banding patterns were reproducible and changed in time and with depth in agreement with changes in photosynthetic bacteria composition. When a taxonomically stable community was followed in time, differences were observed in the intensity but not in the composition of the SDS-PAGE banding pattern. Three environmental variables directly related to the activity of sulfur bacteria (light, oxygen, and sulfide concentrations) had a significant effect on protein banding patterns and explained 33% of the variance. Changes in natural protein profiles of the bacterial blooms agreed with changes in species composition and in the in situ metabolic state of the populations.

  4. A novel approach to the identification of T-cell epitopes in Mycobacterium tuberculosis using human T-lymphocyte clones.

    PubMed Central

    Lamb, J R; Young, D B

    1987-01-01

    Current approaches to the analysis of antigens involved in the cellular immune response to mycobacterial infection rely on the initial identification and isolation of molecular components using monoclonal antibodies. In order to overcome the constraints of this approach, we have utilized a procedure involving T-cell recognition of antigens fractionated by polyacrylamide gel electrophoresis (SDS-PAGE) and added to proliferation assays after blotting onto nitrocellulose membranes. Analysis of human T-cell responses to Mycobacterium tuberculosis and Mycobacterium bovis BCG by this procedure revealed distinctive patterns of reactivity to different molecular weight components indicative of the selective recognition of immunodominant and species-specific determinants. Human T-cell clones were subsequently derived, and SDS-PAGE immunoblotting was used to identify the antigen recognized by each clone. Three epitopes defined by individual T-cell clones were identified on separate polypeptides with molecular weights 16,000-18,000 (clone P53), 18,000-20,000 (clone P57) and 52,000-55,000 (clone P35). This study demonstrates the potential application of T-cell cloning in conjunction with SDS-PAGE immunoblotting for the dissection and analysis of the cellular immune response to pathogenic agents during human infection. Images Figure 1 PMID:2434413

  5. Evaluating Por Nuestra Salud: A Feasibility Study.

    PubMed

    Marshall, Brenda; Gonzales, Gustavo; Kernan, William

    2016-01-01

    This feasibility study evaluated the impact of a culturally consistent diabetes educational program, led by a Latino pastor, on Latino community diabetes knowledge levels. Latino adults were recruited from two churches serving Latino populations, one identified as intervention, the other as nonintervention. Both churches received the American Diabetes Association's booklets on diabetes education, "Four Steps to Control Your Diabetes for Life." The intervention group also received weekly reviews of the booklet's information from the church pastor, who was educated about diabetes by a Latino family nurse practitioner. Pre- and postintervention levels of diabetes knowledge were measured using the Diabetes Knowledge Questionnaire. Mean changes in the Diabetes Knowledge Questionnaire (DKQ-24) survey from pretest to posttest indicated a significant increase in knowledge in both groups. Only the grades for the participants from the pastor-led group, however, moved from failure in knowledge to high levels of passing. One hundred percent of intervention group participants reported having read the literature. This pilot study supports the feasibility of a local Latino pastor, as a culturally consistent diabetes educator, to increase Latino parishioner's knowledge of diabetes. © 2015 Society for Public Health Education.

  6. Characterization of invasive Neisseria meningitidis from Atlantic Canada, 2009 to 2013: With special reference to the nonpolysaccharide vaccine targets (PorA, factor H binding protein, Neisseria heparin-binding antigen and Neisseria adhesin A)

    PubMed Central

    Tsang, Raymond SW; Law, Dennis KS; Gad, Rita R; Mailman, Tim; German, Gregory; Needle, Robert

    2015-01-01

    BACKGROUND: Serogroup B Neisseria meningitidis (MenB) has always been a major cause of invasive meningococcal disease (IMD) in Canada. With the successful implementation of a meningitis C conjugate vaccine, the majority of IMD in Canada is now caused by MenB. OBJECTIVE: To investigate IMD case isolates in Atlantic Canada from 2009 to 2013. Data were analyzed to determine the potential coverage of the newly licensed MenB vaccine. METHODS: Serogroup, serotype and serosubtype antigens were determined from IMD case isolates. Clonal analysis was performed using multilocus sequence typing. The protein-based vaccine antigen genes were sequenced and the predicted peptides were investigated. RESULTS: The majority of the IMD isolates were MenB (82.5%, 33 of 40) and, in particular, sequence type (ST)-154 B:4:P1.4 was responsible for 47.5% (19 of 40) of all IMD case isolates in Atlantic Canada. Isolates of this clone expressed the PorA antigen P1.4 and possessed the nhba genes encoding for Neisseria heparin-binding antigen peptide 2, which together matched exactly with two of the four components of the new four-component meningococcal B vaccine. Nineteen MenB isolates had two antigenic matches, another five MenB and one meningitis Y isolate had one antigenic match. This provided 75.8% (25 of 33) potential coverage for MenB, or a 62.5% (25 of 40) overall potential coverage for IMD. CONCLUSION: From 2009 to 2013, IMD in Atlantic Canada was mainly caused by MenB and, in particular, the B:4:P1.4 ST-154 clone, which accounted for 47.5% of all IMD case isolates. The new four-component meningococcal B vaccine appeared to offer adequate coverage against MenB in Atlantic Canada. PMID:26744586

  7. Characterization of fHbp, nhba (gna2132), nadA, porA, sequence type (ST), and genomic presence of IS1301 in group B meningococcal ST269 clonal complex isolates from England and Wales.

    PubMed

    Lucidarme, Jay; Comanducci, Maurizio; Findlow, Jamie; Gray, Stephen J; Kaczmarski, Edward B; Guiver, Malcolm; Kugelberg, Elisabeth; Vallely, Pamela J; Oster, Philipp; Pizza, Mariagrazia; Bambini, Stefania; Muzzi, Alessandro; Tang, Christoph M; Borrow, Ray

    2009-11-01

    Highly effective glycoconjugate vaccines exist against four of the five major pathogenic groups of meningococci: A, C, W-135, and Y. An equivalent vaccine against group B meningococci (menB) has remained elusive due to the poorly immunogenic capsular polysaccharide. A promising alternative, the investigational recombinant menB (rMenB)- outer membrane vesicle (OMV) vaccine, contains fHBP, NHBA (previously GNA2132), NadA, and outer membrane vesicles (OMVs) from the New Zealand MeNZB vaccine. MenB currently accounts for 90% of meningococcal disease in England and Wales, where the multilocus sequence type (ST) 269 (ST269) clonal complex (cc269) has recently expanded to account for a third of menB cases. To assess the potential cc269 coverage of the rMenB-OMV vaccine, English and Welsh cc269 isolates from the past decade were genetically characterized with respect to fHBP, NHBA, and NadA. All of the isolates harbored fHbp and nhba alleles, while 98% of the cc269 isolates were devoid of nadA. Subvariant profiling of fHbp, nhba, and porA against STs revealed the presence of two broadly distinct and well-defined clusters of isolates, centered around ST269 and ST275, respectively. An additional molecular marker, insertion sequence IS1301, was found to be present in 100% and <2% of isolates of the respective clusters. On the basis of the genetic data, the potential rMenB-OMV coverage of cc269 in England and Wales is high (up to 100%) within both clusters. Expression studies and serum bactericidal antibody assays will serve to enhance predictions of coverage and will augment ongoing studies regarding the significance of IS1301 within the ST269 cluster.

  8. Characterization of fHbp, nhba (gna2132), nadA, porA, Sequence Type (ST), and Genomic Presence of IS1301 in Group B Meningococcal ST269 Clonal Complex Isolates from England and Wales▿

    PubMed Central

    Lucidarme, Jay; Comanducci, Maurizio; Findlow, Jamie; Gray, Stephen J.; Kaczmarski, Edward B.; Guiver, Malcolm; Kugelberg, Elisabeth; Vallely, Pamela J.; Oster, Philipp; Pizza, Mariagrazia; Bambini, Stefania; Muzzi, Alessandro; Tang, Christoph M.; Borrow, Ray

    2009-01-01

    Highly effective glycoconjugate vaccines exist against four of the five major pathogenic groups of meningococci: A, C, W-135, and Y. An equivalent vaccine against group B meningococci (menB) has remained elusive due to the poorly immunogenic capsular polysaccharide. A promising alternative, the investigational recombinant menB (rMenB)- outer membrane vesicle (OMV) vaccine, contains fHBP, NHBA (previously GNA2132), NadA, and outer membrane vesicles (OMVs) from the New Zealand MeNZB vaccine. MenB currently accounts for 90% of meningococcal disease in England and Wales, where the multilocus sequence type (ST) 269 (ST269) clonal complex (cc269) has recently expanded to account for a third of menB cases. To assess the potential cc269 coverage of the rMenB-OMV vaccine, English and Welsh cc269 isolates from the past decade were genetically characterized with respect to fHBP, NHBA, and NadA. All of the isolates harbored fHbp and nhba alleles, while 98% of the cc269 isolates were devoid of nadA. Subvariant profiling of fHbp, nhba, and porA against STs revealed the presence of two broadly distinct and well-defined clusters of isolates, centered around ST269 and ST275, respectively. An additional molecular marker, insertion sequence IS1301, was found to be present in 100% and <2% of isolates of the respective clusters. On the basis of the genetic data, the potential rMenB-OMV coverage of cc269 in England and Wales is high (up to 100%) within both clusters. Expression studies and serum bactericidal antibody assays will serve to enhance predictions of coverage and will augment ongoing studies regarding the significance of IS1301 within the ST269 cluster. PMID:19759227

  9. Chlamydia trachomatis clinical isolates identified as tetracycline resistant do not exhibit resistance in vitro: whole-genome sequencing reveals a mutation in porB but no evidence for tetracycline resistance genes.

    PubMed

    O'Neill, C E; Seth-Smith, H M B; Van Der Pol, B; Harris, S R; Thomson, N R; Cutcliffe, L T; Clarke, I N

    2013-04-01

    Chlamydia trachomatis is the most common bacterial sexually transmitted infection worldwide and the leading cause of preventable blindness in developing countries. Tetracycline is commonly the drug of choice for treating C. trachomatis infections, but cases of antibiotic resistance in clinical isolates have previously been reported. Here, we used antibiotic resistance assays and whole-genome sequencing to interrogate the hypothesis that two clinical isolates (IU824 and IU888) have acquired mechanisms of antibiotic resistance. Immunofluorescence staining was used to identify C. trachomatis inclusions in cell cultures grown in the presence of tetracycline; however, only antibiotic-free control cultures yielded the strong fluorescence associated with the presence of chlamydial inclusions. Infectivity was lost upon passage of harvested cultures grown in the presence of tetracycline into antibiotic-free medium, so we conclude that these isolates were phenotypically sensitive to tetracycline. Comparisons of the genome and plasmid sequences for the two isolates with tetracycline-sensitive strains did not identify regions of low sequence identity that could accommodate horizontally acquired resistance genes, and the tetracycline binding region of the 16S rRNA gene was identical to that of the sensitive control strains. The porB gene of strain IU824, however, was found to contain a premature stop codon not previously identified, which is noteworthy but unlikely to be related to tetracycline resistance. In conclusion, we found no evidence of tetracycline resistance in the two strains investigated, and it seems most likely that the small, aberrant inclusions previously identified resulted from the high chlamydial load used in the original antibiotic resistance assays.

  10. Unprecedented peroxidase-like activity of Rhodnius prolixus nitrophorin 2: identification of the [FeIV=O Por•]+ and [FeIV=O Por](Tyr38•) intermediates and their role(s) in substrate oxidation.

    PubMed

    Singh, Rahul; Berry, Robert E; Yang, Fei; Zhang, Hongjun; Walker, F Ann; Ivancich, Anabella

    2010-10-19

    We have identified a novel enzymatic reaction for nitrophorin 2 (NP2), a heme protein previously characterized as a nitric oxide carrier in the saliva of the Rhodnius prolixus insect. NP2 exhibited levels of peroxidase activity comparable to those of the bifunctional peroxidases (KatGs), despite their heme pocket structural differences (heme ruffling, Tyr38 and Tyr85 in hydrogen bonding interactions with the propionates in NP2). The intermediates of the peroxidase-like reaction of NP2 were identified by Electron Paramagnetic Resonance (EPR) and electronic absorption spectroscopies. The EPR spectrum consistent with an [Fe(IV)=O Por•]+ species was detected at pH <7. At pH ≥ 7, the change from a strong to a weak antiferromagnetic coupling interaction for the [Fe(IV)=O Por•]+ species was accompanied by the subsequent formation of an [Fe(IV)=O Por](Tyr•) intermediate. Tyr38 was shown to be the unique naturally occurring radical site in NP2. The Y38F mutant stabilized the radical on the tyrosine in hydrogen-bonding interaction with the other heme propionate (Tyr85). Kinetic studies using stopped-flow electronic absorption spectrophotometry revealed that the [Fe(IV)=O Por•]+ species reacts with histamine and norepinephrine in a peroxidase-like manner. Our findings demonstrate that NP2 has pH-dependent dual function: at the acidic pH of the insect saliva the protein behaves as a NO carrier, and, if exposed to the higher pH of the tissues and capillaries of the host, NP2 is able to bind histamine or it can efficiently inactivate norepinephrine through a peroxidase-like reaction, in the presence of hydrogen peroxide. Accordingly, the unprecedented peroxidase-like activity of NP2 is concluded to be a key biological function.

  11. Personal Docente del Nivel Primario. Series Estadisticas Basicas: Colombia (Teaching Personnel in Primary Schools. Basic Statistics Series: Colombia).

    ERIC Educational Resources Information Center

    Ministerio de Educacion Nacional, Bogota (Colombia). Instituto Colombiano de Pedagogia.

    This document provides statistical data on the distribution and education of teacher personnel working in Colombian elementary schools between 1940 and 1968. The statistics cover the number of men and women, public and private schools, urban and rural location, and the amount of education of teachers. (VM)

  12. Descripcion y Medida del Bilinguismo a Nivel Colectivo. [Description and Measurement of Bilingualism at the Collective Level

    ERIC Educational Resources Information Center

    Siguan, Miguel

    1976-01-01

    A presentation of a rigorous method allowing an accurate description of collective bilingualism in any given population, including both the speaker's degree of language command and the patterns of linguistic behavior in each of the languages. [In Spanish] (NQ)

  13. Otros amigos, otras culturas: Libro de lectura 2, nivel 2 (Other Friends, Other Cultures: Reader 2, Level 2).

    ERIC Educational Resources Information Center

    Martinez, Emiliano; And Others

    This reading textbook was designed for the elementary school Spanish-speaking student. It contains short passages which provide an initiation into knowledge of other countries and ways of life to encourage development of the senses of friendship, curiosity, and solidarity. See FL 004 069 for the accompanying workbook. (Author/SK)

  14. Las Matematicas: Lenguaje Universal. Nivel 2c: Factores y Multiplos (Mathematics: A Universql Language. Level 2c: Factors and Multiples).

    ERIC Educational Resources Information Center

    Dissemination and Assessment Center for Bilingual Education, Austin, TX.

    This is one of a series of student booklets designed for use in a bilingual mathematics program in grades 6-8. The general format is to present each page in both Spanish and English. The mathematical topics in this booklet include factors, prime and composite numbers, divisibility, and exponents. (MK)

  15. Las Matematicas: Lenguaje Universal. Nivel 1: Numeros y Numeracion (Mathematics: A Universal Language. Level 1: Numbers and Numeration).

    ERIC Educational Resources Information Center

    Dissemination and Assessment Center for Bilingual Education, Austin, TX.

    This is one of a series of student booklets designed for use in a bilingual mathematics program in grades 6-8. The general format is to present each page in both Spanish and English. The mathematical topics in this booklet include graphing on a number line, place value, using exponents, flow charts, and Roman numerals. (MK)

  16. Revenido de un acero bain?tico nanoestructurado: un an?lisis a nivel at?mico

    SciTech Connect

    Caballero, Francesca G.; Miller, Michael K; Mateo, C. Garcia

    2008-01-01

    New developments with bainite have resulted in a steel with an ultimate tensile strength of 1.7-2.5 GPa and a toughness in excess of 30-40 MPa m1/2. The novel microstructure is generated in a high carbon high silicon steel isothermally transformed at temperatures of 200 C-350 C. Iron does not diffuse during the transformation to bainite at this low temperature. As a result, 20-40 nm thick plates of ferrite are generated, giving rise to the exceptional properties. X-ray diffraction results indicated that the carbon concentration in the bainitic ferrite was much higher than that expected from equilibrium thermodynamics between austenite and ferrite. Atom probe tomography demonstrated that a substantial quantity of carbon (7.4 at. % C) was found to be trapped at dislocations in the vicinity of the ferrite/austenite interface. Carbon trapping at dislocations was found to prevent the decarburization of supersaturated ferrite and in some extent to alter the carbide precipitation sequence during low temperature bainite formation. These results provided the first direct observation of carbon Cottrell Atmospheres in bainitic ferrite. There are important differences in the tempering behavior of bainite and martensite. Much of the carbon precipitates as carbides or partitions from the ferrite to the remaining austenite during bainite formation. As a consequence the bainitic microstructure is less sensitive to additional tempering heat treatment. In contrast, the level of carbon in the bainitic ferrite of this novel bainitic steel is unusually high. Redistribution of alloying elements during tempering of this nanocrystalline steel has been analysed by atom probe tomography. Results suggest that retained austenite decomposes during tempering before full equilibrium is reached at the interface. Moreover, cementite precipitates from supersaturated ferrite via a paraequilibrium transformation mechanism.

  17. Las Matematicas: Lenguaje Universal. Nivel 2c: Factores y Multiplos (Mathematics: A Universql Language. Level 2c: Factors and Multiples).

    ERIC Educational Resources Information Center

    Dissemination and Assessment Center for Bilingual Education, Austin, TX.

    This is one of a series of student booklets designed for use in a bilingual mathematics program in grades 6-8. The general format is to present each page in both Spanish and English. The mathematical topics in this booklet include factors, prime and composite numbers, divisibility, and exponents. (MK)

  18. Las Matematicas: Lenguaje Universal. Nivel 1: Numeros y Numeracion (Mathematics: A Universal Language. Level 1: Numbers and Numeration).

    ERIC Educational Resources Information Center

    Dissemination and Assessment Center for Bilingual Education, Austin, TX.

    This is one of a series of student booklets designed for use in a bilingual mathematics program in grades 6-8. The general format is to present each page in both Spanish and English. The mathematical topics in this booklet include graphing on a number line, place value, using exponents, flow charts, and Roman numerals. (MK)

  19. Descripcion y Medida del Bilinguismo a Nivel Colectivo. [Description and Measurement of Bilingualism at the Collective Level

    ERIC Educational Resources Information Center

    Siguan, Miguel

    1976-01-01

    A presentation of a rigorous method allowing an accurate description of collective bilingualism in any given population, including both the speaker's degree of language command and the patterns of linguistic behavior in each of the languages. [In Spanish] (NQ)

  20. Institutos Superiores de Formacion Docente: Profesorado de Nivel Elemental (Higher Institutes for Teacher Training: Elementary School Teachers).

    ERIC Educational Resources Information Center

    Ministerio de Cultura y Educacion, Buenos Aires (Argentina). Centro National de Documentacion e Informacion Educativa.

    This booklet describes and explains the Argentine educational reform, instituted in March 1971, concerning the training of elementary school teachers in institutions of higher learning. The legislation cited here establishes areas of study and qualifications. The document also contains a discussion of previous teacher education and the bases and…