5,830 research outputs found

    Detailed characterization of the solution kinetics and thermodynamics of biotin, biocytin and HABA binding to avidin and streptavidin

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    The high affinity (KD ~ 10−15 M) of biotin for avidin and streptavidin is the essential component in a multitude of bioassays with many experiments using biotin modifications to invoke coupling. Equilibration times suggested for these assays assume that the association rate constant (kon) is approximately diffusion limited (109 M-1s-1) but recent single molecule and surface binding studies indicate that they are slower than expected (105 to 107 M-1s-1). In this study, we asked whether these reactions in solution are diffusion controlled, which reaction model and thermodynamic cycle describes the complex formation, and if there are any functional differences between avidin and streptavidin. We have studied the biotin association by two stopped-flow methodologies using labeled and unlabeled probes: I) fluorescent probes attached to biotin and biocytin; and II) unlabeled biotin and HABA, 2-(4’-hydroxyazobenzene)- benzoic acid. Both native avidin and streptavidin are homo-tetrameric and the association data show no cooperativity between the binding sites. The kon values of streptavidin are faster than avidin but slower than expected for a diffusion limited reaction in both complexes. Moreover, the Arrhenius plots of the kon values revealed strong temperature dependence with large activation energies (6–15 kcal/mol) that do not correspond to a diffusion limited process (3–4 kcal/mol). Accordingly, we propose a simple reaction model with a single transition state for non-immobilized reactants whose forward thermodynamic parameters complete the thermodynamic cycle, in agreement with previously reported studies. Our new understanding and description of the kinetics, thermodynamics, and spectroscopic parameters for these complexes will help to improve purification efficiencies, molecule detection, and drug screening assays or find new applications

    Knowledge and practices of tour guides in Cuzco on the prevention and treatment of traveler's diarrhea

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    Objectives: To describe the knowledge and practices among tour guides in Cuzco, Peru regarding prevention and treatment of traveler's diarrhea. Methods: The main tour guide association in Cuzco organized a mandatory re-certification course for tour guides in February 2004. We invited tour guides attending this course to participate in the study. Those aged 18 to 50 who had worked as a tour guide for at least one year were asked to complete a brief anonymous questionnaire. Results: A total of 173 questionnaires were returned; 137 met the inclusion criteria and were included in the analysis. The median age was 31 years (interquartile range (IQR): 28-34 years), and 56.7% were male. The median number of foreign languages spoken by subjects included was 1, being English (91.9%) the most common, followed by French (17.9%) and Italian (16.8%). The median time working as a tour guide was 4 years (IQR: 2-8 years). Tour guidance was a full-time job for 47.1% of the subjects, and for 82.4% Inca Trail was the most commonly covered route. Traveler's diarrhea was considered a food-borne disease by 85.4%, but only 60.6% considered it a water-borne disease. The majority of subjects identified raw salads (84.4%), cold sauces (81.5%) and tap water (81.1%) as risky products whereas hot soups (77 8%) and bread (75.0%) were mainly considered as safe. Most of the tour guides considered bloody stools (84.8%) and fever (60.6%) as indications to seek medical attention. The medications most frequently recommended by tour guides were oral re-hydration solutions ( 85.1%), trimethoprim-sulphamethoxazole (26.1%) and loperamide (20.1%). Conclusions: Tour guides have a basic knowledge about traveler's diarrhea. However, more training is necessary to improve management while trekking outside of Cuzco The recommendation to seek pharmacists should be particularly addressed

    Geomagnetic anomalies observed at volcano Popocatepetl, Mexico

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    International audienceResults of the ULF geomagnetic monitoring of the volcano Popocatepetl (Mexico) and their analysis are summarized and presented for the period 2003?2006. Our analysis reveals some anomalies which are considered to be of local volcanic origin: the EM background in the vicinity of the volcano was found to be significantly noisier than at other reference stations; sporadic strong noise-like geomagnetic activity was observed in the H-component; some geomagnetic pulsations were observed only at the Tlamacas station (located at 4 km near the volcano). The results are discussed in terms of a physical mechanism involving the presence of a second magmatic chamber within the volcano and, finally, further perspective directions to study volcanic geodynamical processes besides the traditional ones are given

    A step-up converter with large voltage gain and low voltage rating on capacitors

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    Step-up converters are widely used in many applications, such as renewable energy generation with photovoltaic panels and fuel cell stacks. In many cases, the required voltage gain is larger for those applications than a traditional boost converter can achieve. Several large-voltage gain converters have been recently studied. This paper introduces a converter topology in which the voltage gain is larger than a traditional boost converter. The main advantages of the proposed topology are: (i) it provides a large voltage gain without the use of an extreme duty cycle; (ii) its capacitors require a smaller voltage to be sustained compared with other, similar state-of-the-art converters; (iii) the voltage among the ground input and output is not pulsating; and (iv) it can be synthesized with commercial, off-the-shelf half-bridge packed transistors. The proposed converter can be employed in different applications, such as distributed generation and microgrids. This paper presents the steady-state analysis of the proposed converter in the continuous conduction mode, a short comparison with similar topologies, and their voltage on capacitors. Computer-based simulation results are provided to verify the principle of the proposed converter in different operating conditions

    Novel prokaryotic expression of thioredoxin-fused insulinoma associated protein tyrosine phosphatase 2 (IA-2), its characterization and immunodiagnostic application

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    Background The insulinoma associated protein tyrosine phosphatase 2 (IA-2) is one of the immunodominant autoantigens involved in the autoimmune attack to the beta-cell in Type 1 Diabetes Mellitus. In this work we have developed a complete and original process for the production and recovery of the properly folded intracellular domain of IA-2 fused to thioredoxin (TrxIA-2ic) in Escherichia coli GI698 and GI724 strains. We have also carried out the biochemical and immunochemical characterization of TrxIA-2icand design variants of non-radiometric immunoassays for the efficient detection of IA-2 autoantibodies (IA-2A). Results The main findings can be summarized in the following statements: i) TrxIA-2ic expression after 3 h of induction on GI724 strain yielded ≈ 10 mg of highly pure TrxIA-2ic/L of culture medium by a single step purification by affinity chromatography, ii) the molecular weight of TrxIA-2ic (55,358 Da) could be estimated by SDS-PAGE, size exclusion chromatography and mass spectrometry, iii) TrxIA-2ic was properly identified by western blot and mass spectrometric analysis of proteolytic digestions (63.25 % total coverage), iv) excellent immunochemical behavior of properly folded full TrxIA-2ic was legitimized by inhibition or displacement of [35S]IA-2 binding from IA-2A present in Argentinian Type 1 Diabetic patients, v) great stability over time was found under proper storage conditions and vi) low cost and environmentally harmless ELISA methods for IA-2A assessment were developed, with colorimetric or chemiluminescent detection. Conclusions E. coli GI724 strain emerged as a handy source of recombinant IA-2ic, achieving high levels of expression as a thioredoxin fusion protein, adequately validated and applicable to the development of innovative and cost-effective immunoassays for IA-2A detection in most laboratories.Fil: Guerra, Luciano Lucas. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; ArgentinaFil: Faccinetti, Natalia Ines. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; ArgentinaFil: Trabucchi, Aldana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; ArgentinaFil: Rovitto, Bruno David. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; ArgentinaFil: Sabljic, Adriana Victoria. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; ArgentinaFil: Poskus, Edgardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; ArgentinaFil: Iacono, Ruben Francisco. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; ArgentinaFil: Valdez, Silvina Noemi. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; Argentin

    Specificity of Transmembrane Protein Palmitoylation in Yeast

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    Many proteins are modified after their synthesis, by the addition of a lipid molecule to one or more cysteine residues, through a thioester bond. This modification is called S-acylation, and more commonly palmitoylation. This reaction is carried out by a family of enzymes, called palmitoyltransferases (PATs), characterized by the presence of a conserved 50- aminoacids domain called “Asp-His-His-Cys- Cysteine Rich Domain” (DHHC-CRD). There are 7 members of this family in the yeast Saccharomyces cerevisiae, and each of these proteins is thought to be responsible for the palmitoylation of a subset of substrates. Substrate specificity of PATs, however, is not yet fully understood. Several yeast PATs seem to have overlapping specificity, and it has been proposed that the machinery responsible for palmitoylating peripheral membrane proteins in mammalian cells, lacks specificity altogether

    Journal Staff

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    We present the first measurements of the differential cross section d sigma/dp(T)(gamma) for the production of an isolated photon in association with at least two b-quark jets. The measurements consider photons with rapidities vertical bar y(gamma)vertical bar < 1.0 and transverse momenta 30 < p(T)(gamma) < 200 GeV. The b-quark jets are required to have p(T)(jet) > 15 GeVand vertical bar y(jet)vertical bar < 1.5. The ratio of differential production cross sections for gamma + 2 b-jets to gamma + b-jet as a function of p(T)(gamma) is also presented. The results are based on the proton-antiproton collision data at root s = 1.96 TeV collected with the D0 detector at the Fermilab Tevatron Collider. The measured cross sections and their ratios are compared to the next- to- leading order perturbative QCD calculations as well as predictions based on the k(T)- factorization approach and those from the sherpa and pythia Monte Carlo event generators

    Search for Neutral Higgs Bosons in Events with Multiple Bottom Quarks at the Tevatron

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    The combination of searches performed by the CDF and D0 collaborations at the Fermilab Tevatron Collider for neutral Higgs bosons produced in association with b quarks is reported. The data, corresponding to 2.6 fb-1 of integrated luminosity at CDF and 5.2 fb-1 at D0, have been collected in final states containing three or more b jets. Upper limits are set on the cross section multiplied by the branching ratio varying between 44 pb and 0.7 pb in the Higgs boson mass range 90 to 300 GeV, assuming production of a narrow scalar boson. Significant enhancements to the production of Higgs bosons can be found in theories beyond the standard model, for example in supersymmetry. The results are interpreted as upper limits in the parameter space of the minimal supersymmetric standard model in a benchmark scenario favoring this decay mode.Comment: 10 pages, 2 figure
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