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Sample records for seo proteins results

  1. GFP Tagging of Sieve Element Occlusion (SEO) Proteins Results in Green Fluorescent Forisomes

    PubMed Central

    Pélissier, Hélène C.; Peters, Winfried S.; Collier, Ray; van Bel, Aart J. E.; Knoblauch, Michael

    2008-01-01

    Forisomes are Ca2+-driven, ATP-independent contractile protein bodies that reversibly occlude sieve elements in faboid legumes. They apparently consist of at least three proteins; potential candidates have been described previously as ‘FOR’ proteins. We isolated three genes from Medicago truncatula that correspond to the putative forisome proteins and expressed their green fluorescent protein (GFP) fusion products in Vicia faba and Glycine max using the composite plant methodology. In both species, expression of any of the constructs resulted in homogenously fluorescent forisomes that formed sieve tube plugs upon stimulation; no GFP fluorescence occurred elsewhere. Isolated fluorescent forisomes reacted to Ca2+ and chelators by contraction and expansion, respectively, and did not lose fluorescence in the process. Wild-type forisomes showed no affinity for free GFP in vitro. The three proteins shared numerous conserved motifs between themselves and with hypothetical proteins derived from the genomes of M. truncatula, Vitis vinifera and Arabidopsis thaliana. However, they showed neither significant similarities to proteins of known function nor canonical metal-binding motifs. We conclude that ‘FOR’-like proteins are components of forisomes that are encoded by a well-defined gene family with relatives in taxa that lack forisomes. Since the mnemonic FOR is already registered and in use for unrelated genes, we suggest the acronym SEO (sieve element occlusion) for this family. The absence of binding sites for divalent cations suggests that the Ca2+ binding responsible for forisome contraction is achieved either by as yet unidentified additional proteins, or by SEO proteins through a novel, uncharacterized mechanism. PMID:18784195

  2. Calcium powered phloem protein of SEO gene family "Forisome" functions in wound sealing and act as biomimetic smart materials.

    PubMed

    Srivastava, Vineet Kumar; Tuteja, Narendra

    2014-01-01

    Forisomes protein belongs to SEO gene family and is unique to Fabaceae family. These proteins are located in sieve tubes of phloem and function to prevent loss of nutrient-rich photoassimilates, upon mechanical injury/wounding. Forisome protein is also known as ATP independent, mechanically active proteins. Despite the wealth of information role of forisome in plants are not yet fully understood. Recent reports suggest that forisomes protein can act as ideal model to study self assembly mechanism for development of nanotechnological devices like microfluidic system application in space exploration mission. Improvement in micro instrument is highly demanding and has been a key technology by NASA in future space exploration missions. Based on its physical parameters, forisome are found to be ideal biomimetic materials for micro fluidic system because the conformational shifts can be replicated in vitro and are fully reversible over large number of cycles. By the use of protein engineering forisome recombinant protein can be tailored. Due to its unique ability to convert chemical energy into mechanical energy forisome has received much attention. For nanotechnological application and handling biomolecules such as DNA, RNA, protein and cell as a whole microfluidic system will be the most powerful technology. The discovery of new biomimetic smart materials has been a key factor in development of space science and its requirements in such a challenging environment. The field of microfludic, particularly in terms of development of its components along with identification of new biomimetic smart materials, deserves more attention. More biophysical investigation is required to characterize it to make it more suitable under parameters of performance. PMID:25763691

  3. Calcium powered phloem protein of SEO gene family "Forisome" functions in wound sealing and act as biomimetic smart materials.

    PubMed

    Srivastava, Vineet Kumar; Tuteja, Narendra

    2014-01-01

    Forisomes protein belongs to SEO gene family and is unique to Fabaceae family. These proteins are located in sieve tubes of phloem and function to prevent loss of nutrient-rich photoassimilates, upon mechanical injury/wounding. Forisome protein is also known as ATP independent, mechanically active proteins. Despite the wealth of information role of forisome in plants are not yet fully understood. Recent reports suggest that forisomes protein can act as ideal model to study self assembly mechanism for development of nanotechnological devices like microfluidic system application in space exploration mission. Improvement in micro instrument is highly demanding and has been a key technology by NASA in future space exploration missions. Based on its physical parameters, forisome are found to be ideal biomimetic materials for micro fluidic system because the conformational shifts can be replicated in vitro and are fully reversible over large number of cycles. By the use of protein engineering forisome recombinant protein can be tailored. Due to its unique ability to convert chemical energy into mechanical energy forisome has received much attention. For nanotechnological application and handling biomolecules such as DNA, RNA, protein and cell as a whole microfluidic system will be the most powerful technology. The discovery of new biomimetic smart materials has been a key factor in development of space science and its requirements in such a challenging environment. The field of microfludic, particularly in terms of development of its components along with identification of new biomimetic smart materials, deserves more attention. More biophysical investigation is required to characterize it to make it more suitable under parameters of performance.

  4. Calcium powered phloem protein of SEO gene family “Forisome” functions in wound sealing and act as biomimetic smart materials

    PubMed Central

    Srivastava, Vineet Kumar; Tuteja, Narendra

    2014-01-01

    Forisomes protein belongs to SEO gene family and is unique to Fabaceae family. These proteins are located in sieve tubes of phloem and function to prevent loss of nutrient-rich photoassimilates, upon mechanical injury/wounding. Forisome protein is also known as ATP independent, mechanically active proteins. Despite the wealth of information role of forisome in plants are not yet fully understood. Recent reports suggest that forisomes protein can act as ideal model to study self assembly mechanism for development of nanotechnological devices like microfluidic system application in space exploration mission. Improvement in micro instrument is highly demanding and has been a key technology by NASA in future space exploration missions. Based on its physical parameters, forisome are found to be ideal biomimetic materials for micro fluidic system because the conformational shifts can be replicated in vitro and are fully reversible over large number of cycles. By the use of protein engineering forisome recombinant protein can be tailored. Due to its unique ability to convert chemical energy into mechanical energy forisome has received much attention. For nanotechnological application and handling biomolecules such as DNA, RNA, protein and cell as a whole microfluidic system will be the most powerful technology. The discovery of new biomimetic smart materials has been a key factor in development of space science and its requirements in such a challenging environment. The field of microfludic, particularly in terms of development of its components along with identification of new biomimetic smart materials, deserves more attention. More biophysical investigation is required to characterize it to make it more suitable under parameters of performance. PMID:25763691

  5. CEOS SEO and GISS Meeting

    NASA Technical Reports Server (NTRS)

    Killough, Brian; Stover, Shelley

    2008-01-01

    The Committee on Earth Observation Satellites (CEOS) provides a brief to the Goddard Institute for Space Studies (GISS) regarding the CEOS Systems Engineering Office (SEO) and current work on climate requirements and analysis. A "system framework" is provided for the Global Earth Observation System of Systems (GEOSS). SEO climate-related tasks are outlined including the assessment of essential climate variable (ECV) parameters, use of the "systems framework" to determine relevant informational products and science models and the performance of assessments and gap analyses of measurements and missions for each ECV. Climate requirements, including instruments and missions, measurements, knowledge and models, and decision makers, are also outlined. These requirements would establish traceability from instruments to products and services allowing for benefit evaluation of instruments and measurements. Additionally, traceable climate requirements would provide a better understanding of global climate models.

  6. Earth resources applications of the Synchronous Earth Observatory Satellite (SEOS)

    NASA Technical Reports Server (NTRS)

    Lowe, D. S.; Cook, J. J.

    1973-01-01

    The results are presented of a four month study to define earth resource applications which are uniquely suited to data collection by a geosynchronous satellite. While such a satellite could also perform many of the functions of ERTS, or its low orbiting successors, those applications were considered in those situations where requirements for timely observation limit the capability of ERTS or EOS. Thus, the application presented could be used to justify a SEOS.

  7. Synthesis, structure, and characterization of two new polar sodium tungsten selenites: Na2(WO3)3(SeO3)·2H2O and Na6(W6O19)(SeO3)2.

    PubMed

    Nguyen, Sau Doan; Halasyamani, P Shiv

    2013-03-01

    Two new quaternary sodium tungsten selenites, Na2(WO3)3(SeO3)·2H2O (P31c) and Na6(W6O19)(SeO3)2 (C2), have been synthesized and characterized. The former exhibits a hexagonal tungsten oxide layered structure, whereas the latter has a one-dimensional "ribbon" structure. The layers and "ribbons" consist of distorted WO6 and asymmetric SeO3 polyhedra. The layers in Na2(WO3)3(SeO3)·2H2O and the "ribbons" in Na6(W6O19)(SeO3)2 are separated by Na(+) cations. Powder second-harmonic-generation (SHG) measurements on Na2(WO3)3(SeO3)·2H2O and Na6(W6O19)(SeO3)2 using 1064 nm radiation reveal SHG efficiencies of approximately 450× and 20× α-SiO2, respectively. Particle size versus SHG efficiency measurements indicate that the materials are type 1 non-phase-matchable. Converse piezoelectric measurements result in d33 values of approximately 23 and 12 pm/V, whereas pyroelectric measurements reveal coefficients of -0.41 and -1.10 μC/m(2)·K at 60 °C for Na2(WO3)3(SeO3)·2H2O and Na6(W6O19)(SeO3)2, respectively. Frequency-dependent polarization measurements confirm that the materials are nonferroelectric; i.e., the macroscopic polarization is not reversible, or "switchable". IR and UV-vis spectroscopy, thermogravimetric and differential thermal analysis measurements, and electron localization function calculations were also done for the materials. Crystal data: Na2(WO3)3(SeO3)·2H2O, trigonal, space group P31c (No. 159), a = 7.2595(6) Å, b = 7.2595(6) Å, c = 12.4867(13) Å, V = 569.89(9) Å(3), Z = 2; Na6(W6O19)(SeO3)2, monoclinic, space group C2 (No. 5), a = 42.169(8) Å, b = 7.2690(15) Å, c = 6.7494(13) Å, β = 98.48(3)°, V = 2046.2(7) Å(3), Z = 4.

  8. RNA adducts with Na 2SeO 4 and Na 2SeO 3 - Stability and structural features

    NASA Astrophysics Data System (ADS)

    Nafisi, Shohreh; Manouchehri, Firouzeh; Montazeri, Maryam

    2011-12-01

    Selenium compounds are widely available in dietary supplements and have been extensively studied for their antioxidant and anticancer properties. Low blood Se levels were found to be associated with an increased incidence and mortality from various types of cancers. Although many in vivo and clinical trials have been conducted using these compounds, their biochemical and chemical mechanisms of efficacy are the focus of much current research. This study was designed to examine the interaction of Na 2SeO 4 and Na 2SeO 3 with RNA in aqueous solution at physiological conditions, using a constant RNA concentration (6.25 mM) and various sodium selenate and sodium selenite/polynucleotide (phosphate) ratios of 1/80, 1/40, 1/20, 1/10, 1/5, 1/2 and 1/1. Fourier transform infrared, UV-Visible spectroscopic methods were used to determine the drug binding modes, the binding constants, and the stability of Na 2SeO 4 and Na 2SeO 3-RNA complexes in aqueous solution. Spectroscopic evidence showed that Na 2SeO 4 and Na 2SeO 3 bind to the major and minor grooves of RNA ( via G, A and U bases) with some degree of the Se-phosphate (PO 2) interaction for both compounds with overall binding constants of K(Na 2SeO 4-RNA) = 8.34 × 10 3 and K(Na 2SeO 3-RNA) = 4.57 × 10 3 M -1. The order of selenium salts-biopolymer stability was Na 2SeO 4-RNA > Na 2SeO 3-RNA. RNA aggregations occurred at higher selenium concentrations. No biopolymer conformational changes were observed upon Na 2SeO 4 and Na 2SeO 3 interactions, while RNA remains in the A-family structure.

  9. On the dielectric susceptibility calculation in the incommensurate phase of K2SeO4

    NASA Astrophysics Data System (ADS)

    Aslanyan, T. A.

    2010-10-01

    It is shown that the thermodynamic potential of the domain-like incommensurate (IC) phase of the K2SeO4crystal (viewed as a model for the IC-C transition) should be supplemented with a term, taking into account the local, Lorentz electric field. The latter qualitatively changes the result of calculation of the dielectric susceptibility for this IC structure by Nattermann and Trimper, J. Phys. C: Solid State Phys. 14, 1603, (1981), and gives phase transition to the ferroelectric IC phase obtained by Aslanyan, Phys. Rev. B 70, 024102, (2004).

  10. Rich structural chemistry in scandium selenium/tellurium oxides: mixed-valent selenite-selenates, Sc2(SeO3)2(SeO4) and Sc2(TeO3)(SeO3)(SeO4), and ternary tellurite, Sc2(TeO3)3.

    PubMed

    Song, Seung Yoon; Lee, Dong Woo; Ok, Kang Min

    2014-07-01

    Both single crystals and pure bulk phases of three new scandium selenium/tellurium oxides, Sc2(SeO3)2(SeO4), Sc2(TeO3)(SeO3)(SeO4), and Sc2(TeO3)3, have been synthesized through hydrothermal and solid-state reactions. X-ray diffractions were used to determine the structures and confirm the phase purities of the reported materials. Isostructural Sc2(SeO3)2(SeO4) and Sc2(TeO3)(SeO3)(SeO4) reveal three-dimensional frameworks with ScO7 pentagonal bipyramids, SeO3 (and TeO3) trigonal pyramids, and SeO4 tetrahedra. A novel ternary scandium tellurite, Sc2(TeO3)3, also shows a three-dimensional framework that is composed of ScO6 octahedra, ScO7-capped octahedra, and TeO3 trigonal pyramids. All three materials accommodate local asymmetric coordination moieties owing to the lone pairs on Se(4+) and Te(4+) cations. The effect of coordination environments of constituent cations on the frameworks, dimensionalities, and centricities of products is discussed. Thorough characterizations including elemental analyses, infrared and UV-vis diffuse reflectance spectroscopies, thermal analyses, and dipole moment calculations for the reported materials are reported. Crystal data: Sc2(SeO3)2(SeO4), monoclinic, space group P21/c (No. 14), a = 6.5294(2) Å, b = 10.8557(4) Å, c = 12.6281(6) Å, β = 103.543(3)°, V = 870.21(6) Å(3), and Z = 4; Sc2(TeO3)(SeO3)(SeO4), monoclinic, space group P21/c (No. 14), a = 6.5345(12) Å, b = 10.970(2) Å, c = 12.559(2) Å, β = 102.699(10)°, V = 878.3(6) Å(3), and Z = 4; Sc2(TeO3)3, monoclinic, space group P21/n (No. 14), a = 5.2345(3) Å, b = 24.3958(15) Å, c = 6.8636(4) Å, β = 106.948(2)°, V = 838.42(9) Å(3), and Z = 4.

  11. An empirical analysis of health care IPOs and SEOs.

    PubMed

    Brau, James C; Holloway, Jonathan M

    2009-01-01

    This article reviews the extant literature regarding the three new issues phenomena: hot issue markets, first-day underpricing, and poor long-run performance as they apply to the heath care industry. Given the "creeping corporatization" of the heath care industry and the unique influence of nonmarket forces on it, we examine whether the three IPO phenomena exist within the industry. We find that hot issue markets, initial underpricing, and negative long-run abnormal returns and sales growth occur among both heath care IPOs and SEOs. Of particular interest, we find that firms are able to issue during times of excess heath care spending and subsequently underperform the market, apparently exploiting windows of opportunity.

  12. Hydroxocobalamin association during cell culture results in pink therapeutic proteins

    PubMed Central

    Prentice, Kenneth M; Gillespie, Ronald; Lewis, Nathan; Fujimori, Kiyoshi; McCoy, Rebecca; Bach, Julia; Connell-Crowley, Lisa; Eakin, Catherine M

    2013-01-01

    Process control of protein therapeutic manufacturing is central to ensuring the product is both safe and efficacious for patients. In this work, we investigate the cause of pink color variability in development lots of monoclonal antibody (mAb) and Fc-fusion proteins. Results show pink-colored product generated during manufacturing is due to association of hydroxocobalamin (OH-Cbl), a form of vitamin B12. OH-Cbl is not part of the product manufacturing process; however we found cyanocobalamin (CN-Cbl) in cell culture media converts to OH-Cbl in the presence of light. OH-Cbl can be released from mAb and Fc-fusion proteins by conversion with potassium cyanide to CN-Cbl, which does not bind. By exploiting the differential binding of CN-Cbl and OH-Cbl, we developed a rapid and specific assay to accurately measure B12 levels in purified protein. Analysis of multiple products and lots using this technique gives insight into color variability during manufacturing. PMID:23924851

  13. Synthesis of LECBD grown cluster assembled SeO 2 thin films

    NASA Astrophysics Data System (ADS)

    Rath, S.; Das, K.; Sarangi, S. N.; Dash, A. K.; Ray, S. K.; Sahu, S. N.

    2006-12-01

    Cluster assembled selenium oxide (SeO 2) thin films, as a function of oxygen flow pressure (OFP) have been synthesized by a low energy cluster beam deposition (LECBD) technique. The OFP dependent surface morphology leading to well separated nanoclusters (size ranging from 50 to 200 nm) and fractal features are confirmed from transmission electron microscopic (TEM) measurements. A diffusion limited aggregation (DLA) mediated fractal growth with dimension as 1.71 ± 0.01 has been observed for high OFP (60 mbar). Structural analysis by glancing angle X-ray diffraction (GXRD) and selected area diffraction (SAD) studies identify the presence of tetragonal phase SeO 2 in the deposit. Micro-Raman studies indicate the shifts in bending and stretching vibrational phonon modes in cluster assembled SeO 2 as compared to their bulk counter part due to the phonon confinement effect.

  14. Synthesis, structure, and characterization of two new bismuth(III) selenite/tellurite nitrates: [(Bi3O2)(SeO3)2](NO3) and [Bi(TeO3)](NO3)

    NASA Astrophysics Data System (ADS)

    Meng, Chang-Yu; Wei, Ming-Fang; Geng, Lei; Hu, Pei-Qing; Yu, Meng-Xia; Cheng, Wen-Dan

    2016-07-01

    Two new bismuth(III) selenite/tellurite nitrates, [(Bi3O2)(SeO3)2](NO3) and [Bi(TeO3)](NO3), have been synthesized by conventional facile hydrothermal method at middle temperature 200 °C and characterized by single-crystal X-ray diffraction, powder diffraction, UV-vis-NIR optical absorption spectrum, infrared spectrum and thermal analylsis. Both [(Bi3O2)(SeO3)2](NO3) and [Bi(TeO3)](NO3) crystallize in the monoclinic centronsymmetric space group P21/c with a=9.9403(4) Å, b=9.6857(4) Å, c=10.6864(5) Å, β=93.1150(10)° for [(Bi3O2)(SeO3)2](NO3) and a=8.1489(3) Å, b=9.0663(4) Å, c=7.4729(3) Å, β=114.899(2)° for Bi(TeO3)(NO3), respectively. The two compounds, whose structures are composed of three different asymmetric building units, exhibit two different types of structures. The structure of [(Bi3O2)(SeO3)2](NO3) features a three-dimensional (3D) bismuth(III) selenite cationic tunnel structure [(Bi3O2)(SeO3)2] 3∞ with NO3- anion group filling in the 1D tunnel along b axis. The structure of [Bi(TeO3)](NO3) features 2D bismuth(III) tellurite [Bi(TeO3)2]2∞ layers separated by NO3- anion groups. The results of optical diffuse-reflectance spectrum measurements and electronic structure calculations based on density functional theory methods show that the two compounds are wide band-gap semiconductors.

  15. Interactive intoxicating and ameliorating effects of tannic acid, aluminum (Al), copper (Cu), and selenate (SeO) in wheat roots: a descriptive and mathematical assessment.

    PubMed

    Kinraide, Thomas B; Hagermann, Ann E

    2010-05-01

    Tannic acids and tannins are produced by plants and are important components of soil and water organic matter. These polyphenolic compounds form complexes with proteins, metals and soil particulate matter and perform several physiological and ecological functions. The tannic acid (TA) used in our study was a mixture of gallic acid and galloyl glucoses ranging up to nonagalloyl glucose. TA inhibited root elongation in wheat seedlings (Triticum aestivum L. cv. Scout 66) at concentrations >4 mg l(-1); but TA alleviated the toxicity of Al(3+), Cu(2+) and SeO(4)(2-); and Al(3+) and SeO(4)(2-) alleviated the toxicity of TA. The interactions of Al(3+) and TA (each toxic but each alleviating the toxicity of the other) were stoichiometric. Growth was affected as though 1 kg TA bound 2.76 mol Al so strongly that if (mol Al)/(kg TA) <2.76, then free Al approximately 0, and if (mol Al)/(kg TA) >2.76, then free TA approximately 0. This stoichiometry is consistent with one mole of galloyl groups binding approximately 0.5 mol Al. Using this binding scheme, growth was modeled successfully on the basis of free TA and free Al. TA enhanced the negativity of root surfaces and enhanced the binding of Al and Cu there without enhancing their toxicity. These and other interactions among TA, Al(3+), Cu(2+), SeO(4)(2-), Ca(2+), Na(+) and H(+) were quantified with a comprehensive non-linear equation with statistically significant coefficients.

  16. Characterization of five subgroups of the sieve element occlusion gene family in Glycine max reveals genes encoding non-forisome P-proteins, forisomes and forisome tails.

    PubMed

    Zielonka, Sascia; Ernst, Antonia M; Hawat, Susan; Twyman, Richard M; Prüfer, Dirk; Noll, Gundula A

    2014-09-01

    P-proteins are structural phloem proteins discussed to be involved in the rapid sealing of injured sieve elements. P-proteins are found in all dicotyledonous and some monocotyledonous plants, but additional crystalloid P-proteins, known as forisomes, have evolved solely in the Fabaceae. Both types are encoded by members of the sieve element occlusion (SEO) gene family, which comprises seven phylogenetic subgroups. The Fabaceae-specific subgroup 1 contains genes encoding forisome subunits in e.g. Medicago truncatula, Vicia faba, Dipteryx panamensis and Canavalia gladiata whereas basal subgroup 5 encodes P-proteins in Nicotiana tabacum (tobacco) and Arabidopsis thaliana. The function of remaining subgroups is still unknown. We chose Glycine max (soybean) as a model to investigate SEO proteins representing different subgroups in one species. We isolated native P-proteins to determine the SEO protein composition and analyzed the expression pattern, localization and structure of the G. max SEO proteins representing five of the subgroups. We found that subgroup 1 GmSEO genes encode forisome subunits, a member of subgroup 5 encodes a non-forisome P-protein and subgroup 2 GmSEO genes encode the components of forisome tails, which are present in a restricted selection of Fabaceaen species. We therefore present the first molecular characterization of a Fabaceae non-forisome P-protein and the first evidence that forisome tails are encoded by a phylogenetically-distinct branch of the SEO gene family.

  17. Effects of sodium and potassium ions on a novel SeO2-B2O3-SiO2-P2O5-CaO bioactive system

    NASA Astrophysics Data System (ADS)

    Trandafir, D. L.; Ponta, O.; Ciceo-Lucacel, R.; Simon, V.

    2015-01-01

    The study is focused on Na2O and/or K2O influence on a new sol-gel derived SeO2-B2O3-SiO2-P2O5-CaO bioactive system. The structural changes induced by Na2O and/or K2O addition were correlated with the samples behavior in simulated biological media. X-ray diffraction (XRD) and Fourier transform infrared (FTIR) spectroscopy were used to characterize the structure and the type of the chemical bonds. The morphology of the samples was characterized through scanning electron microscopy (SEM). XRD results pointed out a prevalent vitreous structure with an incipient hydroxyapatite (HA) crystalline phase. FTIR results revealed a complex network consisting of silicate, phosphate and borate units, as well as the development of both A- and B-type of carbonate-substituted HA. The bioactivity of the samples was tested in vitro following the evolution of the apatite layers self-assembled on the samples surface in simulated body fluid. Their biocompatibility was investigated after samples surface functionalization with protein. The results indicate that sodium and potassium addition improves the biocompatibility by enhancement of protein adherence on samples surface and without to prevent the samples bioactivity.

  18. Use of vesicular stomatitis virus pseudotypes bearing hantaan or seoul virus envelope proteins in a rapid and safe neutralization test.

    PubMed

    Ogino, Michiko; Ebihara, Hideki; Lee, Byoung-Hee; Araki, Koichi; Lundkvist, Ake; Kawaoka, Yoshihiro; Yoshimatsu, Kumiko; Arikawa, Jiro

    2003-01-01

    A vesicular stomatitis virus (VSV) pseudotype bearing hantavirus envelope glycoproteins was produced and used in a neutralization test as a substitute for native hantavirus. The recombinant VSV, in which the enveloped protein gene (G) was replaced by the green fluorescent protein gene and complemented with G protein expressed in trans (VSVDeltaG*G), was kindly provided by M. A. Whitt. 293T cells were transfected with plasmids for the expression of envelope glycoproteins (G1 and G2) of HTNV or SEOV and were then infected with VSVDeltaG*G. Pseudotype VSV with the Hantaan (VSVDeltaG*-HTN) or Seoul (VSVDeltaG*-SEO) envelope glycoproteins were harvested from the culture supernatant. The number of infectious units (IU) of the pseudotype VSVs ranged from 10(5) to 10(6)/ml. The infectivity of VSVDeltaG*-HTN and VSVDeltaG*-SEO was neutralized with monoclonal antibodies, immune rabbit sera, and sera from patients with hemorrhagic fever with renal syndrome, and the neutralizing titers were similar to those obtained with native hantaviruses. These results show that VSVDeltaG*-HTN and -SEO can be used as a rapid, specific, and safe neutralization test for detecting hantavirus-neutralizing antibodies as an effective substitute for the use of native hantaviruses. Furthermore, the IU of VSVDeltaG*-HTN and -SEO did not decrease by more than 10-fold when stored at 4 degrees C for up to 30 days. The stability of the pseudotype viruses allows distribution of the material to remote areas by using conventional cooling boxes for use as a diagnostic reagent.

  19. Ribosomal Protein Mutations Result in Constitutive p53 Protein Degradation through Impairment of the AKT Pathway

    PubMed Central

    Hermkens, Dorien; Wlodarski, Marcin W.; Da Costa, Lydie; MacInnes, Alyson W.

    2015-01-01

    Mutations in ribosomal protein (RP) genes can result in the loss of erythrocyte progenitor cells and cause severe anemia. This is seen in patients with Diamond-Blackfan anemia (DBA), a pure red cell aplasia and bone marrow failure syndrome that is almost exclusively linked to RP gene haploinsufficiency. While the mechanisms underlying the cytopenia phenotype of patients with these mutations are not completely understood, it is believed that stabilization of the p53 tumor suppressor protein may induce apoptosis in the progenitor cells. In stark contrast, tumor cells from zebrafish with RP gene haploinsufficiency are unable to stabilize p53 even when exposed to acute DNA damage despite transcribing wild type p53 normally. In this work we demonstrate that p53 has a limited role in eliciting the anemia phenotype of zebrafish models of DBA. In fact, we find that RP-deficient embryos exhibit the same normal p53 transcription, absence of p53 protein, and impaired p53 response to DNA damage as RP haploinsufficient tumor cells. Recently we reported that RP mutations suppress activity of the AKT pathway, and we show here that this suppression results in proteasomal degradation of p53. By re-activating the AKT pathway or by inhibiting GSK-3, a downstream modifier that normally represses AKT signaling, we are able to restore the stabilization of p53. Our work indicates that the anemia phenotype of zebrafish models of DBA is dependent on factors other than p53, and may hold clinical significance for both DBA and the increasing number of cancers revealing spontaneous mutations in RP genes. PMID:26132763

  20. Protein networks identify novel symbiogenetic genes resulting from plastid endosymbiosis.

    PubMed

    Méheust, Raphaël; Zelzion, Ehud; Bhattacharya, Debashish; Lopez, Philippe; Bapteste, Eric

    2016-03-29

    The integration of foreign genetic information is central to the evolution of eukaryotes, as has been demonstrated for the origin of the Calvin cycle and of the heme and carotenoid biosynthesis pathways in algae and plants. For photosynthetic lineages, this coordination involved three genomes of divergent phylogenetic origins (the nucleus, plastid, and mitochondrion). Major hurdles overcome by the ancestor of these lineages were harnessing the oxygen-evolving organelle, optimizing the use of light, and stabilizing the partnership between the plastid endosymbiont and host through retargeting of proteins to the nascent organelle. Here we used protein similarity networks that can disentangle reticulate gene histories to explore how these significant challenges were met. We discovered a previously hidden component of algal and plant nuclear genomes that originated from the plastid endosymbiont: symbiogenetic genes (S genes). These composite proteins, exclusive to photosynthetic eukaryotes, encode a cyanobacterium-derived domain fused to one of cyanobacterial or another prokaryotic origin and have emerged multiple, independent times during evolution. Transcriptome data demonstrate the existence and expression of S genes across a wide swath of algae and plants, and functional data indicate their involvement in tolerance to oxidative stress, phototropism, and adaptation to nitrogen limitation. Our research demonstrates the "recycling" of genetic information by photosynthetic eukaryotes to generate novel composite genes, many of which function in plastid maintenance. PMID:26976593

  1. Protein networks identify novel symbiogenetic genes resulting from plastid endosymbiosis

    PubMed Central

    Méheust, Raphaël; Zelzion, Ehud; Bhattacharya, Debashish; Lopez, Philippe; Bapteste, Eric

    2016-01-01

    The integration of foreign genetic information is central to the evolution of eukaryotes, as has been demonstrated for the origin of the Calvin cycle and of the heme and carotenoid biosynthesis pathways in algae and plants. For photosynthetic lineages, this coordination involved three genomes of divergent phylogenetic origins (the nucleus, plastid, and mitochondrion). Major hurdles overcome by the ancestor of these lineages were harnessing the oxygen-evolving organelle, optimizing the use of light, and stabilizing the partnership between the plastid endosymbiont and host through retargeting of proteins to the nascent organelle. Here we used protein similarity networks that can disentangle reticulate gene histories to explore how these significant challenges were met. We discovered a previously hidden component of algal and plant nuclear genomes that originated from the plastid endosymbiont: symbiogenetic genes (S genes). These composite proteins, exclusive to photosynthetic eukaryotes, encode a cyanobacterium-derived domain fused to one of cyanobacterial or another prokaryotic origin and have emerged multiple, independent times during evolution. Transcriptome data demonstrate the existence and expression of S genes across a wide swath of algae and plants, and functional data indicate their involvement in tolerance to oxidative stress, phototropism, and adaptation to nitrogen limitation. Our research demonstrates the “recycling” of genetic information by photosynthetic eukaryotes to generate novel composite genes, many of which function in plastid maintenance. PMID:26976593

  2. Protein networks identify novel symbiogenetic genes resulting from plastid endosymbiosis.

    PubMed

    Méheust, Raphaël; Zelzion, Ehud; Bhattacharya, Debashish; Lopez, Philippe; Bapteste, Eric

    2016-03-29

    The integration of foreign genetic information is central to the evolution of eukaryotes, as has been demonstrated for the origin of the Calvin cycle and of the heme and carotenoid biosynthesis pathways in algae and plants. For photosynthetic lineages, this coordination involved three genomes of divergent phylogenetic origins (the nucleus, plastid, and mitochondrion). Major hurdles overcome by the ancestor of these lineages were harnessing the oxygen-evolving organelle, optimizing the use of light, and stabilizing the partnership between the plastid endosymbiont and host through retargeting of proteins to the nascent organelle. Here we used protein similarity networks that can disentangle reticulate gene histories to explore how these significant challenges were met. We discovered a previously hidden component of algal and plant nuclear genomes that originated from the plastid endosymbiont: symbiogenetic genes (S genes). These composite proteins, exclusive to photosynthetic eukaryotes, encode a cyanobacterium-derived domain fused to one of cyanobacterial or another prokaryotic origin and have emerged multiple, independent times during evolution. Transcriptome data demonstrate the existence and expression of S genes across a wide swath of algae and plants, and functional data indicate their involvement in tolerance to oxidative stress, phototropism, and adaptation to nitrogen limitation. Our research demonstrates the "recycling" of genetic information by photosynthetic eukaryotes to generate novel composite genes, many of which function in plastid maintenance.

  3. Protein crystal growth results from shuttle flight 51-F

    NASA Technical Reports Server (NTRS)

    Bugg, C. E.

    1985-01-01

    The protein crystal growth (PCG) experiments run on 51-F were analyzed. It was found that: (1) sample stability is increased over that observed during the experiments on flight 51-D; (2) the dialysis experiments produced lysozyme crystals that were significantly larger than those obtained in our identical ground-based studies; (3) temperature fluctuations apparently caused problems during the crystallization experiments on 51-F; (4) it is indicated that teflon tape stabilizes droplets on the syringe tips; (5) samples survived during the reentry and landing in glass tips that were not stoppered with plungers; (6) from the ground-based studies, it was expected that equilibration should be complete within 2 to 4 days for all of these vapor-diffusion experiments, thus it appears that the vapor diffusion rates are somewhat slower under microgravity conditions; (7) drop tethering was highly successful, all four of the tethered drops were stable, even though they contained MPD solutions; (8) the PCG experiments on 51-F were done to assess the hardware and experimental procedures that are developed for future flights, when temperature control will be available. Lysozyme crystals obtained by microdialysis are considerably larger than those obtained on the ground, using the identical apparatus and procedures.

  4. Consumption of Milk Protein or Whey Protein Results in a Similar Increase in Muscle Protein Synthesis in Middle Aged Men.

    PubMed

    Mitchell, Cameron J; McGregor, Robin A; D'Souza, Randall F; Thorstensen, Eric B; Markworth, James F; Fanning, Aaron C; Poppitt, Sally D; Cameron-Smith, David

    2015-10-21

    The differential ability of various milk protein fractions to stimulate muscle protein synthesis (MPS) has been previously described, with whey protein generally considered to be superior to other fractions. However, the relative ability of a whole milk protein to stimulate MPS has not been compared to whey. Sixteen healthy middle-aged males ingested either 20 g of milk protein (n = 8) or whey protein (n = 8) while undergoing a primed constant infusion of ring (13)C₆ phenylalanine. Muscle biopsies were obtained 120 min prior to consumption of the protein and 90 and 210 min afterwards. Resting myofibrillar fractional synthetic rates (FSR) were 0.019% ± 0.009% and 0.021% ± 0.018% h(-1) in the milk and whey groups respectively. For the first 90 min after protein ingestion the FSR increased (p < 0.001) to 0.057% ± 0.018% and 0.052% ± 0.024% h(-1) in the milk and whey groups respectively with no difference between groups (p = 0.810). FSR returned to baseline in both groups between 90 and 210 min after protein ingestion. Despite evidence of increased rate of digestion and leucine availability following the ingestion of whey protein, there was similar activation of MPS in middle-aged men with either 20 g of milk protein or whey protein.

  5. Consumption of Milk Protein or Whey Protein Results in a Similar Increase in Muscle Protein Synthesis in Middle Aged Men

    PubMed Central

    Mitchell, Cameron J.; McGregor, Robin A.; D’Souza, Randall F.; Thorstensen, Eric B.; Markworth, James F.; Fanning, Aaron C.; Poppitt, Sally D.; Cameron-Smith, David

    2015-01-01

    The differential ability of various milk protein fractions to stimulate muscle protein synthesis (MPS) has been previously described, with whey protein generally considered to be superior to other fractions. However, the relative ability of a whole milk protein to stimulate MPS has not been compared to whey. Sixteen healthy middle-aged males ingested either 20 g of milk protein (n = 8) or whey protein (n = 8) while undergoing a primed constant infusion of ring 13C6 phenylalanine. Muscle biopsies were obtained 120 min prior to consumption of the protein and 90 and 210 min afterwards. Resting myofibrillar fractional synthetic rates (FSR) were 0.019% ± 0.009% and 0.021% ± 0.018% h−1 in the milk and whey groups respectively. For the first 90 min after protein ingestion the FSR increased (p < 0.001) to 0.057% ± 0.018% and 0.052% ± 0.024% h−1 in the milk and whey groups respectively with no difference between groups (p = 0.810). FSR returned to baseline in both groups between 90 and 210 min after protein ingestion. Despite evidence of increased rate of digestion and leucine availability following the ingestion of whey protein, there was similar activation of MPS in middle-aged men with either 20 g of milk protein or whey protein. PMID:26506377

  6. Toxicity of selenium (Na2SeO3) and mercury (HgCl2) on the planarian Dugesia gonocephala.

    PubMed

    Congiu, A M; Casu, S; Ugazio, G

    1989-10-01

    The toxicity of selenium (Na2SeO3) and mercury (HgCl2) was determined by using a freshwater planarian which is particularly sensitive to pollution, and belongs to a fissiparous breed of Dugesia gonocephala. The mortality and fissiparity frequency of the subjects were studied. They were exposed to intense treatments (48 hours) or for medium to long periods of time (21 days) to either the single compounds or a combination of both, and were fed or fasting. The lethal effect of sodium selenite is correlated to the food intake, whereas the toxicity of mercurous chloride is probably the result of a fixative effect which does not depend on feeding. The 21-day treatment with the first compound has a non-negligible lethal effect which is probably due to an accumulation phenomenon. At doses where an antioxidant effect prevails, fissiparity is stimulated. On the other hand, the second compound reduces reproduction frequency to half the base values. Compared to the Paracentrotus lividus, the Dugesia gonocephala offers various advantages concerning toxicological experiments; besides being easier to handle in the laboratory, it is available all year round and is not subject to seasonal cycles. It is also more susceptible to the toxic effect of mercury, which is a common and highly toxic pollutant, than the sea urchin.

  7. Protein engineering of protein kinase A catalytic subunits results in the acquisition of novel inhibitor sensitivity.

    PubMed

    Niswender, Colleen M; Ishihara, R Wesley; Judge, Luke M; Zhang, Chao; Shokat, Kevan M; McKnight, G Stanley

    2002-08-01

    Analysis of the role of specific protein kinases in signal transduction networks has relied heavily on ATP analog inhibitors. Currently used agents, however, often do not distinguish between kinase family members. Genetic approaches can also be used to inactivate a specific kinase, but these techniques do not afford the rapid kinetics possible with pharmacological inhibitors. To circumvent this problem, modification of the structure of a particular protein kinase can be performed to engineer a drug-target interaction of choice. We have used this method to create protein kinase A (PKA) catalytic subunits with modifications that confer sensitivity to novel ATP analog inhibitors. Mutation of methionine 120 to alanine or glycine in either the Calpha or Cbeta subunits of PKA induces sensitivity to a series of C-3 derivatized pyrazolo[3,4-d]pyrimidine-based inhibitors. Modification of threonine 183 enhances this inhibitor sensitivity. The IC(50) values in cell culture of the most broadly effective agent, 1-NM, ranged from 25 to 200 nm depending upon the combination of modified amino acids and were significantly higher than the potencies observed with H-89. Despite their high sequence conservation, Cbeta enzymes with inhibitor-sensitive amino acids at position 120 showed a substantial loss of overall catalytic activity when used to induce reporter gene transcription in transfected cells. Conversion of position 46 (lysine to isoleucine) rescued the ability of position 120 mutated Cbeta enzymes to induce gene transcription. Application of this combined genetic and pharmacological approach should allow analysis of the specific roles of PKA isoforms in cell culture and in vivo. PMID:12034735

  8. Investigating the importance of Delaunay-based definition of atomic interactions in scoring of protein-protein docking results.

    PubMed

    Jafari, Rahim; Sadeghi, Mehdi; Mirzaie, Mehdi

    2016-05-01

    The approaches taken to represent and describe structural features of the macromolecules are of major importance when developing computational methods for studying and predicting their structures and interactions. This study attempts to explore the significance of Delaunay tessellation for the definition of atomic interactions by evaluating its impact on the performance of scoring protein-protein docking prediction. Two sets of knowledge-based scoring potentials are extracted from a training dataset of native protein-protein complexes. The potential of the first set is derived using atomic interactions extracted from Delaunay tessellated structures. The potential of the second set is calculated conventionally, that is, using atom pairs whose interactions were determined by their separation distances. The scoring potentials were tested against two different docking decoy sets and their performances were compared. The results show that, if properly optimized, the Delaunay-based scoring potentials can achieve higher success rate than the usual scoring potentials. These results and the results of a previous study on the use of Delaunay-based potentials in protein fold recognition, all point to the fact that Delaunay tessellation of protein structure can provide a more realistic definition of atomic interaction, and therefore, if appropriately utilized, may be able to improve the accuracy of pair potentials.

  9. Investigating the importance of Delaunay-based definition of atomic interactions in scoring of protein-protein docking results.

    PubMed

    Jafari, Rahim; Sadeghi, Mehdi; Mirzaie, Mehdi

    2016-05-01

    The approaches taken to represent and describe structural features of the macromolecules are of major importance when developing computational methods for studying and predicting their structures and interactions. This study attempts to explore the significance of Delaunay tessellation for the definition of atomic interactions by evaluating its impact on the performance of scoring protein-protein docking prediction. Two sets of knowledge-based scoring potentials are extracted from a training dataset of native protein-protein complexes. The potential of the first set is derived using atomic interactions extracted from Delaunay tessellated structures. The potential of the second set is calculated conventionally, that is, using atom pairs whose interactions were determined by their separation distances. The scoring potentials were tested against two different docking decoy sets and their performances were compared. The results show that, if properly optimized, the Delaunay-based scoring potentials can achieve higher success rate than the usual scoring potentials. These results and the results of a previous study on the use of Delaunay-based potentials in protein fold recognition, all point to the fact that Delaunay tessellation of protein structure can provide a more realistic definition of atomic interaction, and therefore, if appropriately utilized, may be able to improve the accuracy of pair potentials. PMID:27060891

  10. Tuning of protein-surfactant interaction to modify the resultant structure.

    PubMed

    Mehan, Sumit; Aswal, Vinod K; Kohlbrecher, Joachim

    2015-09-01

    Small-angle neutron scattering and dynamic light scattering studies have been carried out to examine the interaction of bovine serum albumin (BSA) protein with different surfactants under varying solution conditions. We show that the interaction of anionic BSA protein (pH7) with surfactant and the resultant structure are strongly modified by the charge head group of the surfactant, ionic strength of the solution, and mixed surfactants. The protein-surfactant interaction is maximum when two components are oppositely charged, followed by components being similarly charged through the site-specific binding, and no interaction in the case of a nonionic surfactant. This interaction of protein with ionic surfactants is characterized by the fractal structure representing a bead-necklace structure of micellelike clusters adsorbed along the unfolded protein chain. The interaction is enhanced with ionic strength only in the case of site-specific binding of an anionic surfactant with an anionic protein, whereas it is almost unchanged for other complexes of cationic and nonionic surfactants with anionic proteins. Interestingly, the interaction of BSA protein with ionic surfactants is significantly suppressed in the presence of nonionic surfactant. These results with mixed surfactants thus can be used to fold back the unfolded protein as well as to prevent surfactant-induced protein unfolding. For different solution conditions, the results are interpreted in terms of a change in fractal dimension, the overall size of the protein-surfactant complex, and the number of micelles attached to the protein. The interplay of electrostatic and hydrophobic interactions is found to govern the resultant structure of complexes.

  11. Long-term high intake of whole proteins results in renal damage in pigs.

    PubMed

    Jia, Yong; Hwang, Sun Young; House, James D; Ogborn, Malcolm R; Weiler, Hope A; O, Karmin; Aukema, Harold M

    2010-09-01

    Despite evidence of potential antiobesity effects of high-protein (HP) diets, the impact of consuming diets with protein levels at the upper limit of the acceptable macronutrient distribution range (AMDR) on kidney health is unknown. To test whether HP diets affect renal health, whole plant and animal proteins in proportions that mimicked human diets were given to pigs, because their kidneys have a similar anatomy and function to those of humans. Adult female pigs received either normal-protein (NP) or HP (15 or 35% of energy from protein, respectively) isocaloric diets for either 4 or 8 mo. The higher protein in the HP diet was achieved by increasing egg and dairy proteins. Although there were initial differences in body weight and composition, after 8 mo these were similar in pigs consuming the NP and HP diets. The HP compared with NP diet, however, resulted in enlarged kidneys at both 4 and 8 mo. Renal and glomerular volumes were 60-70% higher by the end of the study. These enlarged kidneys had greater evidence of histological damage, with 55% more fibrosis and 30% more glomerulosclerosis. Renal monocyte chemoattractant protein-1 levels also were 22% higher in pigs given the HP diet. Plasma homocysteine levels were higher in the HP pigs at 4 mo and continued to be elevated by 35% at 8 mo of feeding. These findings suggest that long-term intakes of protein at the upper limit of the AMDR from whole protein sources may compromise renal health.

  12. Recent results and new hardware developments for protein crystal growth in microactivity

    NASA Technical Reports Server (NTRS)

    Delucas, L. J.; Long, M. M.; Moore, K. M.; Smith, C.; Carson, M.; Narayana, S. V. L.; Carter, D.; Clark, A. D., Jr.; Nanni, R. G.; Ding, J.

    1993-01-01

    Protein crystal growth experiments have been performed on 16 space shuttle missions since April, 1985. The initial experiments utilized vapor diffusion crystallization techniques similar to those used in laboratories for earth-based experiments. More recent experiments have utilized temperature induced crystallization as an alternative method for growing high quality protein crystals in microgravity. Results from both vapor diffusion and temperature induced crystallization experiments indicate that proteins grown in microgravity may be larger, display more uniform morphologies, and yield diffraction data to significantly higher resolutions than the best crystals of these proteins grown on earth.

  13. 31 CFR 30.3 - Q-3: How are the SEOs and most highly compensated employees identified for purposes of compliance...

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... Finance: Treasury Office of the Secretary of the Treasury TARP STANDARDS FOR COMPENSATION AND CORPORATE GOVERNANCE § 30.3 Q-3: How are the SEOs and most highly compensated employees identified for purposes...

  14. Polar Fixation of Plasmids during Recombinant Protein Production in Bacillus megaterium Results in Population Heterogeneity.

    PubMed

    Münch, Karin M; Müller, Johannes; Wienecke, Sarah; Bergmann, Simone; Heyber, Steffi; Biedendieck, Rebekka; Münch, Richard; Jahn, Dieter

    2015-09-01

    During the past 2 decades, Bacillus megaterium has been systematically developed for the gram-per-liter scale production of recombinant proteins. The plasmid-based expression systems employed use a xylose-controlled promoter. Protein production analyses at the single-cell level using green fluorescent protein as a model product revealed cell culture heterogeneity characterized by a significant proportion of less productive bacteria. Due to the enormous size of B. megaterium, such bistable behavior seen in subpopulations was readily analyzed by time lapse microscopy and flow cytometry. Cell culture heterogeneity was not caused simply by plasmid loss: instead, an asymmetric distribution of plasmids during cell division was detected during the exponential-growth phase. Multicopy plasmids are generally randomly distributed between daughter cells. However, in vivo and in vitro experiments demonstrated that under conditions of strong protein production, plasmids are retained at one of the cell poles. Furthermore, it was found that cells with accumulated plasmids and high protein production ceased cell division. As a consequence, the overall protein production of the culture was achieved mainly by the subpopulation with a sufficient plasmid copy number. Based on our experimental data, we propose a model whereby the distribution of multicopy plasmids is controlled by polar fixation under protein production conditions. Thereby, cell lines with fluctuating plasmid abundance arise, which results in population heterogeneity. Our results provide initial insights into the mechanism of cellular heterogeneity during plasmid-based recombinant protein production in a Bacillus species. PMID:26116677

  15. Polar Fixation of Plasmids during Recombinant Protein Production in Bacillus megaterium Results in Population Heterogeneity

    PubMed Central

    Münch, Karin M.; Müller, Johannes; Wienecke, Sarah; Bergmann, Simone; Heyber, Steffi; Biedendieck, Rebekka; Jahn, Dieter

    2015-01-01

    During the past 2 decades, Bacillus megaterium has been systematically developed for the gram-per-liter scale production of recombinant proteins. The plasmid-based expression systems employed use a xylose-controlled promoter. Protein production analyses at the single-cell level using green fluorescent protein as a model product revealed cell culture heterogeneity characterized by a significant proportion of less productive bacteria. Due to the enormous size of B. megaterium, such bistable behavior seen in subpopulations was readily analyzed by time lapse microscopy and flow cytometry. Cell culture heterogeneity was not caused simply by plasmid loss: instead, an asymmetric distribution of plasmids during cell division was detected during the exponential-growth phase. Multicopy plasmids are generally randomly distributed between daughter cells. However, in vivo and in vitro experiments demonstrated that under conditions of strong protein production, plasmids are retained at one of the cell poles. Furthermore, it was found that cells with accumulated plasmids and high protein production ceased cell division. As a consequence, the overall protein production of the culture was achieved mainly by the subpopulation with a sufficient plasmid copy number. Based on our experimental data, we propose a model whereby the distribution of multicopy plasmids is controlled by polar fixation under protein production conditions. Thereby, cell lines with fluctuating plasmid abundance arise, which results in population heterogeneity. Our results provide initial insights into the mechanism of cellular heterogeneity during plasmid-based recombinant protein production in a Bacillus species. PMID:26116677

  16. Polar Fixation of Plasmids during Recombinant Protein Production in Bacillus megaterium Results in Population Heterogeneity.

    PubMed

    Münch, Karin M; Müller, Johannes; Wienecke, Sarah; Bergmann, Simone; Heyber, Steffi; Biedendieck, Rebekka; Münch, Richard; Jahn, Dieter

    2015-09-01

    During the past 2 decades, Bacillus megaterium has been systematically developed for the gram-per-liter scale production of recombinant proteins. The plasmid-based expression systems employed use a xylose-controlled promoter. Protein production analyses at the single-cell level using green fluorescent protein as a model product revealed cell culture heterogeneity characterized by a significant proportion of less productive bacteria. Due to the enormous size of B. megaterium, such bistable behavior seen in subpopulations was readily analyzed by time lapse microscopy and flow cytometry. Cell culture heterogeneity was not caused simply by plasmid loss: instead, an asymmetric distribution of plasmids during cell division was detected during the exponential-growth phase. Multicopy plasmids are generally randomly distributed between daughter cells. However, in vivo and in vitro experiments demonstrated that under conditions of strong protein production, plasmids are retained at one of the cell poles. Furthermore, it was found that cells with accumulated plasmids and high protein production ceased cell division. As a consequence, the overall protein production of the culture was achieved mainly by the subpopulation with a sufficient plasmid copy number. Based on our experimental data, we propose a model whereby the distribution of multicopy plasmids is controlled by polar fixation under protein production conditions. Thereby, cell lines with fluctuating plasmid abundance arise, which results in population heterogeneity. Our results provide initial insights into the mechanism of cellular heterogeneity during plasmid-based recombinant protein production in a Bacillus species.

  17. Sub-MIC Tylosin Inhibits Streptococcus suis Biofilm Formation and Results in Differential Protein Expression.

    PubMed

    Wang, Shuai; Yang, Yanbei; Zhao, Yulin; Zhao, Honghai; Bai, Jingwen; Chen, Jianqing; Zhou, Yonghui; Wang, Chang; Li, Yanhua

    2016-01-01

    Streptococcus suis (S.suis) is an important zoonotic pathogen that causes severe diseases in humans and pigs. Biofilms of S. suis can induce persistent infections that are difficult to treat. In this study, the effect of tylosin on biofilm formation of S. suis was investigated. 1/2 minimal inhibitory concentration (MIC) and 1/4 MIC of tylosin were shown to inhibit S. suis biofilm formation in vitro. By using the iTRAQ strategy, we compared the protein expression profiles of S. suis grown with sub-MIC tylosin treatment and with no treatment. A total of 1501 proteins were identified by iTRAQ. Ninety-six differentially expressed proteins were identified (Ratio > ±1.5, p < 0.05). Several metabolism proteins (such as phosphoglycerate kinase) and surface proteins (such as ABC transporter proteins) were found to be involved in biofilm formation. Our results indicated that S. suis metabolic regulation, cell surface proteins, and virulence proteins appear to be of importance in biofilm growth with sub-MIC tylosin treatment. Thus, our data revealed the rough regulation of biofilm formation that may provide a foundation for future research into mechanisms and targets. PMID:27065957

  18. Extensive exploration of conformational space improves Rosetta results for short protein domains.

    PubMed

    Li, Yaohang; Bordner, Andrew J; Tian, Yuan; Tao, Xiuping; Gorin, Andrey A

    2008-01-01

    With some simplifications, computational protein folding can be understood as an optimization problem of a potential energy function on a variable space consisting of all conformation for a given protein molecule. It is well known that realistic energy potentials are very "rough" functions, when expressed in the standard variables, and the folding trajectories can be easily trapped in multiple local minima. We have integrated our variation of Parallel Tempering optimization into the protein folding program Rosetta in order to improve its capability to overcome energy barriers and estimate how such improvement will influence the quality of the folded protein domains. Here we report that (1) Parallel Tempering Rosetta (PTR) is significantly better in the exploration of protein structures than previous implementations of the program; (2) systematic improvements are observed across a large benchmark set in the parameters that are normally followed to estimate robustness of the folding; (3) these improvements are most dramatic in the subset of the shortest domains, where high-quality structures have been obtained for >75% of all tested sequences. Further analysis of the results will improve our understanding of protein conformational space and lead to new improvements in the protein folding methodology, while the current PTR implementation should be very efficient for short (up to approximately 80 a.a.) protein domains and therefore may find practical application in system biology studies.

  19. Sub-MIC Tylosin Inhibits Streptococcus suis Biofilm Formation and Results in Differential Protein Expression

    PubMed Central

    Wang, Shuai; Yang, Yanbei; Zhao, Yulin; Zhao, Honghai; Bai, Jingwen; Chen, Jianqing; Zhou, Yonghui; Wang, Chang; Li, Yanhua

    2016-01-01

    Streptococcus suis (S.suis) is an important zoonotic pathogen that causes severe diseases in humans and pigs. Biofilms of S. suis can induce persistent infections that are difficult to treat. In this study, the effect of tylosin on biofilm formation of S. suis was investigated. 1/2 minimal inhibitory concentration (MIC) and 1/4 MIC of tylosin were shown to inhibit S. suis biofilm formation in vitro. By using the iTRAQ strategy, we compared the protein expression profiles of S. suis grown with sub-MIC tylosin treatment and with no treatment. A total of 1501 proteins were identified by iTRAQ. Ninety-six differentially expressed proteins were identified (Ratio > ±1.5, p < 0.05). Several metabolism proteins (such as phosphoglycerate kinase) and surface proteins (such as ABC transporter proteins) were found to be involved in biofilm formation. Our results indicated that S. suis metabolic regulation, cell surface proteins, and virulence proteins appear to be of importance in biofilm growth with sub-MIC tylosin treatment. Thus, our data revealed the rough regulation of biofilm formation that may provide a foundation for future research into mechanisms and targets. PMID:27065957

  20. Casein protein results in higher prandial and exercise induced whole body protein anabolism than whey protein in chronic obstructive pulmonary disease.

    PubMed

    Engelen, Mariëlle P K J; Rutten, Erica P A; De Castro, Carmen L N; Wouters, Emiel F M; Schols, Annemie M W J; Deutz, Nicolaas E P

    2012-09-01

    Exercise is known to improve physical functioning and health status in Chronic Obstructive Pulmonary Disease (COPD). Recently, disturbances in protein turnover and amino acid kinetics have been observed after exercise in COPD. The objective was to investigate which dairy protein is able to positively influence the protein metabolic response to exercise in COPD. 8 COPD patients and 8 healthy subjects performed a cycle test on two days while ingesting casein or whey protein. Whole body protein breakdown (WbPB), synthesis (WbPS), splanchnic amino acid extraction (SPE), and NetWbPS (=WbPS-WbPB) were measured using stable isotope methodology during 20 min of exercise (at 50% peak work load of COPD group). The controls performed a second exercise test at the same relative workload. Exercise was followed by 1 h of recovery. In the healthy group, WbPS, SPE, and NetPS were higher during casein than during whey feeding (P<.01). WbPS and NetPS were higher during exercise, independent of exercise intensity (P<.01). NetPS was higher during casein feeding in COPD due to lower WbPB (P<.05). Higher SPE was found during exercise during casein and whey feeding in COPD (P<.05). Lactate levels during exercise were higher in COPD (P<.05) independent of the protein. Post-exercise, lower NetPS values were found independent of protein type in both groups. Casein resulted in more protein anabolism than whey protein which was maintained during and following exercise in COPD. Optimizing protein intake might be of importance for muscle maintenance during daily physical activities in COPD.

  1. Synthesis, characterization, and structure-property relationships in two new polar oxides: Zn2(MoO4)(SeO3) and Zn2(MoO4)(TeO3).

    PubMed

    Nguyen, Sau Doan; Kim, Sang-Hwan; Halasyamani, P Shiv

    2011-06-01

    Two new noncentrosymmetric (NCS) polar oxide materials, Zn(2)(MoO(4))(AO(3)) (A = Se(4+) or Te(4+)), have been synthesized by hydrothermal and solid-state techniques. Their crystal structures have been determined, and characterization of their functional properties (second-harmonic generation, piezoelectricity, and polarization) has been performed. The isostructural materials exhibit a three-dimensional network consisting of ZnO(4), ZnO(6), MoO(4), and AO(3) polyhedra that share edges and corners. Powder second-harmonic generation (SHG) measurements using 1064 nm radiation indicate the materials exhibit moderate SHG efficiencies of 100 × and 80 × α-SiO(2) for Zn(2)(MoO(4))(SeO(3)) and Zn(2)(MoO(4))(TeO(3)), respectively. Particle size vs SHG efficiency measurements indicate the materials are type 1 non-phase-matchable. Converse piezoelectric measurements resulted in d(33) values of ∼14 and ∼30 pm/V for Zn(2)(MoO(4))(SeO(3)) and Zn(2)(MoO(4))(TeO(3)), respectively, whereas pyroelectric measurements revealed coefficients of -0.31 and -0.64 μC/m(2) K at 55 °C for Zn(2)(MoO(4))(SeO(3)) and Zn(2)(MoO(4))(TeO(3)), respectively. Frequency-dependent polarization measurements confirmed that all of the materials are nonferroelectric; that is, the macroscopic polarization is not reversible, or "switchable". Infrared, UV-vis, thermogravimetric, and differential thermal analysis measurements were also performed. First-principles density functional theory (DFT) electronic structure calculations were also done. Crystal data: Zn(2)(MoO(4))(SeO(3)), monoclinic, space group P2(1) (No. 4), a = 5.1809(4) Å, b = 8.3238(7) Å, c = 7.1541(6) Å, β = 99.413(1)°, V = 305.2(1) Å(3), Z = 2; Zn(2)(MoO(4))(TeO(3)), monoclinic, space group P2(1) (No. 4), a = 5.178(4) Å, b = 8.409(6) Å, c = 7.241(5) Å, β = 99.351(8)°, V = 311.1(4) Å(3), Z = 2. PMID:21557565

  2. Body Characteristics, Dietary Protein and Body Weight Regulation. Reconciling Conflicting Results from Intervention and Observational Studies?

    PubMed Central

    Ankarfeldt, Mikkel Z.; Ängquist, Lars; Stocks, Tanja; Jakobsen, Marianne U.; Overvad, Kim; Halkjær, Jytte; Saris, Wim H. M.; Astrup, Arne; Sørensen, Thorkild I. A.

    2014-01-01

    Background/Objectives Physiological evidence indicates that high-protein diets reduce caloric intake and increase thermogenic response, which may prevent weight gain and regain after weight loss. Clinical trials have shown such effects, whereas observational cohort studies suggest an association between greater protein intake and weight gain. In both types of studies the results are based on average weight changes, and show considerable diversity in both directions. This study investigates whether the discrepancy in the evidence could be due to recruitment of overweight and obese individuals into clinical trials. Subjects/Methods Data were available from the European Diet, Obesity and Genes (DiOGenes) post-weight-loss weight-maintenance trial and the Danish Diet, Cancer and Health (DCH) cohort. Participants of the DCH cohort were matched with participants from the DiOGenes trial on gender, diet, and body characteristics. Different subsets of the DCH-participants, comparable with the trial participants, were analyzed for weight maintenance according to the randomization status (high or low protein) of the matched trial participants. Results Trial participants were generally heavier, had larger waist circumference and larger fat mass than the participants in the entire DCH cohort. A better weight maintenance in the high-protein group compared to the low protein group was observed in the subgroups of the DCH cohort matching body characteristics of the trial participants. Conclusion This modified observational study, minimized the differences between the RCT and observational data with regard to dietary intake, participant characteristics and statistical analysis. Compared with low protein diet the high protein diet was associated with better weight maintenance when individuals with greater body mass index and waist circumference were analyzed. Selecting subsets of large-scale observational cohort studies with similar characteristics as participants in clinical trials

  3. Crystallization Optimum Solubility Screening: using crystallization results to identify the optimal buffer for protein crystal formation

    SciTech Connect

    Collins, Bernard; Stevens, Raymond C.; Page, Rebecca

    2005-12-01

    It is shown how protein crystallization results can be used to identify buffers that improve protein solubility and, in turn, crystallization success. An optimal solubility screen is described that uses the results of crystallization trials to identify buffers that improve protein solubility and, in turn, crystallization success. This screen is useful not only for standard crystallization experiments, but also can easily be implemented into any high-throughput structure-determination pipeline. As a proof of principle, the predicted novel-fold protein AF2059 from Archaeoglobus fulgidus, which was known to precipitate in most buffers and particularly during concentration experiments, was selected. Using the crystallization results of 192 independent crystallization trials, it was possible to identify a buffer containing 100 mM CHES pH 9.25 that significantly improves its solubility. After transferring AF2059 into this ‘optimum-solubility’ buffer, the protein was rescreened for crystal formation against these same 192 conditions. Instead of extensive precipitation, as observed initially, it was found that 24 separate conditions produced crystals and the exchange of AF2059 into CHES buffer significantly improved crystallization success. Fine-screen optimization of these conditions led to the production of a crystal suitable for high-resolution (2.2 Å) structure determination.

  4. Small structural differences of targeted anti-tumor toxins result in strong variation of protein expression.

    PubMed

    Gilabert-Oriol, Roger; Thakur, Mayank; Weise, Christoph; Dernedde, Jens; von Mallinckrodt, Benedicta; Fuchs, Hendrik; Weng, Alexander

    2013-09-01

    Targeted anti-tumor toxins consist of a toxic functional moiety that is chemically linked or recombinantly fused to a cell-directing ligand. Ribosome-inactivating proteins (RIPs), especially type I RIPs such as saporin or dianthin, are commonly used as toxin components. Although expression of type I RIP-based fusion proteins is well reported, the achievement of higher protein yields in heterologous expression systems through innovative strategies is of major interest. In the present study, the targeted toxins (his)saporin-EGF (SE) and (his)dianthin-EGF (DE) were expressed as fusion proteins under identical expression conditions. However, the total amount of DE was nearly two-times higher than SE. The identity of the heterologously expressed targeted toxins was confirmed by mass spectrometric studies. Their biological specific activity, monitored in real time, was almost equal. Sequence alignment shows 84% identity and a structural comparison revealed five major differences, two of which affect the secondary structure resulting in a loop (SE) to β-strand (DE) conversion and one introduces a gap in SE (after position 57). In conclusion, these structural variations resulted in different protein expression levels while codon usage and toxicity to bacteria were excluded as a cause. Minor structural differences identified in this study may be considered responsible for the protection of DE from bacterial proteases and therefore may serve as a lead to modify certain domains in type I RIP-based targeted toxins.

  5. Arenavirus budding resulting from viral-protein-associated cell membrane curvature

    PubMed Central

    Schley, David; Whittaker, Robert J.; Neuman, Benjamin W.

    2013-01-01

    Viral replication occurs within cells, with release (and onward infection) primarily achieved through two alternative mechanisms: lysis, in which virions emerge as the infected cell dies and bursts open; or budding, in which virions emerge gradually from a still living cell by appropriating a small part of the cell membrane. Virus budding is a poorly understood process that challenges current models of vesicle formation. Here, a plausible mechanism for arenavirus budding is presented, building on recent evidence that viral proteins embed in the inner lipid layer of the cell membrane. Experimental results confirm that viral protein is associated with increased membrane curvature, whereas a mathematical model is used to show that localized increases in curvature alone are sufficient to generate viral buds. The magnitude of the protein-induced curvature is calculated from the size of the amphipathic region hypothetically removed from the inner membrane as a result of translation, with a change in membrane stiffness estimated from observed differences in virion deformation as a result of protein depletion. Numerical results are based on experimental data and estimates for three arenaviruses, but the mechanisms described are more broadly applicable. The hypothesized mechanism is shown to be sufficient to generate spontaneous budding that matches well both qualitatively and quantitatively with experimental observations. PMID:23864502

  6. Differential Nanos 2 protein stability results in selective germ cell accumulation in the sea urchin.

    PubMed

    Oulhen, Nathalie; Wessel, Gary M

    2016-10-01

    Nanos is a translational regulator required for the survival and maintenance of primordial germ cells. In the sea urchin, Strongylocentrotus purpuratus (Sp), Nanos 2 mRNA is broadly transcribed but accumulates specifically in the small micromere (sMic) lineage, in part because of the 3'UTR element GNARLE leads to turnover in somatic cells but retention in the sMics. Here we found that the Nanos 2 protein is also selectively stabilized; it is initially translated throughout the embryo but turned over in the future somatic cells and retained only in the sMics, the future germ line in this animal. This differential stability of Nanos protein is dependent on the open reading frame (ORF), and is independent of the sumoylation and ubiquitylation pathways. Manipulation of the ORF indicates that 68 amino acids in the N terminus of the Nanos protein are essential for its stability in the sMics whereas a 45 amino acid element adjacent to the zinc fingers targets its degradation. Further, this regulation of Nanos protein is cell autonomous, following formation of the germ line. These results are paradigmatic for the unique presence of Nanos in the germ line by a combination of selective RNA retention, distinctive translational control mechanisms (Oulhen et al., 2013), and now also by defined Nanos protein stability.

  7. Differential Nanos 2 protein stability results in selective germ cell accumulation in the sea urchin.

    PubMed

    Oulhen, Nathalie; Wessel, Gary M

    2016-10-01

    Nanos is a translational regulator required for the survival and maintenance of primordial germ cells. In the sea urchin, Strongylocentrotus purpuratus (Sp), Nanos 2 mRNA is broadly transcribed but accumulates specifically in the small micromere (sMic) lineage, in part because of the 3'UTR element GNARLE leads to turnover in somatic cells but retention in the sMics. Here we found that the Nanos 2 protein is also selectively stabilized; it is initially translated throughout the embryo but turned over in the future somatic cells and retained only in the sMics, the future germ line in this animal. This differential stability of Nanos protein is dependent on the open reading frame (ORF), and is independent of the sumoylation and ubiquitylation pathways. Manipulation of the ORF indicates that 68 amino acids in the N terminus of the Nanos protein are essential for its stability in the sMics whereas a 45 amino acid element adjacent to the zinc fingers targets its degradation. Further, this regulation of Nanos protein is cell autonomous, following formation of the germ line. These results are paradigmatic for the unique presence of Nanos in the germ line by a combination of selective RNA retention, distinctive translational control mechanisms (Oulhen et al., 2013), and now also by defined Nanos protein stability. PMID:27424271

  8. Pokeweed antiviral protein alters splicing of HIV-1 RNAs, resulting in reduced virus production.

    PubMed

    Zhabokritsky, Alice; Mansouri, Sheila; Hudak, Katalin A

    2014-08-01

    Processing of HIV-1 transcripts results in three populations in the cytoplasm of infected cells: full-length RNA, singly spliced, and multiply spliced RNAs. Rev, regulator of virion expression, is an essential regulatory protein of HIV-1 required for transporting unspliced and singly spliced viral transcripts from the nucleus to the cytoplasm. Export allows these RNAs to be translated and the full-length RNA to be packaged into virus particles. In our study, we investigate the activity of pokeweed antiviral protein (PAP), a glycosidase isolated from the pokeweed plant Phytolacca americana, on the processing of viral RNAs. We show that coexpression of PAP with a proviral clone alters the splicing ratio of HIV-1 RNAs. Specifically, PAP causes the accumulation of multiply spliced 2-kb RNAs at the expense of full-length 9-kb and singly spliced 4-kb RNAs. The change in splicing ratio is due to a decrease in activity of Rev. We show that PAP depurinates the rev open reading frame and that this damage to the viral RNA inhibits its translation. By decreasing Rev expression, PAP indirectly reduces the availability of full-length 9-kb RNA for packaging and translation of the encoded structural proteins required for synthesis of viral particles. The decline we observe in virus protein expression is not due to cellular toxicity as PAP did not diminish translation rate. Our results describing the reduced activity of a regulatory protein of HIV-1, with resulting change in virus mRNA ratios, provides new insight into the antiviral mechanism of PAP.

  9. Phylogenetic continuum indicates "galaxies" in the protein universe: preliminary results on the natural group structures of proteins.

    PubMed

    Ladunga, I

    1992-04-01

    The markedly nonuniform, even systematic distribution of sequences in the protein "universe" has been analyzed by methods of protein taxonomy. Mapping of the natural hierarchical system of proteins has revealed some dense cores, i.e., well-defined clusterings of proteins that seem to be natural structural groupings, possibly seeds for a future protein taxonomy. The aim was not to force proteins into more or less man-made categories by discriminant analysis, but to find structurally similar groups, possibly of common evolutionary origin. Single-valued distance measures between pairs of superfamilies from the Protein Identification Resource were defined by two chi 2-like methods on tripeptide frequencies and the variable-length subsequence identity method derived from dot-matrix comparisons. Distance matrices were processed by several methods of cluster analysis to detect phylogenetic continuum between highly divergent proteins. Only well-defined clusters characterized by relatively unique structural, intracellular environmental, organismal, and functional attribute states were selected as major protein groups, including subsets of viral and Escherichia coli proteins, hormones, inhibitors, plant, ribosomal, serum and structural proteins, amino acid synthases, and clusters dominated by certain oxidoreductases and apolar and DNA-associated enzymes. The limited repertoire of functional patterns due to small genome size, the high rate of recombination, specific features of the bacterial membranes, or of the virus cycle canalize certain proteins of viruses and Gram-negative bacteria, respectively, to organismal groups.

  10. Interaction of lysozyme protein with different sized silica nanoparticles and their resultant structures

    NASA Astrophysics Data System (ADS)

    Yadav, Indresh; Aswal, V. K.; Kohlbrecher, J.

    2016-05-01

    The interaction of model protein-lysozyme with three different sized anionic silica nanoparticles has been studied by UV-vis spectroscopy, dynamic light scattering (DLS) and small-angle neutron scattering (SANS). The surface area and curvature of the nanoparticles change with size, which significantly influence their interaction with protein. The lysozyme adsorbs on the surface of the nanoparticles due to electrostatic attraction and leads to the phase transformation from one phase (clear) to two-phase (turbid) of the nanoparticle-protein system. The dominance of lysozyme induced short-range attraction over long-range electrostatic repulsion between nanoparticles is responsible for phase transformation and modeled by the two-Yukawa potential. The magnitude of the attractive interaction increases with the size of the nanoparticles as a result the phase transformation commences relatively at lower concentration of lysozyme. The structure of the nanoparticle-protein system in two-phase is characterized by the diffusion limited aggregate type of mass fractal morphology.

  11. Aging results in an unusual expression of Drosophila heat shock proteins

    SciTech Connect

    Fleming, J.E.; Walton, J.K.; Dubitsky, R.; Bensch, K.G. )

    1988-06-01

    The authors used high-resolution two-dimensional polyacrylamide gel electrophoresis to evaluate the effect of aging on the heat shock response in Drosophila melanogaster. Although the aging process is not well understood at the molecular level, recent observations suggest that quantitative changes in gene expression occur as these fruit flies approach senescence. Such genetic alterations are in accord with our present data, which clearly show marked differences in the synthesis of heat shock proteins between young and old fruit flies. In 10-day-old flies, a heat shock of 20 min results in the expression of 14 new proteins as detectable by two-dimensional electrophoresis of ({sup 35}S)methionine-labeled polypeptides, whereas identical treatment of 45-day-old flies leads to the expression of at least 50 new or highly up-regulated proteins. In addition, there is also a concomitant increase in the rate of synthesis of a number of the normal proteins in the older animals. Microdensitometric determinations of the low molecular weight heat shock polypeptides on autoradiographs of five age groups revealed that their maximum expression occurs at 47 days for a population of flies with a mean life span of 33.7 days. Moreover, a heat shock effect similar to that observed in senescent flies occurs in young flies fed canavanine, an arginine analogue, before heat shock.

  12. Diurnal Rhythms Result in Significant Changes in the Cellular Protein Complement in the Cyanobacterium Cyanothece 51142

    SciTech Connect

    Stockel, Jana; Jacobs, Jon M.; Elvitigala, Thanura R.; Liberton, Michelle L.; Welsh, Eric A.; Polpitiya, Ashoka D.; Gritsenko, Marina A.; Nicora, Carrie D.; Koppenaal, David W.; Smith, Richard D.; Pakrasi, Himadri B.

    2011-02-22

    Cyanothece sp. ATCC 51142 is a diazotrophic cyanobacterium notable for its ability to perform oxygenic photosynthesis and dinitrogen fixation in the same single cell. Previous transcriptional analysis revealed that the existence of these incompatible cellular processes largely depends on tightly synchronized expression programs involving ,30% of genes in the genome. To expand upon current knowledge, we have utilized sensitive proteomic approaches to examine the impact of diurnal rhythms on the protein complement in Cyanothece 51142. We found that 250 proteins accounting for,5% of the predicted ORFs from the Cyanothece 51142 genome and 20% of proteins detected under alternating light/dark conditions exhibited periodic oscillations in their abundances. Our results suggest that altered enzyme activities at different phases during the diurnal cycle can be attributed to changes in the abundance of related proteins and key compounds. The integration of global proteomics and transcriptomic data further revealed that post-transcriptional events are important for temporal regulation of processes such as photosynthesis in Cyanothece 51142. This analysis is the first comprehensive report on global quantitative proteomics in a unicellular diazotrophic cyanobacterium and uncovers novel findings about diurnal rhythms.

  13. Protein crystal growth results from the United States Microgravity Laboratory-1 mission

    NASA Technical Reports Server (NTRS)

    Delucas, Lawrence J.; Moore, K. M.; Vanderwoerd, M.; Bray, T. L.; Smith, C.; Carson, M.; Narayana, S. V. L.; Rosenblum, W. M.; Carter, D.; Clark, A. D, Jr.

    1994-01-01

    Protein crystal growth experiments have been performed by this laboratory on 18 Space Shuttle missions since April, 1985. In addition, a number of microgravity experiments also have been performed and reported by other investigators. These Space Shuttle missions have been used to grow crystals of a variety of proteins using vapor diffusion, liquid diffusion, and temperature-induced crystallization techniques. The United States Microgravity Laboratory - 1 mission (USML-1, June 25 - July 9, 1992) was a Spacelab mission dedicated to experiments involved in materials processing. New protein crystal growth hardware was developed to allow in orbit examination of initial crystal growth results, the knowledge from which was used on subsequent days to prepare new crystal growth experiments. In addition, new seeding hardware and techniques were tested as well as techniques that would prepare crystals for analysis by x-ray diffraction, a capability projected for the planned Space Station. Hardware that was specifically developed for the USML-1 mission will be discussed along with the experimental results from this mission.

  14. Molecular alterations resulting from frameshift mutations in peripheral myelin protein 22: implications for neuropathy severity.

    PubMed

    Johnson, J S; Roux, K J; Fletcher, B S; Fortun, J; Notterpek, L

    2005-12-15

    Alterations in peripheral myelin protein 22 (PMP22) expression are associated with a heterogeneous group of hereditary demyelinating peripheral neuropathies. Two mutations at glycine 94, a single guanine insertion or deletion in PMP22, result in different reading frameshifts and, consequently, an extended G94fsX222 or a truncated G94fsX110 protein, respectively. Both of these autosomal dominant mutations alter the second half of PMP22 and yet are linked to clinical phenotypes with distinct severities. The G94fsX222 is associated with hereditary neuropathy with liability to pressure palsies, whereas G94fsX110 causes severe neuropathy diagnosed as Dejerine-Sottas disease or Charcot-Marie-Tooth disease type IA. To investigate the subcellular changes associated with the G94 frameshift mutations, we expressed epitope-tagged forms in primary rat Schwann cells. Biochemical and immunolabeling studies indicate that, unlike the wild-type protein, which is targeted for the plasma membrane, frameshift PMP22s are retained in the cell, prior to reaching the medial Golgi compartment. Similar to Wt-PMP22, both frameshift mutants are targeted for proteasomal degradation and accumulate in detergent-insoluble, ubiquitin-containing aggregates upon inhibition of this pathway. The extended frameshift PMP22 shows the ability to form spontaneous aggregates in the absence of proteasome inhibition. On the other hand, Schwann cells expressing the truncated protein proliferate at a significantly higher rate than Schwann cells expressing the wild-type or the extended PMP22. In summary, these results suggest that a greater potential for PMP22 aggregation is associated with a less severe phenotype, whereas dysregulation of Schwann cell proliferation is linked to severe neuropathy. PMID:16273544

  15. Divergent Evolution of CHD3 Proteins Resulted in MOM1 Refining Epigenetic Control in Vascular Plants

    PubMed Central

    Čaikovski, Marian; Yokthongwattana, Chotika; Habu, Yoshiki; Nishimura, Taisuke; Mathieu, Olivier; Paszkowski, Jerzy

    2008-01-01

    Arabidopsis MOM1 is required for the heritable maintenance of transcriptional gene silencing (TGS). Unlike many other silencing factors, depletion of MOM1 evokes transcription at selected loci without major changes in DNA methylation or histone modification. These loci retain unusual, bivalent chromatin properties, intermediate to both euchromatin and heterochromatin. The structure of MOM1 previously suggested an integral nuclear membrane protein with chromatin-remodeling and actin-binding activities. Unexpected results presented here challenge these presumed MOM1 activities and demonstrate that less than 13% of MOM1 sequence is necessary and sufficient for TGS maintenance. This active sequence encompasses a novel Conserved MOM1 Motif 2 (CMM2). The high conservation suggests that CMM2 has been the subject of strong evolutionary pressure. The replacement of Arabidopsis CMM2 by a poplar motif reveals its functional conservation. Interspecies comparison suggests that MOM1 proteins emerged at the origin of vascular plants through neo-functionalization of the ubiquitous eukaryotic CHD3 chromatin remodeling factors. Interestingly, despite the divergent evolution of CHD3 and MOM1, we observed functional cooperation in epigenetic control involving unrelated protein motifs and thus probably diverse mechanisms. PMID:18725928

  16. Single color FRET based measurements of conformational changes of proteins resulting from translocation inside cells.

    PubMed

    Gahl, Robert F; Tekle, Ephrem; Tjandra, Nico

    2014-03-15

    Translocation of proteins to different parts of the cell is necessary for many cellular mechanisms as a means for regulation and a variety of other functions. Identifying how these proteins undergo conformational changes or interact with various partners during these events is critical to understanding how these mechanisms are executed. A protocol is presented that identifies conformational changes in a protein that occur during translocation while overcoming challenges in extracting distance information in very different environments of a living cell. Only two samples are required to be prepared and are observed with one optical setup. Live-cell FRET imaging has been applied to identify conformational changes between two native cysteines in Bax, a member of the Bcl-2 family of proteins that regulates apoptosis. Bax exists in the cytosol and translocates to the mitochondria outer membrane upon apoptosis induction. The distance, r, between the two native cysteines in the cytosolic structure of Bax necessitates the use of a FRET donor-accepter pair with R0~r as the most sensitive probe for identifying structural changes at these positions. Alexa Fluor 546 and Dabcyl, a dark acceptor, were used as FRET pairs - resulting in single color intensity variations of Alexa-546 as a measure of FRET efficiency. An internal reference, conjugated to Bax, was employed to normalize changes in fluorescence intensity of Alexa Fluor 546 due to inherent inhomogeneities in the living cell. This correction allowed the true FRET effects to be measured with increased precision during translocation. Normalization of intensities to the internal reference identified a FRET efficiency of 0.45±0.14 in the cytosol and 0.11±0.20 in the mitochondria. The procedure for the conjugation of the internal reference and FRET probes as well as the data analysis is presented.

  17. Glucocerebrosidase Deficiency in Drosophila Results in α-Synuclein-Independent Protein Aggregation and Neurodegeneration

    PubMed Central

    Thomas, Ruth E.; Yu, Selina; Germanos, Alexandre A.; Whitley, Brittany N.; Sardi, Sergio Pablo; Montine, Thomas J.; Pallanck, Leo J.

    2016-01-01

    Mutations in the glucosidase, beta, acid (GBA1) gene cause Gaucher’s disease, and are the most common genetic risk factor for Parkinson’s disease (PD) and dementia with Lewy bodies (DLB) excluding variants of low penetrance. Because α-synuclein-containing neuronal aggregates are a defining feature of PD and DLB, it is widely believed that mutations in GBA1 act by enhancing α-synuclein toxicity. To explore this hypothesis, we deleted the Drosophila GBA1 homolog, dGBA1b, and compared the phenotypes of dGBA1b mutants in the presence and absence of α-synuclein expression. Homozygous dGBA1b mutants exhibit shortened lifespan, locomotor and memory deficits, neurodegeneration, and dramatically increased accumulation of ubiquitinated protein aggregates that are normally degraded through an autophagic mechanism. Ectopic expression of human α-synuclein in dGBA1b mutants resulted in a mild enhancement of dopaminergic neuron loss and increased α-synuclein aggregation relative to controls. However, α-synuclein expression did not substantially enhance other dGBA1b mutant phenotypes. Our findings indicate that dGBA1b plays an important role in the metabolism of protein aggregates, but that the deleterious consequences of mutations in dGBA1b are largely independent of α-synuclein. Future work with dGBA1b mutants should reveal the mechanism by which mutations in dGBA1b lead to accumulation of protein aggregates, and the potential influence of this protein aggregation on neuronal integrity. PMID:27019408

  18. Glucocerebrosidase Deficiency in Drosophila Results in α-Synuclein-Independent Protein Aggregation and Neurodegeneration.

    PubMed

    Davis, Marie Y; Trinh, Kien; Thomas, Ruth E; Yu, Selina; Germanos, Alexandre A; Whitley, Brittany N; Sardi, Sergio Pablo; Montine, Thomas J; Pallanck, Leo J

    2016-03-01

    Mutations in the glucosidase, beta, acid (GBA1) gene cause Gaucher's disease, and are the most common genetic risk factor for Parkinson's disease (PD) and dementia with Lewy bodies (DLB) excluding variants of low penetrance. Because α-synuclein-containing neuronal aggregates are a defining feature of PD and DLB, it is widely believed that mutations in GBA1 act by enhancing α-synuclein toxicity. To explore this hypothesis, we deleted the Drosophila GBA1 homolog, dGBA1b, and compared the phenotypes of dGBA1b mutants in the presence and absence of α-synuclein expression. Homozygous dGBA1b mutants exhibit shortened lifespan, locomotor and memory deficits, neurodegeneration, and dramatically increased accumulation of ubiquitinated protein aggregates that are normally degraded through an autophagic mechanism. Ectopic expression of human α-synuclein in dGBA1b mutants resulted in a mild enhancement of dopaminergic neuron loss and increased α-synuclein aggregation relative to controls. However, α-synuclein expression did not substantially enhance other dGBA1b mutant phenotypes. Our findings indicate that dGBA1b plays an important role in the metabolism of protein aggregates, but that the deleterious consequences of mutations in dGBA1b are largely independent of α-synuclein. Future work with dGBA1b mutants should reveal the mechanism by which mutations in dGBA1b lead to accumulation of protein aggregates, and the potential influence of this protein aggregation on neuronal integrity.

  19. Glucocerebrosidase Deficiency in Drosophila Results in α-Synuclein-Independent Protein Aggregation and Neurodegeneration.

    PubMed

    Davis, Marie Y; Trinh, Kien; Thomas, Ruth E; Yu, Selina; Germanos, Alexandre A; Whitley, Brittany N; Sardi, Sergio Pablo; Montine, Thomas J; Pallanck, Leo J

    2016-03-01

    Mutations in the glucosidase, beta, acid (GBA1) gene cause Gaucher's disease, and are the most common genetic risk factor for Parkinson's disease (PD) and dementia with Lewy bodies (DLB) excluding variants of low penetrance. Because α-synuclein-containing neuronal aggregates are a defining feature of PD and DLB, it is widely believed that mutations in GBA1 act by enhancing α-synuclein toxicity. To explore this hypothesis, we deleted the Drosophila GBA1 homolog, dGBA1b, and compared the phenotypes of dGBA1b mutants in the presence and absence of α-synuclein expression. Homozygous dGBA1b mutants exhibit shortened lifespan, locomotor and memory deficits, neurodegeneration, and dramatically increased accumulation of ubiquitinated protein aggregates that are normally degraded through an autophagic mechanism. Ectopic expression of human α-synuclein in dGBA1b mutants resulted in a mild enhancement of dopaminergic neuron loss and increased α-synuclein aggregation relative to controls. However, α-synuclein expression did not substantially enhance other dGBA1b mutant phenotypes. Our findings indicate that dGBA1b plays an important role in the metabolism of protein aggregates, but that the deleterious consequences of mutations in dGBA1b are largely independent of α-synuclein. Future work with dGBA1b mutants should reveal the mechanism by which mutations in dGBA1b lead to accumulation of protein aggregates, and the potential influence of this protein aggregation on neuronal integrity. PMID:27019408

  20. Correlating labeling chemistry and in-vitro test results with the biological behavior of radiolabeled proteins

    SciTech Connect

    Srivastava, S.C.; Meinken, G.E.

    1985-01-01

    Monoclonal antibodies possess enormous potential for delivery of therapeutic amounts of radionuclides to target antigens in vivo, in particular for tumor imaging and therapy. Translation of this concept into practice has encountered numerous problems. Specifically whereas general protein radiolabeling methods are applicable to antibodies, immunological properties of the antibodies are often compromised resulting in reduced in-vivo specificity for the target antigens. The bifunctional chelating agent approach shows the most promise, however, development of other agents will be necessary for widespread usefulness of this technique. The effects of labeling chemistry on the in-vivo behavior of several monoclonal antibodies are described. 30 refs., 4 figs., 10 tabs.

  1. HIV-1 Tat Protein Increases Microglial Outward K+ Current and Resultant Neurotoxic Activity

    PubMed Central

    Liu, Jianuo; Xu, Peng; Collins, Cory; Liu, Han; Zhang, Jingdong; Keblesh, James P.; Xiong, Huangui

    2013-01-01

    Microglia plays a crucial role in the pathogenesis of HIV-1-associated neurocognitive disorders. Increasing evidence indicates the voltage-gated potassium (Kv) channels are involved in the regulation of microglia function, prompting us to hypothesize Kv channels may also be involved in microglia-mediated neurotoxic activity in HIV-1-infected brain. To test this hypothesis, we investigated the involvement of Kv channels in the response of microglia to HIV-1 Tat protein. Treatment of rat microglia with HIV-1 Tat protein (200 ng/ml) resulted in pro-inflammatory microglial activation, as indicated by increases in TNF-α, IL-1β, reactive oxygen species, and nitric oxide, which were accompanied by enhanced outward K+ current and Kv1.3 channel expression. Suppression of microglial Kv1.3 channel activity, either with Kv1.3 channel blockers Margatoxin, 5-(4-Phenoxybutoxy)psoralen, or broad-spectrum K+ channel blocker 4-Aminopyridine, or by knockdown of Kv1.3 expression via transfection of microglia with Kv1.3 siRNA, was found to abrogate the neurotoxic activity of microglia resulting from HIV-1 Tat exposure. Furthermore, HIV-1 Tat-induced neuronal apoptosis was attenuated with the application of supernatant collected from K+ channel blocker-treated microglia. Lastly, the intracellular signaling pathways associated with Kv1.3 were investigated and enhancement of microglial Kv1.3 was found to correspond with an increase in Erk1/2 mitogen-activated protein kinase activation. These data suggest targeting microglial Kv1.3 channels may be a potential new avenue of therapy for inflammation-mediated neurological disorders. PMID:23738010

  2. Cooking Chicken Breast Reduces Dialyzable Iron Resulting from Digestion of Muscle Proteins

    PubMed Central

    Gokhale, Aditya S.; Mahoney, Raymond R.

    2014-01-01

    The purpose of this research was to study the effect of cooking chicken breast on the production of dialyzable iron (an in vitro indicator of bioavailable iron) from added ferric iron. Chicken breast muscle was cooked by boiling, baking, sautéing, or deep-frying. Cooked samples were mixed with ferric iron and either extracted with acid or digested with pepsin and pancreatin. Total and ferrous dialyzable iron was measured after extraction or digestion and compared to raw chicken samples. For uncooked samples, dialyzable iron was significantly enhanced after both extraction and digestion. All cooking methods led to markedly reduced levels of dialyzable iron both by extraction and digestion. In most cooked, digested samples dialyzable iron was no greater than the iron-only (no sample) control. Cooked samples showed lower levels of histidine and sulfhydryls but protein digestibility was not reduced, except for the sautéed sample. The results showed that, after cooking, little if any dialyzable iron results from digestion of muscle proteins. Our research indicates that, in cooked chicken, residual acid-extractable components are the most important source of dialyzable iron. PMID:26904627

  3. Characterization of Factors Affecting Nanoparticle Tracking Analysis Results With Synthetic and Protein Nanoparticles.

    PubMed

    Krueger, Aaron B; Carnell, Pauline; Carpenter, John F

    2016-04-01

    In many manufacturing and research areas, the ability to accurately monitor and characterize nanoparticles is becoming increasingly important. Nanoparticle tracking analysis is rapidly becoming a standard method for this characterization, yet several key factors in data acquisition and analysis may affect results. Nanoparticle tracking analysis is prone to user input and bias on account of a high number of parameters available, contains a limited analysis volume, and individual sample characteristics such as polydispersity or complex protein solutions may affect analysis results. This study systematically addressed these key issues. The integrated syringe pump was used to increase the sample volume analyzed. It was observed that measurements recorded under flow caused a reduction in total particle counts for both polystyrene and protein particles compared to those collected under static conditions. In addition, data for polydisperse samples tended to lose peak resolution at higher flow rates, masking distinct particle populations. Furthermore, in a bimodal particle population, a bias was seen toward the larger species within the sample. The impacts of filtration on an agitated intravenous immunoglobulin sample and operating parameters including "MINexps" and "blur" were investigated to optimize the method. Taken together, this study provides recommendations on instrument settings and sample preparations to properly characterize complex samples. PMID:27019960

  4. Distinct stress conditions result in aggregation of proteins with similar properties

    PubMed Central

    Weids, Alan J.; Ibstedt, Sebastian; Tamás, Markus J.; Grant, Chris M.

    2016-01-01

    Protein aggregation is the abnormal association of proteins into larger aggregate structures which tend to be insoluble. This occurs during normal physiological conditions and in response to age or stress-induced protein misfolding and denaturation. In this present study we have defined the range of proteins that aggregate in yeast cells during normal growth and after exposure to stress conditions including an oxidative stress (hydrogen peroxide), a heavy metal stress (arsenite) and an amino acid analogue (azetidine-2-carboxylic acid). Our data indicate that these three stress conditions, which work by distinct mechanisms, promote the aggregation of similar types of proteins probably by lowering the threshold of protein aggregation. The proteins that aggregate during physiological conditions and stress share several features; however, stress conditions shift the criteria for protein aggregation propensity. This suggests that the proteins in aggregates are intrinsically aggregation-prone, rather than being proteins which are affected in a stress-specific manner. We additionally identified significant overlaps between stress aggregating yeast proteins and proteins that aggregate during ageing in yeast and C. elegans. We suggest that similar mechanisms may apply in disease- and non-disease settings and that the factors and components that control protein aggregation may be evolutionary conserved. PMID:27086931

  5. Preparation and characterization of SeO2/TiO2 composite photocatalyst with excellent performance for sunset yellow azo dye degradation under natural sunlight illumination.

    PubMed

    Rajamanickam, D; Dhatshanamurthi, P; Shanthi, M

    2015-03-01

    To improve the solar light induced photocatalytic application performances of TiO2, in this study, the SeO2 modified TiO2 composite photocatalysts with various ratios of SeO2 to TiO2 were prepared by sol-gel method. The catalyst was characterized by X-ray diffraction (XRD), high resolution scanning electron microscope (HR-SEM), energy dispersive spectra (EDS), diffuse reflectance spectra (DRS), photoluminescence spectra (PL), X-ray photoelectron spectroscopy (XPS) and Brunauer-Emmett-Teller (BET) surface area measurement methods. The photocatalytic activity of SeO2/TiO2 was investigated for the degradation of sunset yellow (SY) in aqueous solution using solar light. The SeO2/TiO2 is found to be more efficient than prepared TiO2 and TiO2-P25 at pH 7 for the mineralization of SY. The effects of operational parameters such as the amount of photocatalyst, dye concentration and initial pH on photo mineralization of SY have been analyzed. The degradation was strongly enhanced in the presence of electron acceptors such as oxone, KIO4 and KBrO3. The kinetics of SY photodegradation was found to follow the pseudo-first order rate law and could be described in terms of Langmuir-Hinshelwood model. The mineralization of SY has been confirmed by COD measurements. The catalyst is found to be reusable.

  6. Preparation and characterization of SeO2/TiO2 composite photocatalyst with excellent performance for sunset yellow azo dye degradation under natural sunlight illumination

    NASA Astrophysics Data System (ADS)

    Rajamanickam, D.; Dhatshanamurthi, P.; Shanthi, M.

    2015-03-01

    To improve the solar light induced photocatalytic application performances of TiO2, in this study, the SeO2 modified TiO2 composite photocatalysts with various ratios of SeO2 to TiO2 were prepared by sol-gel method. The catalyst was characterized by X-ray diffraction (XRD), high resolution scanning electron microscope (HR-SEM), energy dispersive spectra (EDS), diffuse reflectance spectra (DRS), photoluminescence spectra (PL), X-ray photoelectron spectroscopy (XPS) and Brunauer-Emmett-Teller (BET) surface area measurement methods. The photocatalytic activity of SeO2/TiO2 was investigated for the degradation of sunset yellow (SY) in aqueous solution using solar light. The SeO2/TiO2 is found to be more efficient than prepared TiO2 and TiO2-P25 at pH 7 for the mineralization of SY. The effects of operational parameters such as the amount of photocatalyst, dye concentration and initial pH on photo mineralization of SY have been analyzed. The degradation was strongly enhanced in the presence of electron acceptors such as oxone, KIO4 and KBrO3. The kinetics of SY photodegradation was found to follow the pseudo-first order rate law and could be described in terms of Langmuir-Hinshelwood model. The mineralization of SY has been confirmed by COD measurements. The catalyst is found to be reusable.

  7. Sonic hedgehog shedding results in functional activation of the solubilized protein.

    PubMed

    Ohlig, Stefanie; Farshi, Pershang; Pickhinke, Ute; van den Boom, Johannes; Höing, Susanne; Jakuschev, Stanislav; Hoffmann, Daniel; Dreier, Rita; Schöler, Hans R; Dierker, Tabea; Bordych, Christian; Grobe, Kay

    2011-06-14

    All Hedgehog (Hh) proteins are released from producing cells despite being synthesized as N- and C-terminally lipidated, membrane-tethered molecules. Thus, a cellular mechanism is needed for Hh solubilization. We previously suggested that a disintegrin and metalloprotease (ADAM)-mediated shedding of Sonic hedgehog (ShhNp) from its lipidated N and C termini results in protein solubilization. This finding, however, seemed at odds with the established role of N-terminal palmitoylation for ShhNp signaling activity. We now resolve this paradox by showing that N-palmitoylation of ShhNp N-terminal peptides is required for their proteolytic removal during solubilization. These peptides otherwise block ShhNp zinc coordination sites required for ShhNp binding to its receptor Patched (Ptc), explaining the essential yet indirect role of N-palmitoylation for ShhNp function. We suggest a functional model in which membrane-tethered multimeric ShhNp is at least partially autoinhibited in trans but is processed into fully active, soluble multimers upon palmitoylation-dependent cleavage of inhibitory N-terminal peptides. PMID:21664575

  8. Results.

    ERIC Educational Resources Information Center

    Zemsky, Robert; Shaman, Susan; Shapiro, Daniel B.

    2001-01-01

    Describes the Collegiate Results Instrument (CRI), which measures a range of collegiate outcomes for alumni 6 years after graduation. The CRI was designed to target alumni from institutions across market segments and assess their values, abilities, work skills, occupations, and pursuit of lifelong learning. (EV)

  9. SANS study of interaction of silica nanoparticles with BSA protein and their resultant structure

    NASA Astrophysics Data System (ADS)

    Yadav, Indresh; Aswal, V. K.; Kohlbrecher, J.

    2014-04-01

    Small angle neutron scattering (SANS) has been carried out to study the interaction of anionic silica nanoparticles (88 Å) with globular protein Bovine Serum Albumin (BSA) (M.W. 66.4 kD) in aqueous solution. The measurements have been carried out on fixed concentration (1 wt %) of Ludox silica nanoparticles with varying concentration of BSA (0-5 wt %) at pH7. Results show that silica nanoparticles and BSA coexist as individual entities at low concentration of BSA where electrostatic repulsive interactions between them prevent their aggregation. However, as the concentration of BSA increases (≥ 0.5 wt %), it induces the attractive depletion interaction among nanoparticles leading to finally their aggregation at higher BSA concentration (2 wt %). The aggregates are found to be governed by the diffusion limited aggregation (DLA) morphology of fractal nature having fractal dimension about 2.4.

  10. SANS study of interaction of silica nanoparticles with BSA protein and their resultant structure

    SciTech Connect

    Yadav, Indresh Aswal, V. K.; Kohlbrecher, J.

    2014-04-24

    Small angle neutron scattering (SANS) has been carried out to study the interaction of anionic silica nanoparticles (88 Å) with globular protein Bovine Serum Albumin (BSA) (M.W. 66.4 kD) in aqueous solution. The measurements have been carried out on fixed concentration (1 wt %) of Ludox silica nanoparticles with varying concentration of BSA (0–5 wt %) at pH7. Results show that silica nanoparticles and BSA coexist as individual entities at low concentration of BSA where electrostatic repulsive interactions between them prevent their aggregation. However, as the concentration of BSA increases (≥ 0.5 wt %), it induces the attractive depletion interaction among nanoparticles leading to finally their aggregation at higher BSA concentration (2 wt %). The aggregates are found to be governed by the diffusion limited aggregation (DLA) morphology of fractal nature having fractal dimension about 2.4.

  11. Lack of Plasma Protein Hemopexin Results in Increased Duodenal Iron Uptake

    PubMed Central

    Fiorito, Veronica; Geninatti Crich, Simonetta; Silengo, Lorenzo; Aime, Silvio; Altruda, Fiorella; Tolosano, Emanuela

    2013-01-01

    Purpose The body concentration of iron is regulated by a fine equilibrium between absorption and losses of iron. Iron can be absorbed from diet as inorganic iron or as heme. Hemopexin is an acute phase protein that limits iron access to microorganisms. Moreover, it is the plasma protein with the highest binding affinity for heme and thus it mediates heme-iron recycling. Considering its involvement in iron homeostasis, it was postulated that hemopexin may play a role in the physiological absorption of inorganic iron. Methods and Results Hemopexin-null mice showed elevated iron deposits in enterocytes, associated with higher duodenal H-Ferritin levels and a significant increase in duodenal expression and activity of heme oxygenase. The expression of heme-iron and inorganic iron transporters was normal. The rate of iron absorption was assessed by measuring the amount of 57Fe retained in tissues from hemopexin-null and wild-type animals after administration of an oral dose of 57FeSO4 or of 57Fe-labelled heme. Higher iron retention in the duodenum of hemopexin-null mice was observed as compared with normal mice. Conversely, iron transfer from enterocytes to liver and bone marrow was unaffected in hemopexin-null mice. Conclusions The increased iron level in hemopexin-null duodenum can be accounted for by an increased iron uptake by enterocytes and storage in ferritins. These data indicate that the lack of hemopexin under physiological conditions leads to an enhanced duodenal iron uptake thus providing new insights to our understanding of body iron homeostasis. PMID:23826373

  12. Influence of protonation, tautomeric, and stereoisomeric states on protein-ligand docking results.

    PubMed

    ten Brink, Tim; Exner, Thomas E

    2009-06-01

    In this work, we present a systematical investigation of the influence of ligand protonation states, stereoisomers, and tautomers on results obtained with the two protein-ligand docking programs GOLD and PLANTS. These different states were generated with a fully automated tool, called SPORES (Structure PrOtonation and Recognition System). First, the most probable protonations, as defined by this rule based system, were compared to the ones stored in the well-known, manually revised CCDC/ASTEX data set. Then, to investigate the influence of the ligand protonation state on the docking results, different protonation states were created. Redocking and virtual screening experiments were conducted demonstrating that both docking programs have problems in identifying the correct protomer for each complex. Therefore, a preselection of plausible protomers or the improvement of the scoring functions concerning their ability to rank different molecules/states is needed. Additionally, ligand stereoisomers were tested for a subset of the CCDC/ASTEX set, showing similar problems regarding the ranking of these stereoisomers as the ranking of the protomers.

  13. Influence of protonation, tautomeric, and stereoisomeric states on protein-ligand docking results.

    PubMed

    ten Brink, Tim; Exner, Thomas E

    2009-06-01

    In this work, we present a systematical investigation of the influence of ligand protonation states, stereoisomers, and tautomers on results obtained with the two protein-ligand docking programs GOLD and PLANTS. These different states were generated with a fully automated tool, called SPORES (Structure PrOtonation and Recognition System). First, the most probable protonations, as defined by this rule based system, were compared to the ones stored in the well-known, manually revised CCDC/ASTEX data set. Then, to investigate the influence of the ligand protonation state on the docking results, different protonation states were created. Redocking and virtual screening experiments were conducted demonstrating that both docking programs have problems in identifying the correct protomer for each complex. Therefore, a preselection of plausible protomers or the improvement of the scoring functions concerning their ability to rank different molecules/states is needed. Additionally, ligand stereoisomers were tested for a subset of the CCDC/ASTEX set, showing similar problems regarding the ranking of these stereoisomers as the ranking of the protomers. PMID:19453150

  14. Immunohistochemistry staining for mismatch repair proteins: the endoscopic biopsy material provides useful and coherent results.

    PubMed

    Vilkin, Alex; Leibovici-Weissman, Ya'ara; Halpern, Marisa; Morgenstern, Sara; Brazovski, Eli; Gingold-Belfer, Rachel; Wasserberg, Nir; Brenner, Baruch; Niv, Yaron; Sneh-Arbib, Orly; Levi, Zohar

    2015-11-01

    Immunohistochemistry (IHC) testing for mismatch repair proteins (MMRP) in patients with colorectal cancer can be performed on endoscopic biopsy material or the surgical resection material. Data are continuing to accumulate regarding the deleterious effect of neoadjuvant chemoradiation on MMRP expression. However, despite continuing rise in the use of endoscopic biopsies for IHC, most pathology departments still use mainly the surgical materials for IHC testing. In this study we compared the quality of stains among 96 colon cancer subjects with paired endoscopic and surgical material available for MLH1, MSH2, MSH6, and PMS2 stains (96 × 4, yielding 384 paired stains). Each slide received both a quantitative score (immunoreactivity [0-3] × percent positivity [0-4]) and a qualitative score (absent; weak and focal; strong). The quantitative scores of all MMRP were significantly higher among the endoscopic material (P<.001 for all). In 358 pairs (93.2%), both the endoscopic and operative material stained either strong (322, 83.9%) or absent (36, 9.4%). In 26 pairs (6.8%), the endoscopic material stained strong, whereas the operative material stained focal and weak. No endoscopic biopsy materials stained focal and weak. Our findings indicate that the biopsy material may provide more coherent results. Although these results may indicate that biopsy material provides coherent and useful results, it is yet to be determined if the demonstrated differences pose a real clinical problem in interpreting final results of IHC staining of such kind. Hence, we suggest that when available, the endoscopic material rather than the operative one should serve as the primary substrate for IHC staining.

  15. Expression of Huntington's disease protein results in apoptotic neurons in the brains of cloned transgenic pigs

    PubMed Central

    Yang, Dongshan; Wang, Chuan-En; Zhao, Bentian; Li, Wei; Ouyang, Zhen; Liu, Zhaoming; Yang, Huaqiang; Fan, Pei; O'Neill, Ashley; Gu, Weiwang; Yi, Hong; Li, Shihua; Lai, Liangxue; Li, Xiao-Jiang

    2010-01-01

    Neurodegeneration is a hallmark of many neurological diseases, including Alzheimer's, Parkinson's and the polyglutamine diseases, which are all caused by misfolded proteins that accumulate in neuronal cells of the brain. Although apoptosis is believed to contribute to neurodegeneration in these cases, genetic mouse models of these diseases often fail to replicate apoptosis and overt neurodegeneration in the brain. Using nuclear transfer, we generated transgenic Huntington's disease (HD) pigs that express N-terminal (208 amino acids) mutant huntingtin with an expanded polyglutamine tract (105Q). Postnatal death, dyskinesia and chorea-like movement were observed in some transgenic pigs that express mutant huntingtin. Importantly, the transgenic HD pigs, unlike mice expressing the same transgene, displayed typical apoptotic neurons with DNA fragmentation in their brains. Also, expression of mutant huntingtin resulted in more neurons with activated caspase-3 in transgenic pig brains than that in transgenic mouse brains. Our findings suggest that species differences determine neuropathology and underscore the importance of large mammalian animals for modeling neurological disorders. PMID:20660116

  16. The Structural Biology Center 19ID undulator beamline: facility specifications and protein crystallographic results

    PubMed Central

    Rosenbaum, Gerd; Alkire, Randy W.; Evans, Gwyndaf; Rotella, Frank J.; Lazarski, Krzystof; Zhang, Rong-Guang; Ginell, Stephan L.; Duke, Norma; Naday, Istvan; Lazarz, Jack; Molitsky, Michael J.; Keefe, Lisa; Gonczy, John; Rock, Larry; Sanishvili, Ruslan; Walsh, Martin A.; Westbrook, Edwin; Joachimiak, Andrzej

    2008-01-01

    The 19ID undulator beamline of the Structure Biology Center has been designed and built to take full advantage of the high flux, brilliance and quality of X-ray beams delivered by the Advanced Photon Source. The beamline optics are capable of delivering monochromatic X-rays with photon energies from 3.5 to 20 keV (3.5–0.6 Å wavelength) with fluxes up to 8–18 × 1012 photons s−1 (depending on photon energy) onto cryogenically cooled crystal samples. The size of the beam (full width at half-maximum) at the sample position can be varied from 2.2 mm × 1.0 mm (horizontal × vertical, unfocused) to 0.083 mm × 0.020 mm in its fully focused configuration. Specimen-to-detector distances of between 100 mm and 1500 mm can be used. The high flexibility, inherent in the design of the optics, coupled with a κ-geometry goniometer and beamline control software allows optimal strategies to be adopted in protein crystallographic experiments, thus maximizing the chances of their success. A large-area mosaic 3 × 3 CCD detector allows high-quality diffraction data to be measured rapidly to the crystal diffraction limits. The beamline layout and the X-ray optical and endstation components are described in detail, and the results of representative crystallographic experiments are presented. PMID:16371706

  17. Reduced functionality of PSE-like chicken breast meat batter resulting from alterations in protein conformation.

    PubMed

    Li, K; Zhao, Y Y; Kang, Z L; Wang, P; Han, M Y; Xu, X L; Zhou, G H

    2015-01-01

    The objectives of this study were to evaluate protein thermal stability, water-protein interaction, microstructure, and protein conformation between PSE-like and normal chicken breast meat batters. Sixty pale, soft, and exudative (PSE)-like (L*>53, pH24 h<5.7) and 60 normal (46protein and 2% salt, and they were analyzed for the protein changes and the microstructure using differential scanning calorimetry, low-field (LF)-NMR, SEM, and Raman spectroscopy. PSE-like meat batter had lower gel strength, water-holding capacity, and salt-soluble protein extraction (P<0.05). Heated PSE-like meat batter formed an aggregated gel matrix, while normal meat batter produced a compact gel network with fine, cross-linked strands by many protein filaments. LF-NMR revealed an increase in the water mobility in heated PSE-like meat batter with an increasing amount of loosely bound water (P<0.05). No significant changes were observed in the electrophoretic patterns of salt-soluble protein extracts by SDS-PAGE. However, differential scanning calorimetry showed that PSE-like meat had greater myosin and sarcoplasmic proteins/collagen denaturation (P<0.05). In PSE-like meat, actin denaturation was particular evident after salt addition (P<0.05) using differential scanning calorimetry. Moreover, Raman spectroscopy indicated that PSE-like meat batter had less unfolded α-helix and β-sheet structure formation, reduced exposure of hydrophobic and tyrosine residues (P<0.05), and changes in the microenvironment of aliphatic residues and tryptophan, which affected salt-soluble protein extraction, gel properties, and water-holding capacity. In conclusion, the inferior functional properties of PSE-like meat were attributed to not only myosin denaturation, but also actin denaturation after salt addition and different

  18. Next-generation protein-rich potato expressing the seed protein gene AmA1 is a result of proteome rebalancing in transgenic tuber.

    PubMed

    Chakraborty, Subhra; Chakraborty, Niranjan; Agrawal, Lalit; Ghosh, Sudip; Narula, Kanika; Shekhar, Shubhendu; Naik, Prakash S; Pande, P C; Chakrborti, Swarup Kumar; Datta, Asis

    2010-10-12

    Protein deficiency is the most crucial factor that affects physical growth and development and that increases morbidity and mortality especially in developing countries. Efforts have been made to improve protein quality and quantity in crop plants but with limited success. Here, we report the development of transgenic potatoes with enhanced nutritive value by tuber-specific expression of a seed protein, AmA1 (Amaranth Albumin 1), in seven genotypic backgrounds suitable for cultivation in different agro-climatic regions. Analyses of the transgenic tubers revealed up to 60% increase in total protein content. In addition, the concentrations of several essential amino acids were increased significantly in transgenic tubers, which are otherwise limited in potato. Moreover, the transgenics also exhibited enhanced photosynthetic activity with a concomitant increase in total biomass. These results are striking because this genetic manipulation also resulted in a moderate increase in tuber yield. The comparative protein profiling suggests that the proteome rebalancing might cause increased protein content in transgenic tubers. Furthermore, the data on field performance and safety evaluation indicate that the transgenic potatoes are suitable for commercial cultivation. In vitro and in vivo studies on experimental animals demonstrate that the transgenic tubers are also safe for human consumption. Altogether, these results emphasize that the expression of AmA1 is a potential strategy for the nutritional improvement of food crops.

  19. Chimeric spider silk proteins mediated by intein result in artificial hybrid silks.

    PubMed

    Lin, Senzhu; Chen, Gefei; Liu, Xiangqin; Meng, Qing

    2016-07-01

    Hybrid silks hold a great potential as specific biomaterials due to its controlled mechanical properties. To produce fibers with tunable properties, here we firstly made chimeric proteins in vitro, called W2C4CT and W2C8CT, with ligation of MaSp repetitive modules (C) with AcSp modules (W) by intein trans splicing technology from smaller precursors without final yield reduction. Intein mediated chimeric proteins form fibers at a low concentration of 0.4 mg/mL in 50 mM K3 PO4 pH 7.5 just drawn by hand. Hybrid fibers show smoother surface, and also have stronger chemical resistance as compared with fibers from W2CT (W fibers) and mixture of W2CT/C8CT (MHF8 fibers). Fibers from chimeric protein W2C4CT (HFH4) have improved mechanical properties than W fibers; however, with more C modules W2C8CT fibers (HFH8) properties decreased, indicates the length proportion of various modules is very important and should be optimized for fibers with specific properties. Generally, hybrid silks generated via chimeric proteins, which can be simplified by intein trans splicing, has greater potential to produce fibers with tunable properties. Our research shows that intein mediated directional protein ligation is a novel way to make large chimeric spider silk proteins and hybrid silks. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 385-392, 2016. PMID:26948769

  20. Committee on Earth Observation Satellites (CEOS) Systems Engineering Office (SEO). Ocean Surface Topography (OST) Workshop, Ruedesheim an Rhein, Germany. [CEOS SEO Status Report

    NASA Technical Reports Server (NTRS)

    Killough, Brian D., Jr.

    2008-01-01

    The CEOS Systems Engineering Office will present a 2007 status report of the CEOS constellation process, present a new systems engineering framework, and analysis results from the GEO Societal Benefit Area (SBA) assessment and the OST constellation requirements assessment.

  1. Changes in structural characteristics of antioxidative soy protein hydrolysates resulting from scavenging of hydroxyl radicals.

    PubMed

    Zhao, Jing; Xiong, Youling L; McNear, Dave H

    2013-02-01

    Antioxidant activity of soy protein (SP) and its hydrolyzed peptides has been widely reported. During scavenging of radicals, these antioxidative compounds would be oxidatively modified, but their fate is not understood. The objective of this study was to evaluate the structural characteristics of SP hydrolysates (SPHs), compared to intact SP, when used to neutralize hydroxyl radicals (•OH). SPHs with degree of hydrolysis (DH) 1 to 5 were prepared with Alcalase. Antioxidant activity of SPHs was confirmed by lipid oxidation inhibition measured with thiobarbituric acid-reactive substances, ability to scavenge 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) radicals, and ferrous ion chelation capability. Oxidation of SPHs was initiated by reaction with •OH generated from 0.1 mM FeCl(3) , 20 mM H(2) O(2) , and 1.0 mM ascorbate. After oxidative stress, carbonyl content of SPHs increased by 2- to 3-fold and sulfhydryl groups decreased by up to 42% compared to nonoxidized samples (P < 0.05). Methionine, histidine, and lysine residues were significantly reduced as a result of inactivating •OH (P < 0.05). Attenuated total reflectance-Fourier transform infrared and circular dichroism spectroscopy suggested the conversion of helical structure to strands and turns. Oxidatively modified SPHs had a lower intrinsic fluorescence intensity but similar solubility when compared to nonoxidized samples. These structural changes due to •OH stress may impact the ingredient interaction and functionality of SPHs in food products. PMID:23331209

  2. Targeted modification of storage protein content resulting in improved amino acid composition of barley grain.

    PubMed

    Sikdar, Md S I; Bowra, S; Schmidt, D; Dionisio, G; Holm, P B; Vincze, E

    2016-02-01

    C-hordein in barley and ω-gliadins in wheat are members of the prolamins protein families. Prolamins are the major component of cereal storage proteins and composed of non-essential amino acids (AA) such as proline and glutamine therefore have low nutritional value. Using double stranded RNAi silencing technology directed towards C-hordein we obtained transgenic barley lines with up to 94.7% reduction in the levels of C-hordein protein relative to the parental line. The composition of the prolamin fraction of the barley parental line cv. Golden Promise was resolved using SDS-PAGE electrophoresis, the protein band were excised and the proteins identified by quadrupole-time-of-flight mass spectrometry. Subsequent SDS-PAGE separation and analysis of the prolamin fraction of the transgenic lines revealed a reduction in the amounts of C-hordeins and increases in the content of other hordein family members. Analysis of the AA composition of the transgenic lines showed that the level of essential amino acids increased with a concomitant reduction in proline and glutamine. Both the barley C-hordein and wheat ω-gliadin genes proved successful for RNAi-gene mediated suppression of barley C-hordein level. All transgenic lines that exhibited a reduction for C-hordein showed off-target effects: the lines exhibited increased level of B/γ-hordein while D-hordein level was reduced. Furthermore, the multicopy insertions correlated negatively with silencing.

  3. Results of a screening programme to identify plants or plant extracts that inhibit ruminal protein degradation.

    PubMed

    Selje, N; Hoffmann, E M; Muetzel, S; Ningrat, R; Wallace, R J; Becker, K

    2007-07-01

    One aim of the EC Framework V project, 'Rumen-up' (QLK5-CT-2001-00 992), was to find plants or plant extracts that would inhibit the nutritionally wasteful degradation of protein in the rumen. A total of 500 samples were screened in vitro using 14C-labelled casein in a 30-min incubation with ruminal digesta. Eight were selected for further investigation using a batch fermentation system and soya protein and bovine serum albumin as proteolysis substrates; proteolysis was monitored over 12 h by the disappearance of soluble protein and the production of branched SCFA and NH3. Freeze-dried, ground foliage of Peltiphyllum peltatum, Helianthemum canum, Arbutus unedo, Arctostaphylos uva-ursi and Knautia arvensis inhibited proteolysis (P < 0.05), while Daucus carota, Clematis vitalba and Erica arborea had little effect. Inhibition by the first four samples appeared to be caused by the formation of insoluble tannin-protein complexes. The samples were rich in phenolics and inhibition was reversed by polyethyleneglycol. In contrast, K. arvensis contained low concentrations of phenolics and no tannins, had no effect in the 30-min assay, yet inhibited the degradation rate of soluble protein (by 14 %, P < 0.0001) and the production of branched SCFA (by 17 %, P < 0.05) without precipitating protein in the 12-h batch fermentation. The effects showed some resemblance to those obtained in parallel incubations containing 3 mum-monensin, suggesting that K. arvensis may be a plant-derived feed additive that can suppress growth and activity of key proteolytic ruminal micro-organisms in a manner similar to that already well known for monensin. PMID:17445338

  4. Transcriptional activation by TAL1 and FUS-CHOP proteins expressed in acute malignancies as a result of chromosomal abnormalities.

    PubMed Central

    Sánchez-García, I; Rabbitts, T H

    1994-01-01

    Proteins that appear to participate in transcriptional control of gene expression are increasingly implicated in leukemias and malignant solid tumors. We report here that the N-terminal domains of the proteins TAL1 (ectopically activated in T-cell acute leukemias after chromosomal abnormalities caused by V-D-J recombinase error) (V, variable; D, diversity; J, joining) and FUS-CHOP (a liposarcoma tumor-specific fusion protein that is produced as a result of a chromosomal translocation) can function as transcription activators of specific responsive reporter genes. The result with TAL1 provides evidence that transcriptional activation can be mediated by a gene activated by translocation in T-cell acute leukemias. In the case of the liposarcoma, transactivation by the FUS-CHOP protein occurs because the FUS transcriptional activation domain is added to the DNA-binding CHOP protein normally lacking such activity. Therefore, the association of transcriptional activation and DNA-binding elements is a common consequence in proteins activated or newly created as fusion proteins after chromosomal translocations in acute leukemias and in malignant solid tumors. Images PMID:8058726

  5. Internal intensity standards for heme protein UV resonance Raman studies: excitation profiles of cacodylic acid and sodium selenate.

    PubMed

    Song, S H; Asher, S A

    1991-02-01

    We examine the utility of SO4(2-), ClO4-, cacodylic acid, and SeO4(2-) as internal intensity standards for Raman spectral measurements of protein structure. We find that 0.1 M SO4(2-) and ClO4- perturb the protein tertiary structure of aquomethemoglobin (met-Hb) and its fluoride (met-HbF) and azide (met-HbN3) complexes. Changes occur for the tryptophan near-UV absorption bands, the iron spin state is altered, and the fluoride ligand affinity decreases. Concentrations of ClO4- and SO4(2-) as low as 0.1 M suppress the met-HbF quaternary R----T transition induced by the allosteric effector inositol hexaphosphate (IHP). In contrast, similar concentrations of cacodylic acid and SeO4(2-) show little effect on the hemoglobin tertiary or quaternary protein structures or upon the R----T transition induced by IHP. We measure the Raman cross sections of cacodylic acid and SeO4(2-) between 218 and 514.5 nm and find that for UV excitation they are ca. 5-fold larger than ClO4- or SO4(2-). Thus, cacodylic acid and selenate can be used at lower concentrations. Cacodylic acid and SeO4(2-) are superior Raman internal intensity standards for protein structural studies.

  6. Magnetic, resonance, and optical properties of Cu3Sm (SeO3)2O2Cl : A rare-earth francisite compound

    NASA Astrophysics Data System (ADS)

    Zakharov, K. V.; Zvereva, E. A.; Markina, M. M.; Stratan, M. I.; Kuznetsova, E. S.; Dunaev, S. F.; Berdonosov, P. S.; Dolgikh, V. A.; Olenev, A. V.; Klimin, S. A.; Mazaev, L. S.; Kashchenko, M. A.; Ahmed, Md. A.; Banerjee, A.; Bandyopadhyay, S.; Iqbal, A.; Rahaman, B.; Saha-Dasgupta, T.; Vasiliev, A. N.

    2016-08-01

    In this combined experimental and theoretical paper, we study the properties of Cu3Sm (SeO3)2O2Cl belonging to the francisite family of compounds, which are novel frustrated layered compounds. Cu3Sm (SeO3)2O2Cl is synthesized through a solid state reaction. Characterizations through measurements of magnetization, specific heat, X-band electron spin resonance, and rare-earth optical spectroscopy, establish that the compound orders antiferromagnetically at TN=35 K and undergoes a spin-reorientation phase transition at TC=8.5 K due to the interplay of anisotropies in transition metal and rare-earth subsystems. The ground state Kramers doublet of Sm is found to split only at T SeO3)2O2Cl .

  7. Simultaneous removal of SO2 and trace SeO2 from flue gas: effect of product layer on mass transfer.

    PubMed

    Li, Yuzhong; Tong, Huiling; Zhuo, Yuqun; Chen, Changhe; Xu, Xuchang

    2006-07-01

    Sulfur dioxide (SO2) and trace elements are all pollutants derived from coal combustion. This study relates to the simultaneous removal of sulfur and trace selenium dioxide (SeO2) by calcium oxide (CaO) adsorption in the medium temperature range, especially the mass transfer effect of sulfate product layer on trace elements. Through experiments on CaO adsorbing different concentrations of SO2 gases, conclusions can be drawn that although the product layer introduces extra mass transfer resistance into the sorbent-gas reaction process, the extent of CaO adsorption ability loss due to this factor decreases with decreasing SO2 concentration. When the gas concentration is at trace level, the loss of CaO adsorption ability can be neglected. Subsequent experiments on CaO adsorbing trace SeO2 gas suggest that the sulfate product layer, whether it is thick or thin, has no obvious effect on the CaO ability to adsorb trace SeO2 gas.

  8. Native-sized recombinant spider silk protein produced in metabolically engineered Escherichia coli results in a strong fiber

    PubMed Central

    Xia, Xiao-Xia; Qian, Zhi-Gang; Ki, Chang Seok; Park, Young Hwan; Kaplan, David L.; Lee, Sang Yup

    2010-01-01

    Spider dragline silk is a remarkably strong fiber that makes it attractive for numerous applications. Much has thus been done to make similar fibers by biomimic spinning of recombinant dragline silk proteins. However, success is limited in part due to the inability to successfully express native-sized recombinant silk proteins (250–320 kDa). Here we show that a 284.9 kDa recombinant protein of the spider Nephila clavipes is produced and spun into a fiber displaying mechanical properties comparable to those of the native silk. The native-sized protein, predominantly rich in glycine (44.9%), was favorably expressed in metabolically engineered Escherichia coli within which the glycyl-tRNA pool was elevated. We also found that the recombinant proteins of lower molecular weight versions yielded inferior fiber properties. The results provide insight into evolution of silk protein size related to mechanical performance, and also clarify why spinning lower molecular weight proteins does not recapitulate the properties of native fibers. Furthermore, the silk expression, purification, and spinning platform established here should be useful for sustainable production of natural quality dragline silk, potentially enabling broader applications. PMID:20660779

  9. Deletion of potD, encoding a putative spermidine-binding protein, results in a complex phenotype in Legionella pneumophila.

    PubMed

    Nasrallah, Gheyath K; Abdelhady, Hany; Tompkins, Nicholas P; Carson, Kaitlyn R; Garduño, Rafael A

    2014-07-01

    L. pneumophila is an intracellular pathogen that replicates in a membrane-bound compartment known as the Legionella-containing vacuole (LCV). We previously observed that the polyamine spermidine, produced by host cells or added exogenously, enhances the intracellular growth of L. pneumophila. To study this enhancing effect and determine whether polyamines are used as nutrients, we deleted potD from L. pneumophila strain JR32. The gene potD encodes a spermidine-binding protein that in other bacteria is essential for the function of the PotABCD polyamine transporter. Deletion of potD did not affect L. pneumophila growth in vitro in the presence or absence of spermidine and putrescine, suggesting that PotD plays a redundant or no role in polyamine uptake. However, deletion of potD resulted in a puzzlingly complex phenotype that included defects in L. pneumophila's ability to form filaments, tolerate Na(+), associate with macrophages and amoeba, recruit host vesicles to the LCV, and initiate intracellular growth. Moreover, the ΔpotD mutant was completely unable to grow in L929 cells treated with a pharmacological inhibitor of spermidine synthesis. These complex and disparate effects suggest that the L. pneumophila potD encodes either: (i) a multifunctional protein, (ii) a protein that interacts with, or regulates a, multifunctional protein, or (iii) a protein that contributes (directly or indirectly) to a regulatory network. Protein function studies with the L. pneumophila PotD protein are thus warranted. PMID:24928741

  10. Native-sized recombinant spider silk protein produced in metabolically engineered Escherichia coli results in a strong fiber.

    PubMed

    Xia, Xiao-Xia; Qian, Zhi-Gang; Ki, Chang Seok; Park, Young Hwan; Kaplan, David L; Lee, Sang Yup

    2010-08-10

    Spider dragline silk is a remarkably strong fiber that makes it attractive for numerous applications. Much has thus been done to make similar fibers by biomimic spinning of recombinant dragline silk proteins. However, success is limited in part due to the inability to successfully express native-sized recombinant silk proteins (250-320 kDa). Here we show that a 284.9 kDa recombinant protein of the spider Nephila clavipes is produced and spun into a fiber displaying mechanical properties comparable to those of the native silk. The native-sized protein, predominantly rich in glycine (44.9%), was favorably expressed in metabolically engineered Escherichia coli within which the glycyl-tRNA pool was elevated. We also found that the recombinant proteins of lower molecular weight versions yielded inferior fiber properties. The results provide insight into evolution of silk protein size related to mechanical performance, and also clarify why spinning lower molecular weight proteins does not recapitulate the properties of native fibers. Furthermore, the silk expression, purification, and spinning platform established here should be useful for sustainable production of natural quality dragline silk, potentially enabling broader applications. PMID:20660779

  11. UU/UA dinucleotide frequency reduction in coding regions results in increased mRNA stability and protein expression.

    PubMed

    Al-Saif, Maher; Khabar, Khalid S A

    2012-05-01

    UU and UA dinucleotides are rare in mammalian genes and may offer natural selection against endoribonuclease-mediated mRNA decay. This study hypothesized that reducing UU and UA (UW) dinucleotides in the mRNA-coding sequence, including the codons and the dicodon boundaries, may promote resistance to mRNA decay, thereby increasing protein production. Indeed, protein expression from UW-reduced coding regions of enhanced green fluorescent protein (EGFP), luciferase, interferon-α, and hepatitis B surface antigen (HBsAg) was higher when compared to the wild-type protein expression. The steady-state level of UW-reduced EGFP mRNA was higher and the mRNA half-life was also longer. Ectopic expression of the endoribonuclease, RNase L, did not reduce the wild type or UW-reduced mRNA. A mutant form of the mRNA decay-promoting protein, tristetraprolin (TTP/ZFP36), which has a point mutation in the zinc-finger domain (C124R), was used. The wild-type EGFP mRNA but not the UW-reduced mRNA responded to the dominant negative action of the C124R ZFP36/TTP mutant. The results indicate the efficacy of the described rational approach to formulate a general scheme for boosting recombinant protein production in mammalian cells.

  12. Lack of the Lysosomal Membrane Protein, GLMP, in Mice Results in Metabolic Dysregulation in Liver

    PubMed Central

    Kong, Xiang Yi; Kase, Eili Tranheim; Herskedal, Anette; Schjalm, Camilla; Damme, Markus; Nesset, Cecilie Kasi; Thoresen, G. Hege; Rustan, Arild C.; Eskild, Winnie

    2015-01-01

    Ablation of glycosylated lysosomal membrane protein (GLMP, formerly known as NCU-G1) has been shown to cause chronic liver injury which progresses into liver fibrosis in mice. Both lysosomal dysfunction and chronic liver injury can cause metabolic dysregulation. Glmpgt/gt mice (formerly known as Ncu-g1gt/gtmice) were studied between 3 weeks and 9 months of age. Body weight gain and feed efficiency of Glmpgt/gt mice were comparable to wild type siblings, only at the age of 9 months the Glmpgt/gt siblings had significantly reduced body weight. Reduced size of epididymal fat pads was accompanied by hepatosplenomegaly in Glmpgt/gt mice. Blood analysis revealed reduced levels of blood glucose, circulating triacylglycerol and non-esterified fatty acids in Glmpgt/gt mice. Increased flux of glucose, increased de novo lipogenesis and lipid accumulation were detected in Glmpgt/gt primary hepatocytes, as well as elevated triacylglycerol levels in Glmpgt/gt liver homogenates, compared to hepatocytes and liver from wild type mice. Gene expression analysis showed an increased expression of genes involved in fatty acid uptake and lipogenesis in Glmpgt/gt liver compared to wild type. Our findings are in agreement with the metabolic alterations observed in other mouse models lacking lysosomal proteins, and with alterations characteristic for advanced chronic liver injury. PMID:26047317

  13. Translational infidelity-induced protein stress results from a deficiency in Trm9-catalyzed tRNA modifications.

    PubMed

    Patil, Ashish; Chan, Clement T Y; Dyavaiah, Madhu; Rooney, John P; Dedon, Peter C; Begley, Thomas J

    2012-07-01

    Correct codon-anticodon pairing promotes translational fidelity, with these interactions greatly facilitated by modified nucleosides found in tRNA. We hypothesized that wobble uridine modifications catalyzed by tRNA methyltransferase 9 (Trm9) are essential for translational fidelity. In support, we have used phenotypic, reporter and protein-based assays to demonstrate increased translational infidelity in trm9Δ Saccharomyces cerevisiae cells. Codon reengineering studies suggest that Trm9-catalyzed tRNA modifications promote fidelity during the translation of specific genes, those rich in arginine and glutamic acid codons from mixed boxes. Using quantitative tRNA modification analysis, we determined that trm9Δ cells are only deficient in 2 of 23 tRNA modifications, with those 2, 5-methoxycarbonylmethyluridine (mcm ( 5) U) and 5-methoxycarbonylmethyl-2-thiouridine (mcm ( 5) s ( 2) U), classified as key determinants of translational fidelity. We also show that in the absence of mcm ( 5) U and mcm ( 5) s ( 2) U, the resulting translational infidelity promotes protein errors and activation of unfolded protein and heat shock responses. These data support a model in which Trm9-catalyzed tRNA modifications promote fidelity during the translation of specific transcripts, with decreased wobble base modification leading to translational infidelity, protein errors and activation of protein stress response pathways. PMID:22832247

  14. Analysis of proteins in bronchoalveolar lavage fluids during pulmonary edema resulting from nitrogen dioxide and cadmium exposure

    SciTech Connect

    Gurley, L.R.; London, J.E.; Dethloff, L.A.; Lehnert, B.E.

    1988-01-01

    We have developed a new HPLC method by which quantitative measurements can be made on the biochemical constituents of the extracellular fluid lining of the lung as sampled by bronchoalveolar lavage. Nine of the fractions are proteins, two are phospholipids, and two fractions remained unidentified. Rats were subjected to the intrapulmonary deposition of cadmium, a treatment model known to induce pulmonary edema and cause a translocation of blood compartment proteins into the lung's alveolar space compartment. Resulting pulmonary edema was hallmarked by /approximately/25-fold increases in three major blood compartment-derived HPLC protein fractions, two of which have been identified as albumin and immunoglobulin(s). Analysis of lavage fluid from rats exposed to 100 ppM NO/sub 2/ for 15 min, an exposure regimen which also produces pulmonary edema, indicated that the three blood compartment proteins in the lavage fluids were elevated 35- to 72-fold over controls 24 h after exposure. These results demonstrate that HPLC can be used to provide a highly sensitive method for detection and quantitation of pulmonary edema that can occur in acute lung injuries resulting from environmental insults.

  15. The selective inhibition of protein phosphatase-1 results in mitotic catastrophe and impaired tumor growth.

    PubMed

    Winkler, Claudia; De Munter, Sofie; Van Dessel, Nele; Lesage, Bart; Heroes, Ewald; Boens, Shannah; Beullens, Monique; Van Eynde, Aleyde; Bollen, Mathieu

    2015-12-15

    The serine/threonine protein phosphatase-1 (PP1) complex is a key regulator of the cell cycle. However, the redundancy of PP1 isoforms and the lack of specific inhibitors have hampered studies on the global role of PP1 in cell cycle progression in vertebrates. Here, we show that the overexpression of nuclear inhibitor of PP1 (NIPP1; also known as PPP1R8) in HeLa cells culminated in a prometaphase arrest, associated with severe spindle-formation and chromosome-congression defects. In addition, the spindle assembly checkpoint was activated and checkpoint silencing was hampered. Eventually, most cells either died by apoptosis or formed binucleated cells. The NIPP1-induced mitotic arrest could be explained by the inhibition of PP1 that was titrated away from other mitotic PP1 interactors. Consistent with this notion, the mitotic-arrest phenotype could be rescued by the overexpression of PP1 or the inhibition of the Aurora B kinase, which acts antagonistically to PP1. Finally, we demonstrate that the overexpression of NIPP1 also hampered colony formation and tumor growth in xenograft assays in a PP1-dependent manner. Our data show that the selective inhibition of PP1 can be used to induce cancer cell death through mitotic catastrophe. PMID:26542020

  16. The selective inhibition of protein phosphatase-1 results in mitotic catastrophe and impaired tumor growth.

    PubMed

    Winkler, Claudia; De Munter, Sofie; Van Dessel, Nele; Lesage, Bart; Heroes, Ewald; Boens, Shannah; Beullens, Monique; Van Eynde, Aleyde; Bollen, Mathieu

    2015-12-15

    The serine/threonine protein phosphatase-1 (PP1) complex is a key regulator of the cell cycle. However, the redundancy of PP1 isoforms and the lack of specific inhibitors have hampered studies on the global role of PP1 in cell cycle progression in vertebrates. Here, we show that the overexpression of nuclear inhibitor of PP1 (NIPP1; also known as PPP1R8) in HeLa cells culminated in a prometaphase arrest, associated with severe spindle-formation and chromosome-congression defects. In addition, the spindle assembly checkpoint was activated and checkpoint silencing was hampered. Eventually, most cells either died by apoptosis or formed binucleated cells. The NIPP1-induced mitotic arrest could be explained by the inhibition of PP1 that was titrated away from other mitotic PP1 interactors. Consistent with this notion, the mitotic-arrest phenotype could be rescued by the overexpression of PP1 or the inhibition of the Aurora B kinase, which acts antagonistically to PP1. Finally, we demonstrate that the overexpression of NIPP1 also hampered colony formation and tumor growth in xenograft assays in a PP1-dependent manner. Our data show that the selective inhibition of PP1 can be used to induce cancer cell death through mitotic catastrophe.

  17. CSF Tau proteins reduce misdiagnosis of sporadic Creutzfeldt-Jakob disease suspected cases with inconclusive 14-3-3 result.

    PubMed

    Leitão, M J; Baldeiras, I; Almeida, M R; Ribeiro, M H; Santos, A C; Ribeiro, M; Tomás, J; Rocha, S; Santana, I; Oliveira, C R

    2016-09-01

    Cerebrospinal fluid (CSF) 14-3-3 protein supports sporadic Creutzfeldt-Jakob (sCJD) diagnosis, but often leads to weak-positive results and lacks standardization. In this study, we explored the added diagnostic value of Total Tau (t-Tau) and phosphorylated Tau (p-Tau) in sCJD diagnosis, particularly in the cases with inconclusive 14-3-3 result. 95 definite sCJD and 287 patients without prion disease (non-CJD) were included in this study. CSF samples were collected in routine clinical diagnosis and analysed for 14-3-3 detection by Western blot (WB). CSF t-Tau and p-Tau were quantified by commercial ELISA kits and PRNP and APOE genotyping assessed by PCR-RFLP. In a regression analysis of the whole cohort, 14-3-3 protein revealed an overall accuracy of 82 % (sensitivity = 96.7 %; specificity = 75.6 %) for sCJD. Regarding 14-3-3 clear positive results, we observed no added value either of t-Tau alone or p-Tau/t-Tau ratio in the model. On the other hand, considering 14-3-3 weak-positive cases, t-Tau protein increased the overall accuracy of 14-3-3 alone from 91 to 94 % and specificity from 74 to 93 % (p < 0.05), with no sensitivity improvement. However, inclusion of p-Tau/t-Tau ratio did not significantly improve the first model (p = 0.0595). Globally, t-Tau protein allowed a further discrimination of 65 % within 14-3-3 inconclusive results. Furthermore, PRNP MV genotype showed a trend to decrease 14-3-3 sensitivity (p = 0.051), but such effect was not seen on t-Tau protein. In light of these results, we suggest that t-Tau protein assay is of significant importance as a second marker in identifying 14-3-3 false-positive results among sCJD probable cases.

  18. CSF Tau proteins reduce misdiagnosis of sporadic Creutzfeldt-Jakob disease suspected cases with inconclusive 14-3-3 result.

    PubMed

    Leitão, M J; Baldeiras, I; Almeida, M R; Ribeiro, M H; Santos, A C; Ribeiro, M; Tomás, J; Rocha, S; Santana, I; Oliveira, C R

    2016-09-01

    Cerebrospinal fluid (CSF) 14-3-3 protein supports sporadic Creutzfeldt-Jakob (sCJD) diagnosis, but often leads to weak-positive results and lacks standardization. In this study, we explored the added diagnostic value of Total Tau (t-Tau) and phosphorylated Tau (p-Tau) in sCJD diagnosis, particularly in the cases with inconclusive 14-3-3 result. 95 definite sCJD and 287 patients without prion disease (non-CJD) were included in this study. CSF samples were collected in routine clinical diagnosis and analysed for 14-3-3 detection by Western blot (WB). CSF t-Tau and p-Tau were quantified by commercial ELISA kits and PRNP and APOE genotyping assessed by PCR-RFLP. In a regression analysis of the whole cohort, 14-3-3 protein revealed an overall accuracy of 82 % (sensitivity = 96.7 %; specificity = 75.6 %) for sCJD. Regarding 14-3-3 clear positive results, we observed no added value either of t-Tau alone or p-Tau/t-Tau ratio in the model. On the other hand, considering 14-3-3 weak-positive cases, t-Tau protein increased the overall accuracy of 14-3-3 alone from 91 to 94 % and specificity from 74 to 93 % (p < 0.05), with no sensitivity improvement. However, inclusion of p-Tau/t-Tau ratio did not significantly improve the first model (p = 0.0595). Globally, t-Tau protein allowed a further discrimination of 65 % within 14-3-3 inconclusive results. Furthermore, PRNP MV genotype showed a trend to decrease 14-3-3 sensitivity (p = 0.051), but such effect was not seen on t-Tau protein. In light of these results, we suggest that t-Tau protein assay is of significant importance as a second marker in identifying 14-3-3 false-positive results among sCJD probable cases. PMID:27357003

  19. Biochemical and biophysical characterization of the selenium-binding and reducing site in Arabidopsis thaliana homologue to mammals selenium-binding protein 1.

    PubMed

    Schild, Florie; Kieffer-Jaquinod, Sylvie; Palencia, Andrés; Cobessi, David; Sarret, Géraldine; Zubieta, Chloé; Jourdain, Agnès; Dumas, Renaud; Forge, Vincent; Testemale, Denis; Bourguignon, Jacques; Hugouvieux, Véronique

    2014-11-14

    The function of selenium-binding protein 1 (SBP1), present in almost all organisms, has not yet been established. In mammals, SBP1 is known to bind the essential element selenium but the binding site has not been identified. In addition, the SBP family has numerous potential metal-binding sites that may play a role in detoxification pathways in plants. In Arabidopsis thaliana, AtSBP1 over-expression increases tolerance to two toxic compounds for plants, selenium and cadmium, often found as soil pollutants. For a better understanding of AtSBP1 function in detoxification mechanisms, we investigated the chelating properties of the protein toward different ligands with a focus on selenium using biochemical and biophysical techniques. Thermal shift assays together with inductively coupled plasma mass spectrometry revealed that AtSBP1 binds selenium after incubation with selenite (SeO3(2-)) with a ligand to protein molar ratio of 1:1. Isothermal titration calorimetry confirmed the 1:1 stoichiometry and revealed an unexpectedly large value of binding enthalpy suggesting a covalent bond between selenium and AtSBP1. Titration of reduced Cys residues and comparative mass spectrometry on AtSBP1 and the purified selenium-AtSBP1 complex identified Cys(21) and Cys(22) as being responsible for the binding of one selenium. These results were validated by site-directed mutagenesis. Selenium K-edge x-ray absorption near edge spectroscopy performed on the selenium-AtSBP1 complex demonstrated that AtSBP1 reduced SeO3(2-) to form a R-S-Se(II)-S-R-type complex. The capacity of AtSBP1 to bind different metals and selenium is discussed with respect to the potential function of AtSBP1 in detoxification mechanisms and selenium metabolism. PMID:25274629

  20. Biochemical and Biophysical Characterization of the Selenium-binding and Reducing Site in Arabidopsis thaliana Homologue to Mammals Selenium-binding Protein 1*

    PubMed Central

    Schild, Florie; Kieffer-Jaquinod, Sylvie; Palencia, Andrés; Cobessi, David; Sarret, Géraldine; Zubieta, Chloé; Jourdain, Agnès; Dumas, Renaud; Forge, Vincent; Testemale, Denis; Bourguignon, Jacques; Hugouvieux, Véronique

    2014-01-01

    The function of selenium-binding protein 1 (SBP1), present in almost all organisms, has not yet been established. In mammals, SBP1 is known to bind the essential element selenium but the binding site has not been identified. In addition, the SBP family has numerous potential metal-binding sites that may play a role in detoxification pathways in plants. In Arabidopsis thaliana, AtSBP1 over-expression increases tolerance to two toxic compounds for plants, selenium and cadmium, often found as soil pollutants. For a better understanding of AtSBP1 function in detoxification mechanisms, we investigated the chelating properties of the protein toward different ligands with a focus on selenium using biochemical and biophysical techniques. Thermal shift assays together with inductively coupled plasma mass spectrometry revealed that AtSBP1 binds selenium after incubation with selenite (SeO32−) with a ligand to protein molar ratio of 1:1. Isothermal titration calorimetry confirmed the 1:1 stoichiometry and revealed an unexpectedly large value of binding enthalpy suggesting a covalent bond between selenium and AtSBP1. Titration of reduced Cys residues and comparative mass spectrometry on AtSBP1 and the purified selenium-AtSBP1 complex identified Cys21 and Cys22 as being responsible for the binding of one selenium. These results were validated by site-directed mutagenesis. Selenium K-edge x-ray absorption near edge spectroscopy performed on the selenium-AtSBP1 complex demonstrated that AtSBP1 reduced SeO32− to form a R-S-Se(II)-S-R-type complex. The capacity of AtSBP1 to bind different metals and selenium is discussed with respect to the potential function of AtSBP1 in detoxification mechanisms and selenium metabolism. PMID:25274629

  1. An extensively hydrolysed rice protein-based formula in the management of infants with cow's milk protein allergy: preliminary results after 1 month

    PubMed Central

    Vandenplas, Yvan; De Greef, Elisabeth; Hauser, Bruno

    2014-01-01

    Background Guidelines recommend extensively hydrolysed cow's milk protein formulas (eHF) in the treatment of infants diagnosed with cow's milk protein allergy (CMPA). Extensively hydrolysed rice protein infant formulas (eRHFs) have recently become available, and could offer a valid alternative. Methods A prospective trial was performed to evaluate the clinical tolerance of a new eRHF in infants with a confirmed CMPA. Patients were followed for 1 month. Clinical tolerance of the eRHF was evaluated with a symptom-based score (SBS) and growth (weight and length) was monitored. Results Thirty-nine infants (mean age 3.4 months, range 0.5–6 months) diagnosed with CMPA were enrolled. All infants tolerated the eRHF and experienced a normal growth. Conclusions In accordance with current guidelines, this eRHF is tolerated by more than 90% of children with proven CMPA with a 95% CI, and is an adequate alternative to cow's milk-based eHF. Trial registration number ClinicalTrials.gov NCT01998074. PMID:24914098

  2. Binding of lithium and boron to human plasma proteins II: results for a bipolar patient not on lithium therapy.

    PubMed

    Clarke, W Brian; Guscott, Richard; Lindstrom, Richard M

    2004-02-01

    We report further measurements of lithium and boron bound to human plasma proteins using the techniques of gel chromatography, thermal-neutron activation, and high-sensitivity helium isotope mass spectrometry. The plasma sample was donated by a bipolar patient who had never been on lithium therapy. The plasma lithium-binding pattern for the bipolar patient is distinctly different from that previously observed in this laboratory for plasma donated by a normal individual. In the bipolar case, virtually all of the lithium is bound to low-molecular-weight proteins (approx 1000 amu), whereas in the normal case, most of the lithium eluted from the gel column was bound to five high-molecular-weight proteins (approx 50,000 amu to approx 1,000,000 amu). The gel elution profiles for boron were roughly similar for the normal and bipolar cases. The lithium results are in agreement with our previous speculation that lithium-binding plasma proteins are missing or exist in very low concentrations in some individuals suffering from affective disorders.

  3. Loss of Daxx, a promiscuously interacting protein, results in extensive apoptosis in early mouse development

    PubMed Central

    Michaelson, Jennifer S.; Bader, Debra; Kuo, Frank; Kozak, Christine; Leder, Philip

    1999-01-01

    The mammalian Daxx gene has been identified in a diverse set of yeast interaction trap experiments. Although a facilitating role for Daxx in Fas-induced apoptosis has been suggested, Daxx’s physiologic function remains unknown. To elucidate the in vivo role of Daxx, we have generated Daxx-deficient mice. Surprisingly, rather than a hyperproliferative disorder expected from the loss of a pro-apoptotic gene, mutation of Daxx results in extensive apoptosis and embryonic lethality. These findings argue against a role for Daxx in promoting Fas-induced cell death and suggest that Daxx either directly or indirectly suppresses apoptosis in the early embryo. PMID:10444590

  4. Transduction of the TAT-FLIP fusion protein results in transient resistance to Fas-induced apoptosis in vivo.

    PubMed

    Krautwald, Stefan; Ziegler, Ekkehard; Tiede, Karen; Pust, Rainer; Kunzendorf, Ulrich

    2004-10-15

    Although tightly regulated programmed cell death (apoptosis) possesses great importance for tissue homeostasis, several pathologic processes are associated with organ failure due to adversely activated cell apoptosis. Transient increase in apoptosis has been shown to cause organ damage during fulminant hepatitis B, autoimmune diseases, ischemia-reperfusion injury, sepsis, or allograft rejection. A defined and temporary inhibition of cell apoptosis may therefore be of high clinical relevance. Activation of death receptors results in caspase-8 recruitment to the death-inducing signaling complex, which initiates the apoptotic process through cleavage of caspase-8 and downstream substrates. This initial step may be inhibited by the caspase-8 inhibitor FLIP (FLICE inhibitory protein). To specifically inhibit the initiation of death receptor-mediated apoptosis we constructed a fusion protein containing FLIP fused N-terminally to the human immunodeficiency virus TAT domain. This TAT domain allows the fusion protein to cross the cell membrane and thus makes the FLIP domain able to interfere with the death-inducing signaling complex inside of the cell. We observed that incubation of lymphocytic Jurkat or BJAB cells with TAT-FLIPS proteins significantly inhibits Fas-induced activation of procaspase-8 and downstream caspases, preventing cells from undergoing apoptosis. Systemic application of TAT-FLIPS prolongs survival and reduces multi-organ failure due to Fas-receptor-mediated lethal apoptosis in mice. Therefore, application of cellular FLIPS in the form of a TAT fusion protein may open a promising, easily applicable new tool for providing protection against transient, pathologically increased apoptosis in various diseases. PMID:15304499

  5. Dihydrotestosterone treatment rescues the decline in protein synthesis as a result of sarcopenia in isolated mouse skeletal muscle fibres

    PubMed Central

    Wendowski, Oskar; Redshaw, Zoe

    2016-01-01

    Abstract Background Sarcopenia, the progressive decline in skeletal muscle mass and function with age, is a debilitating condition. It leads to inactivity, falls, and loss of independence. Despite this, its cause(s) and the underlying mechanism(s) are still poorly understood. Methods In this study, small skeletal muscle fibre bundles isolated from the extensor digitorum longus (a fast‐twitch muscle) and the soleus (a slow‐twitch muscle) of adult mice of different ages (range 100–900 days old) were used to investigate the effects of ageing and dihydrotestosterone (DHT) treatment on protein synthesis as well as the expression and function of two amino acid transporters; the sodium‐coupled neutral amino acid transporter (SNAT) 2, and the sodium‐independent L‐type amino‐acid transporter (LAT) 2. Results At all ages investigated, protein synthesis was always higher in the slow‐twitch than in the fast‐twitch muscle fibres and decreased with age in both fibre types. However, the decline was greater in the fast‐twitch than in the slow‐twitch fibres and was accompanied by a reduction in the expression of SNAT2 and LAT2 at the protein level. Again, the decrease in the expression of the amino acid transporters was greater in the fast‐twitch than in the slow‐twitch fibres. In contrast, ageing had no effect on SNAT2 and LAT2 expressions at the mRNA level. Treating the muscle fibre bundles with physiological concentrations (~2 nM) of DHT for 1 h completely reversed the effects of ageing on protein synthesis and the expression of SNAT2 and LAT2 protein in both fibre types. Conclusion From the observations that ageing is accompanied by a reduction in protein synthesis and transporter expression and that these effects are reversed by DHT treatment, we conclude that sarcopenia arises from an age‐dependent reduction in protein synthesis caused, in part, by the lack of or by the low bioavailability of the male sex steroid, DHT. PMID:27239418

  6. In vivo introduction of unpreferred synonymous codons into the Drosophila Adh gene results in reduced levels of ADH protein.

    PubMed Central

    Carlini, David B; Stephan, Wolfgang

    2003-01-01

    The evolution of codon bias, the unequal usage of synonymous codons, is thought to be due to natural selection for the use of preferred codons that match the most abundant species of isoaccepting tRNA, resulting in increased translational efficiency and accuracy. We examined this hypothesis by introducing 1, 6, and 10 unpreferred codons into the Drosophila alcohol dehydrogenase gene (Adh). We observed a significant decrease in ADH protein production with number of unpreferred codons, confirming the importance of natural selection as a mechanism leading to codon bias. We then used this empirical relationship to estimate the selection coefficient (s) against unpreferred synonymous mutations and found the value (s >or= 10(-5)) to be approximately one order of magnitude greater than previous estimates from population genetics theory. The observed differences in protein production appear to be too large to be consistent with current estimates of the strength of selection on synonymous sites in D. melanogaster. PMID:12586711

  7. Proteins.

    ERIC Educational Resources Information Center

    Doolittle, Russell F.

    1985-01-01

    Examines proteins which give rise to structure and, by virtue of selective binding to other molecules, make genes. Binding sites, amino acids, protein evolution, and molecular paleontology are discussed. Work with encoding segments of deoxyribonucleic acid (exons) and noncoding stretches (introns) provides new information for hypotheses. (DH)

  8. Protein

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Proteins are the major structural and functional components of all cells in the body. They are macromolecules that comprise 1 or more chains of amino acids that vary in their sequence and length and are folded into specific 3-dimensional structures. The sizes and conformations of proteins, therefor...

  9. Single Amino Acid Deletion in Kindlin-1 Results in Partial Protein Degradation Which Can Be Rescued by Chaperone Treatment.

    PubMed

    Maier, Kristin; He, Yinghong; Esser, Philipp R; Thriene, Kerstin; Sarca, Daniela; Kohlhase, Jürgen; Dengjel, Jörn; Martin, Ludovic; Has, Cristina

    2016-05-01

    Kindler syndrome, a distinct type of epidermolysis bullosa, is a rare disorder caused by mutations in FERMT1, encoding kindlin-1. Most FERMT1 mutations lead to premature termination codons and absence of kindlin-1. Here we investigated the molecular and cellular consequences of a naturally occurring FERMT1 mutation, c.299_301del resulting in a single amino acid deletion, p.R100del. The mutation led to a 50% reduction of FERMT1 mRNA and 90% reduction of kindlin-1 protein in keratinocytes derived from the patient, as compared with control cells. The misfolded p.R100del kindlin-1 mutant was lysosomally degraded and launched a homeostatic unfolded protein response. Sodium-phenylbutyrate significantly increased kindlin-1 mRNA and protein levels and the area of mutant cells, acting as a chemical chaperone and probably also as a histone deacetylase inhibitor. In a recombinant system, low levels of wild-type or p.R100del mutant kindlin-1 were sufficient to improve the cellular phenotype in respect of spreading and proliferation as compared with kindlin-1 negative keratinocytes. The study of this hypomorphic mutation provides evidence that low amounts of kindlin-1 are sufficient to improve the epidermal architecture and Kindler syndrome cellular phenotype and proposes a personalized chaperone therapy for the patient.

  10. Resistance of Dynamin-related Protein 1 Oligomers to Disassembly Impairs Mitophagy, Resulting in Myocardial Inflammation and Heart Failure.

    PubMed

    Cahill, Thomas J; Leo, Vincenzo; Kelly, Matthew; Stockenhuber, Alexander; Kennedy, Nolan W; Bao, Leyuan; Cereghetti, Grazia; Harper, Andrew R; Czibik, Gabor; Lao, Chunyan; Bellahcene, Mohamed; Steeples, Violetta; Ghaffari, Safar; Yavari, Arash; Mayer, Alice; Poulton, Joanna; Ferguson, David J P; Scorrano, Luca; Hettiarachchi, Nishani T; Peers, Chris; Boyle, John; Hill, R Blake; Simmons, Alison; Watkins, Hugh; Dear, T Neil; Ashrafian, Houman

    2015-10-23

    We have reported previously that a missense mutation in the mitochondrial fission gene Dynamin-related protein 1 (Drp1) underlies the Python mouse model of monogenic dilated cardiomyopathy. The aim of this study was to investigate the consequences of the C452F mutation on Drp1 protein function and to define the cellular sequelae leading to heart failure in the Python monogenic dilated cardiomyopathy model. We found that the C452F mutation increased Drp1 GTPase activity. The mutation also conferred resistance to oligomer disassembly by guanine nucleotides and high ionic strength solutions. In a mouse embryonic fibroblast model, Drp1 C452F cells exhibited abnormal mitochondrial morphology and defective mitophagy. Mitochondria in C452F mouse embryonic fibroblasts were depolarized and had reduced calcium uptake with impaired ATP production by oxidative phosphorylation. In the Python heart, we found a corresponding progressive decline in oxidative phosphorylation with age and activation of sterile inflammation. As a corollary, enhancing autophagy by exposure to a prolonged low-protein diet improved cardiac function in Python mice. In conclusion, failure of Drp1 disassembly impairs mitophagy, leading to a downstream cascade of mitochondrial depolarization, aberrant calcium handling, impaired ATP synthesis, and activation of sterile myocardial inflammation, resulting in heart failure. PMID:26370078

  11. Novel mutations in 3-phosphoglycerate dehydrogenase (PHGDH) are distributed throughout the protein and result in altered enzyme kinetics.

    PubMed

    Tabatabaie, L; de Koning, T J; Geboers, A J J M; van den Berg, I E T; Berger, R; Klomp, L W J

    2009-05-01

    Three-phosphoglycerate dehydrogenase (3-PGDH) deficiency is a rare recessive inborn error in the biosynthesis of the amino acid L-serine characterized clinically by congenital microcephaly, psychomotor retardation, and intractable seizures. The biochemical abnormalities associated with this disorder are low concentrations of L-serine, D-serine, and glycine in cerebrospinal fluid (CSF). Only two missense mutations (p.V425M and p.V490M) have been identified in PHGDH, the gene encoding 3-PGDH, but it is currently unclear how these mutations in the carboxy-terminal regulatory domain of the protein affect enzyme function. We now describe five novel mutations in five patients with 3-PGDH deficiency; one frameshift mutation (p.G238fsX), and four missense mutations (p.R135W, p.V261M, p.A373T, and p.G377S). The missense mutations were located in the nucleotide binding and regulatory domains of 3-PGDH and did not affect steady-state expression, protein stability, and protein degradation rates. Patients' fibroblasts displayed a significant, but incomplete, reduction in maximal enzyme activities associated with all missense mutations. In transient overexpression studies in HEK293T cells, the p.A373T, p.V425M, and p.V490M mutations resulted in almost undetectable enzyme activities. Molecular modeling of the p.R135W and p.V261M mutations onto the partial crystal structure of 3-PGDH predicted that these mutations affect substrate and cofactor binding. This prediction was confirmed by the results of kinetic measurements in fibroblasts and transiently transfected HEK293T cells, which revealed a markedly decreased V(max) and an increase in K(m) values, respectively. Taken together, these data suggest that missense mutations associated with 3-PGDH deficiency either primarily affect substrate binding or result in very low residual enzymatic activity. PMID:19235232

  12. Prototype integration of protein electrophoresis laboratory results in an information warehouse to improve workflow and data analysis.

    PubMed

    Liu, Jianhua; Silvey, Scott A; Bissell, Michael G; Saltz, Joel H; Kamal, Jyoti

    2006-01-01

    This poster demonstrates our efforts to enhance workflow and clinical analysis of protein electrophoresis (PEP) data through integration with the Information Warehouse (IW) at The Ohio State University Medical Center (OSUMC). A new desktop application has been developed with the aim of enabling more efficient and accurate gel analysis by clinical pathologists. This tool gives the pathologists the ability to perform their analysis conveniently from anywhere on the OSUMC network along with the aid of numerical analysis algorithms, image enhancement techniques, and access to historical PEP results for the given patient.

  13. Chromothripsis in healthy individuals affects multiple protein-coding genes and can result in severe congenital abnormalities in offspring.

    PubMed

    de Pagter, Mirjam S; van Roosmalen, Markus J; Baas, Annette F; Renkens, Ivo; Duran, Karen J; van Binsbergen, Ellen; Tavakoli-Yaraki, Masoumeh; Hochstenbach, Ron; van der Veken, Lars T; Cuppen, Edwin; Kloosterman, Wigard P

    2015-04-01

    Chromothripsis represents an extreme class of complex chromosome rearrangements (CCRs) with major effects on chromosomal architecture. Although recent studies have associated chromothripsis with congenital abnormalities, the incidence and pathogenic effects of this phenomenon require further investigation. Here, we analyzed the genomes of three families in which chromothripsis rearrangements were transmitted from a mother to her child. The chromothripsis in the mothers resulted in completely balanced rearrangements involving 8-23 breakpoint junctions across three to five chromosomes. Two mothers did not show any phenotypic abnormalities, although 3-13 protein-coding genes were affected by breakpoints. Unbalanced but stable transmission of a subset of the derivative chromosomes caused apparently de novo complex copy-number changes in two children. This resulted in gene-dosage changes, which are probably responsible for the severe congenital phenotypes of these two children. In contrast, the third child, who has a severe congenital disease, harbored all three chromothripsis chromosomes from his healthy mother, but one of the chromosomes acquired de novo rearrangements leading to copy-number changes. These results show that the human genome can tolerate extreme reshuffling of chromosomal architecture, including breakage of multiple protein-coding genes, without noticeable phenotypic effects. The presence of chromothripsis in healthy individuals affects reproduction and is expected to substantially increase the risk of miscarriages, abortions, and severe congenital disease.

  14. Vitiligo-inducing phenols activate the unfolded protein response in melanocytes resulting in upregulation of IL6 and IL8.

    PubMed

    Toosi, Siavash; Orlow, Seth J; Manga, Prashiela

    2012-11-01

    Vitiligo is characterized by depigmented skin patches caused by loss of epidermal melanocytes. Oxidative stress may have a role in vitiligo onset, while autoimmunity contributes to disease progression. In this study, we sought to identify mechanisms that link disease triggers and spreading of lesions. A hallmark of melanocytes at the periphery of vitiligo lesions is dilation of the endoplasmic reticulum (ER). We hypothesized that oxidative stress results in redox disruptions that extend to the ER, causing accumulation of misfolded peptides, which activates the unfolded protein response (UPR). We used 4-tertiary butyl phenol and monobenzyl ether of hydroquinone, known triggers of vitiligo. We show that expression of key UPR components, including the transcription factor X-box-binding protein 1 (XBP1), is increased following exposure of melanocytes to phenols. XBP1 activation increases production of immune mediators IL6 and IL8. Co-treatment with XBP1 inhibitors reduced IL6 and IL8 production induced by phenols, while overexpression of XBP1 alone increased their expression. Thus, melanocytes themselves produce cytokines associated with activation of an immune response following exposure to chemical triggers of vitiligo. These results expand our understanding of the mechanisms underlying melanocyte loss in vitiligo and pathways linking environmental stressors and autoimmunity.

  15. Recombinant envelope protein (rgp90) ELISA for equine infectious anemia virus provides comparable results to the agar gel immunodiffusion.

    PubMed

    Reis, Jenner K P; Diniz, Rejane S; Haddad, João P A; Ferraz, Isabella B F; Carvalho, Alex F; Kroon, Erna G; Ferreira, Paulo C P; Leite, Rômulo C

    2012-03-01

    Equine infectious anemia (EIA) is an important viral infection affecting horses worldwide. The course of infection is accompanied generally by three characteristic stages: acute, chronic and inapparent. There is no effective EIA vaccine or treatment, and the control of the disease is based currently on identification of EIAV inapparent carriers by laboratory tests. Recombinant envelope protein (rgp90) was expressed in Escherichia coli and evaluated via enzyme-linked immunosorbent assay (ELISA). There was an excellent agreement (95.42%) between the ELISA results using rgp90 and agar gel immunodiffusion test results. AGID is considered the "gold-standard" serologic test for equine infectious anemia (EIA). After 1160 serum samples were tested, the relative sensitivity and specificity of the ELISA were 96.1% and 96.4%, respectively. Moreover, analysis diagnostic accuracy of the ELISA was performed. The ELISA proved robust. Furthermore, good reproducibility was observed for the negative controls and, positive controls for all plates tested.

  16. Proteins

    NASA Astrophysics Data System (ADS)

    Regnier, Fred E.; Gooding, Karen M.

    Because of the complexity of cellular material and body fluids, it is seldom possible to analyze a natural product directly. Qualitative and quantitative analyses must often be preceded by some purification step that separates the molecular species being examined from interfering materials. In the case of proteins, column liquid chromatography has been used extensively for these fractionations. With the advent of gel permeation, cation exchange, anion exchange, hydrophobic, and affinity chromatography, it became possible to resolve proteins through their fundamental properties of size, charge, hydrophobicity, and biological affinity. The chromatographic separations used in the early isolation and characterization of many proteins later became analytical tools in their routine analysis. Unfortunately, these inherently simple and versatile column chromatographic techniques introduced in the 50s and 60s have a severe limitation in routine analysis-separation time. It is common to encounter 1-24 h separation times with the classical gel-type supports.

  17. Molecular structure, vibrational spectra, MEP, HOMO-LUMO and NBO analysis of Hf(SeO3)(SeO4)(H2O)4

    NASA Astrophysics Data System (ADS)

    Yankova, Rumyana; Genieva, Svetlana; Halachev, Nenko; Dimitrova, Ginka

    2016-02-01

    Hf(SeO3)(SeO4)(H2O)4 was obtained with the hydrothermal synthesis. The geometry optimization of this molecule was done by Density Functional Theory (DFT/B3LYP) method with 6-31G(d) basis set and LANL2DZ for Hf. The experimental infrared spectrum was compared with calculated and complete vibrational assignment was provided. The bond orders and the electronic properties of the molecule were calculated. The natural bond orbital analysis (NBO) was performed in order to study the intramolecular bonding interactions among bonds and delocalization of unpaired electrons. The calculated highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) with frontier orbital gap were presented. The electrostatic potential was calculated in order to investigate the reaction properties of the molecule. The thermodynamic properties of the studied compound at different temperatures were calculated.

  18. Life-cycle and genetic characterization of Astiotrema odhneri Bhalerao, 1936 sensu Cho & Seo 1977 from the Primorsky Region (Russian Far East).

    PubMed

    Besprozvannykh, V V; Atopkin, D M; Ermolenko, A V; Kharitonova, A V; Khamatova, A Yu

    2015-12-01

    Adult Astiotrema odhneri Bhalerao, 1936 sensu Cho & Seo 1977 were found in the intestine of a freshwater turtle, Pelodiscus sinensis (Wiegmann), from the Komissarovka River Basin, Primorsky Region, Russia. It was established that the first intermediate host of this parasite is a snail, Anisus centrifugops, and that the second intermediate hosts include the snails, Helicorbis sujfunensis and A. centrifugops, tadpoles of the frog Rana dybowskii, and the fish Perccottus glenii. The development of A. odhneri includes the formation of sporocyst and xiphidiocercariae, which is typical for species belonging to Plagiorchioidea. Phylogenetic analysis based on 28S rRNA gene sequences showed that A. odhneri, together with Astiotrema monticellii, form a monophyletic clade that was closer to Opisthorchioidea than to any other taxon represented in the tree. However, phylogenetic analysis without outgroup taxon indicated a high degree of differentiation of Astiotrema from both Plagiorchioidea and Opisthorchioidea.

  19. Protein attachment onto silica surfaces--a survey of molecular fundamentals, resulting effects and novel preventive strategies in CE.

    PubMed

    Stutz, Hanno

    2009-06-01

    This review addresses the fundamentals governing the adsorption of individual protein molecules onto the surface of fused-silica capillaries, the protein aggregation to adsorbate clusters and their final accretion to monolayers with subsequent stratification to protein multilayers. The attention in CE protein separation has primarily been focused on (i) tuning the BGE including the buffer type, ionic strength, pH and additives, (ii) tailored post-rinse procedures to detach adhered protein residues and (iii) the optimization of capillary wall shielding in order to reduce protein attachment. Improvements in protein separation as well as related adverse effects are mainly discussed on the basis of parameters known to become deteriorated in case of protein adhesion, e.g. repeatability of the EOF and of migration times, peak width, theoretical plate numbers, resolution and asymmetry factor. However, knowledge of the molecular principles controlling protein adsorption onto silica surfaces is indispensable for separation optimization. Furthermore, it facilitates troubleshooting and the interpretation of undesired concomitant phenomena. This review comprehensively discusses protein adsorption models derived from surface chemistry primarily in terms of their relevance for CE, clearly showing that the adsorption process in its complexity is only partially revealed by models, which address single or binary protein solutions. In a further section theoretical concepts and surface models are related to surface phenomena encountered in CE. The final part of the review surveys recent concepts for prevention of protein adhesion, thereby addressing capillary treatment, favorable buffer types, dynamic and adhesive semi-permanent coating strategies covering the literature from 2000-2008.

  20. Developmental methamphetamine exposure results in short- and long-term alterations in hypothalamic-pituitary-adrenal axis-associated proteins

    PubMed Central

    Zuloaga, Damian G.; Siegel, Jessica A.; Acevedo, Summer F.; Agam, Maayan; Raber, Jacob

    2013-01-01

    Developmental exposure to methamphetamine (MA) causes long-term behavioral and cognitive deficits. One pathway through which MA might induce these deficits is by elevating glucocorticoid levels. Glucocorticoid overexposure during brain development can lead to long-term disruptions in the hypothalamic-pituitary-adrenal (HPA) axis. These disruptions affect the regulation of stress responses and may contribute to behavioral and cognitive deficits reported following developmental MA exposure. Furthermore, alterations in proteins associated with the HPA axis, including vasopressin, oxytocin, and glucocorticoid receptors (GR), are correlated with disruptions in mood and cognition. We therefore hypothesized that early MA exposure will result in short- and long-term alterations in the expression of HPA axis-associated proteins. Male mice were treated with MA (5 mg/kg daily) or Saline from postnatal day (P) 11–20. At P20 and P90, mice were perfused and their brains processed for vasopressin, oxytocin, and GR-immunoreactivity within HPA axis-associated regions. At P20, there was a significant decrease in the number of vasopressin-immunoreactive cells and area occupied by vasopressin-immunoreactiviy in the paraventricular nucleus (PVN) of MA-treated mice, but no difference in oxytocin-immunoreactivity in the PVN, or GR-immunoreactivity in the hippocampus or PVN. In the central nucleus of the amygdala, area occupied by GR-immunoreactivity was decreased by MA. At P90, the number of vasopressin-immunoreactive cells was still decreased, but the area occupied by vasopressin-immunoreactivity no longer differed from Saline controls. No effects of MA were found on oxytocin or GR-immunoreactivity at P90. Thus developmental MA exposure has short- and long-term effects on vasopressin-immunoreactivity and short-term effects on GR-immunoreactivity. PMID:23860125

  1. Reduced Sweetness of a Monellin (MNEI) Mutant Results from Increased Protein Flexibility and Disruption of a Distant Poly-(L-Proline) II Helix

    PubMed Central

    Templeton, Catherine M.; Ostovar pour, Saeideh; Hobbs, Jeanette R.; Blanch, Ewan W.; Munger, Steven D.

    2011-01-01

    Monellin is a highly potent sweet-tasting protein but relatively little is known about how it interacts with the sweet taste receptor. We determined X-ray crystal structures of 3 single-chain monellin (MNEI) proteins with alterations at 2 core residues (G16A, V37A, and G16A/V37A) that induce 2- to 10-fold reductions in sweetness relative to the wild-type protein. Surprisingly, no changes were observed in the global protein fold or the positions of surface amino acids important for MNEI sweetness that could explain these differences in protein activity. Differential scanning calorimetry showed that while the thermal stability of each mutant MNEI was reduced, the least sweet mutant, G16A-MNEI, was not the least stable protein. In contrast, solution spectroscopic measurements revealed that changes in protein flexibility and the C-terminal structure correlate directly with protein activity. G16A mutation-induced disorder in the protein core is propagated via changes to hydrophobic interactions that disrupt the formation and/or position of a critical C-terminal poly-(L-proline) II helix. These findings suggest that MNEI interaction with the sweet taste receptor is highly sensitive to the relative positions of key residues across its protein surface and that loss of sweetness in G16A-MNEI may result from an increased entropic cost of binding. PMID:21343241

  2. The Antagonistic Effect of Selenium on Lead-Induced Inflammatory Factors and Heat Shock Proteins mRNA Expression in Chicken Livers.

    PubMed

    Wang, Hao; Li, Shu; Teng, Xiaohua

    2016-06-01

    The aim of this study was to investigate the effect of lead (Pb) poisoning on nitric oxide (NO) content, inducible nitric oxide synthase (iNOS) activity, the messenger RNA (mRNA) levels of inflammatory factors (nuclear factor-kappa B (NF-κB), tumor necrosis factor-α (TNF-α), cyclooxygenase-2 (COX-2), prostaglandin E synthases (PTGEs), and iNOS), heat shock proteins (HSPs) (HSP27, HSP40, HSP60, HSP70, and HSP90), and the antagonistic effect of selenium (Se) on Pb in chicken livers. One hundred eighty 7-day-old male chickens were randomly divided into four groups and were fed commercial diet and drinking water, Na2SeO3-added commercial diet and drinking water, commercial diet and (CH3OO)2Pb-added drinking water, and Na2SeO3-added commercial diet and (CH3OO)2Pb-added drinking water, respectively, for 30, 60, and 90 days. Then, NO content, iNOS activity, and the mRNA levels of NF-κB, TNF-α, COX-2, PTGEs, iNOS, HSP27, HSP40, HSP60, HSP70, and HSP90 were examined in chicken livers. The results showed that Pb poisoning induced NO content, iNOS activity, and mRNA expression of inflammation factors and HSPs in chicken livers. In addition, Se alleviated Pb-induced increase of inflammation factor and HSP expression in chicken livers. PMID:26470710

  3. Plasmodium falciparum spermidine synthase inhibition results in unique perturbation-specific effects observed on transcript, protein and metabolite levels

    PubMed Central

    2010-01-01

    Background Plasmodium falciparum, the causative agent of severe human malaria, has evolved to become resistant to previously successful antimalarial chemotherapies, most notably chloroquine and the antifolates. The prevalence of resistant strains has necessitated the discovery and development of new chemical entities with novel modes-of-action. Although much effort has been invested in the creation of analogues based on existing drugs and the screening of chemical and natural compound libraries, a crucial shortcoming in current Plasmodial drug discovery efforts remains the lack of an extensive set of novel, validated drug targets. A requirement of these targets (or the pathways in which they function) is that they prove essential for parasite survival. The polyamine biosynthetic pathway, responsible for the metabolism of highly abundant amines crucial for parasite growth, proliferation and differentiation, is currently under investigation as an antimalarial target. Chemotherapeutic strategies targeting this pathway have been successfully utilized for the treatment of Trypanosomes causing West African sleeping sickness. In order to further evaluate polyamine depletion as possible antimalarial intervention, the consequences of inhibiting P. falciparum spermidine synthase (PfSpdSyn) were examined on a morphological, transcriptomic, proteomic and metabolic level. Results Morphological analysis of P. falciparum 3D7 following application of the PfSpdSyn inhibitor cyclohexylamine confirmed that parasite development was completely arrested at the early trophozoite stage. This is in contrast to untreated parasites which progressed to late trophozoites at comparable time points. Global gene expression analyses confirmed a transcriptional arrest in the parasite. Several of the differentially expressed genes mapped to the polyamine biosynthetic and associated metabolic pathways. Differential expression of corresponding parasite proteins involved in polyamine biosynthesis was

  4. Phenotypic Consequences Resulting from a Methionine-to-Valine Substitution at Position 48 in the HPr Protein of Streptococcus salivarius

    PubMed Central

    Plamondon, Pascale; Brochu, Denis; Thomas, Suzanne; Fradette, Julie; Gauthier, Lucie; Vaillancourt, Katy; Buckley, Nicole; Frenette, Michel; Vadeboncoeur, Christian

    1999-01-01

    In gram-positive bacteria, the HPr protein of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) can be phosphorylated on a histidine residue at position 15 (His15) by enzyme I (EI) of the PTS and on a serine residue at position 46 (Ser46) by an ATP-dependent protein kinase (His∼P and Ser-P, respectively). We have isolated from Streptococcus salivarius ATCC 25975, by independent selection from separate cultures, two spontaneous mutants (Ga3.78 and Ga3.14) that possess a missense mutation in ptsH (the gene encoding HPr) replacing the methionine at position 48 by a valine. The mutation did not prevent the phosphorylation of HPr at His15 by EI nor the phosphorylation at Ser46 by the ATP-dependent HPr kinase. The levels of HPr(Ser-P) in glucose-grown cells of the parental and mutant Ga3.78 were virtually the same. However, mutant cells growing on glucose produced two- to threefold less HPr(Ser-P)(His∼P) than the wild-type strain, while the levels of free HPr and HPr(His∼P) were increased 18- and 3-fold, respectively. The mutants grew as well as the wild-type strain on PTS sugars (glucose, fructose, and mannose) and on the non-PTS sugars lactose and melibiose. However, the growth rate of both mutants on galactose, also a non-PTS sugar, decreased rapidly with time. The M48V substitution had only a minor effect on the repression of α-galactosidase, β-galactosidase, and galactokinase by glucose, but this mutation abolished diauxie by rendering cells unable to prevent the catabolism of a non-PTS sugar (lactose, galactose, and melibiose) when glucose was available. The results suggested that the capacity of the wild-type cells to preferentially metabolize glucose over non-PTS sugars resulted mainly from inhibition of the catabolism of these secondary energy sources via a HPr-dependent mechanism. This mechanism was activated following glucose but not lactose metabolism, and it did not involve HPr(Ser-P) as the only regulatory molecule. PMID:10559156

  5. Deficiency in macrophage-stimulating protein results in spontaneous intestinal inflammation and increased susceptibility toward epithelial damage in zebrafish.

    PubMed

    Witte, Merlijn; Huitema, Leonie F A; Nieuwenhuis, Edward E S; Brugman, Sylvia

    2014-12-01

    Several genome-wide association studies have identified the genes encoding for macrophage-stimulating protein (MSP) and its receptor RON (Recepteur d'Origine Nantais) as possible susceptibility factors in inflammatory bowel disease. While it has been shown that the MSP-RON signaling pathway is involved in tissue injury responses, current mouse models for MSP and RON deficiency have not clearly demonstrated a role of MSP-RON signaling in the context of intestinal inflammation. In this study, we report that the recently identified zebrafish Msp mutant (msp(t34230)) develops spontaneous intestinal inflammation over time. From 14 to 28 weeks postfertilization Msp-deficient zebrafish show intestinal eosinophilia, increased intestinal expression of inflammatory marker mmp9, and activation of intestinal goblet cells. Moreover, these Msp mutant zebrafish are more susceptible toward ethanol-induced epithelial damage, which resulted in increased infiltration and proliferation of immune cells within the lamina propria and prolonged intestinal proinflammatory cytokine responses in some mutant fish. In light of the recent development of many tools to visualize, monitor, and genetically modify zebrafish, these Msp-deficient zebrafish will enable in-depth in vivo analysis of epithelial and macrophage-specific MSP-RON signaling in the context of intestinal inflammation.

  6. Deficiency in macrophage-stimulating protein results in spontaneous intestinal inflammation and increased susceptibility toward epithelial damage in zebrafish.

    PubMed

    Witte, Merlijn; Huitema, Leonie F A; Nieuwenhuis, Edward E S; Brugman, Sylvia

    2014-12-01

    Several genome-wide association studies have identified the genes encoding for macrophage-stimulating protein (MSP) and its receptor RON (Recepteur d'Origine Nantais) as possible susceptibility factors in inflammatory bowel disease. While it has been shown that the MSP-RON signaling pathway is involved in tissue injury responses, current mouse models for MSP and RON deficiency have not clearly demonstrated a role of MSP-RON signaling in the context of intestinal inflammation. In this study, we report that the recently identified zebrafish Msp mutant (msp(t34230)) develops spontaneous intestinal inflammation over time. From 14 to 28 weeks postfertilization Msp-deficient zebrafish show intestinal eosinophilia, increased intestinal expression of inflammatory marker mmp9, and activation of intestinal goblet cells. Moreover, these Msp mutant zebrafish are more susceptible toward ethanol-induced epithelial damage, which resulted in increased infiltration and proliferation of immune cells within the lamina propria and prolonged intestinal proinflammatory cytokine responses in some mutant fish. In light of the recent development of many tools to visualize, monitor, and genetically modify zebrafish, these Msp-deficient zebrafish will enable in-depth in vivo analysis of epithelial and macrophage-specific MSP-RON signaling in the context of intestinal inflammation. PMID:25353089

  7. A Loss in Cellular Protein Partners Promotes α-Synuclein Aggregation in Cells Resulting from Oxidative Stress

    PubMed Central

    Guo, Yuanjian; Scarlata, Suzanne

    2015-01-01

    There is a consensus that oxidative stress promotes neurodegeneration and may be linked to plaque formation. α-Synuclein is the main component of neurodegenerative plaques. We have found that α-synuclein binds strongly to the enzyme phospholipase Cβ1 (PLCβ1) in vitro and in cells affecting both its G protein activation and its degradation. Because PLCβ1 binds to α-synuclein in cells, we tested whether decreasing its level would promote α-synuclein aggregation and whether overproducing PLCβ1 would inhibit aggregation. By imaging fluorescent α-synuclein in living HEK293, PC12, and SK-H-SH cells, we find that α-synuclein aggregation is directly related to the level of PLCβ1. Importantly, we found that oxidative stress does not affect the cellular levels of α-synuclein but results in the down-regulation of PLCβ1 thereby promoting α-synuclein aggregation. A peptide that mimics part of the α-synuclein binding site to PLCβ prevents aggregation. Our studies indicate that PLCβ1 can reduce cell damage under oxidative stress and offers a potential site that might be exploited to prevent α-synuclein aggregation. PMID:23659438

  8. Repeated exposures to roadside particulate matter extracts suppresses pulmonary defense mechanisms, resulting in lipid and protein oxidative damage.

    PubMed

    Pardo, Michal; Porat, Ziv; Rudich, Assaf; Schauer, James J; Rudich, Yinon

    2016-03-01

    Exposure to particulate matter (PM) pollution in cities and urban canyons can be harmful to the exposed population. However, the underlying mechanisms that lead to health effects are not yet elucidated. It is postulated that exposure to repeated, small, environmentally relevant concentrations can affect lung homeostasis. This study examines the impact of repeated exposures to urban PM on mouse lungs with focus on inflammatory and oxidative stress parameters. Aqueous extracts from collected urban PM were administered to mice by 5 repeated intra-tracheal instillations (IT). Multiple exposures, led to an increase in cytokine levels in both bronchoalveolar lavage fluid and in the blood serum, indicating a systemic reaction. Lung mRNA levels of antioxidant/phase II detoxifying enzymes decreased by exposure to the PM extract, but not when metals were removed by chelation. Finally, disruption of lung tissue oxidant-inflammatory/defense balance was evidenced by increased levels of lipid and protein oxidation. Unlike response to a single IT exposure to the same dose and source of extract, multiple exposures result in lung oxidative damage and a systemic inflammatory reaction. These could be attributed to compromised capacity to activate the protective Nrf2 tissue defense system. It is suggested that water-soluble metals present in urban PM, potentially from break and tire wear, may constitute major drivers of the pulmonary and systemic responses to multiple exposure to urban PM.

  9. Deletion of GSTA4-4 results in increased mitochondrial post-translational modification of proteins by reactive aldehydes following chronic ethanol consumption in mice

    PubMed Central

    Shearn, Colin T.; Fritz, Kristofer S.; Shearn, Alisabeth H.; Saba, Laura M.; Mercer, Kelly E.; Engi, Bridgette; Galligan, James J.; Zimniak, Piotr; Orlicky, David J.; Ronis, Martin J.; Petersen, Dennis R.

    2015-01-01

    Chronic alcohol consumption induces hepatic oxidative stress resulting in production of highly reactive electrophilic α/β-unsaturated aldehydes that have the potential to modify proteins. A primary mechanism of reactive aldehyde detoxification by hepatocytes is through GSTA4-driven enzymatic conjugation with GSH. Given reports that oxidative stress initiates GSTA4 translocation to the mitochondria, we hypothesized that increased hepatocellular damage in ethanol (EtOH)-fed GSTA4−/− mice is due to enhanced mitochondrial protein modification by reactive aldehydes. Chronic ingestion of EtOH increased hepatic protein carbonylation in GSTA4−/− mice as evidenced by increased 4-HNE and MDA immunostaining in the hepatic periportal region. Using mass spectrometric analysis of biotin hydrazide conjugated carbonylated proteins, a total of 829 proteins were identified in microsomal, cytosolic and mitochondrial fractions. Of these, 417 were novel to EtOH models. Focusing on mitochondrial fractions, 1.61-fold more carbonylated proteins were identified in EtOH-fed GSTA4−/− mice compared to their respective WT mice ingesting EtOH. Bioinformatic KEGG pathway analysis of carbonylated proteins from the mitochondrial fractions revealed an increased propensity for modification of proteins regulating oxidative phosphorylation, glucose, fatty acid, glutathione and amino acid metabolic processes in GSTA4−/− mice. Additional analysis revealed sites of reactive aldehyde protein modification on 26 novel peptides/proteins isolated from either SV/GSTA4−/− PF or EtOH fed mice. Among the peptides/proteins identified, ACSL, ACOX2, MTP, and THIKB contribute to regulation of fatty acid metabolism and ARG1, ARLY, and OAT, which regulate nitrogen and ammonia metabolism having direct relevance to ethanol-induced liver injury. These data define a role for GSTA4-4 in buffering hepatic oxidative stress associated with chronic alcohol consumption and that this GST isoform plays an

  10. Spaceflight results in increase of thick filament but not thin filament proteins in the paramyosin mutant of Caenorhabditis elegans

    NASA Astrophysics Data System (ADS)

    Adachi, R.; Takaya, T.; Kuriyama, K.; Higashibata, A.; Ishioka, N.; Kagawa, H.

    We have investigated the effect of microgravity during spaceflight on body-wall muscle fiber size and muscle proteins in the paramyosin mutant of Caenorhabditis elegans. Both mutant and wild-type strains were subjected to 10 days of microgravity during spaceflight and compared to ground control groups. No significant change in muscle fiber size or quantity of the protein was observed in wild-type worms; where as atrophy of body-wall muscle and an increase in thick filament proteins were observed in the paramyosin mutant unc-15(e73) animals after spaceflight. We conclude that the mutant with abnormal muscle responded to microgravity by increasing the total amount of muscle protein in order to compensate for the loss of muscle function.

  11. Mechanisms of cross-talk between G-protein-coupled receptors resulting in enhanced release of intracellular Ca2+.

    PubMed Central

    Werry, Tim D; Wilkinson, Graeme F; Willars, Gary B

    2003-01-01

    Alteration in [Ca(2+)](i) (the intracellular concentration of Ca(2+)) is a key regulator of many cellular processes. To allow precise regulation of [Ca(2+)](i) and a diversity of signalling by this ion, cells possess many mechanisms by which they are able to control [Ca(2+)](i) both globally and at the subcellular level. Among these are many members of the superfamily of GPCRs (G-protein-coupled receptors), which are characterized by the presence of seven transmembrane domains. Typically, those receptors able to activate PLC (phospholipase C) enzymes cause release of Ca(2+) from intracellular stores and influence Ca(2+) entry across the plasma membrane. It has been well documented that Ca(2+) signalling by one type of GPCR can be influenced by stimulation of a different type of GPCR. Indeed, many studies have demonstrated heterologous desensitization between two different PLC-coupled GPCRs. This is not surprising, given our current understanding of negative-feedback regulation and the likely shared components of the signalling pathway. However, there are also many documented examples of interactions between GPCRs, often coupling preferentially to different signalling pathways, which result in a potentiation of Ca(2+) signalling. Such interactions have important implications for both the control of cell function and the interpretation of in vitro cell-based assays. However, there is currently no single mechanism that adequately accounts for all examples of this type of cross-talk. Indeed, many studies either have not addressed this issue or have been unable to determine the mechanism(s) involved. This review seeks to explore a range of possible mechanisms to convey their potential diversity and to provide a basis for further experimental investigation. PMID:12790797

  12. Bortezomib-induced unfolded protein response increases oncolytic HSV-1 replication resulting in synergistic, anti-tumor effects

    PubMed Central

    Yoo, Ji Young; Hurwitz, Brian S; Bolyard, Chelsea; Yu, Jun-Ge; Zhang, Jianying; Selvendiran, Karuppaiyah; Rath, Kellie S; He, Shun; Bailey, Zachary; Eaves, David; Cripe, Timothy P; Parris, Deborah S.; Caligiuri, Michael A.; Yu, Jianhua; Old, Matthew; Kaur, Balveen

    2014-01-01

    Background Bortezomib is an FDA-approved proteasome inhibitor, and oncolytic HSV-1 (oHSV) is a promising therapeutic approach for cancer. We tested the impact of combining bortezomib with oHSV for anti-tumor efficacy. Methods The synergistic interaction between oHSV and bortezomib was calculated using Chou-Talalay analysis. Viral replication was evaluated using plaque assay and immune fluorescence. Western-blot assays were used to evaluate induction of ER stress and unfolded protein response (UPR). Inhibitors targeting Hsp90 were utilized to investigate the mechanism of cell killing. Anti-tumor efficacy in vivo was evaluated using subcutaneous and intracranial tumor xenografts of glioma and head and neck cancer. Survival was analyzed by Kaplan-Meier curves and two-sided log rank test. Results Combination treatment with bortezomib and oHSV, 34.5ENVE, displayed strong synergistic interaction in ovarian cancer, head & neck cancer, glioma, and malignant peripheral nerve sheath tumor (MPNST) cells. Bortezomib treatment induced ER stress, evident by strong induction of Grp78, CHOP, PERK and IRE1α (western blot analysis) and the UPR (induction of hsp40, 70 and 90). Bortezomib treatment of cells at both sublethal and lethal doses increased viral replication (p value <0.001), but inhibition of Hsp90 ablated this response, reducing viral replication and synergistic cell killing. The combination of bortezomib and 34.5ENVE significantly enhanced anti-tumor efficacy in multiple different tumor models in vivo. Conclusions The dramatic synergy of bortezomib and 34.5ENVE is mediated by bortezomib- induced UPR and warrants future clinical testing in patients. PMID:24815720

  13. Strong improvement of interfacial properties can result from slight structural modifications of proteins: the case of native and dry-heated lysozyme.

    PubMed

    Desfougères, Yann; Saint-Jalmes, Arnaud; Salonen, Anniina; Vié, Véronique; Beaufils, Sylvie; Pezennec, Stéphane; Desbat, Bernard; Lechevalier, Valérie; Nau, Françoise

    2011-12-20

    Identification of the key physicochemical parameters of proteins that determine their interfacial properties is still incomplete and represents a real stake challenge, especially for food proteins. Many studies have thus consisted in comparing the interfacial behavior of different proteins, but it is difficult to draw clear conclusions when the molecules are completely different on several levels. Here the adsorption process of a model protein, the hen egg-white lysozyme, and the same protein that underwent a thermal treatment in the dry state, was characterized. The consequences of this treatment have been previously studied: net charge and hydrophobicity increase and lesser protein stability, but no secondary and tertiary structure modification (Desfougères, Y.; Jardin, J.; Lechevalier, V.; Pezennec, S.; Nau, F. Biomacromolecules 2011, 12, 156-166). The present study shows that these slight modifications dramatically increase the interfacial properties of the protein, since the adsorption to the air-water interface is much faster and more efficient (higher surface pressure). Moreover, a thick and strongly viscoelastic multilayer film is created, while native lysozyme adsorbs in a fragile monolayer film. Another striking result is that completely different behaviors were observed between two molecular species, i.e., native and native-like lysozyme, even though these species could not be distinguished by usual spectroscopic methods. This suggests that the air-water interface could be considered as a useful tool to reveal very subtle differences between protein molecules. PMID:22040020

  14. Genetic Ablation of the CDP/Cux Protein C Terminus Results in Hair Cycle Defects and Reduced Male Fertility

    PubMed Central

    Luong, Mai X.; van der Meijden, Caroline M.; Xing, DongXia; Hesselton, Ruth; Monuki, Edwin S.; Jones, Stephen N.; Lian, Jane B.; Stein, Janet L.; Stein, Gary S.; Neufeld, Ellis J.; van Wijnen, Andre J.

    2002-01-01

    Murine CDP/Cux, a homologue of the Drosophila Cut homeoprotein, modulates the promoter activity of cell cycle-related and cell-type-specific genes. CDP/Cux interacts with histone gene promoters as the DNA binding subunit of a large nuclear complex (HiNF-D). CDP/Cux is a ubiquitous protein containing four conserved DNA binding domains: three Cut repeats and a homeodomain. In this study, we analyzed genetically targeted mice (Cutl1tm2Ejn, referred to as ΔC) that express a mutant CDP/Cux protein with a deletion of the C terminus, including the homeodomain. In comparison to the wild-type protein, indirect immunofluorescence showed that the mutant protein exhibited significantly reduced nuclear localization. Consistent with these data, DNA binding activity of HiNF-D was lost in nuclear extracts derived from mouse embryonic fibroblasts (MEFs) or adult tissues of homozygous mutant (ΔC−/−) mice, indicating the functional loss of CDP/Cux protein in the nucleus. No significant difference in growth characteristics or total histone H4 mRNA levels was observed between wild-type and ΔC−/− MEFs in culture. However, specific histone genes (H4.1 and H1) containing CDP/Cux binding sites have reduced expression levels in homozygous mutant MEFs. Stringent control of growth and differentiation appears to be compromised in vivo. Homozygous mutant mice have stunted growth (20 to 50% weight reduction), a high postnatal death rate of 60 to 70%, sparse abnormal coat hair, and severely reduced fertility. The deregulated hair cycle and severely diminished fertility in Cutl1tm2Ejn/tm2Ejn mice suggest that CDP/Cux is required for the developmental control of dermal and reproductive functions. PMID:11839809

  15. Quantification of bovine milk protein composition and coagulation properties using infrared spectroscopy and chemometrics: A result of collinearity among reference variables.

    PubMed

    Eskildsen, C E; Skov, T; Hansen, M S; Larsen, L B; Poulsen, N A

    2016-10-01

    Predicting protein fractions and coagulation properties in bovine milk using Fourier transform infrared (FT-IR) measurements is desirable. However, such predictions may rely on correlations with total protein content. The aim of this study was to show how correlations between total protein content, protein fractions, and coagulation properties are responsible for the successful prediction of protein fractions and rennet-induced coagulation properties in milk samples. This study comprised 832 bovine milk samples from 2 breeds (426 Holstein and 406 Jersey). Holstein samples were collected from 20 Danish dairy herds from October to December 2009; Jersey samples were collected from 22 Danish dairy herds from February to April 2010. All samples were from conventional herds and taken while cows were housed. The results showed that κ-CN, αS1-CN, αS1-CN with 8 phosphorylated groups attached (αS1-CN 8P), and curd firming rate could be predicted from FT-IR measurements of the milk samples (with coefficients of determination between 0.66 and 0.71). However, the success of these FT-IR-based predictions was based on indirect relationships with total protein content. Hence, the FT-IR predictions relied on covariance structures with total protein content rather than absorption bands directly associated with the protein fractions and coagulation properties. If covariance structures between the protein fractions, coagulation properties, and total protein content used to calibrate partial least squares models were not conserved in future samples, these samples would show incorrect predictions of the protein fractions and coagulation properties. We demonstrated this using samples from 1 breed to calibrate and samples from the other breed to validate partial least squares models for β-CN. The 2 breeds had different covariance structures between β-CN and total protein content, and the validation samples yielded incorrect predictions. This finding may limit the usefulness of FT

  16. A comprehensive platform to investigate protein-metal ion interactions by affinity capillary electrophoresis.

    PubMed

    Alhazmi, Hassan A; Nachbar, Markus; Albishri, Hassan M; Abd El-Hady, Deia; Redweik, Sabine; El Deeb, Sami; Wätzig, Hermann

    2015-03-25

    In this work, the behavior of several metal ions with different globular proteins was investigated by affinity capillary electrophoresis. Screening was conducted by applying a proper rinsing protocol developed by our group. The use of 0.1M EDTA in the rinsing solution successfully desorbs metal ions from the capillary wall. The mobility ratio was used to evaluate the precision of the method. Excellent precision for repeated runs was achieved for different protein metal ion interactions (RSD% of 0.05-1.0%). Run times were less than 6 min for all of the investigated interactions. The method has been successfully applied for the interaction study of Li(+), Na(+), Mg(2+), Ca(2+), Ba(2+), Al(3+), Ga(3+), La(3+), Pd(2+), Ir(3+), Ru(3+), Rh(3+), Pt(2+), Pt(4+), Os(3+), Au(3+), Au(+), Ag(+), Cu(1+), Cu(2+), Fe(2+), Fe(3+), Co(2+), Ni(2+), Cr(3+), V(3+), MoO4(2-) and SeO3(2-) with bovine serum albumin, ovalbumin, β-lactoglobulin and myoglobin. Different interaction values were obtained for most of the tested metal ions even for that in the same metal group. Results were discussed and compared in view of metal and semimetal group's interaction behavior with the tested proteins. The calculated normalized difference of mobility ratios for each protein-metal ion interaction and its sign (positive and negative) has been successfully used to detect the interaction and estimate further coordination of the bound metal ion, respectively. The comprehensive platform summarizes all the obtained interaction results, and is valuable for any future protein-metal ion investigation.

  17. Single amino acid exchange in bacteriophage HK620 tailspike protein results in thousand-fold increase of its oligosaccharide affinity.

    PubMed

    Broeker, Nina K; Gohlke, Ulrich; Müller, Jürgen J; Uetrecht, Charlotte; Heinemann, Udo; Seckler, Robert; Barbirz, Stefanie

    2013-01-01

    Bacteriophage HK620 recognizes and cleaves the O-antigen polysaccharide of Escherichia coli serogroup O18A1 with its tailspike protein (TSP). HK620TSP binds hexasaccharide fragments with low affinity, but single amino acid exchanges generated a set of high-affinity mutants with submicromolar dissociation constants. Isothermal titration calorimetry showed that only small amounts of heat were released upon complex formation via a large number of direct and solvent-mediated hydrogen bonds between carbohydrate and protein. At room temperature, association was both enthalpy- and entropy-driven emphasizing major solvent rearrangements upon complex formation. Crystal structure analysis showed identical protein and sugar conformers in the TSP complexes regardless of their hexasaccharide affinity. Only in one case, a TSP mutant bound a different hexasaccharide conformer. The extended sugar binding site could be dissected in two regions: first, a hydrophobic pocket at the reducing end with minor affinity contributions. Access to this site could be blocked by a single aspartate to asparagine exchange without major loss in hexasaccharide affinity. Second, a region where the specific exchange of glutamate for glutamine created a site for an additional water molecule. Side-chain rearrangements upon sugar binding led to desolvation and additional hydrogen bonding which define this region of the binding site as the high-affinity scaffold.

  18. B-lymphocyte Specific loss of Ric-8A Results in a Gα Protein Deficit and Severe Humoral Immunodeficiency

    PubMed Central

    Boularan, Cedric; Hwang, Il-Young; Kamenyeva, Olena; Park, Chung; Harrison, Kathleen; Huang, Zhen; Kehrl, John H.

    2015-01-01

    Resistance to inhibitors of cholinesterase 8A (Ric-8A) is a highly evolutionarily conserved cytosolic protein initially identified in C. elegans, where it was assigned a regulatory role in asymmetric cell divisions. It functions as a guanine nucleotide exchange factor for Gαi, Gαq, and Gα12/13 and as a molecular chaperone required for the initial association of nascent Gα subunits with cellular membranes in embryonic stem cell lines. To test its role in hematopoiesis and B lymphocytes specifically, we generated ric8fl/flvav1-cre and ric8fl/flmb1-cre mice. The major hematopoietic cell lineages developed in the ric8fl/flvav1-cre mice, notwithstanding severe reduction in Gαi2/3, Gαq, and Gα13 proteins. B lymphocyte specific loss of Ric-8A did not compromise bone marrow B lymphopoiesis, but splenic marginal zone B cell development failed, and B cells underpopulated lymphoid organs. The ric8fl/flmb1-cre B cells exhibited poor responses to chemokines, abnormal trafficking, improper in situ positioning, and loss of polarity components during B cell differentiation. The ric8fl/flmb1-cre mice had a severely disrupted lymphoid architecture and poor primary and secondary antibody responses. In B lymphocytes, Ric-8A is essential for normal Gα protein levels; and is required for B cell differentiation, trafficking, and antibody responses. PMID:26232433

  19. NCYM, a Cis-Antisense Gene of MYCN, Encodes a De Novo Evolved Protein That Inhibits GSK3β Resulting in the Stabilization of MYCN in Human Neuroblastomas

    PubMed Central

    Suenaga, Yusuke; Islam, S. M. Rafiqul; Alagu, Jennifer; Kaneko, Yoshiki; Kato, Mamoru; Tanaka, Yukichi; Kawana, Hidetada; Hossain, Shamim; Matsumoto, Daisuke; Yamamoto, Mami; Shoji, Wataru; Itami, Makiko; Shibata, Tatsuhiro; Nakamura, Yohko; Ohira, Miki; Haraguchi, Seiki; Takatori, Atsushi; Nakagawara, Akira

    2014-01-01

    The rearrangement of pre-existing genes has long been thought of as the major mode of new gene generation. Recently, de novo gene birth from non-genic DNA was found to be an alternative mechanism to generate novel protein-coding genes. However, its functional role in human disease remains largely unknown. Here we show that NCYM, a cis-antisense gene of the MYCN oncogene, initially thought to be a large non-coding RNA, encodes a de novo evolved protein regulating the pathogenesis of human cancers, particularly neuroblastoma. The NCYM gene is evolutionally conserved only in the taxonomic group containing humans and chimpanzees. In primary human neuroblastomas, NCYM is 100% co-amplified and co-expressed with MYCN, and NCYM mRNA expression is associated with poor clinical outcome. MYCN directly transactivates both NCYM and MYCN mRNA, whereas NCYM stabilizes MYCN protein by inhibiting the activity of GSK3β, a kinase that promotes MYCN degradation. In contrast to MYCN transgenic mice, neuroblastomas in MYCN/NCYM double transgenic mice were frequently accompanied by distant metastases, behavior reminiscent of human neuroblastomas with MYCN amplification. The NCYM protein also interacts with GSK3β, thereby stabilizing the MYCN protein in the tumors of the MYCN/NCYM double transgenic mice. Thus, these results suggest that GSK3β inhibition by NCYM stabilizes the MYCN protein both in vitro and in vivo. Furthermore, the survival of MYCN transgenic mice bearing neuroblastoma was improved by treatment with NVP-BEZ235, a dual PI3K/mTOR inhibitor shown to destabilize MYCN via GSK3β activation. In contrast, tumors caused in MYCN/NCYM double transgenic mice showed chemo-resistance to the drug. Collectively, our results show that NCYM is the first de novo evolved protein known to act as an oncopromoting factor in human cancer, and suggest that de novo evolved proteins may functionally characterize human disease. PMID:24391509

  20. Parageorgbokiite, β-Cu5O2(SeO3)2Cl2, a new mineral species from volcanic exhalations, Kamchatka Peninsula, Russia

    NASA Astrophysics Data System (ADS)

    Vergasova, L. P.; Krivovichev, S. V.; Filatov, S. K.; Britvin, S. N.; Burns, P. C.; Anan'ev, V. V.

    2007-12-01

    Parageorgbokiite, β-Cu5O2(SeO3)2Cl2, has been found at the second cinder cone of the Great Fissure Tolbachik Eruption, Kamchatka Peninsula, Russia. Ralstonite, tolbachite, melanothallite, chalcocyanite, euchlorine, Fe oxides, tenorite, native gold, sophiite, Na, Ca, and Mg sulfates, cotunnite, and some copper oxoselenites are associated minerals. The estimated temperature of the mineral formation is 400-625°C. The color is green, with a vitreous luster; the streak is light green. The mineral is brittle, with the Mohs hardness ranging from 3 to 4. Cleavage is not observed. The calculated density is 4.70 g/cm3. Parageorgbokiite is biaxial (+); α = 2.05(1), β = 2.05(1), and γ = 2.08(1); 2 V (meas.) is ˜03, and 2 V (calc.) = 0(5)°. The optical orientation is X = a; other details remain unclear. The mineral is pleochroic, from grass green on X and Y to yellowish green on Z. The empirical formula calculated on the basis of O + Cl = 10 is Cu4.91Pb0.02O1.86(ScO3)2Cl2.14. The simplified formula is Cu5O2(ScO3)2Cl2. Parageorgbokiite pertains to a new structural type of inorganic compounds. Its name points out its dimorphism with georgbokiite, which was named in honor of G.B. Bokii, the prominent Russian crystal chemist (1909-2000).

  1. Ligand binding to an Allergenic Lipid Transfer Protein Enhances Conformational Flexibility resulting in an Increase in Susceptibility to Gastroduodenal Proteolysis.

    PubMed

    Abdullah, Syed Umer; Alexeev, Yuri; Johnson, Philip E; Rigby, Neil M; Mackie, Alan R; Dhaliwal, Balvinder; Mills, E N Clare

    2016-01-01

    Non-specific lipid transfer proteins (LTPs) are a family of lipid-binding molecules that are widely distributed across flowering plant species, many of which have been identified as allergens. They are highly resistant to simulated gastroduodenal proteolysis, a property that may play a role in determining their allergenicity and it has been suggested that lipid binding may further increase stability to proteolysis. It is demonstrated that LTPs from wheat and peach bind a range of lipids in a variety of conditions, including those found in the gastroduodenal tract. Both LTPs are initially cleaved during gastroduodenal proteolysis at three major sites between residues 39-40, 56-57 and 79-80, with wheat LTP being more resistant to cleavage than its peach ortholog. The susceptibility of wheat LTP to proteolyic cleavage increases significantly upon lipid binding. This enhanced digestibility is likely to be due to the displacement of Tyr79 and surrounding residues from the internal hydrophobic cavity upon ligand binding to the solvent exposed exterior of the LTP, facilitating proteolysis. Such knowledge contributes to our understanding as to how resistance to digestion can be used in allergenicity risk assessment of novel food proteins, including GMOs. PMID:27458082

  2. Human C4b-binding protein selectively interacts with Neisseria gonorrhoeae and results in species-specific infection.

    PubMed

    Ngampasutadol, Jutamas; Ram, Sanjay; Blom, Anna M; Jarva, Hanna; Jerse, Ann E; Lien, Egil; Goguen, Jon; Gulati, Sunita; Rice, Peter A

    2005-11-22

    Neisseria gonorrhoeae is the causative agent of gonorrhea, a disease that is restricted to humans. Complement forms a key arm of the innate immune system that combats gonococcal infections. N. gonorrhoeae uses its outer membrane porin (Por) molecules to bind the classical pathway of complement down-regulatory protein C4b-binding protein (C4bp) to evade killing by human complement. Strains of N. gonorrhoeae that resisted killing by human serum complement were killed by serum from rodent, lagomorph, and primate species, which cannot be readily infected experimentally with this organism and whose C4bp molecules did not bind to N. gonorrhoeae. In contrast, we found that Yersinia pestis, an organism that can infect virtually all mammals, bound species-specific C4bp and uniformly resisted serum complement-mediated killing by these species. Serum resistance of gonococci was restored in these sera by human C4bp. An exception was serotype Por1B-bearing gonococcal strains that previously had been used successfully in a chimpanzee model of gonorrhea that simulates human disease. Por1B gonococci bound chimpanzee C4bp and resisted killing by chimpanzee serum, providing insight into the host restriction of gonorrhea and addressing why Por1B strains, but not Por1A strains, have been successful in experimental chimpanzee infection. Our findings may lead to the development of better animal models for gonorrhea and may also have implications in the choice of complement sources to evaluate neisserial vaccine candidates.

  3. Alternative processing of H-2Dd pre-mRNAs results in membrane expression of differentially phosphorylated protein products.

    PubMed Central

    McCluskey, J; Boyd, L F; Maloy, W L; Coligan, J E; Margulies, D H

    1986-01-01

    Two distinct mRNA species encoding the mouse major histocompatibility antigen H-2Dd have been identified in BALB/c spleen cells as well as in cultured cell lines expressing this cell surface glycoprotein. The alternate transcripts of H-2Dd arise from either removal or inclusion of exon VII (encoding I2) during pre-mRNA processing. The relative levels of each kind of H-2Dd transcript varied considerably between different cell types, and in all cells examined both forms of alloantigen were expressed on the cell membrane. Antigen derived from both types of transcript reacted with H-2Dd-specific monoclonal antibodies, whereas only protein lacking the 13 amino acids of I2 reacted with a specific antiserum raised against a predicted exon VI/VIII fusion peptide. Those H-2Dd proteins translated from full length, but not smaller, transcripts were phosphorylated in resting and phorbol myristate acetate-stimulated BALB/c spleen cells, suggesting that the major site of in vivo phosphorylation is within the highly conserved sequence encoded by exon VII. Thus alternative splicing of pre-mRNA transcripts is a mechanism which leads to membrane expression of two forms of H-2Dd, one of which lacks a major site of phosphorylation. Images Fig. 1. Fig. 2. Fig. 4. PMID:3640710

  4. Protein C Thr315Ala variant results in gain of function but manifests as type II deficiency in diagnostic assays

    PubMed Central

    Ding, Qiulan; Yang, Likui; Dinarvand, Peyman

    2015-01-01

    Protein C (PC) is a vitamin K–dependent plasma glycoprotein, which upon activation by thrombin in complex with thrombomodulin (TM), regulates the coagulation cascade through a feedback loop inhibition mechanism. PC deficiency is associated with an increased risk of venous thromboembolism (VTE). A recent cohort study aimed at establishing a normal PC range identified a healthy PC-deficient subject whose PC antigen level of 65% and activity levels of 50% (chromogenic assay) and 36% (clotting assay) were markedly low. The proband has a negative family history of VTE. Genetic analysis revealed the proband has a heterozygous missense mutation in which Thr-315 of the PC heavy chain has been substituted with Ala. We expressed this mutant in HEK-293 cells and purified it to homogeneity. A similar decrease in both anticoagulant and anti-inflammatory activities of the activated protein C mutant was observed in plasma- and cell-based assays. Interestingly, we discovered if functional assays were coupled to PC activation by the thrombin-TM complex, the variant exhibits improved activities in all assays. Sequence analysis revealed Thr-315 is a consensus N-linked glycosylation site for Asn-313 and that its elimination significantly (∼four- to fivefold) improves the maximum velocity of PC activation by the thrombin-TM complex, explaining the basis for the proband’s negative VTE pedigree. PMID:25651845

  5. Protein C Thr315Ala variant results in gain of function but manifests as type II deficiency in diagnostic assays.

    PubMed

    Ding, Qiulan; Yang, Likui; Dinarvand, Peyman; Wang, Xuefeng; Rezaie, Alireza R

    2015-04-01

    Protein C (PC) is a vitamin K-dependent plasma glycoprotein, which upon activation by thrombin in complex with thrombomodulin (TM), regulates the coagulation cascade through a feedback loop inhibition mechanism. PC deficiency is associated with an increased risk of venous thromboembolism (VTE). A recent cohort study aimed at establishing a normal PC range identified a healthy PC-deficient subject whose PC antigen level of 65% and activity levels of 50% (chromogenic assay) and 36% (clotting assay) were markedly low. The proband has a negative family history of VTE. Genetic analysis revealed the proband has a heterozygous missense mutation in which Thr-315 of the PC heavy chain has been substituted with Ala. We expressed this mutant in HEK-293 cells and purified it to homogeneity. A similar decrease in both anticoagulant and anti-inflammatory activities of the activated protein C mutant was observed in plasma- and cell-based assays. Interestingly, we discovered if functional assays were coupled to PC activation by the thrombin-TM complex, the variant exhibits improved activities in all assays. Sequence analysis revealed Thr-315 is a consensus N-linked glycosylation site for Asn-313 and that its elimination significantly (∼four- to fivefold) improves the maximum velocity of PC activation by the thrombin-TM complex, explaining the basis for the proband's negative VTE pedigree. PMID:25651845

  6. Ligand binding to an Allergenic Lipid Transfer Protein Enhances Conformational Flexibility resulting in an Increase in Susceptibility to Gastroduodenal Proteolysis

    PubMed Central

    Abdullah, Syed Umer; Alexeev, Yuri; Johnson, Philip E.; Rigby, Neil M.; Mackie, Alan R.; Dhaliwal, Balvinder; Mills, E. N. Clare

    2016-01-01

    Non-specific lipid transfer proteins (LTPs) are a family of lipid-binding molecules that are widely distributed across flowering plant species, many of which have been identified as allergens. They are highly resistant to simulated gastroduodenal proteolysis, a property that may play a role in determining their allergenicity and it has been suggested that lipid binding may further increase stability to proteolysis. It is demonstrated that LTPs from wheat and peach bind a range of lipids in a variety of conditions, including those found in the gastroduodenal tract. Both LTPs are initially cleaved during gastroduodenal proteolysis at three major sites between residues 39–40, 56–57 and 79–80, with wheat LTP being more resistant to cleavage than its peach ortholog. The susceptibility of wheat LTP to proteolyic cleavage increases significantly upon lipid binding. This enhanced digestibility is likely to be due to the displacement of Tyr79 and surrounding residues from the internal hydrophobic cavity upon ligand binding to the solvent exposed exterior of the LTP, facilitating proteolysis. Such knowledge contributes to our understanding as to how resistance to digestion can be used in allergenicity risk assessment of novel food proteins, including GMOs. PMID:27458082

  7. Introduction of Caveolae Structural Proteins into the Protozoan Toxoplasma Results in the Formation of Heterologous Caveolae but Not Caveolar Endocytosis

    PubMed Central

    Lige, Bao; Sonda, Sabrina; Joiner, Keith A.; Coppens, Isabelle

    2012-01-01

    Present on the plasma membrane of most metazoans, caveolae are specialized microdomains implicated in several endocytic and trafficking mechanisms. Caveolins and the more recently discovered cavins are the major protein components of caveolae. Previous studies reported that caveolar invaginations can be induced de novo on the surface of caveolae-negative mammalian cells upon heterologous expression of caveolin-1. However, it remains undocumented whether other components in the transfected cells participate in caveolae formation. To address this issue, we have exploited the protozoan Toxoplasma as a heterologous expression system to provide insights into the minimal requirements for caveogenesis and caveolar endocytosis. Upon expression of caveolin-1, Toxoplasma accumulates prototypical exocytic caveolae ‘precursors’ in the cytoplasm. Toxoplasma expressing caveolin-1 alone, or in conjunction with cavin-1, neither develops surface-located caveolae nor internalizes caveolar ligands. These data suggest that the formation of functional caveolae at the plasma membrane in Toxoplasma and, by inference in all non-mammalian cells, requires effectors other than caveolin-1 and cavin-1. Interestingly, Toxoplasma co-expressing caveolin-1 and cavin-1 displays an impressive spiraled network of membranes containing the two proteins, in the cytoplasm. This suggests a synergistic activity of caveolin-1 and cavin-1 in the morphogenesis and remodeling of membranes, as illustrated for Toxoplasma. PMID:23272165

  8. [Changes in heat shock protein synthesis and thermotolerance of Arabidopsis thaliana seedlings as a result of inhibition of Hsp90 by geldanamycin].

    PubMed

    Kozeko, L G

    2014-01-01

    The influence of geldanamycin (GA), which is a specific inhibitor of heat shock protein Hsp90 activities, on synthesis of Hsp70 and Hsp90 and thermotolerance of Arabidopsis thaliana seedlings has been studied. Incubation of seedlings with GA was shown to induce synthesis of these stress proteins under normal conditions. Treatment of seeds with the Hsp90 inhibitor resulted in the elevated constitutive levels of Hsp70 and Hsp90 in seedlings as well as increased induction of their synthesis under heat shock, at that the effect of GA increased with its concentration. These up-regulation of Hsp promoted thermotolerance of seedlings. The obtained results are considered as evidence for autoregulation of heat shock protein synthesis and regulation of plant tolerance by Hsp90.

  9. Overexpression of the Saccharomyces cerevisiae mannosylphosphodolichol synthase-encoding gene in Trichoderma reesei results in an increased level of protein secretion and abnormal cell ultrastructure.

    PubMed

    Kruszewska, J S; Butterweck, A H; Kurzatkowski, W; Migdalski, A; Kubicek, C P; Palamarczyk, G

    1999-06-01

    Production of extracellular proteins plays an important role in the physiology of Trichoderma reesei and has potential industrial application. To improve the efficiency of protein secretion, we overexpressed in T. reesei the DPM1 gene of Saccharomyces cerevisiae, encoding mannosylphosphodolichol (MPD) synthase, under homologous, constitutively acting expression signals. Four stable transformants, each with different copy numbers of tandemly integrated DPM1, exhibited roughly double the activity of MPD synthase in the respective endoplasmic reticulum membrane fraction. On a dry-weight basis, they secreted up to sevenfold-higher concentrations of extracellular proteins during growth on lactose, a carbon source promoting formation of cellulases. Northern blot analysis showed that the relative level of the transcript of cbh1, which encodes the major cellulase (cellobiohydrolase I [CBH I]), did not increase in the transformants. On the other hand, the amount of secreted CBH I and, in all but one of the transformants, intracellular CBH I was elevated. Our results suggest that posttranscriptional processes are responsible for the increase in CBH I production. The carbohydrate contents of the extracellular proteins were comparable in the wild type and in the transformants, and no hyperglycosylation was detected. Electron microscopy of the DPM1-amplified strains revealed amorphous structure of the cell wall and over three times as many mitochondria as in the control. Our data indicate that molecular manipulation of glycan biosynthesis in Trichoderma can result in improved protein secretion.

  10. Carbonic anhydrase related protein 8 mutation results in aberrant synaptic morphology and excitatory synaptic function in the cerebellum

    PubMed Central

    Hirasawa, Michiru; Xu, Xinjie; Trask, Robert B.; Maddatu, Terry P.; Johnson, Britt A; Naggert, Jürgen K.; Nishina, Patsy M.; Ikeda, Akihiro

    2007-01-01

    Carbonic anhydrase related protein 8 (Car8) is known to be abundantly expressed in Purkinje cells (PCs), and its genetic mutation causes a motor coordination defect. To determine the underlying mechanism, we analyzed the mouse cerebellum carrying a Car8 mutation. Electrophysiological analysis showed that spontaneous excitatory transmission was largely diminished while paired pulse ratio at parallel fiber-PC synapses was comparable to wild-type, suggesting functional synapses have normal release probability but the number of functional synapses may be lower in mutants. Light microscopic study revealed an abnormal extension of climbing fibers to the distal PC dendrites. At ultrastructural level, we found numerous PC spines not forming synapses primarily in distal dendrites and occasionally multiple spines contacting a single varicosity. These abnormalities of parallel fiber-PC synapses may underlie the functional defect in excitatory transmission. Thus, Car8 plays a critical role in synaptogenesis and/or maintenance of proper synaptic morphology and function in the cerebellum. PMID:17376701

  11. Conformational changes of hapten-protein conjugates resulting in improved broad-specificity and sensitivity of an ELISA for organophosphorus pesticides

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The type of hapten linkage to the carrier protein can play an important role in determining the nature of the resulting antibody response. Generic haptens using three types of linkers were synthesized (a monocarboxylic acid, an unsaturated hydrocarbon, and a carboxamido spacer). These haptens were...

  12. Ablation of the 14-3-3gamma Protein Results in Neuronal Migration Delay and Morphological Defects in the Developing Cerebral Cortex.

    PubMed

    Wachi, Tomoka; Cornell, Brett; Marshall, Courtney; Zhukarev, Vladimir; Baas, Peter W; Toyo-oka, Kazuhito

    2016-06-01

    14-3-3 proteins are ubiquitously-expressed and multifunctional proteins. There are seven isoforms in mammals with a high level of homology, suggesting potential functional redundancy. We previously found that two of seven isoforms, 14-3-3epsilon and 14-3-3zeta, are important for brain development, in particular, radial migration of pyramidal neurons in the developing cerebral cortex. In this work, we analyzed the function of another isoform, the protein 14-3-3gamma, with respect to neuronal migration in the developing cortex. We found that in utero 14-3-3gamma-deficiency resulted in delays in neuronal migration as well as morphological defects. Migrating neurons deficient in 14-3-3gamma displayed a thicker leading process stem, and the basal ends of neurons were not able to reach the boundary between the cortical plate and the marginal zone. Consistent with the results obtained from in utero electroporation, time-lapse live imaging of brain slices revealed that the ablation of the 14-3-3gamma proteins in pyramidal neurons slowed down their migration. In addition, the 14-3-3gamma deficient neurons showed morphological abnormalities, including increased multipolar neurons with a thicker leading processes stem during migration. These results indicate that the 14-3-3gamma proteins play an important role in radial migration by regulating the morphology of migrating neurons in the cerebral cortex. The findings underscore the pathological phenotypes of brain development associated with the disruption of different 14-3-3 proteins and will advance the preclinical data regarding disorders caused by neuronal migration defects.

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

    PubMed

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

    2012-12-01

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

  14. Two methods for proteomic analysis of formalin-fixed, paraffin embedded tissue result in differential protein identification, data quality, and cost.

    PubMed

    Luebker, Stephen A; Wojtkiewicz, Melinda; Koepsell, Scott A

    2015-11-01

    Formalin-fixed paraffin-embedded (FFPE) tissue is a rich source of clinically relevant material that can yield important translational biomarker discovery using proteomic analysis. Protocols for analyzing FFPE tissue by LC-MS/MS exist, but standardization of procedures and critical analysis of data quality is limited. This study compared and characterized data obtained from FFPE tissue using two methods: a urea in-solution digestion method (UISD) versus a commercially available Qproteome FFPE Tissue Kit method (Qkit). Each method was performed independently three times on serial sections of homogenous FFPE tissue to minimize pre-analytical variations and analyzed with three technical replicates by LC-MS/MS. Data were evaluated for reproducibility and physiochemical distribution, which highlighted differences in the ability of each method to identify proteins of different molecular weights and isoelectric points. Each method replicate resulted in a significant number of new protein identifications, and both methods identified significantly more proteins using three technical replicates as compared to only two. UISD was cheaper, required less time, and introduced significant protein modifications as compared to the Qkit method, which provided more precise and higher protein yields. These data highlight significant variability among method replicates and type of method used, despite minimizing pre-analytical variability. Utilization of only one method or too few replicates (both method and technical) may limit the subset of proteomic information obtained. PMID:26306679

  15. Amino acid substitutions in the coat protein result in loss of insect transmissibility of a plant virus.

    PubMed Central

    Atreya, P L; Atreya, C D; Pirone, T P

    1991-01-01

    Amino acids near the N terminus of the coat protein of tobacco vein mottling virus were deleted or altered by site-directed mutagenesis to determine the effect on aphid transmissibility of the virus. Deletion of a three amino acid sequence Asp-Ala-Gly, which is conserved in aphid-transmissible potyvirus isolates, abolished transmission. The mutation Ala----Thr in this triplet drastically reduced transmission, whereas the mutation Asp----Asn had no effect, and the mutation Asp----Lys consistently reverted to the wild-type residue. The mutation Lys----Glu, in the residue adjacent to the glycine of the triplet, drastically reduced transmission, whereas the mutation Gln----Pro, seven residues downstream from the glycine had no effect. Comparison of the sequences of other potyviruses suggests that the presence of a glycine residue at the third position of the Asp-Ala-Gly triplet is critical for aphid transmissibility and that certain changes in the residues adjacent to this position abolish or greatly reduce aphid transmissibility. PMID:1881922

  16. The human cytomegalovirus UL11 protein interacts with the receptor tyrosine phosphatase CD45, resulting in functional paralysis of T cells.

    PubMed

    Gabaev, Ildar; Steinbrück, Lars; Pokoyski, Claudia; Pich, Andreas; Stanton, Richard J; Schwinzer, Reinhard; Schulz, Thomas F; Jacobs, Roland; Messerle, Martin; Kay-Fedorov, Penelope C

    2011-12-01

    Human cytomegalovirus (CMV) exerts diverse and complex effects on the immune system, not all of which have been attributed to viral genes. Acute CMV infection results in transient restrictions in T cell proliferative ability, which can impair the control of the virus and increase the risk of secondary infections in patients with weakened or immature immune systems. In a search for new immunomodulatory proteins, we investigated the UL11 protein, a member of the CMV RL11 family. This protein family is defined by the RL11 domain, which has homology to immunoglobulin domains and adenoviral immunomodulatory proteins. We show that pUL11 is expressed on the cell surface and induces intercellular interactions with leukocytes. This was demonstrated to be due to the interaction of pUL11 with the receptor tyrosine phosphatase CD45, identified by mass spectrometry analysis of pUL11-associated proteins. CD45 expression is sufficient to mediate the interaction with pUL11 and is required for pUL11 binding to T cells, indicating that pUL11 is a specific CD45 ligand. CD45 has a pivotal function regulating T cell signaling thresholds; in its absence, the Src family kinase Lck is inactive and signaling through the T cell receptor (TCR) is therefore shut off. In the presence of pUL11, several CD45-mediated functions were inhibited. The induction of tyrosine phosphorylation of multiple signaling proteins upon TCR stimulation was reduced and T cell proliferation was impaired. We therefore conclude that pUL11 has immunosuppressive properties, and that disruption of T cell function via inhibition of CD45 is a previously unknown immunomodulatory strategy of CMV. PMID:22174689

  17. Nonablative skin rejuvenation devices and the role of heat shock protein 70: results of a human skin explant model

    NASA Astrophysics Data System (ADS)

    Helbig, Doris; Moebius, Anne; Simon, Jan C.; Paasch, Uwe

    2010-05-01

    Nonablative thermal laser therapy with a 1540-nm laser induces controlled, spatially determined thermal damage, allowing subsequent collagen remodeling while preserving the epidermis. A photorejuvenation effect using nonthermal nonablative stimulation of cells with low energy and narrow band light has been termed photomodulation. Light emitting diodes (LEDs) are narrow band emitters that lead to photomodulation via stimulation of mitochondrial cell organelles. In a previous study, we demonstrated in a human skin explant model that heat shock protein 70 (HSP70) plays a pivotal role in the initiation of skin remodeling after ablative fractional photothermolysis. To test its importance in nonablative laser therapy and photomodulation, the spatio-temporal expression of HSP70 is investigated in response to a 1540-nm laser treatment and six different LED therapies. An Er:glass laser is used with a 1-Hz repetition rate, 30-J/cm2 fluence, and a hand piece with a 2-mm spot size. Nonthermal nonablative treatment is performed using two LED (LEDA SCR red light: 635 nm, 40 to 120 W/cm2, 40 to 120 J/cm2 LEDA SCR yellow light: 585 nm, 16 to 35 W/cm2, 20 to 100 J/cm2 spot size 16×10 cm). Immediate responses as well as responses 1, 3, or 7 days postprocedure are studied; untreated skin explants serve as control. Immunohistochemical investigation (HSP70) is performed in all native, nontreated, and Er:glass laser- or LED-treated samples (n=175). Nonablative laser therapy leads to a clear time-dependent induction of epidermally expressed HSP70, peaking between one to three days post-treatment. In contrast, none of the various LED treatments up-regulated the HSP70 expression in our skin explant model. HSP70 is up-regulated by nonablative but thermal laser devices, but does not seem to play a significant role in the induction of skin remodeling induced by photomodulation. The maximum of HSP70 expression is reached later after Er:glass laser intervention compared to ablative fractional

  18. Exposure to vehicle emissions results in altered blood brain barrier permeability and expression of matrix metalloproteinases and tight junction proteins in mice

    PubMed Central

    2013-01-01

    Background Traffic-generated air pollution-exposure is associated with adverse effects in the central nervous system (CNS) in both human exposures and animal models, including neuroinflammation and neurodegeneration. While alterations in the blood brain barrier (BBB) have been implicated as a potential mechanism of air pollution-induced CNS pathologies, pathways involved have not been elucidated. Objectives To determine whether inhalation exposure to mixed vehicle exhaust (MVE) mediates alterations in BBB permeability, activation of matrix metalloproteinases (MMP) -2 and −9, and altered tight junction (TJ) protein expression. Methods Apolipoprotein (Apo) E−/− and C57Bl6 mice were exposed to either MVE (100 μg/m3 PM) or filtered air (FA) for 6 hr/day for 30 days and resulting BBB permeability, expression of ROS, TJ proteins, markers of neuroinflammation, and MMP activity were assessed. Serum from study mice was applied to an in vitro BBB co-culture model and resulting alterations in transport and permeability were quantified. Results MVE-exposed Apo E−/− mice showed increased BBB permeability, elevated ROS and increased MMP-2 and −9 activity, compared to FA controls. Additionally, cerebral vessels from MVE-exposed mice expressed decreased levels of TJ proteins, occludin and claudin-5, and increased levels of inducible nitric oxide synthase (iNOS) and interleukin (IL)-1β in the parenchyma. Serum from MVE-exposed animals also resulted in increased in vitro BBB permeability and altered P-glycoprotein transport activity. Conclusions These data indicate that inhalation exposure to traffic-generated air pollutants promotes increased MMP activity and degradation of TJ proteins in the cerebral vasculature, resulting in altered BBB permeability and expression of neuroinflammatory markers. PMID:24344990

  19. Changes in intake of protein foods, carbohydrate amount and quality, and long-term weight change: results from 3 prospective cohorts1234

    PubMed Central

    Hou, Tao; Ludwig, David S; Rimm, Eric B; Willett, Walter; Hu, Frank B; Mozaffarian, Dariush

    2015-01-01

    Background: Dietary guidelines recommend interchanging protein foods (e.g., chicken for red meat), but they may be exchanged for carbohydrate-rich foods varying in quality [glycemic load (GL)]. Whether such exchanges occur and how they influence long-term weight gain are not established. Objective: Our objective was to determine how changes in intake of protein foods, GL, and their interrelationship influence long-term weight gain. Design: We investigated the association between 4-y changes in consumption of protein foods, GL, and their interaction with 4-y weight change over a 16- to 24-y follow-up, adjusted for other lifestyle changes (smoking, physical activity, television watching, sleep duration), body mass index, and all dietary factors simultaneously in 3 prospective US cohorts (Nurses’ Health Study, Nurses’ Health Study II, and Health Professionals Follow-Up Study) comprising 120,784 men and women free of chronic disease or obesity at baseline. Results: Protein foods were not interchanged with each other (intercorrelations typically <|0.05|) but with carbohydrate (negative correlation as low as −0.39). Protein foods had different relations with long-term weight gain, with positive associations for meats, chicken with skin, and regular cheese (per increased serving/d, 0.13–1.17 kg; P = 0.02 to P < 0.001); no association for milk, legumes, peanuts, or eggs (P > 0.40 for each); and relative weight loss for yogurt, peanut butter, walnuts, other nuts, chicken without skin, low-fat cheese, and seafood (−0.14 to −0.71 kg; P = 0.01 to P < 0.001). Increases in GL were independently associated with a 0.42-kg greater weight gain per 50-unit increase (P < 0.001). Significant interactions (P-interaction < 0.05) between changes in protein foods and GL were identified; for example, increased cheese intake was associated with weight gain when GL increased, with weight stability when GL did not change, and with weight loss when exchanged for GL (i.e., decrease

  20. Feeding soy protein isolate prevents impairment of bone acquisition by western diets as a result of insulin signaling in bone

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Excessive consumption of high fat/high cholesterol “Western” diets during postnatal life results in increased energy intake, development of obesity and systemic insulin resistance. However, how this diet impairs bone development and remodeling is not well understood, and no effective dietary interve...

  1. Over-expression of rice leucine-rich repeat protein results in activation of defense response, thereby enhancing resistance to bacterial soft rot in Chinese cabbage.

    PubMed

    Park, Young Ho; Choi, Changhyun; Park, Eun Mi; Kim, Hyo Sun; Park, Hong Jae; Bae, Shin Cheol; Ahn, Ilpyung; Kim, Min Gab; Park, Sang Ryeol; Hwang, Duk-Ju

    2012-10-01

    Pectobacterium carotovorum subsp. carotovorum causes soft rot disease in various plants, including Chinese cabbage. The simple extracellular leucine-rich repeat (eLRR) domain proteins have been implicated in disease resistance. Rice leucine-rich repeat protein (OsLRP), a rice simple eLRR domain protein, is induced by pathogens, phytohormones, and salt. To see whether OsLRP enhances disease resistance to bacterial soft rot, OsLRP was introduced into Chinese cabbage by Agrobacterium-mediated transformation. Two independent transgenic lines over-expressing OsLRP were generated and further analyzed. Transgenic lines over-expressing OsLRP showed enhanced disease resistance to bacterial soft rot compared to non-transgenic control. Bacterial growth was retarded in transgenic lines over-expressing OsLRP compared to non-transgenic controls. We propose that OsLRP confers enhanced resistance to bacterial soft rot. Monitoring expression of defense-associated genes in transgenic lines over-expressing OsLRP, two different glucanases and Brassica rapa polygalacturonase inhibiting protein 2, PDF1 were constitutively activated in transgenic lines compared to non-transgenic control. Taken together, heterologous expression of OsLRP results in the activation of defense response and enhanced resistance to bacterial soft rot.

  2. Altered levels of the Taraxacum kok-saghyz (Russian dandelion) small rubber particle protein, TkSRPP3, result in qualitative and quantitative changes in rubber metabolism.

    PubMed

    Collins-Silva, Jillian; Nural, Aise Taban; Skaggs, Amanda; Scott, Deborah; Hathwaik, Upul; Woolsey, Rebekah; Schegg, Kathleen; McMahan, Colleen; Whalen, Maureen; Cornish, Katrina; Shintani, David

    2012-07-01

    Several proteins have been identified and implicated in natural rubber biosynthesis, one of which, the small rubber particle protein (SRPP), was originally identified in Hevea brasiliensis as an abundant protein associated with cytosolic vesicles known as rubber particles. While previous in vitro studies suggest that SRPP plays a role in rubber biosynthesis, in vivo evidence is lacking to support this hypothesis. To address this issue, a transgene approach was taken in Taraxacum kok-saghyz (Russian dandelion or Tk) to determine if altered SRPP levels would influence rubber biosynthesis. Three dandelion SRPPs were found to be highly abundant on dandelion rubber particles. The most abundant particle associated SRPP, TkSRPP3, showed temporal and spatial patterns of expression consistent with patterns of natural rubber accumulation in dandelion. To confirm its role in rubber biosynthesis, TkSRPP3 expression was altered in Russian dandelion using over-expression and RNAi methods. While TkSRPP3 over-expressing lines had slightly higher levels of rubber in their roots, relative to the control, TkSRPP3 RNAi lines showed significant decreases in root rubber content and produced dramatically lower molecular weight rubber than the control line. Not only do results here provide in vivo evidence of TkSRPP proteins affecting the amount of rubber in dandelion root, but they also suggest a function in regulating the molecular weight of the cis-1, 4-polyisoprene polymer.

  3. Accumulation of PrLeg, a Perilla legumin protein in potato tuber results in enhanced level of sulphur-containing amino acids.

    PubMed

    Goo, Young-Min; Kim, Tae-Won; Lee, Min-Kyung; Lee, Shin-Woo

    2013-09-01

    Potato is the fourth staple food in the world, following rice, wheat, and maize, whereas tubers contain high quality of starch, relatively high amounts of vitamin C and many other important substances. It also contains relatively good quality of protein (about 3 to 6% of the dried weight) and patatin, and 11S globulin is a major storage protein with high level of lysine. However, tuber protein contains relatively low amounts of sulphur-containing amino acids, which may result in low nutritional value. Recently, we cloned a gene encoding PrLeg polypeptide, a seed storage protein from Perilla, which contains relatively higher levels of sulphur-containing amino acids. We transformed PrLeg cDNA into a potato plant to over-express under the direction of the tuber-specific promoter, patatin. Most of the transgenic lines identified through PCR and RT-PCR analyses were able to accumulate high amount of prLeg transcript in their tuber tissue, while very little or no transcript that were detected in their leaf tissues. The level of methionine content was elevated up to three-fold compared to non-transgenic parental line, without any significant changes in other amino acids, suggesting that further research is required to get a deeper insight into their nutritional value. PMID:24161240

  4. Accumulation of radium in ferruginous protein bodies formed in lung tissue: association of resulting radiation hotspots with malignant mesothelioma and other malignancies

    PubMed Central

    Nakamura, Eizo; Makishima, Akio; Hagino, Kyoko; Okabe, Kazunori

    2009-01-01

    While exposure to fibers and particles has been proposed to be associated with several different lung malignancies including mesothelioma, the mechanism for the carcinogenesis is not fully understood. Along with mineralogical observation, we have analyzed forty-four major and trace elements in extracted asbestos bodies (fibers and proteins attached to them) with coexisting fiber-free ferruginous protein bodies from extirpative lungs of individuals with malignant mesothelioma. These observations together with patients’ characteristics suggest that inhaled iron-rich asbestos fibers and dust particles, and excess iron deposited by continuous cigarette smoking would induce ferruginous protein body formation resulting in ferritin aggregates in lung tissue. Chemical analysis of ferruginous protein bodies extracted from lung tissues reveals anomalously high concentrations of radioactive radium, reaching millions of times higher concentration than that of seawater. Continuous and prolonged internal exposure to hotspot ionizing radiation from radium and its daughter nuclides could cause strong and frequent DNA damage in lung tissue, initiate different types of tumour cells, including malignant mesothelioma cells, and may cause cancers. PMID:19644223

  5. soaPDB: a web application for searching the Protein Data Bank, organizing results, and receiving automatic email alerts.

    PubMed

    Lesburg, Charles A; Duca, José S

    2008-07-01

    soaPDB is a web application that allows generation and organization of saved PDB searches, and offers automatic email alerts. This tool is used from a web interface to store PDB searches and results in a backend relational database. Written using the Ruby on Rails open-source web framework, soaPDB is easy to deploy, maintain and customize. soaPDB is freely available upon request for local installation and is also available at http://soapdb.dyndns.org:3000. PMID:18487276

  6. Acute leukemias of different lineages have similar MLL gene fusions encoding related chimeric proteins resulting from chromosomal translocation

    SciTech Connect

    Corral, J.; Forster, A.; Thompson, S.; Rabbitts, T.H. ); Lampert, F. ); Kaneko, Y. ); Slater, R.; Kroes, W.G. ); Van Der Schoot, C.E. ); Ludwig, W.D. ); Karpas, A. ); Pocock, C.; Cotter, F. )

    1993-09-15

    The MLL gene, on human chromosome 11q23, undergoes chromosomal translocation in acute leukemias, resulting in gene fusion with AF4 (chromosome 4) and ENL (chromosome 19). The authors report here translocation of MLL with nine different chromosomes and two paracentric chromosome 11 deletions in early B cell, B- or T-cell lineage, or nonlymphocytic acute leukemias. The mRNA translocation junction from 22t(4;11) patients, including six adult leukemias, and nine t(11;19) tumors reveals a remarkable conservation of breakpoints within MLL, AF4, or ENL genes, irrespective of tumor phenotype. Typically, the breakpoints are upstream of the zinc-finger region of MLL, and deletion of this region can accompany translocation, supporting the der(11) chromosome as the important component in leukemogenesis. Partial sequence of a fusion between MLL and the AFX1 gene from chromosome X shows the latter to be rich in Ser/Pro codons, like the ENL mRNA. These data suggest that the heterogeneous 11q23 abnormalities might cause attachment of Ser/Pro-rich segments to the NH[sub 2] terminus of MLL, lacking the zinc-finger region, and that translocation occurs in early hematopoietic cells, before commitment to distinct lineages. 36 refs., 2 figs.

  7. Ras transformation results in cleavage of reticulon protein Nogo-B that is associated with impairment of IFN response

    PubMed Central

    Ahn, Dae-Gyun; Sharif, Tanveer; Chisholm, Kenneth; Pinto, Devanand M; Gujar, Shashi A; Lee, Patrick WK

    2015-01-01

    Dysregulation of Ras signaling is the major cause of various cancers. Aberrant Ras signaling, however, provides a favorable environment for many viruses, making them suitable candidates as cancer-killing therapeutic agents. Susceptibility of cancer cells to such viruses is mainly due to impaired type I interferon (IFN) response, often as a result of activated Ras/ERK signaling in these cells. In this study, we searched for cellular factors modulated by Ras signaling and their potential involvement in promoting viral oncolysis. We found that upon Ras transformation of NIH-3T3 cells, the N-terminus of Nogo-B (reticulon 4) was proteolytically cleaved. Interestingly, Nogo knockdown (KD) in non-transformed and Ras-transformed cells both enhanced virus-induced IFN response, suggesting that both cleaved and uncleaved Nogo can suppress IFN response. However, pharmacological blockade of Nogo cleavage in Ras-transformed cells significantly enhanced virus-induced IFN response, suggesting that cleaved Nogo contributes to enhanced IFN suppression in these cells. We further showed that IFN suppression associated with Ras-induced Nogo-B cleavage was distinct from but synergistic with that associated with an activated Ras/ERK pathway. Our study therefore reveals an important and novel role of Nogo-B and its cleavage in the suppression of anti-viral immune responses by oncogenic Ras transformation. PMID:25946643

  8. Partial loss of the DNA repair scaffolding protein, Xrcc1, results in increased brain damage and reduced recovery from ischemic stroke in mice.

    PubMed

    Ghosh, Somnath; Canugovi, Chandrika; Yoon, Jeong Seon; Wilson, David M; Croteau, Deborah L; Mattson, Mark P; Bohr, Vilhelm A

    2015-07-01

    Oxidative DNA damage is mainly repaired by base excision repair (BER). Previously, our laboratory showed that mice lacking the BER glycosylases 8-oxoguanine glycosylase 1 (Ogg1) or nei endonuclease VIII-like 1 (Neil1) recover more poorly from focal ischemic stroke than wild-type mice. Here, a mouse model was used to investigate whether loss of 1 of the 2 alleles of X-ray repair cross-complementing protein 1 (Xrcc1), which encodes a nonenzymatic scaffold protein required for BER, alters recovery from stroke. Ischemia and reperfusion caused higher brain damage and lower functional recovery in Xrcc1(+/-) mice than in wild-type mice. Additionally, a greater percentage of Xrcc1(+/-) mice died as a result of the stroke. Brain samples from human individuals who died of stroke and individuals who died of non-neurological causes were assayed for various steps of BER. Significant losses of thymine glycol incision, abasic endonuclease incision, and single nucleotide incorporation activities were identified, as well as lower expression of XRCC1 and NEIL1 proteins in stroke brains compared with controls. Together, these results suggest that impaired BER is a risk factor in ischemic brain injury and contributes to its recovery.

  9. Partial loss of the DNA repair scaffolding protein, Xrcc1, results in increased brain damage and reduced recovery from ischemic stroke in mice.

    PubMed

    Ghosh, Somnath; Canugovi, Chandrika; Yoon, Jeong Seon; Wilson, David M; Croteau, Deborah L; Mattson, Mark P; Bohr, Vilhelm A

    2015-07-01

    Oxidative DNA damage is mainly repaired by base excision repair (BER). Previously, our laboratory showed that mice lacking the BER glycosylases 8-oxoguanine glycosylase 1 (Ogg1) or nei endonuclease VIII-like 1 (Neil1) recover more poorly from focal ischemic stroke than wild-type mice. Here, a mouse model was used to investigate whether loss of 1 of the 2 alleles of X-ray repair cross-complementing protein 1 (Xrcc1), which encodes a nonenzymatic scaffold protein required for BER, alters recovery from stroke. Ischemia and reperfusion caused higher brain damage and lower functional recovery in Xrcc1(+/-) mice than in wild-type mice. Additionally, a greater percentage of Xrcc1(+/-) mice died as a result of the stroke. Brain samples from human individuals who died of stroke and individuals who died of non-neurological causes were assayed for various steps of BER. Significant losses of thymine glycol incision, abasic endonuclease incision, and single nucleotide incorporation activities were identified, as well as lower expression of XRCC1 and NEIL1 proteins in stroke brains compared with controls. Together, these results suggest that impaired BER is a risk factor in ischemic brain injury and contributes to its recovery. PMID:25971543

  10. Disruption of the A-Kinase Anchoring Domain in Flagellar Radial Spoke Protein 3 Results in Unregulated Axonemal cAMP-dependent Protein Kinase Activity and Abnormal Flagellar Motility

    PubMed Central

    Gaillard, Anne R.; Fox, Laura A.; Rhea, Jeanne M.; Craige, Branch

    2006-01-01

    Biochemical studies of Chlamydomonas flagellar axonemes revealed that radial spoke protein (RSP) 3 is an A-kinase anchoring protein (AKAP). To determine the physiological role of PKA anchoring in the axoneme, an RSP3 mutant, pf14, was transformed with an RSP3 gene containing a mutation in the PKA-binding domain. Analysis of several independent transformants revealed that the transformed cells exhibit an unusual phenotype: a fraction of the cells swim normally; the remainder of the cells twitch feebly or are paralyzed. The abnormal/paralyzed motility is not due to an obvious deficiency of radial spoke assembly, and the phenotype cosegregates with the mutant RSP3. We postulated that paralysis was due to failure in targeting and regulation of axonemal cAMP-dependent protein kinase (PKA). To test this, reactivation experiments of demembranated cells were performed in the absence or presence of PKA inhibitors. Importantly, motility in reactivated cell models mimicked the live cell phenotype with nearly equal fractions of motile and paralyzed cells. PKA inhibitors resulted in a twofold increase in the number of motile cells, rescuing paralysis. These results confirm that flagellar RSP3 is an AKAP and reveal that a mutation in the PKA binding domain results in unregulated axonemal PKA activity and inhibition of normal motility. PMID:16571668

  11. Coupling Peptide Antigens to Virus-Like Particles or to Protein Carriers Influences the Th1/Th2 Polarity of the Resulting Immune Response.

    PubMed

    Pomwised, Rattanaruji; Intamaso, Uraiwan; Teintze, Martin; Young, Mark; Pincus, Seth H

    2016-01-01

    We have conjugated the S9 peptide, a mimic of the group B streptococcal type III capsular polysaccharide, to different carriers in an effort to elicit an optimal immune response. As carriers, we utilized the soluble protein keyhole limpet hemocyanin and virus-like particles (VLPs) from two plant viruses, Cowpea Chlorotic Mottle Virus and Cowpea Mosaic Virus. We have found that coupling the peptide to the soluble protein elicits a Th2 immune response, as evidenced by the production of the peptide-specific IgG1 antibody and IL-4/IL-10 production in response to antigen stimulation, whereas the peptide conjugated to VLPs elicited a Th1 response (IgG2a, IFN-γ). Because the VLPs used as carriers package RNA during the assembly process, we hypothesize that this effect may result from the presence of nucleic acid in the immunogen, which affects the Th1/Th2 polarity of the response. PMID:27164150

  12. Coupling Peptide Antigens to Virus-Like Particles or to Protein Carriers Influences the Th1/Th2 Polarity of the Resulting Immune Response

    PubMed Central

    Pomwised, Rattanaruji; Intamaso, Uraiwan; Teintze, Martin; Young, Mark; Pincus, Seth H.

    2016-01-01

    We have conjugated the S9 peptide, a mimic of the group B streptococcal type III capsular polysaccharide, to different carriers in an effort to elicit an optimal immune response. As carriers, we utilized the soluble protein keyhole limpet hemocyanin and virus-like particles (VLPs) from two plant viruses, Cowpea Chlorotic Mottle Virus and Cowpea Mosaic Virus. We have found that coupling the peptide to the soluble protein elicits a Th2 immune response, as evidenced by the production of the peptide-specific IgG1 antibody and IL-4/IL-10 production in response to antigen stimulation, whereas the peptide conjugated to VLPs elicited a Th1 response (IgG2a, IFN-γ). Because the VLPs used as carriers package RNA during the assembly process, we hypothesize that this effect may result from the presence of nucleic acid in the immunogen, which affects the Th1/Th2 polarity of the response. PMID:27164150

  13. Cationic motions and phase transitions in [(CH 3) 4N] 2SO 4·4H 2O, [(CH 3) 4N] 2SO 4, and [(CH 3) 4N] 2SeO 4 as studied by 1H NMR, differential thermal analysis, and X-ray powder diffraction techniques

    NASA Astrophysics Data System (ADS)

    Sato, Setsuko; Endo, Midori; Hara, Naoki; Nakamura, Daiyu; Ikeda, Ryuichi

    1995-02-01

    Cationic reorientations have been studied in solid [(CH 3) 4N] 2SO 4·4H 2O, [(CH 3) 4N] 2SO 4, and [(CH 3) 4N] 2SeO 4 by measuring 1H NMR spin-lattice relaxation times, T1. These motions have been discussed in association with the crystal structures and the phase transitions examined by X-ray powder diffraction and differential thermal analysis, respectively. In crystals of [(CH 3) 4N] 2SO 4·4H 2O, there are two kinds of cations distorted from regular tetrahedra. T1 is calculated according to the interpretation that two T1 minima are due to the two inequivalent (NH 3) 4N + ions reorienting at different frequencies. The result shows that at the phase transition temperatures, the correlation times to those cationic reorientations are very different from each other in this compound in contrast with (NH 4) 2SO 4 and [(CH 3) 4N] 2CdX 4 (X = Cl and Br). For the sulfate and selenate, there is a single kind of cation which can be considered tetrahedral, and the phase transitions occur in the temperature region where the narrowing of the resonance line owing to the overall cationic reorientations starts.

  14. First results on label-free detection of DNA and protein molecules using a novel integrated sensor technology based on gravimetric detection principles.

    PubMed

    Gabl, R; Feucht, H-D; Zeininger, H; Eckstein, G; Schreiter, M; Primig, R; Pitzer, D; Wersing, W

    2004-01-15

    A novel integrated bio-sensor technology based on thin-film bulk acoustic wave resonators on silicon is presented and the feasibility of detecting DNA and protein molecules proofed. The detection principle of these sensors is label-free and relies on a resonance frequency shift caused by mass loading of an acoustic resonator, a principle very well known from quartz crystal micro balances. Integrated ZnO bulk acoustic wave resonators with resonance frequencies around 2 GHz have been fabricated, employing an acoustic mirror for isolation from the silicon substrate. DNA oligos have been thiol-coupled to the gold electrode by on-wafer dispensing. In a further step, samples have either been hybridised or alternatively a protein has been coupled to the receptor. The measurement results show the new bio-sensor being capable of both, detecting proteins as well as the DNA hybridisation without using a label. Due to the substantially higher oscillation frequency, these sensors already show much higher sensitivity and resolution comparable to quartz crystal micro balances. The potential for these sensors and sensors arrays as well as technological challenges will be discussed in detail.

  15. Differences in folate-protein interactions result in differing inhibition of native rat liver and recombinant glycine N-methyltransferase by 5-methyltetrahydrofolate

    SciTech Connect

    Luka, Zigmund; Pakhomova, Svetlana; Loukachevitch, Lioudmila V; Newcomer, Marcia E; Wagner, Conrad

    2012-06-27

    Glycine N-methyltransferase (GNMT) is a key regulatory enzyme in methyl group metabolism. In mammalian liver it reduces S-adenosylmethionine levels by using it to methylate glycine, producing N-methylglycine (sarcosine) and S-adenosylhomocysteine. GNMT is inhibited by binding two molecules of 5-methyltetrahydrofolate (mono- or polyglutamate forms) per tetramer of the active enzyme. Inhibition is sensitive to the status of the N-terminal valine of GNMT and to polyglutamation of the folate inhibitor. It is inhibited by pentaglutamate form more efficiently compared to monoglutamate form. The native rat liver GNMT contains an acetylated N-terminal valine and is inhibited much more efficiently compared to the recombinant protein expressed in E. coli where the N-terminus is not acetylated. In this work we used a protein crystallography approach to evaluate the structural basis for these differences. We show that in the folate-GNMT complexes with the native enzyme, two folate molecules establish three and four hydrogen bonds with the protein. In the folate-recombinant GNMT complex only one hydrogen bond is established. This difference results in more effective inhibition by folate of the native liver GNMT activity compared to the recombinant enzyme.

  16. Differences in folate-protein interactions result in differing inhibition of native rat liver and recombinant glycine N-methyltransferase by 5-methyltetrahydrofolate

    PubMed Central

    Luka, Zigmund; Pakhomova, Svetlana; Loukachevitch, Lioudmila V.; Newcomer, Marcia E.; Wagner, Conrad

    2011-01-01

    Glycine N-methyltransferase (GNMT) is a key regulatory enzyme in methyl group metabolism. In mammalian liver it reduces S-adenosylmethionine levels by using it to methylate glycine, producing N-methylglycine (sarcosine) and S-adenosylhomocysteine. GNMT is inhibited by binding two molecules of 5-methyltetrahydrofolate (mono- or polyglutamate forms) per tetramer of the active enzyme. Inhibition is sensitive to the status of the N-terminal valine of GNMT and to polyglutamation of the folate inhibitor. It is inhibited by pentaglutamate form more efficiently compared to monoglutamate form. The native rat liver GNMT contains an acetylated N-terminal valine and is inhibited much more efficiently compared to the recombinant protein expressed in E. coli where the N-terminus is not acetylated. In this work we used a protein crystallography approach to evaluate the structural basis for these differences. We show that in the folate-GNMT complexes with the native enzyme, two folate molecules establish three and four hydrogen bonds with the protein. In the folate-recombinant GNMT complex only one hydrogen bond is established. This difference results in more effective inhibition by folate of the native liver GNMT activity compared to the recombinant enzyme. PMID:22037183

  17. Agricultural illustrations of 19th century Korea: 'Imwon gyeongjeji' (Treatises on Management of Forest and Garden) by Seo Yugu.

    PubMed

    Chung, Hyung-Min

    2011-01-01

    The generative relationship between text and image has long been established. Its structure evolved historically as a result of varying understandings of the functions of art and technology. Agriculture illustration, which emerged in China during the Song dynasty, is a prime example of this creative dialogue in which aspects of both disciplines were combined. Political, technological, and aesthetic concerns informed the reformulations of this new genre. This paper will address agricultural illustrations on nineteenth-century Korea, when notable changes occurred in the visualization of agricultural texts. It will explore changes in the understanding of the roles of agriculture, technology, and labor through an analysis of shifts in modes of illustration and the texts selected. The relationship between technology and visual representations during late Joseon Korea will be contextualized through an exploration of the evolution of technical drawing in East Asia. This paper will suggest that the recognition of imagery's ability to convey textual and technical information provided an important alternative paradigm for the presentation and use of knowledge. PMID:22171414

  18. Improving docking results via reranking of ensembles of ligand poses in multiple X-ray protein conformations with MM-GBSA.

    PubMed

    Greenidge, P A; Kramer, C; Mozziconacci, J-C; Sherman, W

    2014-10-27

    There is a tendency in the literature to be critical of scoring functions when docking programs perform poorly. The assumption is that existing scoring functions need to be enhanced or new ones developed in order to improve the performance of docking programs for tasks such as pose prediction and virtual screening. However, failures can result from either sampling or scoring (or a combination of the two), although less emphasis tends to be given to the former. In this work, we use the programs GOLD and Glide on a high-quality data set to explore whether failures in pose prediction and binding affinity estimation can be attributable more to sampling or scoring. We show that identification of the correct pose (docking power) can be improved by incorporating ligand strain into the scoring function or rescoring an ensemble of diverse docking poses with MM-GBSA in a postprocessing step. We explore the use of nondefault docking settings and find that enhancing ligand sampling also improves docking power, again suggesting that sampling is more limiting than scoring for the docking programs investigated in this work. In cross-docking calculations (docking a ligand to a noncognate receptor structure) we observe a significant reduction in the accuracy of pose ranking, as expected and has been reported by others; however, we demonstrate that these alternate poses may in fact be more complementary between the ligand and the rigid receptor conformation, emphasizing that treating the receptor rigidly is an artificial constraint on the docking problem. We simulate protein flexibility by the use of multiple crystallographic conformations of a protein and demonstrate that docking results can be improved with this level of protein sampling. This work indicates the need for better sampling in docking programs, especially for the receptor. This study also highlights the variable descriptive value of RMSD as the sole arbiter of pose replication quality. It is shown that ligand poses

  19. Lack of the H-NS Protein Results in Extended and Aberrantly Positioned DNA during Chromosome Replication and Segregation in Escherichia coli

    PubMed Central

    Helgesen, Emily; Fossum-Raunehaug, Solveig

    2016-01-01

    ABSTRACT The architectural protein H-NS binds nonspecifically to hundreds of sites throughout the chromosome and can multimerize to stiffen segments of DNA as well as to form DNA-protein-DNA bridges. H-NS has been suggested to contribute to the orderly folding of the Escherichia coli chromosome in the highly compacted nucleoid. In this study, we investigated the positioning and dynamics of the origins, the replisomes, and the SeqA structures trailing the replication forks in cells lacking the H-NS protein. In H-NS mutant cells, foci of SeqA, replisomes, and origins were irregularly positioned in the cell. Further analysis showed that the average distance between the SeqA structures and the replisome was increased by ∼100 nm compared to that in wild-type cells, whereas the colocalization of SeqA-bound sister DNA behind replication forks was not affected. This result may suggest that H-NS contributes to the folding of DNA along adjacent segments. H-NS mutant cells were found to be incapable of adopting the distinct and condensed nucleoid structures characteristic of E. coli cells growing rapidly in rich medium. It appears as if H-NS mutant cells adopt a “slow-growth” type of chromosome organization under nutrient-rich conditions, which leads to a decreased cellular DNA content. IMPORTANCE It is not fully understood how and to what extent nucleoid-associated proteins contribute to chromosome folding and organization during replication and segregation in Escherichia coli. In this work, we find in vivo indications that cells lacking the nucleoid-associated protein H-NS have a lower degree of DNA condensation than wild-type cells. Our work suggests that H-NS is involved in condensing the DNA along adjacent segments on the chromosome and is not likely to tether newly replicated strands of sister DNA. We also find indications that H-NS is required for rapid growth with high DNA content and for the formation of a highly condensed nucleoid structure under such

  20. Integration of Known DNA, RNA and Protein Biomarkers Provides Prediction of Anti-TNF Response in Rheumatoid Arthritis: Results from the COMBINE Study

    PubMed Central

    Folkersen, Lasse; Brynedal, Boel; Diaz-Gallo, Lina Marcela; Ramsköld, Daniel; Shchetynsky, Klementy; Westerlind, Helga; Sundström, Yvonne; Schepis, Danika; Hensvold, Aase; Vivar, Nancy; Eloranta, Maija-Leena; Rönnblom, Lars; Brunak, Søren; Malmström, Vivianne; Catrina, Anca; Moerch, Ulrik GW; Klareskog, Lars; Padyukov, Leonid; Berg, Louise

    2016-01-01

    OBJECTIVE: In rheumatoid arthritis (RA) several recent efforts have sought to discover means of predicting which patients would benefit from treatment. However, results have been discrepant with few successful replications. Our objective was to build a biobank with DNA, RNA and protein measurements to test the claim that the current state-of-the-art precision medicine will benefit RA patients. METHODS: We collected 451 blood samples from 61 healthy individuals and 185 RA patients initiating treatment, before treatment initiation and at a 3 month follow-up time. All samples were subjected to high-throughput RNA sequencing, DNA genotyping, extensive proteomics and flow cytometry measurements, as well as comprehensive clinical phenotyping. Literature review identified 2 proteins, 52 single-nucleotide polymorphisms (SNPs) and 72 gene-expression biomarkers that had previously been proposed as predictors of Tumor Necrosis Factor (TNF) inhibitor response (ΔDAS28-CRP). RESULTS: From these published TNFi biomarkers we found that 2 protein, 2 SNP and 8 mRNA biomarkers could be replicated in the 59 TNF initiating patients. Combining these replicated biomarkers into a single signature we found that we could explain 51% of the variation in ΔDAS28-CRP. This corresponds to a sensitivity of 0.73 and specificity of 0.78 for the prediction of three month ΔDAS28-CRP better than –1.2. CONCLUSIONS: The COMBINE biobank is currently the largest collection of multi-omics data from RA patients with high potential for discovery and replication. Taking advantage of this we surveyed the current state-of-the-art of drug-response stratification in RA, and identified a small set of previously published biomarkers available in peripheral blood which predicts clinical response to TNF blockade in this independent cohort. PMID:27532898

  1. Should the Amounts of Fat and Protein Be Taken into Consideration to Calculate the Lunch Prandial Insulin Bolus? Results from a Randomized Crossover Trial

    PubMed Central

    González-Rodriguez, María; Pazos-Couselo, Marcos; Gude, Francisco; Prieto-Tenreiro, Alma; Casanueva, Felipe

    2013-01-01

    Abstract Background Concerning continuous subcutaneous insulin infusion (CSII), there are controversial results related to changes in glycemic response according to the meal composition and bolus design. Our aim is to determine whether the presence of protein and fat in a meal could involve a different postprandial glycemic response than that obtained with only carbohydrates (CHs). Subjects and Methods This was a crossover, randomized clinical trial. Seventeen type 1 diabetes (T1D) patients on CSII wore a blinded continuous glucose monitoring system sensor for 3 days. They ingested two meals (meal 1 vs. meal 2) with the same CH content (50 g) but different fat (8.9 g vs. 37.4 g) and protein (3.3 g vs. 28.9 g) contents. A single-wave insulin bolus was used, and the interstitial glucose values were measured every 30 min for 3 h. We evaluated the different postprandial glycemic response between meal 1 and meal 2 by using mixed-effects models. Results The postmeal glucose increase was 22 mg/dL for meal 1 and 31 mg/dL for meal 2. In univariate analysis, at different times not statistically significant differences in glucose levels between meals occurred. In mixed-model analysis, a time×meal interaction was found, indicating a different response between treatments along the time. However, most of the patients remained in the normoglycemic range (70–180 mg/dL) during the 3-h postmeal period (84.4% for meal 1 and 93.1% for meal 2). Conclusions The presence of balanced amounts of protein and fat determined a different glycemic response from that obtained with only CH up to 3 h after eating. The clinical relevance of this finding remains to be elucidated. PMID:23259764

  2. DNA-binding and transcriptional activation properties of the EWS-FLI-1 fusion protein resulting from the t(11;22) translocation in Ewing sarcoma.

    PubMed Central

    Bailly, R A; Bosselut, R; Zucman, J; Cormier, F; Delattre, O; Roussel, M; Thomas, G; Ghysdael, J

    1994-01-01

    The 5' half of the EWS gene has recently been described to be fused to the 3' regions of genes encoding the DNA-binding domain of several transcriptional regulators, including ATF1, FLI-1, and ERG, in several human tumors. The most frequent occurrence of this situation results from the t(11;22)(q24;q12) chromosome translocation specific for Ewing sarcoma (ES) and related tumors which joins EWS sequences to the 3' half of FLI-1, which encodes a member of the Ets family of transcriptional regulators. We show here that this chimeric gene encodes an EWS-FLI-1 nuclear protein which binds DNA with the same sequence specificity as the wild-type parental FLI-1 protein. We further show that EWS-FLI-1 is an efficient sequence-specific transcriptional activator of model promoters containing FLI-1 (Ets)-binding sites, a property which is strictly dependent on the presence of its EWS domain. Comparison of the properties of the N-terminal activation domain of FLI-1 to those of the EWS domain of the fusion protein indicates that EWS-FLI-1 has altered transcriptional activation properties compared with FLI-1. These results suggest that EWS-FLI-1 contributes to the transformed phenotype of ES tumor cells by inducing the deregulated and/or unscheduled activation of genes normally responsive to FLI-1 or to other close members of the Ets family. ES and related tumors are characterized by an elevated level of c-myc expression. We show that EWS-FLI-1 is a transactivator of the c-myc promoter, suggesting that upregulation of c-myc expression is under control of EWS-FLI-1. Images PMID:8164678

  3. Graphite grains studded with silver nanoparticles: description and application in promoting direct biocatalysis between heme protein and the resulting carbon paste electrode.

    PubMed

    ElKaoutit, Mohammed; Naggar, Ahmad H; Naranjo-Rodríguez, Ignacio; de Cisneros, José L Hidalgo-Hidalgo

    2012-04-01

    The impregnation of graphite grains with silver nanoparticles (AgNPs) is proposed for making a novel carbon paste electrode (CPE). The resulting material promotes direct electron transfer and direct biocatalysis of embedded heme protein. It is demonstrated that the impregnation of graphite grains with AgNPs of 16-25 nm, incorporated in a CPE, can promote measurable bio-electrochemical phenomena involving hemoglobin and myoglobin. Unlike other biosensors prepared with simple carbon, those based on carbon grains studded with AgNPs show well-defined and quasi-reversible voltammetric peak with heterogeneous electron transfer rate k(s) of approximately 0.037±0.007 and 0.013±0.005s(-1) for hemoglobin and myoglobin, respectively. The embedded proteins also retain their bio-catalytical activity for hydrogen peroxide and nitrite reduction with linear ranges of 0.5-3000 μM and 30-150 μM, sensitivities of 73.6±0.6nA μM(-1) and 5.72±0.11 nA μM(-1), and detection limits close to 0.08 μM and 5.80 μM, for these two analytes respectively. These results support the viability of this preliminary approach for the development of advanced third-generation biosensors. PMID:22154098

  4. Characterization of the non-sexual flocculation of fission yeast cells that results from the deletion of ribosomal protein L32.

    PubMed

    Liu, Zhonghua; Li, Rongpeng; Dong, Qing; Bian, Lezhi; Li, Xuesong; Yuan, Sheng

    2015-05-01

    We recently reported that deleting either of the two paralogous rpl32 genes resulted in non-sexual flocculation in fission yeast. This study represents the first report that these non-sexually flocculating fission yeast cells exhibit a thicker cell wall, an increased wall protein content with smeared glycosylated wall proteins, and increased cell wall polysaccharide content and adhesin-binding sugar residues (i.e. glucose, mannose and galactose). These changes reflect the wall features of flocculating cells that mediate recognition and connections between cells. Furthermore, this study demonstrates that this non-sexual flocculation is an adhesin-mediated process: (a) the transcription levels of several members of the Mam3/Map4 family of adhesins (i.e. PFL3, PFL7 and PFL6) and a Flo11-like adhesin protein are upregulated in rpl32-1Δ and rpl32-2Δ cells; (b) this non-sexual flocculation of rpl32-1Δ and rpl32-2Δ cells was eliminated by heating or enzyme digestion; (c) this non-sexual flocculation of rpl32-1Δ and rpl32-2Δ cells was enhanced by Ca(2+) and some other divalent metal ions, which stabilize the active conformation of adhesins; and (d) this non-sexual flocculation of rpl32-1Δ and rpl32-2Δ cells was competitively inhibited by glucose, galactose or mannose rather than only by galactose, as reported previously. Although different adhesin genes are selectively expressed under particular physiological or environmental conditions, the functions of these adhesins are the same and are interchangeable.

  5. Rapamycin and Chloroquine: The In Vitro and In Vivo Effects of Autophagy-Modifying Drugs Show Promising Results in Valosin Containing Protein Multisystem Proteinopathy

    PubMed Central

    Nalbandian, Angèle; Llewellyn, Katrina J.; Nguyen, Christopher; Yazdi, Puya G.; Kimonis, Virginia E.

    2015-01-01

    Mutations in the valosin containing protein (VCP) gene cause hereditary Inclusion body myopathy (hIBM) associated with Paget disease of bone (PDB), frontotemporal dementia (FTD), more recently termed multisystem proteinopathy (MSP). Affected individuals exhibit scapular winging and die from progressive muscle weakness, and cardiac and respiratory failure, typically in their 40s to 50s. Histologically, patients show the presence of rimmed vacuoles and TAR DNA-binding protein 43 (TDP-43)-positive large ubiquitinated inclusion bodies in the muscles. We have generated a VCPR155H/+ mouse model which recapitulates the disease phenotype and impaired autophagy typically observed in patients with VCP disease. Autophagy-modifying agents, such as rapamycin and chloroquine, at pharmacological doses have previously shown to alter the autophagic flux. Herein, we report results of administration of rapamycin, a specific inhibitor of the mechanistic target of rapamycin (mTOR) signaling pathway, and chloroquine, a lysosomal inhibitor which reverses autophagy by accumulating in lysosomes, responsible for blocking autophagy in 20-month old VCPR155H/+ mice. Rapamycin-treated mice demonstrated significant improvement in muscle performance, quadriceps histological analysis, and rescue of ubiquitin, and TDP-43 pathology and defective autophagy as indicated by decreased protein expression levels of LC3-I/II, p62/SQSTM1, optineurin and inhibiting the mTORC1 substrates. Conversely, chloroquine-treated VCPR155H/+ mice revealed progressive muscle weakness, cytoplasmic accumulation of TDP-43, ubiquitin-positive inclusion bodies and increased LC3-I/II, p62/SQSTM1, and optineurin expression levels. Our in vitro patient myoblasts studies treated with rapamycin demonstrated an overall improvement in the autophagy markers. Targeting the mTOR pathway ameliorates an increasing list of disorders, and these findings suggest that VCP disease and related neurodegenerative multisystem proteinopathies can

  6. An HLA-A2-restricted tyrosinase antigen on melanoma cells results from posttranslational modification and suggests a novel pathway for processing of membrane proteins

    PubMed Central

    1996-01-01

    T lymphocytes recognize antigens consisting of peptides presented by class I and II major histocompatibility complex (MHC) molecules. The peptides identified so far have been predictable from the amino acid sequences of proteins. We have identified the natural peptide target of a CTL clone that recognizes the tyrosinase gene product on melanoma cells. The peptide results from posttranslational conversion of asparagine to aspartic acid. This change is of central importance for peptide recognition by melanoma-specific T cells, but has no impact on peptide binding to the MHC molecule. This posttranslational modification has not been previously described for any MHC-associated peptide and represents the first demonstration of posttranslational modification of a naturally processed class I-associated peptide. This observation is relevant to the identification and prediction of potential peptide antigens. The most likely mechanism for production of this peptide leads to the suggestion that antigenic peptides can be derived from proteins that are translated into the endoplasmic reticulum. PMID:8627164

  7. Overexpression of the Gene Encoding the Multidrug Resistance-Associated Protein Results in Increased ATP-Dependent Glutathione S-Conjugate Transport

    NASA Astrophysics Data System (ADS)

    Muller, Michael; Meijer, Coby; Zaman, Guido J. R.; Borst, Piet; Scheper, Rik J.; Mulder, Nanno H.; de Vries, Elisabeth G. E.; Jansen, Peter L. M.

    1994-12-01

    The multidrug resistance-associated protein (MRP) is a 180- to 195-kDa glycoprotein associated with multidrug resistance of human tumor cells. MRP is mainly located in the plasma membrane and it confers resistance by exporting natural product drugs out of the cell. Here we demonstrate that overexpression of the MRP gene in human cancer cells increases the ATP-dependent glutathione S-conjugate carrier activity in plasma membrane vesicles isolated from these cells. The glutathione S-conjugate export carrier is known to mediate excretion of bivalent anionic conjugates from mammalian cells and is thought to play a role in the elimination of conjugated xenobiotics. Our results suggest that MRP can cause multidrug resistance by promoting the export of drug modification products from cells and they shed light on the reported link between drug resistance and cellular glutathione and glutathione S-transferase levels.

  8. Measurement of multisite oxidation kinetics reveals an active site conformational change in Spo0F as a result of protein oxidation.

    PubMed

    Sharp, Joshua S; Sullivan, Daniel M; Cavanagh, John; Tomer, Kenneth B

    2006-05-23

    When most proteins undergo oxidative damage, they yield a variety of products containing oxidative damage at a large number of sites, most of which are modified substoichiometrically. The resulting complex mixture of products is not amenable to high-resolution structural analyses. The previous methods of structural analysis have relied upon either very generalized structural analyses such as circular dichroism or the creation of a battery of mutants to try to isolate single-residue damage effects. We present a methodology using mass spectrometry to measure the kinetics of oxidation at many sites simultaneously. Previous studies have shown that these kinetics are determined by the chemical nature of the damage site and by the accessibility of that site to the radical. By measuring deviations in the rate of oxidation from the expected pseudo-zero-order kinetics, we can detect and characterize local structural changes due to the oxidative damage. We demonstrate the application of this new technique to the Spo0F protein, a regulator of sporulation in Bacillus subtilis. Circular dichroism studies suggest a partial loss of helical structure of Spo0F as a result of oxidative damage. We report that oxidation causes a three-stage conformational change in Spo0F. Furthermore, we find the dramatic structural changes affect only the region surrounding the active site, while the remainder of the structure remains relatively unperturbed. Finally, we are able to determine that the specific oxidation event that triggers the conformational change at the active site of Spo0F occurs at Met81, a partially conserved methionine in the CheY superfamily.

  9. ValidatorDB: database of up-to-date validation results for ligands and non-standard residues from the Protein Data Bank.

    PubMed

    Sehnal, David; Svobodová Vařeková, Radka; Pravda, Lukáš; Ionescu, Crina-Maria; Geidl, Stanislav; Horský, Vladimír; Jaiswal, Deepti; Wimmerová, Michaela; Koča, Jaroslav

    2015-01-01

    Following the discovery of serious errors in the structure of biomacromolecules, structure validation has become a key topic of research, especially for ligands and non-standard residues. ValidatorDB (freely available at http://ncbr.muni.cz/ValidatorDB) offers a new step in this direction, in the form of a database of validation results for all ligands and non-standard residues from the Protein Data Bank (all molecules with seven or more heavy atoms). Model molecules from the wwPDB Chemical Component Dictionary are used as reference during validation. ValidatorDB covers the main aspects of validation of annotation, and additionally introduces several useful validation analyses. The most significant is the classification of chirality errors, allowing the user to distinguish between serious issues and minor inconsistencies. Other such analyses are able to report, for example, completely erroneous ligands, alternate conformations or complete identity with the model molecules. All results are systematically classified into categories, and statistical evaluations are performed. In addition to detailed validation reports for each molecule, ValidatorDB provides summaries of the validation results for the entire PDB, for sets of molecules sharing the same annotation (three-letter code) or the same PDB entry, and for user-defined selections of annotations or PDB entries. PMID:25392418

  10. Overview of the HUPO Plasma Proteome Project: Results from the pilot phase with 35 collaborating laboratories and multiple analytical groups, generating a core dataset of 3020 proteins and a publicly-available database

    SciTech Connect

    Omenn, Gilbert; States, David J.; Adamski, Marcin; Blackwell, Thomas W.; Menon, Rajasree; Hermjakob, Henning; Apweiler, Rolf; Haab, Brian B.; Simpson, Richard; Eddes, James; Kapp, Eugene; Moritz, Rod; Chan, Daniel W.; Rai, Alex J.; Admon, Arie; Aebersold, Ruedi; Eng, Jimmy K.; Hancock, William S.; Hefta, Stanley A.; Meyer, Helmut; Paik, Young-Ki; Yoo, Jong-Shin; Ping, Peipei; Pounds, Joel G.; Adkins, Joshua N.; Qian, Xiaohong; Wang, Rong; Wasinger, Valerie; Wu, Chi Yue; Zhao, Xiaohang; Zeng, Rong; Archakov, Alexander; Tsugita, Akira; Beer, Ilan; Pandey, Akhilesh; Pisano, Michael; Andrews, Philip; Tammen, Harald; Speicher, David W.; Hanash, Samir M.

    2005-08-13

    . These PPP results on complexity, dynamic range, incomplete sampling, false-positive matches, and integration of diverse datasets for plasma and serum proteins lay a foundation for development and validation of circulating protein biomarkers in health and disease.

  11. Moderate hypoxia followed by reoxygenation results in blood-brain barrier breakdown via oxidative stress-dependent tight-junction protein disruption.

    PubMed

    Zehendner, Christoph M; Librizzi, Laura; Hedrich, Jana; Bauer, Nina M; Angamo, Eskedar A; de Curtis, Marco; Luhmann, Heiko J

    2013-01-01

    Re-canalization of cerebral vessels in ischemic stroke is pivotal to rescue dysfunctional brain areas that are exposed to moderate hypoxia within the penumbra from irreversible cell death. Goal of the present study was to evaluate the effect of moderate hypoxia followed by reoxygenation (MHR) on the evolution of reactive oxygen species (ROS) and blood-brain barrier (BBB) integrity in brain endothelial cells (BEC). BBB integrity was assessed in BEC in vitro and in microvessels of the guinea pig whole brain in situ preparation. Probes were exposed to MHR (2 hours 67-70 mmHg O2, 3 hours reoxygenation, BEC) or towards occlusion of the arteria cerebri media (MCAO) with or without subsequent reperfusion in the whole brain preparation. In vitro BBB integrity was evaluated using trans-endothelial electrical resistance (TEER) and transwell permeability assays. ROS in BEC were evaluated using 2',7'-dichlorodihydrofluorescein diacetate (DCF), MitoSox and immunostaining for nitrotyrosine. Tight-junction protein (TJ) integrity in BEC, stainings for nitrotyrosine and FITC-albumin extravasation in the guinea pig brain preparation were assessed by confocal microscopy. Diphenyleneiodonium (DPI) was used to investigate NADPH oxidase dependent ROS evolution and its effect on BBB parameters in BEC. MHR impaired TJ proteins zonula occludens 1 (ZO-1) and claudin 5 (Cl5), decreased TEER, and significantly increased cytosolic ROS in BEC. These events were blocked by the NADPH oxidase inhibitor DPI. MCAO with or without subsequent reoxygenation resulted in extravasation of FITC-albumin and ROS generation in the penumbra region of the guinea pig brain preparation and confirmed BBB damage. BEC integrity may be impaired through ROS in MHR on the level of TJ and the BBB is also functionally impaired in moderate hypoxic conditions followed by reperfusion in a complex guinea pig brain preparation. These findings suggest that the BBB is susceptible towards MHR and that ROS play a key role in this

  12. Acute kidney injury induced by protein-overload nephropathy down-regulates gene expression of hepatic cerebroside sulfotransferase in mice, resulting in reduction of liver and serum sulfatides.

    PubMed

    Zhang, Xiaowei; Nakajima, Takero; Kamijo, Yuji; Li, Gang; Hu, Rui; Kannagi, Reiji; Kyogashima, Mamoru; Aoyama, Toshifumi; Hara, Atsushi

    2009-12-25

    Sulfatides, possible antithrombotic factors belonging to sphingoglycolipids, are widely distributed in mammalian tissues and serum. We recently found that the level of serum sulfatides was significantly lower in hemodialysis patients than that in normal subjects, and that the serum level closely correlated to the incidence of cardiovascular disease. These findings suggest a relationship between the level of serum sulfatides and kidney function; however, the molecular mechanism underlying this relationship remains unclear. In the present study, the influence of kidney dysfunction on the metabolism of sulfatides was examined using an established murine model of acute kidney injury, protein-overload nephropathy in mice. Protein-overload treatment caused severe proximal tubular injuries within 4days, and this treatment obviously decreased both serum and hepatic sulfatide levels. The sphingoid composition of serum sulfatides was very similar to that of hepatic ones at each time point, suggesting that the serum sulfatide level is dependent on the hepatic secretory ability of sulfatides. The treatment also decreased hepatic expression of cerebroside sulfotransferase (CST), a key enzyme in sulfatide metabolism, while it scarcely influenced the expression of the other sulfatide-metabolizing enzymes, including arylsulfatase A, ceramide galactosyltransferase, and galactosylceramidase. Pro-inflammatory responses were not detected in the liver of these mice; however, potential oxidative stress was increased. These results suggest that down-regulation of hepatic CST expression, probably affected by oxidative stress from kidney injury, causes reduction in liver and serum sulfatide levels. This novel mechanism, indicating the crosstalk between kidney injury and specific liver function, may prove useful for helping to understand the situation where human hemodialysis patients have low levels of serum sulfatides.

  13. Gene transfer of master autophagy regulator TFEB results in clearance of toxic protein and correction of hepatic disease in alpha-1-anti-trypsin deficiency

    PubMed Central

    Pastore, Nunzia; Blomenkamp, Keith; Annunziata, Fabio; Piccolo, Pasquale; Mithbaokar, Pratibha; Maria Sepe, Rosa; Vetrini, Francesco; Palmer, Donna; Ng, Philip; Polishchuk, Elena; Iacobacci, Simona; Polishchuk, Roman; Teckman, Jeffrey; Ballabio, Andrea; Brunetti-Pierri, Nicola

    2013-01-01

    Alpha-1-anti-trypsin deficiency is the most common genetic cause of liver disease in children and liver transplantation is currently the only available treatment. Enhancement of liver autophagy increases degradation of mutant, hepatotoxic alpha-1-anti-trypsin (ATZ). We investigated the therapeutic potential of liver-directed gene transfer of transcription factor EB (TFEB), a master gene that regulates lysosomal function and autophagy, in PiZ transgenic mice, recapitulating the human hepatic disease. Hepatocyte TFEB gene transfer resulted in dramatic reduction of hepatic ATZ, liver apoptosis and fibrosis, which are key features of alpha-1-anti-trypsin deficiency. Correction of the liver phenotype resulted from increased ATZ polymer degradation mediated by enhancement of autophagy flux and reduced ATZ monomer by decreased hepatic NFκB activation and IL-6 that drives ATZ gene expression. In conclusion, TFEB gene transfer is a novel strategy for treatment of liver disease of alpha-1-anti-trypsin deficiency. This study may pave the way towards applications of TFEB gene transfer for treatment of a wide spectrum of human disorders due to intracellular accumulation of toxic proteins. PMID:23381957

  14. Increased adenosine levels in mice expressing mutant glial fibrillary acidic protein in astrocytes result in failure of induction of LTP reversal (depotentiation) in hippocampal CA1 neurons.

    PubMed

    Fujii, Satoshi; Tanaka, Kenji F; Ikenaka, Kazuhiro; Yamazaki, Yoshihiko

    2014-08-26

    Astrocytes regulate the activity of neighboring neurons by releasing chemical transmitters, including ATP. Adenosine levels in the cerebrospinal fluid of mice that express a mutant human glial fibrillary acidic protein in astrocytes are slightly elevated compared to those in wild type mice and this might result from the observed increased release by mutant astrocytes of ATP, which can be used to produce adenosine. Using hippocampal slices from these mutant mice, we examined whether the increased endogenous adenosine levels in the hippocampus modulate the reversal of long-term potentiation (LTP), i.e. depotentiation (DP), in CA1 neurons. In hippocampal slices from wild type mice, a stable LTP was induced by tetanic stimulation consisting of 100 pulses at 100 Hz, and this was reversed by a train of low frequency stimulation (LFS) of 500 pulses at 1 Hz applied 30 min later. This induction of DP was inhibited by application of either 100 nM adenosine or 0.5 nM N(6)-cyclopentyladenosine, an adenosine A1 receptor agonist, during LFS, indicating that the increase in extracellular adenosine levels attenuated DP induction by acting on adenosine A1 receptors. In contrast, although a stable LTP was also induced in hippocampal slices from mutant mice, induction of DP was inhibited, but DP could be induced by application, during LFS, of 50 nM 8-cyclopentyltheophylline, an adenosine A1 receptor antagonist. These results suggest that a small increase in extracellular adenosine levels resulting from increased ATP release by astrocytes results in attenuation of DP in hippocampal CA1 neurons in the mutant mice.

  15. Staphylococcus aureus protein A binding to osteoblast tumour necrosis factor receptor 1 results in activation of nuclear factor kappa B and release of interleukin-6 in bone infection.

    PubMed

    Claro, Tânia; Widaa, Amro; McDonnell, Cormac; Foster, Timothy J; O'Brien, Fergal J; Kerrigan, Steven W

    2013-01-01

    Staphylococcus aureus is the major pathogen among the staphylococci and the most common cause of bone infections. These infections are mainly characterized by bone destruction and inflammation, and are often debilitating and very difficult to treat. Previously we demonstrated that S. aureus protein A (SpA) can bind to osteoblasts, which results in inhibition of osteoblast proliferation and mineralization, apoptosis, and activation of osteoclasts. In this study we used small interfering RNA (siRNA) to demonstrate that osteoblast tumour necrosis factor receptor-1 (TNFR-1) is responsible for the recognition of and binding to SpA. TNFR-1 binding to SpA results in the activation of nuclear factor kappa B (NFκB). In turn, NFκB translocates to the nucleus of the osteoblast, which leads to release of interleukin 6 (IL-6). Silencing TNFR-1 in osteoblasts or disruption of the spa gene in S. aureus prevented both NFκB activation and IL-6 release. As well as playing a key role in proinflammatory reactions, IL-6 is also an important osteotropic factor. Release of IL-6 from osteoblasts results in the activation of the bone-resorbing cells, the osteoclasts. Consistent with our results described above, both silencing TNFR-1 in osteoblasts and disruption of spa in S. aureus prevented osteoclast activation. These studies are the first to demonstrate the importance of the TNFR-1-SpA interaction in bone infection, and may help explain the mechanism through which osteoclasts become overactivated, leading to bone destruction. Anti-inflammatory drug therapy could be used either alone or in conjunction with antibiotics to treat osteomyelitis or for prophylaxis in high-risk patients.

  16. Increased adenosine levels in mice expressing mutant glial fibrillary acidic protein in astrocytes result in failure of induction of LTP reversal (depotentiation) in hippocampal CA1 neurons.

    PubMed

    Fujii, Satoshi; Tanaka, Kenji F; Ikenaka, Kazuhiro; Yamazaki, Yoshihiko

    2014-08-26

    Astrocytes regulate the activity of neighboring neurons by releasing chemical transmitters, including ATP. Adenosine levels in the cerebrospinal fluid of mice that express a mutant human glial fibrillary acidic protein in astrocytes are slightly elevated compared to those in wild type mice and this might result from the observed increased release by mutant astrocytes of ATP, which can be used to produce adenosine. Using hippocampal slices from these mutant mice, we examined whether the increased endogenous adenosine levels in the hippocampus modulate the reversal of long-term potentiation (LTP), i.e. depotentiation (DP), in CA1 neurons. In hippocampal slices from wild type mice, a stable LTP was induced by tetanic stimulation consisting of 100 pulses at 100 Hz, and this was reversed by a train of low frequency stimulation (LFS) of 500 pulses at 1 Hz applied 30 min later. This induction of DP was inhibited by application of either 100 nM adenosine or 0.5 nM N(6)-cyclopentyladenosine, an adenosine A1 receptor agonist, during LFS, indicating that the increase in extracellular adenosine levels attenuated DP induction by acting on adenosine A1 receptors. In contrast, although a stable LTP was also induced in hippocampal slices from mutant mice, induction of DP was inhibited, but DP could be induced by application, during LFS, of 50 nM 8-cyclopentyltheophylline, an adenosine A1 receptor antagonist. These results suggest that a small increase in extracellular adenosine levels resulting from increased ATP release by astrocytes results in attenuation of DP in hippocampal CA1 neurons in the mutant mice. PMID:25017946

  17. Immuno-spin trapping detection of antioxidant/pro-oxidant properties of zinc or selenium on DNA and protein radical formation via hydrogen peroxide.

    PubMed

    Deletioglu, Vedia; Tuncay, Erkan; Toy, Aysegul; Atalay, Mustafa; Turan, Belma

    2015-11-01

    Trace elements can participate in the catalysis of group-transfer reactions and can serve as their structural components. However, most of them including zinc and selenium have multifunctional roles in biological environments such as antioxidant and/or pro-oxidant effects, as concentration-dependent manner. Although it has been demonstrated the antioxidant actions of either selenium or zinc compounds, there are several documents pointing out their pro-oxidant/oxidant roles in biological systems. Here we have used ELISA-based immuno-spin trapping, a method for detection of free radical formation, to detect whether or not a zinc compound, Zn3(PO4)2, or a selenium compound, Na2SeO3, has antioxidant and/or pro-oxidant effect on 5,5-Dimethyl-1-Pyrroline-N-Oxide (DMPO)-DNA nitrone adducts induced with Cu(II)-H2O2-oxidizing system in in vitro preparations. Second, we examined whether this technique is capable to demonstrate the different DMPO-protein nitrone adduct productions in isolated protein crude of hearts from normal rats (CON) or rats with metabolic syndrome (MetS). Our data demonstrated that either Zn(2+) (100 µM) or SeO3(-2) (50 nM) has very strong antioxidant action against 200 µM H2O2-induced DMPO-DNA nitrone adduct production, whereas their higher concentrations have apparent pro-oxidant actions. We also used verification by Western blotting analysis whether immuno-spin trapping can be used to assess H2O2-induced DMPO-protein nitrone adducts in heart protein crudes. Our Western blot data further confirmed the ELISA-data from proteins and demonstrated how Zn(2+) or SeO3(-2) are dual-functioning ions such as antioxidant at lower concentrations while pro-oxidant at higher concentrations. Particularly, our present data with SeO3(-2) in DMPO-protein nitrone adducts, being in line with our previous observation on its dual-actions in ischemia/reperfusion-induced damaged heart, have shown that this ion has higher pro-oxidant actions over 50 nM in Met

  18. Suppression of telomere-binding protein TPP1 resulted in telomere dysfunction and enhanced radiation sensitivity in telomerase-negative osteosarcoma cell line

    SciTech Connect

    Qiang, Weiguang; Wu, Qinqin; Zhou, Fuxiang; Xie, Conghua; Wu, Changping; Zhou, Yunfeng

    2014-03-07

    Highlights: • Down-regulation of TPP1 shortened telomere length in telomerase-negative cells. • Down-regulation of TPP1 induced cell apoptosis in telomerase-negative cells. • Down-regulation of TPP1 increased radiosensitivity in telomerase-negative cells. - Abstract: Mammalian telomeres are protected by the shelterin complex that contains the six core proteins POT1, TPP1, TIN2, TRF1, TRF2 and RAP1. TPP1, formerly known as TINT1, PTOP, and PIP1, is a key factor that regulates telomerase recruitment and activity. In addition to this, TPP1 is required to mediate the shelterin assembly and stabilize telomere. Previous work has found that TPP1 expression was elevated in radioresistant cells and that overexpression of TPP1 led to radioresistance and telomere lengthening in telomerase-positive cells. However, the exact effects and mechanism of TPP1 on radiosensitivity are yet to be precisely defined in the ALT cells. Here we report on the phenotypes of the conditional deletion of TPP1 from the human osteosarcoma U2OS cells using ALT pathway to extend the telomeres.TPP1 deletion resulted in telomere shortening, increased apoptosis and radiation sensitivity enhancement. Together, our findings show that TPP1 plays a vital role in telomere maintenance and protection and establish an intimate relationship between TPP1, telomere and cellular response to ionizing radiation, but likely has the specific mechanism yet to be defined.

  19. Providing a diet containing only maintenance levels of energy and protein during the latter stages of pregnancy resulted in a prolonged delivery time during parturition in rats.

    PubMed

    Tanaka, Y; Kadokawa, H

    2012-01-01

    In mammals, a prolonged delivery time during parturition is dangerous for both mother and fetus, although the mechanisms that prolong delivery are unclear. To investigate whether nutrition affects delivery time, we administered two feeds containing maintenance (L-feed) or higher (H-feed) levels of energy and protein at different points during the latter half of pregnancy and compared the effects of the various treatments on delivery time in rats. After the rats had been maintained on the L-feed and then copulated on pro-oestrus (Day 0), pregnant females were randomly allocated to one of three groups: (1) the no-improvement group, which was fed L-feed throughout gestation; (2) the early group, which was fed L-feed until Day 11 of gestation and then switched to H-feed; and (3) the late group, which was fed L-feed until Day 16 of gestation and then switched to H-feed. There was no significant difference in the number of pups among the three groups. However, delivery time was significantly longer in the no-improvement group (73.7±5.2 min) than the early (46.9±5.6 min) and late (55.4±5.5 min) groups. Consuming a maintenance diet during the latter half of pregnancy resulted in a prolonged delivery time.

  20. Disease Phenotype, Activity and Clinical Course Prediction Based on C-Reactive Protein Levels at Diagnosis in Patients with Crohn’s Disease: Results from the CONNECT Study

    PubMed Central

    Kwon, Jee Hye; Im, Jong Pil; Ye, Byong Duk; Cheon, Jae Hee; Jang, Hyun Joo; Lee, Kang Moon; Kim, You Sun; Kim, Sang Wook; Kim, Young Ho; Song, Geun Am; Han, Dong Soo; Kim, Won Ho; Kim, Joo Sung

    2016-01-01

    Background/Aims C-reactive protein (CRP) is an easily measured index of disease activity, but its ability to predict clinical course is controversial. We therefore designed a study to determine whether the CRP level at Crohn’s disease (CD) diagnosis is a valuable indicator of the disease phenotype, activity, and clinical course. Methods We retrospectively analyzed 705 CD patients from 32 institutions. The patients were classified into two groups according to CRP level. The patients’ demographic and clinical characteristics and their use of immunosuppressive or biological agents were recorded. Disease location and behavior, hospitalization, and surgery were analyzed. Results A high CRP was associated with younger age, steroid use, colonic or ileocolonic location, high CD activity index, and active inflammation at colonoscopy (p<0.001). As the disease progressed, patients with high CRP were more likely to exhibit strictures (p=0.027). There were significant differences in the use of 5-aminosalicylic acid, antibiotics, corticosteroids, azathioprine, and infliximab (p<0.001, p<0.001, p<0.001, p<0.001, and p=0.023, respectively). Hospitalization was also more frequent in patients with high CRP. Conclusions The CRP level at diagnosis is useful for evaluating the phenotype, activity, and clinical course of CD. Closer follow-up strategies, with early aggressive treatment, could be considered for patients with high CRP. PMID:27021506

  1. Infection of a Single Cell Line with Distinct Strains of Human Cytomegalovirus Can Result in Large Variations in Virion Production and Facilitate Efficient Screening of Virus Protein Function

    PubMed Central

    Zavala, Anamaria G.; O'Dowd, John M.

    2015-01-01

    ABSTRACT Previously, we reported that the absence of the ataxia telangiectasia mutated (ATM) kinase, a critical DNA damage response (DDR) signaling component for double-strand breaks, caused no change in HCMV Towne virion production. Later, others reported decreased AD169 viral titers in the absence of ATM. To address this discrepancy, human foreskin fibroblasts (HFF) and three ATM− lines (GM02530, GM05823, and GM03395) were infected with both Towne and AD169. Two additional ATM− lines (GM02052 and GM03487) were infected with Towne. Remarkably, both previous studies' results were confirmed. However, the increased number of cell lines and infections with both lab-adapted strains confirmed that ATM was not necessary to produce wild-type-level titers in fibroblasts. Instead, interactions between individual virus strains and the cellular microenvironment of the individual ATM− line determined efficiency of virion production. Surprisingly, these two commonly used lab-adapted strains produced drastically different titers in one ATM− cell line, GM05823. The differences in titer suggested a rapid method for identifying genes involved in differential virion production. In silico comparison of the Towne and AD169 genomes determined a list of 28 probable candidates responsible for the difference. Using serial iterations of an experiment involving virion entry and input genome nuclear trafficking with a panel of related strains, we reduced this list to four (UL129, UL145, UL147, and UL148). As a proof of principle, reintroduction of UL148 largely rescued genome trafficking. Therefore, use of a battery of related strains offers an efficient method to narrow lists of candidate genes affecting various virus life cycle checkpoints. IMPORTANCE Human cytomegalovirus (HCMV) infection of multiple cell lines lacking ataxia telangiectasia mutated (ATM) protein produced wild-type levels of infectious virus. Interactions between virus strains and the microenvironment of individual

  2. Prasugrel Results in Higher Decrease in High-Sensitivity C-Reactive Protein Level in Patients Undergoing Percutaneous Coronary Intervention Comparing to Clopidogrel

    PubMed Central

    Hajsadeghi, Shokoufeh; Chitsazan, Mandana; Chitsazan, Mitra; Salehi, Negar; Amin, Ahmad; Bidokhti, Arash Amin; Babaali, Nima; Bordbar, Armin; Hejrati, Maral; Moghadami, Samar

    2016-01-01

    OBJECTIVES A growing body of clinical and laboratory evidence indicates that inflammation plays a crucial role in atherosclerosis. In the present study, we compared the effects of clopidogrel and prasugrel on high-sensitivity C-reactive protein (hs-CRP) in patients undergoing percutaneous coronary intervention (PCI). METHODS The present randomized, double-blind clinical trial included 120 patients who underwent PCI. Eligible patients were randomly assigned 2:1 to one of the two groups: 80 patients in the first group received clopidogrel (Plavix®; loading dose and maintenance dose of 300 and 75 mg daily, respectively) and 40 patients in the second group received prasugrel (Effient®; loading dose and maintenance dose of 60 and 10 mg, respectively) for 12 weeks. The hs-CRP levels between baseline and 12th week were compared. RESULTS Of the 120 patients, 69 patients (57.5%) were male. Pretreatment hs-CRP level was statistically comparable in clopidogrel (median, 15.10 mg/dL; interquartile range [IQR], 9.62–23.75 mg/dL) and prasugrel groups (median, 18 mg/dL; IQR, 14.25–22 mg/dL; P = 0.06). Patients taking clopidogrel showed a significant reduction in hs-CRP level compared with the baseline values (P < 0.001). Prasugrel administration also resulted in a significant reduction in hs-CRP level (P < 0.001). A significant 73% overall reduction in the hs-CRP level was seen with prasugrel compared with 39% overall reduction in hs-CRP level with clopidogrel (P = 0.002). CONCLUSION Prasugrel seems to be superior to clopidogrel in the reduction of hs-CRP in patients undergoing PCI.

  3. N-Octanoyl Dopamine Treatment of Endothelial Cells Induces the Unfolded Protein Response and Results in Hypometabolism and Tolerance to Hypothermia

    PubMed Central

    Stamellou, Eleni; Fontana, Johann; Wedel, Johannes; Ntasis, Emmanouil; Sticht, Carsten; Becker, Anja; Pallavi, Prama; Wolf, Kerstin; Krämer, Bernhard K.; Hafner, Mathias; van Son, Willem J.; Yard, Benito A.

    2014-01-01

    Aim N-acyl dopamines (NADD) are gaining attention in the field of inflammatory and neurological disorders. Due to their hydrophobicity, NADD may have access to the endoplasmic reticulum (ER). We therefore investigated if NADD induce the unfolded protein response (UPR) and if this in turn influences cell behaviour. Methods Genome wide gene expression profiling, confirmatory qPCR and reporter assays were employed on human umbilical vein endothelial cells (HUVEC) to validate induction of UPR target genes and UPR sensor activation by N-octanoyl dopamine (NOD). Intracellular ATP, apoptosis and induction of thermotolerance were used as functional parameters to assess adaptation of HUVEC. Results NOD, but not dopamine dose dependently induces the UPR. This was also found for other synthetic NADD. Induction of the UPR was dependent on the redox activity of NADD and was not caused by selective activation of a particular UPR sensor. UPR induction did not result in cell apoptosis, yet NOD strongly impaired cell proliferation by attenuation of cells in the S-G2/M phase. Long-term treatment of HUVEC with low NOD concentration showed decreased intracellular ATP concentration paralleled with activation of AMPK. These cells were significantly more resistant to cold inflicted injury. Conclusions We provide for the first time evidence that NADD induce the UPR in vitro. It remains to be assessed if UPR induction is causally associated with hypometabolism and thermotolerance. Further pharmacokinetic studies are warranted to address if the NADD concentrations used in vitro can be obtained in vivo and if this in turn shows therapeutic efficacy. PMID:24926788

  4. Prasugrel Results in Higher Decrease in High-Sensitivity C-Reactive Protein Level in Patients Undergoing Percutaneous Coronary Intervention Comparing to Clopidogrel

    PubMed Central

    Hajsadeghi, Shokoufeh; Chitsazan, Mandana; Chitsazan, Mitra; Salehi, Negar; Amin, Ahmad; Bidokhti, Arash Amin; Babaali, Nima; Bordbar, Armin; Hejrati, Maral; Moghadami, Samar

    2016-01-01

    OBJECTIVES A growing body of clinical and laboratory evidence indicates that inflammation plays a crucial role in atherosclerosis. In the present study, we compared the effects of clopidogrel and prasugrel on high-sensitivity C-reactive protein (hs-CRP) in patients undergoing percutaneous coronary intervention (PCI). METHODS The present randomized, double-blind clinical trial included 120 patients who underwent PCI. Eligible patients were randomly assigned 2:1 to one of the two groups: 80 patients in the first group received clopidogrel (Plavix®; loading dose and maintenance dose of 300 and 75 mg daily, respectively) and 40 patients in the second group received prasugrel (Effient®; loading dose and maintenance dose of 60 and 10 mg, respectively) for 12 weeks. The hs-CRP levels between baseline and 12th week were compared. RESULTS Of the 120 patients, 69 patients (57.5%) were male. Pretreatment hs-CRP level was statistically comparable in clopidogrel (median, 15.10 mg/dL; interquartile range [IQR], 9.62–23.75 mg/dL) and prasugrel groups (median, 18 mg/dL; IQR, 14.25–22 mg/dL; P = 0.06). Patients taking clopidogrel showed a significant reduction in hs-CRP level compared with the baseline values (P < 0.001). Prasugrel administration also resulted in a significant reduction in hs-CRP level (P < 0.001). A significant 73% overall reduction in the hs-CRP level was seen with prasugrel compared with 39% overall reduction in hs-CRP level with clopidogrel (P = 0.002). CONCLUSION Prasugrel seems to be superior to clopidogrel in the reduction of hs-CRP in patients undergoing PCI. PMID:27597810

  5. Technical decision making with higher order structure data: utilization of differential scanning calorimetry to elucidate critical protein structural changes resulting from oxidation.

    PubMed

    Arthur, Kelly K; Dinh, Nikita; Gabrielson, John P

    2015-04-01

    Differential scanning calorimetry (DSC) is a useful tool for monitoring thermal stability of the molecular conformation of proteins. Here, we present an example of the sensitivity of DSC to changes in stability arising from a common chemical degradation pathway, oxidation. This Note is part of a series of industry case studies demonstrating the application of higher order structure data for technical decision making. For this study, six protein products from three structural classes were evaluated at multiple levels of oxidation. For each protein, the melting temperature (Tm ) decreased linearly as a function of oxidation; however, differences in the rate of change in Tm , as well as differences in domain Tm stability were observed across and within structural classes. For one protein, analysis of the impact of oxidation on protein function was also performed. For this protein, DSC was shown to be a leading indicator of decreased antigen binding suggesting a subtle conformation change may be underway that can be detected using DSC prior to any observable impact on product potency. Detectable changes in oxidized methionine by mass spectrometry (MS) occurred at oxidation levels below those with a detectable conformational or functional impact. Therefore, by using MS, DSC, and relative potency methods in concert, the intricate relationship between a primary structural modification, changes in conformational stability, and functional impact can be elucidated.

  6. Long-acting recombinant coagulation factor IX albumin fusion protein (rIX-FP) in hemophilia B: results of a phase 3 trial

    PubMed Central

    Martinowitz, Uri; Lissitchkov, Toshko; Pan-Petesch, Brigitte; Hanabusa, Hideji; Oldenburg, Johannes; Boggio, Lisa; Negrier, Claude; Pabinger, Ingrid; von Depka Prondzinski, Mario; Altisent, Carmen; Castaman, Giancarlo; Yamamoto, Koji; Álvarez-Roman, Maria-Teresa; Voigt, Christine; Blackman, Nicole; Jacobs, Iris

    2016-01-01

    A global phase 3 study evaluated the pharmacokinetics, efficacy, and safety of recombinant fusion protein linking coagulation factor IX with albumin (rIX-FP) in 63 previously treated male patients (12-61 years) with severe hemophilia B (factor IX [FIX] activity ≤2%). The study included 2 groups: group 1 patients received routine prophylaxis once every 7 days for 26 weeks, followed by either 7-, 10-, or 14-day prophylaxis regimen for a mean of 50, 38, or 51 weeks, respectively; group 2 patients received on-demand treatment of bleeding episodes for 26 weeks and then switched to a 7-day prophylaxis regimen for a mean of 45 weeks. The mean terminal half-life of rIX-FP was 102 hours, 4.3-fold longer than previous FIX treatment. Patients maintained a mean trough of 20 and 12 IU/dL FIX activity on prophylaxis with rIX-FP 40 IU/kg weekly and 75 IU/kg every 2 weeks, respectively. There was 100% reduction in median annualized spontaneous bleeding rate (AsBR) and 100% resolution of target joints when subjects switched from on-demand to prophylaxis treatment with rIX-FP (P < .0001). The median AsBR was 0.00 for all prophylaxis regimens. Overall, 98.6% of bleeding episodes were treated successfully, including 93.6% that were treated with a single injection. No patient developed an inhibitor, and no safety concerns were identified. These results indicate rIX-FP is safe and effective for preventing and treating bleeding episodes in patients with hemophilia B at dosing regimens of 40 IU/kg weekly and 75 IU/kg every 2 weeks. This trial was registered at www.clinicaltrials.gov as #NCT0101496274. PMID:26755710

  7. Long-acting recombinant coagulation factor IX albumin fusion protein (rIX-FP) in hemophilia B: results of a phase 3 trial.

    PubMed

    Santagostino, Elena; Martinowitz, Uri; Lissitchkov, Toshko; Pan-Petesch, Brigitte; Hanabusa, Hideji; Oldenburg, Johannes; Boggio, Lisa; Negrier, Claude; Pabinger, Ingrid; von Depka Prondzinski, Mario; Altisent, Carmen; Castaman, Giancarlo; Yamamoto, Koji; Álvarez-Roman, Maria-Teresa; Voigt, Christine; Blackman, Nicole; Jacobs, Iris

    2016-04-01

    A global phase 3 study evaluated the pharmacokinetics, efficacy, and safety of recombinant fusion protein linking coagulation factor IX with albumin (rIX-FP) in 63 previously treated male patients (12-61 years) with severe hemophilia B (factor IX [FIX] activity ≤2%). The study included 2 groups: group 1 patients received routine prophylaxis once every 7 days for 26 weeks, followed by either 7-, 10-, or 14-day prophylaxis regimen for a mean of 50, 38, or 51 weeks, respectively; group 2 patients received on-demand treatment of bleeding episodes for 26 weeks and then switched to a 7-day prophylaxis regimen for a mean of 45 weeks. The mean terminal half-life of rIX-FP was 102 hours, 4.3-fold longer than previous FIX treatment. Patients maintained a mean trough of 20 and 12 IU/dL FIX activity on prophylaxis with rIX-FP 40 IU/kg weekly and 75 IU/kg every 2 weeks, respectively. There was 100% reduction in median annualized spontaneous bleeding rate (AsBR) and 100% resolution of target joints when subjects switched from on-demand to prophylaxis treatment with rIX-FP (P< .0001). The median AsBR was 0.00 for all prophylaxis regimens. Overall, 98.6% of bleeding episodes were treated successfully, including 93.6% that were treated with a single injection. No patient developed an inhibitor, and no safety concerns were identified. These results indicate rIX-FP is safe and effective for preventing and treating bleeding episodes in patients with hemophilia B at dosing regimens of 40 IU/kg weekly and 75 IU/kg every 2 weeks. This trial was registered at www.clinicaltrials.gov as #NCT0101496274.

  8. Delivery Mode, Duration of Labor, and Cord Blood Adiponectin, Leptin, and C-Reactive Protein: Results of the Population-Based Ulm Birth Cohort Studies

    PubMed Central

    Logan, Chad A.; Thiel, Larissa; Bornemann, Rebecca; Koenig, Wolfgang; Reister, Frank; Brenner, Hermann; Rothenbacher, Dietrich; Genuneit, Jon

    2016-01-01

    Background Numerous studies have reported associations between delivery mode and health outcomes in infancy and later life. Previous smaller studies indicated a relationship between delivery mode and newborn inflammation potentially constituting a mediating factor. We aimed to determine the influence of delivery mode and duration of labor on cord blood concentrations of adiponectin, leptin, and high-sensitivity C-reactive protein (hs-CRP). Methods In the Ulm SPATZ Health Study, 934 singleton newborns and their mothers were recruited during their hospital stay in the University Medical Center Ulm, Southern Germany, from 04/2012-05/2013. Inflammatory biomarkers were measured by ELISAs (n = 836). Delivery mode was analyzed categorically (elective cesarean (reference), active labor delivery: emergency cesarean, assisted vaginal, and spontaneous vaginal); duration of labor continuously. Following log-transformation, linear regression was used to estimate geometric means ratios (GMR) adjusted for potential confounders for the effects of delivery mode and duration of labor on each biomarker separately. Independent replication was sought in the similarly conducted Ulm Birth Cohort Study recruited from 11/2000-11/2001. Results Individually, active labor delivery modes as well as increasing duration of labor were associated with higher leptin and hs-CRP concentrations. After mutual adjustment, the associations with delivery modes were attenuated but those for duration of labor remained statistically significant (GMR (95%CI) 1.10 (1.00; 1.21) and 1.15 (1.04; 1.27) for leptin and hs-CRP per hour of labor, respectively). No significant adjusted associations were observed between delivery modes and adiponectin concentrations. These findings were replicated in an independent birth cohort study. Conclusions Cord blood leptin and hs-CRP concentrations were associated with duration of labor rather than delivery mode. Further research is warranted to investigate these associations

  9. Learning and memory deficits consequent to reduction of the fragile X mental retardation protein result from metabotropic glutamate receptor-mediated inhibition of cAMP signaling in Drosophila.

    PubMed

    Kanellopoulos, Alexandros K; Semelidou, Ourania; Kotini, Andriana G; Anezaki, Maria; Skoulakis, Efthimios M C

    2012-09-19

    Loss of the RNA-binding fragile X protein [fragile X mental retardation protein (FMRP)] results in a spectrum of cognitive deficits, the fragile X syndrome (FXS), while aging individuals with decreased protein levels present with a subset of these symptoms and tremor. The broad range of behavioral deficits likely reflects the ubiquitous distribution and multiple functions of the protein. FMRP loss is expected to affect multiple neuronal proteins and intracellular signaling pathways, whose identity and interactions are essential in understanding and ameliorating FXS symptoms. We used heterozygous mutants and targeted RNA interference-mediated abrogation in Drosophila to uncover molecular pathways affected by FMRP reduction. We present evidence that FMRP loss results in excess metabotropic glutamate receptor (mGluR) activity, attributable at least in part to elevation of the protein in affected neurons. Using high-resolution behavioral, genetic, and biochemical analyses, we present evidence that excess mGluR upon FMRP attenuation is linked to the cAMP decrement reported in patients and models, and underlies olfactory associative learning and memory deficits. Furthermore, our data indicate positive transcriptional regulation of the fly fmr1 gene by cAMP, via protein kinase A, likely through the transcription factor CREB. Because the human Fmr1 gene also contains CREB binding sites, the interaction of mGluR excess and cAMP signaling defects we present suggests novel combinatorial pharmaceutical approaches to symptom amelioration upon FMRP attenuation.

  10. Human Gene and Protein Database (HGPD): a novel database presenting a large quantity of experiment-based results in human proteomics.

    PubMed

    Maruyama, Yukio; Wakamatsu, Ai; Kawamura, Yoshifumi; Kimura, Kouichi; Yamamoto, Jun-ichi; Nishikawa, Tetsuo; Kisu, Yasutomo; Sugano, Sumio; Goshima, Naoki; Isogai, Takao; Nomura, Nobuo

    2009-01-01

    Completion of human genome sequencing has greatly accelerated functional genomic research. Full-length cDNA clones are essential experimental tools for functional analysis of human genes. In one of the projects of the New Energy and Industrial Technology Development Organization (NEDO) in Japan, the full-length human cDNA sequencing project (FLJ project), nucleotide sequences of approximately 30 000 human cDNA clones have been analyzed. The Gateway system is a versatile framework to construct a variety of expression clones for various experiments. We have constructed 33 275 human Gateway entry clones from full-length cDNAs, representing to our knowledge the largest collection in the world. Utilizing these clones with a highly efficient cell-free protein synthesis system based on wheat germ extract, we have systematically and comprehensively produced and analyzed human proteins in vitro. Sequence information for both amino acids and nucleotides of open reading frames of cDNAs cloned into Gateway entry clones and in vitro expression data using those clones can be retrieved from the Human Gene and Protein Database (HGPD, http://www.HGPD.jp). HGPD is a unique database that stores the information of a set of human Gateway entry clones and protein expression data and helps the user to search the Gateway entry clones.

  11. A serine-to-threonine substitution in the triazine herbicide-binding protein in potato cells results in atrazine resistance without impairing productivity.

    PubMed Central

    Smeda, R J; Hasegawa, P M; Goldsbrough, P B; Singh, N K; Weller, S C

    1993-01-01

    A mutation of the psbA gene was identified in photoautotrophic potato (Solanum tuberosum L. cv Superior x U.S. Department of Agriculture line 66-142) cells selected for resistance to 6-chloro-N-ethyl-N'-(1-methylethyl)-1,3,5-triazine-2,4-diamine (atrazine). Photoaffinity labeling with 6-azido-N-ethyl-N'-(1-methylethyl)-1,3,5-triazine-2,4-diamine detected a thylakoid membrane protein with a M(r) of 32,000 in susceptible, but not in resistant, cells. This protein was identified as the secondary quinone acceptor of photosystem II (QB) protein. Atrazine resistance in selected cells was attributable to a mutation from AGT (serine) to ACT (threonine) in codon 264 of the psbA gene that encodes the QB protein. Although the mutant cells exhibited extreme levels of resistance to atrazine, no concomitant reductions in photosynthetic electron transport or cell growth rates compared to the unselected cells were detected. This is in contrast with the losses in productivity observed in atrazine-resistant mutants that contain a glycine-264 alteration. PMID:8022941

  12. Formation of high-molecular-weight angiotensinogen during pregnancy is a result of competing redox reactions with the proform of eosinophil major basic protein.

    PubMed

    Kløverpris, Søren; Skov, Louise L; Glerup, Simon; Pihl, Kasper; Christiansen, Michael; Oxvig, Claus

    2013-01-01

    The plasma concentration of the placentally derived proMBP (proform of eosinophil major basic protein) increases in pregnancy, and three different complexes containing proMBP have been isolated from pregnancy plasma and serum: a 2:2 complex with the metalloproteinase, PAPP-A (pregnancy-associated plasma protein-A), a 2:2 complex with AGT (angiotensinogen) and a 2:2:2 complex with AGT and complement C3dg. In the present study we show that during human pregnancy, all of the circulating proMBP exists in covalent complexes, bound to either PAPP-A or AGT. We also show that the proMBP-AGT complex constitutes the major fraction of circulating HMW (high-molecular weight) AGT in late pregnancy, and that this complex is able to further associate with complement C3 derivatives post-sampling. Clearance experiments in mice suggest that complement C3-based complexes are removed faster from the circulation compared to monomeric AGT and the proMBP-AGT complex. Furthermore, we have used recombinant proteins to analyse the formation of the proMBP-PAPP-A and the proMBP-AGT complexes, and we demonstrate that they are competing reactions, depending on the same cysteine residue of proMBP, but differentially on the redox potential, potentially important for the relative amounts of the complexes in vivo. These findings may be important physiologically, since the biochemical properties of the proteins change as a consequence of complex formation.

  13. MART-1 peptide vaccination plus IMP321 (LAG-3Ig fusion protein) in patients receiving autologous PBMCs after lymphodepletion: results of a Phase I trial

    PubMed Central

    2014-01-01

    Background Immunotherapy offers a promising novel approach for the treatment of cancer and both adoptive T-cell transfer and immune modulation lead to regression of advanced melanoma. However, the potential synergy between these two strategies remains unclear. Methods We investigated in 12 patients with advanced stage IV melanoma the effect of multiple MART-1 analog peptide vaccinations with (n = 6) or without (n = 6) IMP321 (LAG-3Ig fusion protein) as an adjuvant in combination with lymphodepleting chemotherapy and adoptive transfer of autologous PBMCs at day (D) 0 (Trial registration No: NCT00324623). All patients were selected on the basis of ex vivo detectable MART-1-specific CD8 T-cell responses and immunized at D0, 8, 15, 22, 28, 52, and 74 post-reinfusion. Results After immunization, a significant expansion of MART-1-specific CD8 T cells was measured in 83% (n = 5/6) and 17% (n = 1/6) of patients from the IMP321 and control groups, respectively (P < 0.02). Compared to the control group, the mean fold increase of MART-1-specific CD8 T cells in the IMP321 group was respectively >2-, >4- and >6-fold higher at D15, D30 and D60 (P < 0.02). Long-lasting MART-1-specific CD8 T-cell responses were significantly associated with IMP321 (P < 0.02). At the peak of the response, MART-1-specific CD8 T cells contained higher proportions of effector (CCR7− CD45RA+/−) cells in the IMP321 group (P < 0.02) and showed no sign of exhaustion (i.e. were mostly PD1−CD160−TIM3−LAG3−2B4+/−). Moreover, IMP321 was associated with a significantly reduced expansion of regulatory T cells (P < 0.04); consistently, we observed a negative correlation between the relative expansion of MART-1-specific CD8 T cells and of regulatory T cells. Finally, although there were no confirmed responses as per RECIST criteria, a transient, 30-day partial response was observed in a patient from the IMP321 group. Conclusions Vaccination with IMP321 as an

  14. Research Results

    NASA Astrophysics Data System (ADS)

    2011-12-01

    Research on Global Carbon Emission and Sequestration NSFC Funded Project Made Significant Progress in Quantum Dynamics Functional Human Blood Protein Obtained from Rice How Giant Pandas Thrive on a Bamboo Diet New Evidence of Interpersonal Violence from 129,000 Years Ago Found in China Aptamer-Mediated Efficient Capture and Release of T Lymphocytes on Nanostructured Surfaces BGI Study Results on Resequencing 50 Accessions of Rice Cast New Light on Molecular Breeding BGI Reports Study Results on Frequent Mutation of Genes Encoding UMPP Components in Kidney Cancer Research on Habitat Shift Promoting Species Diversification

  15. Novel Cell-Ess ® supplement used as a feed or as an initial boost to CHO serum free media results in a significant increase in protein yield and production.

    PubMed

    Elhofy, Adam

    2016-01-01

    Many metrics, including metabolic profiles, have been used to analyze cell health and optimize productivity. In this study, we investigated the ability of a lipid supplement to increase protein yield. At a concentration of 1% (v/v) the lipid supplement caused a significant increase in protein titer (1118 ± 65.4 ng 10(5) cells(- 1) days(- 1)) when compared to cultures grown in the absence of supplementation (819.3 ± 38.1 ng 10(5) cells(- 1) days(- 1); p < 0.05). This equated to a 37% increase in productivity. Furthermore, metabolic profiles of ammonia, glutamate, lactate, and glucose were not significantly altered by the polar lipid supplement. In a separate set of experiments, using the supplement as a feed resulted in 2 notable effects. The first was a 25% increase in protein titer. The second was an extension of peak protein production from 1 day to 2 days. These results suggest that lipid supplementation is a promising avenue for enhancing protein production. In addition, our results also suggest that an increase in protein production may not necessarily require a change in the metabolic state of the cells. PMID:27594979

  16. A rhizobium selenitireducens protein showing selenite reductase activity

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Biobarriers remove, via precipitation, the metalloid selenite (SeO3–2) from groundwater; a process that involves the biological reduction of soluble SeO3–2 to insoluble elemental red selenium (Se0). The enzymes associated with this reduction process are poorly understood. In Rhizobium selenitiredu...

  17. Male-specific beta-cell dysfunction and diabetes resulting from increased expression of a syngeneic MHC class I protein in the pancreata of transgenic mice.

    PubMed

    Martin, L; Keller, G A; Liggitt, D; Oakley, H; Pitts-Meek, S L; Siegel, M W; Terrell, T; Stewart, T A

    1990-12-01

    It is well established that insulin-dependent diabetes (IDDM) is an autoimmune disease with a strong genetic link to the HLA locus. It is less well understood, however, how the destruction of the insulin-producing beta cells is effected and why neighboring non-beta islet cells are spared. Also incompletely explained are the observations that, unlike other autoimmune diseases such as multiple sclerosis, IDDM does not preferentially affect females, the incidence of the disease is highest among young adults, and there are temporal correlations between the onset of the disease and emotional trauma. We have addressed some of these questions by using transgenic mice that constitutively express the MHC class I antigen Dd in the beta cells of the pancreas. Although both male and female Ins.Dd mice expressed equivalent amounts of the Dd protein only the males developed diabetes. The diabetes in the males could be reversed by castration, and the normoglycemic females became diabetic following either ovariectomy and the implantation of a slow-release pellet containing testosterone or the inclusion of dexamethasone in the drinking water. In contrast, transgenic mice that expressed the herpes simplex virus type 1 glycoprotein D in the pancreatic beta cells were normoglycemic and showed no obvious histopathological consequences. The observation that the beta-cell dysfunction by the increased expression of the MHC class I protein Dd cannot be induced by the herpes viral protein suggests that the cellular damage is related to a specific structure or function of the MHC proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

  18. Numerical impact simulation of gradually increased kinetic energy transfer has the potential to break up folded protein structures resulting in cytotoxic brain tissue edema.

    PubMed

    von Holst, Hans; Li, Xiaogai

    2013-07-01

    Although the consequences of traumatic brain injury (TBI) and its treatment have been improved, there is still a substantial lack of understanding the mechanisms. Numerical simulation of the impact can throw further lights on site and mechanism of action. A finite element model of the human head and brain tissue was used to simulate TBI. The consequences of gradually increased kinetic energy transfer was analyzed by evaluating the impact intracranial pressure (ICP), strain level, and their potential influences on binding forces in folded protein structures. The gradually increased kinetic energy was found to have the potential to break apart bonds of Van der Waals in all impacts and hydrogen bonds at simulated impacts from 6 m/s and higher, thereby superseding the energy in folded protein structures. Further, impacts below 6 m/s showed none or very slight increase in impact ICP and strain levels, whereas impacts of 6 m/s or higher showed a gradual increase of the impact ICP and strain levels reaching over 1000 KPa and over 30%, respectively. The present simulation study shows that the free kinetic energy transfer, impact ICP, and strain levels all have the potential to initiate cytotoxic brain tissue edema by unfolding protein structures. The definition of mild, moderate, and severe TBI should thus be looked upon as the same condition and separated only by a gradual severity of impact.

  19. Interactive intoxicating and ameliorating effects of tannic acid, aluminum (Al3+), copper (Cu2+), and selenate (SeO42-) in wheat roots. A descriptive and mathematical assessment

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Tannic acids and tannins are polyphenolic compounds produced by plants and are important components of soil and water organic matter. Tannic acids and tannins form complexes with proteins, metals, and soil particulate matter and perform several physiological and ecological functions. The tannic ac...

  20. First-principles study of the magnetic ground state and magnetization process of the kagome francisites Cu3Bi (SeO3)2 O2X (X =Cl ,Br )

    NASA Astrophysics Data System (ADS)

    Nikolaev, S. A.; Mazurenko, V. V.; Tsirlin, A. A.; Mazurenko, V. G.

    2016-10-01

    We explore the magnetic behavior of the kagome francisites Cu3Bi (SeO3)2 O2X (X =Cl ,Br ) by using first-principles electronic structure calculations. To this end, we propose an approach based on the effective Hubbard model in the Wannier functions basis constructed on the level of local-density approximation. The ground-state spin configuration is determined by a mean-field Hartree-Fock solution of the Hubbard model both in zero magnetic field and in applied magnetic fields. Additionally, parameters of an effective spin Hamiltonian are obtained by taking into account hybridization effects and spin-orbit coupling. We show that only the former approach based on the Hartree-Fock approximation allows for a complete description of the anisotropic magnetization process. While our calculations confirm that the canted zero-field ground state arises from a competition between ferromagnetic nearest-neighbor and antiferromagnetic next-nearest-neighbor couplings in the kagome planes, weaker anisotropic terms are crucial for fixing spin directions and for the strong anisotropy of the magnetization. We show that the Hartree-Fock solution of an electronic Hamiltonian is a viable alternative to the analysis of effective spin Hamiltonians when magnetic ground states and their evolution in external field are concerned.

  1. Rats fed soy protein isolate (SPI) have impaired hepatic CYP1A1 induction by polycyclic aromatic hydrocarbons as a result of interference with aryl hydrocarbon receptor signaling.

    PubMed

    Singhal, Rohit; Badger, Thomas M; Ronis, Martin J

    2008-03-01

    Consumption of soy diets has been found to reduce cancer incidence in animals and is associated with reduced cancer risk in humans. Previously, we have demonstrated that female Sprague-Dawley rats fed purified AIN-93G diets with soy protein isolate (SPI) as the sole protein source had reduced CYP1A1 induction and basal aryl hydrocarbon receptor (AhR) levels relative to those fed the same diet containing casein (CAS). In the present study, the molecular mechanisms underlying reduced AhR expression have been studied. The SPI-effect on AhR was not observed after feeding diets containing the purified soy isoflavones genistein or daidzein. Rat hepatoma FGC-4 cells were treated with the serum obtained from rats fed CAS- or SPI-containing diets. Reduced AhR levels (P<0.05) were observed after 24 h exposure to SPI-serum without any changes in the overall expression of chaperone proteins--HSP90 and XAP2. SPI-serum-stimulated AhR degradation was inhibited by treating the cells with the proteasome inhibitor, MG132, and was observed to be preceded by ubiquitination of the receptor. A reduced association of XAP2 with the immunoprecipitated AhR complex was observed. SPI-serum-mediated AhR degradation was preceded by nuclear translocation of the receptor. However, the translocated receptor was found to be unable to heterodimerize with ARNT or to bind to XRE elements on the CYP1A1 enhancer. These data suggest that feeding SPI-containing diets antagonizes AhR signaling by a novel mechanism which differs from those established for known AhR antagonists.

  2. Genotypic Variation under Fe Deficiency Results in Rapid Changes in Protein Expressions and Genes Involved in Fe Metabolism and Antioxidant Mechanisms in Tomato Seedlings (Solanum lycopersicum L.)

    PubMed Central

    Muneer, Sowbiya; Jeong, Byoung Ryong

    2015-01-01

    To investigate Fe deficiency tolerance in tomato cultivars, quantification of proteins and genes involved in Fe metabolism and antioxidant mechanisms were performed in “Roggusanmaru” and “Super Doterang”. Fe deficiency (Moderate, low and –Fe) significantly decreased the biomass, total, and apoplastic Fe concentration of “Roggusanmaru”, while a slight variation was observed in “Super Doterang” cultivar. The quantity of important photosynthetic pigments such as total chlorophyll and carotenoid contents significantly decreased in “Roggusanmaru” than “Super Doterang” cultivar. The total protein profile in leaves and roots determines that “Super Doterang” exhibited an optimal tolerance to Fe deficiency compared to “Roggusanmaru” cultivar. A reduction in expression of PSI (photosystem I), PSII (photosystem II) super-complexes and related thylakoid protein contents were detected in “Roggusanmaru” than “Super Doterang” cultivar. Moreover, the relative gene expression of SlPSI and SlPSII were well maintained in “Super Doterang” than “Roggusanmaru” cultivar. The relative expression of genes involved in Fe-transport (SlIRT1 and SlIRT2) and Fe(III) chelates reductase oxidase (SlFRO1) were relatively reduced in “Roggusanmaru”, while increased in “Super Doterang” cultivar under Fe deficient conditions. The H+-ATPase relative gene expression (SlAHA1) in roots were maintained in “Super Doterang” compared to “Roggusanmaru”. Furthermore, the gene expressions involved in antioxidant defense mechanisms (SlSOD, SlAPX and SlCAT) in leaves and roots showed that these genes were highly increased in “Super Doterang”, whereas decreased in “Roggusanmaru” cultivar under Fe deficiency. The present study suggested that “Super Doterang” is better tomato cultivar than “Roggusanmaru” for calcareous soils. PMID:26602920

  3. Rats fed soy protein isolate (SPI) have impaired hepatic CYP1A1 induction by polycyclic aromatic hydrocarbons as a result of interference with aryl hydrocarbon receptor signaling

    SciTech Connect

    Singhal, Rohit; Badger, Thomas M.; Ronis, Martin J.

    2008-03-01

    Consumption of soy diets has been found to reduce cancer incidence in animals and is associated with reduced cancer risk in humans. Previously, we have demonstrated that female Sprague-Dawley rats fed purified AIN-93G diets with soy protein isolate (SPI) as the sole protein source had reduced CYP1A1 induction and basal aryl hydrocarbon receptor (AhR) levels relative to those fed the same diet containing casein (CAS). In the present study, the molecular mechanisms underlying reduced AhR expression have been studied. The SPI-effect on AhR was not observed after feeding diets containing the purified soy isoflavones genistein or daidzein. Rat hepatoma FGC-4 cells were treated with the serum obtained from rats fed CAS- or SPI-containing diets. Reduced AhR levels (P < 0.05) were observed after 24 h exposure to SPI-serum without any changes in the overall expression of chaperone proteins-HSP90 and XAP2. SPI-serum-stimulated AhR degradation was inhibited by treating the cells with the proteasome inhibitor, MG132, and was observed to be preceded by ubiquitination of the receptor. A reduced association of XAP2 with the immunoprecipitated AhR complex was observed. SPI-serum-mediated AhR degradation was preceded by nuclear translocation of the receptor. However, the translocated receptor was found to be unable to heterodimerize with ARNT or to bind to XRE elements on the CYP1A1 enhancer. These data suggest that feeding SPI-containing diets antagonizes AhR signaling by a novel mechanism which differs from those established for known AhR antagonists.

  4. Single Point Mutations Result in the Miss-Sorting of Glut4 to a Novel Membrane Compartment Associated with Stress Granule Proteins

    PubMed Central

    Song, XiaoMei; Lichti, Cheryl F.; Townsend, R. Reid; Mueckler, Mike

    2013-01-01

    Insulin increases cellular glucose uptake and metabolism in the postprandial state by acutely stimulating the translocation of the Glut4 glucose transporter from intracellular membrane compartments to the cell surface in muscle and fat cells. The intracellular targeting of Glut4 is dictated by specific structural motifs within cytoplasmic domains of the transporter. We demonstrate that two leucine residues at the extreme C-terminus of Glut4 are critical components of a motif (IRM, insulin responsive motif) involved in the sorting of the transporter to insulin responsive vesicles in 3T3L1 adipocytes. Light microscopy, immunogold electron microscopy, subcellular fractionation, and sedimentation analysis indicate that mutations in the IRM cause the aberrant targeting of Glut4 to large dispersed membrane vesicles that are not insulin responsive. Proteomic characterization of rapidly and slowly sedimenting membrane vesicles (RSVs and SSVs) that were highly enriched by immunoadsorption for either wild-type Glut4 or an IRM mutant revealed that the major vesicle fraction containing the mutant transporter (IRM-RSVs) possessed a relatively small and highly distinct protein population that was enriched for proteins associated with stress granules. We suggest that the IRM is critical for an early step in the sorting of Glut4 to insulin-responsive subcellular membrane compartments and that IRM mutants are miss-targeted to relatively large, amorphous membrane vesicles that may be involved in a degradation pathway for miss-targeted or miss-folded proteins or represent a transitional membrane compartment that Glut4 traverses en route to insulin responsive storage compartments. PMID:23874650

  5. Neonatal exposure to PFOS and PFOA in mice results in changes in proteins which are important for neuronal growth and synaptogenesis in the developing brain.

    PubMed

    Johansson, Niclas; Eriksson, Per; Viberg, Henrik

    2009-04-01

    Perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) belong to the family of perfluorinated compounds. They are used in industrial and consumer applications, e.g., clothing fabrics, carpets, and food packaging. PFOS and PFOA are present in the environment and are found in dust and human milk, which implies that newborns and toddlers can be directly exposed to these agents during brain development. Recently, we reported that PFOS and PFOA can cause neurobehavioral defects and changes in the cholinergic system, in the adult animal, when given directly to neonatal mice, and thereby showing similarities with other investigated persistent organic pollutants, such as dichloro-diphenyl-trichloroethan, polychlorinated biphenyls, and polybrominated diphenyl ethers (PBDEs). In recent studies, we have also seen that highly brominated PBDEs can affect the levels of proteins that are important for neuronal growth and synaptogenesis in the neonatal mouse brain. The present study shows that a single oral dose of either 21 micromol PFOS or PFOA/kg body weight (11.3 or 8.70 mg), given directly to the neonatal mice on postnatal day 10, significantly increased the levels of CaMKII, GAP-43, and synaptophysin in the hippocampus of the neonatal mouse. Both compounds significantly increased the levels of synaptophysin and tau in cerebral cortex, and PFOA also increased the levels of tau in hippocampus. These proteins are important for normal brain development, and altered levels of these proteins during a critical period of the brain growth spurts could be one of the mechanisms behind earlier reported behavioral defects. PMID:19211617

  6. Differing features of proteins in membranes may result in antioxidant or prooxidant action: opposite effects on lipid peroxidation of alcohol dehydrogenase and albumin in liposomal systems.

    PubMed

    Riedl, A; Shamsi, Z; Anderton, M; Goldfarb, P; Wiseman, A

    1996-02-01

    The influence of 3 thiol-containing compounds, bovine serum albumin (fatty acid free: BSA), glutathione (GSH) and yeast alcohol dehydrogenase (YADH) on lipid peroxidation in multilamellar liposomes, prepared from ox-brain phospholipid, was investigated. Thiol-compounds were added either before liposome formation, or after liposome formation; and their effects compared to a positive control. Bovine serum albumin (BSA), an acidic hydrophilic protein, displays a small, concentration dependent, antioxidant effect when added to preformed liposomes. A much larger antioxidant effect was observed when the BSA was entrapped inside the liposome, by adding BSA just prior to liposome preparation. In contrast, a Zn(2+) containing redox enzyme, YADH, a basic hydrophobic membrane-associating protein, displays a large pro-oxidant effect at much lower concentrations especially when entrapped inside the liposome. This was observed also with GSH; but per mole of -SH, YADH was about 18 times as powerful a pro-oxidant perhaps because of structural changes to the membrane. Oxidized glutathione and N-acetylcysteine were also pro-oxidant (cysteine and cystine showed little effect). Formation of thiyl radicals may occur in the presence of iron ions with these pro-oxidant sulphur-containing compounds. Partial protection against lipid peroxidation was observed with EDTA, desferrioxamine and protoporphyrin (IX), potent iron-chelating agents.

  7. Selection for low or high primary dormancy in Lolium rigidum Gaud seeds results in constitutive differences in stress protein expression and peroxidase activity

    PubMed Central

    Goggin, Danica E.; Powles, Stephen B.; Steadman, Kathryn J.

    2011-01-01

    Seed dormancy in wild Lolium rigidum Gaud (annual ryegrass) populations is highly variable and not well characterized at the biochemical level. To identify some of the determinants of dormancy level in these seeds, the proteomes of subpopulations selected for low and high levels of primary dormancy were compared by two-dimensional polyacrylamide gel electrophoresis of extracts from mature, dry seeds. High-dormancy seeds showed higher expression of small heat shock proteins, enolase, and glyoxalase I than the low-dormancy seeds. The functional relevance of these differences in protein expression was confirmed by the fact that high-dormancy seeds were more tolerant to high temperatures imposed at imbibition and had consistently higher glyoxalase I activity over 0–42 d dark stratification. Higher expression of a putative glutathione peroxidase in low-dormancy seeds was not accompanied by higher activity, but these seeds had a slightly more oxidized glutathione pool and higher total peroxidase activity. Overall, these biochemical and physiological differences suggest that L. rigidum seeds selected for low dormancy are more prepared for rapid germination via peroxidase-mediated cell wall weakening, whilst seeds selected for high dormancy are constitutively prepared to survive environmental stresses, even in the absence of stress during seed development. PMID:20974739

  8. Up-regulation of Translation Eukaryotic Initiation Factor 4E in Nucleophosmin 1 Haploinsufficient Cells Results in Changes in CCAAT Enhancer-binding Protein α Activity

    PubMed Central

    Khanna-Gupta, Arati; Abayasekara, Nirmalee; Levine, Michelle; Sun, Hong; Virgilio, Maria; Nia, Navid; Halene, Stephanie; Sportoletti, Paolo; Jeong, Jee-Yeong; Pandolfi, Pier Paolo; Berliner, Nancy

    2012-01-01

    NPM1 is a ubiquitously expressed nucleolar phosphoprotein, the gene for which maps to chromosome 5q35 in close proximity to a commonly deleted region associated with (del)5q, a type of myelodysplastic syndrome (MDS). This region is also a frequent target of deletions in de novo and therapy-related MDS/acute myeloid leukemia. Previous studies have shown that Npm1+/− mice develop an MDS-like disease that transforms to acute myeloid leukemia over time. To better understand the mechanism by which NPM1 haploinsufficiency causes an MDS phenotype, we generated factor-dependent myeloid cell lines from the bone marrow of Npm1+/+ and Npm1+/− mice and demonstrated compromised neutrophil-specific gene expression in the MNPM1+/− cells. We attribute these observations to increased levels of the shorter, dominant negative leukemogenic isoform (p30) of CCAAT enhancer-binding protein α (C/EBPα). We show that this increase is caused, in part, by elevated levels of the activated translation initiation factor eIF4E, overexpression of which also increases translation of C/EBPαp30 in HEK293 cells. In a positive feedback loop, eIF4E expression is further elevated both at the mRNA and protein levels by C/EBPαp30 but not by the full-length C/EBPαp42. Re-expression of C/EBPαp42 or NPM1 but not C/EBPαp30 in MNPM1+/− cells partially rescues the myeloid phenotype. Our observations suggest that the aberrant feed-forward pathway that keeps eIF4E and C/EBPαp30 elevated in NPM1+/− cells contributes to the MDS phenotype associated with NPM1 deficiency. PMID:22851180

  9. Consumption of a healthy dietary pattern results in significant reductions in C-reactive protein levels in adults: a meta-analysis.

    PubMed

    Neale, E P; Batterham, M J; Tapsell, L C

    2016-05-01

    Consumption of healthy dietary patterns has been associated with reduced risk of cardiovascular disease and metabolic syndrome. Dietary intervention targets disease prevention, so studies increasingly use biomarkers of underlying inflammation and metabolic syndrome progression to examine the diet-health relationship. The extent to which these biomarkers contribute to the body of evidence on healthy dietary patterns is unknown. The aim of this meta-analysis was to determine the effect of healthy dietary patterns on biomarkers associated with adiposity, insulin resistance, and inflammation in adults. A systematic search of Scopus, PubMed, Web of Science, and Cochrane Central Register of Controlled Trials (all years to April 2015) was conducted. Inclusion criteria were randomized controlled trials; effects of dietary patterns assessed on C-reactive protein (CRP), total adiponectin, high-molecular-weight adiponectin, tumor necrosis factor-α, adiponectin:leptin, resistin, or retinol binding protein 4. Random effects meta-analyses were conducted to assess the weighted mean differences in change or final mean values for each outcome. Seventeen studies were included in the review. These reflected research on dietary patterns associated with the Mediterranean diet, Nordic diet, Tibetan diet, and the Dietary Approaches to Stop Hypertension diet. Consumption of a healthy dietary pattern was associated with significant reductions in CRP (weighted mean difference, -0.75 [-1.16, -0.35]; P = .0003). Non-significant changes were found for all other biomarkers. This analysis found evidence for favorable effects of healthy dietary patterns on CRP, with limited evidence for other biomarkers. Future research should include additional randomized controlled trials incorporating a greater range of dietary patterns and biomarkers. PMID:27101757

  10. Sustained augmentation of cardiac alpha1A-adrenergic drive results in pathological remodeling with contractile dysfunction, progressive fibrosis and reactivation of matricellular protein genes.

    PubMed

    Chaulet, H; Lin, F; Guo, J; Owens, W A; Michalicek, J; Kesteven, S H; Guan, Z; Prall, O W; Mearns, B M; Feneley, M P; Steinberg, S F; Graham, R M

    2006-04-01

    We previously reported that transgenic (TG) mice with cardiac-restricted alpha(1A)-adrenergic receptor (alpha(1A)-AR)-overexpression showed enhanced contractility, but no hypertrophy. Since chronic inotropic enhancement may be deleterious, we investigated if long-term, cardiac function and longevity are compromised. alpha(1A)-TG mice, but not their non-TG littermates (NTLs), showed progressive loss of left ventricular (LV) hypercontractility (dP/dt(max): 14,567+/-603 to 11,610+/-915 mmHg/s, P<0.05, A1A1 line: 170-fold overexpression; and 13,625+/-826 to 8322+/-682 mmHg/s, respectively, P<0.05, A1A4 line: 112-fold overexpression, at 2 and 6 months, respectively). Both TG lines developed LV fibrosis, but not LV dilatation or hypertrophy, despite activation of hypertrophic signaling pathways. Microarray and real time RT-PCR analyses revealed activation of matricellular protein genes, including those for thrombospondin 1, connective tissue growth factor and tenascin C, but not transforming growth factor beta1. Life-span was markedly shortened (mean age at death: 155 days, A1A1 line; 224 days, A1A4 line compared with NTLs: >300 days). Telemetric electrocardiography revealed that death in the alpha(1A)-AR TG mice was due to cardiac standstill preceded by a progressive diminution in QRS amplitude, but not by arrhythmias. The QRS changes and sudden death could be mimicked by alpha(1)-AR activation, and reversed preterminally by alpha(1)-AR blockade, suggesting a relationship to stress- or activity-associated catecholamine release. Thus, long-term augmentation of cardiac alpha(1A)-adrenergic drive leads to premature death and progressive LV fibrosis with reactivation of matricellular protein genes. To our knowledge this is the first evidence in vivo for a role of the alpha(1A)-AR in ventricular fibrosis and in pathological cardiac remodeling.

  11. Enhanced expression of dihydrofolate reductase by bovine kidney epithelial cells results in altered cell morphology, IGF-I responsiveness, and IGF binding protein-3 expression.

    PubMed

    Cohick, W S; Clemmons, D R

    1994-10-01

    The kidney epithelial cell line (MDBK) secretes primarily insulin-like growth factor binding protein (IGFBP)-2 under basal conditions, but exposure to forskolin decreases the synthesis of and induces IGFBP-3. Since IGFBP-3 has been shown to both potentiate and inhibit insulin-like growth factor (IGF) bioactivity, MDBK cells were transfected with an expression vector containing bovine IGFBP-3 cDNA and the dihydrofolate reductase (DHFR) gene as a selectable marker, with the goal of obtaining an epithelial cell line which constitutively secreted IGFBP-3. Stable clones which secreted greater than 100 ng/ml of IGFBP-3 were obtained and designated MDBKpMONBP-3. Northern blotting indicated that endogenous IGFBP-3 mRNA, which was undetectable in wild-type (WT) MDBK cells, was expressed in MDBKpMONBP-3 cells while the IGFBP-3 transgene did not appear to be expressed. DHFR mRNA transcripts were also expressed by MDBKp-MONBP-3 cells, whereas these transcripts were not detected in WT MDBK cells, suggesting that gene amplification of DHFR may have allowed cells to survive in methotrexate (MTX) without taking up the expression vector. In addition to the altered pattern of IGFBP-3 secretion, a marked alteration in cell morphology was observed. MDBKpMONBP-3 cells grew in distinct islands and exhibited dome formation (a characteristic of differentiated epithelial cells) whereas the WT cells did not. The alterations in morphology and IGFBP-3 expression were irreversible, since MDBKpMONBP-3 cells failed to revert to the WT phenotype upon removal of MTX and dialyzed serum. Since vectorial secretion of proteins is often associated with epithelial cell differentiation, cells were plated on tissue culture inserts which allowed conditioned media (CM) to be collected from both the apical and basal surfaces of confluent monolayers. Release of IGFBP-2 was approximately equal from apical and basal surfaces in WT MDBK cells. In contrast, release of both IGFBP-2 and IGFBP-3 was greater (3

  12. Serotonin via 5-HT7 receptors activates p38 mitogen-activated protein kinase and protein kinase C epsilon resulting in interleukin-6 synthesis in human U373 MG astrocytoma cells.

    PubMed

    Lieb, Klaus; Biersack, Lisa; Waschbisch, Anne; Orlikowski, Sonja; Akundi, Ravi Shankar; Candelario-Jalil, Eduardo; Hüll, Michael; Fiebich, Bernd L

    2005-05-01

    Serotonin [5-hydroxytryptamine (5-HT)] is a widely distributed neurotransmitter which is involved in neuroimmunomodulatory processes. Previously, it has been demonstrated that 5-HT may induce interleukin (IL)-6 expression in primary rat hippocampal astrocytes. The present study was undertaken to investigate the molecular pathways underlying this induction of IL-6 synthesis. As a model system, we used the human astrocytoma cell line U373 MG, which synthesizes IL-6 upon stimulation with various inducers. 5-HT dose- and time-dependently induced IL-6 protein synthesis. We identified several 5-HT receptors to be expressed on U373 MG cells, including the 5-HT1D, 5-HT2A, 5-HT3 and 5-HT7 receptors. In this report, we show that the 5-HT-induced IL-6 release is mediated by the 5-HT7 receptor based on several agonist/antagonists that were used. 5-HT-induced IL-6 synthesis is inhibited by the partially selective 5-HT7 receptor antagonist, pimozide, and the selective antagonist SB269970. Furthermore, IL-6 synthesis was induced by the 5-HT7 receptor agonist carboxamidotryptamin. In addition, we found p38 MAPKs and protein kinase C (PKC) epsilon to be involved in 5-HT-induced IL-6 synthesis as specific inhibitors of these enzymes (SB202190 and RO-31-8425, respectively) blocked 5-HT-induced IL-6 synthesis. Furthermore, 5-HT mediated the phosphorylation of both p38 MAPK as well as the PKC epsilon isoform. The p42/44 MAPKs, however, were not involved in 5-HT-induced IL-6 synthesis. This study shows, for the first time, a central role of 5-HT7 receptor linked to p38 MAPK and PKC epsilon for the induction of cytokine synthesis in astrocytic cells. PMID:15836614

  13. Targeted disruption of the M(r) 46,000 mannose 6-phosphate receptor gene in mice results in misrouting of lysosomal proteins.

    PubMed Central

    Köster, A; Saftig, P; Matzner, U; von Figura, K; Peters, C; Pohlmann, R

    1993-01-01

    Lysosomal enzymes containing mannose 6-phosphate recognition markers are sorted to lysosomes by mannose 6-phosphate receptors (MPRs). The physiological importance of this targeting mechanism is illustrated by I-cell disease, a fatal lysosomal storage disorder caused by the absence of mannose 6-phosphate residues in lysosomal enzymes. Most mammalian cells express two MPRs. Although the binding specificities, subcellular distribution and expression pattern of the two receptors can be differentiated, their coexpression is not understood. The larger of the two receptors with an M(r) of approximately 300,000 (MPR300), which also binds IGFII, appears to have a dominant role in lysosomal enzyme targeting, while the function of the smaller receptor with an M(r) of 46,000 (MPR46) is less clear. To investigate the in vivo function of the MPR46, we generated MPR46-deficient mice using gene targeting in embryonic stem cells. Reduced intracellular retention of newly synthesized lysosomal proteins in cells from MPR46 -/- mice demonstrated an essential sorting function of MPR46. The phenotype of MPR46 -/- mice was normal, indicating mechanisms that compensate the MPR46 deficiency in vivo. Images PMID:8262064

  14. Placental amino acid transport may be regulated by maternal vitamin D and vitamin D-binding protein: results from the Southampton Women's Survey.

    PubMed

    Cleal, J K; Day, P E; Simner, C L; Barton, S J; Mahon, P A; Inskip, H M; Godfrey, K M; Hanson, M A; Cooper, C; Lewis, R M; Harvey, N C

    2015-06-28

    Both maternal 25-hydroxyvitamin D (25(OH)D) concentrations during pregnancy and placental amino acid transporter gene expression have been associated with development of the offspring in terms of body composition and bone structure. Several amino acid transporter genes have vitamin D response elements in their promoters suggesting the possible linkage of these two mechanisms. We aimed to establish whether maternal 25(OH)D and vitamin D-binding protein (VDBP) levels relate to expression of placental amino acid transporters. RNA was extracted from 102 placental samples collected in the Southampton Women's Survey, and gene expression was analysed using quantitative real-time PCR. Gene expression data were normalised to the geometric mean of three housekeeping genes, and related to maternal factors and childhood body composition. Maternal serum 25(OH)D and VDBP levels were measured by radioimmunoassay. Maternal 25(OH)D and VDBP levels were positively associated with placental expression of specific genes involved in amino acid transport. Maternal 25(OH)D and VDBP concentrations were correlated with the expression of specific placental amino acid transporters, and thus may be involved in the regulation of amino acid transfer to the fetus. The positive correlation of VDBP levels and placental transporter expression suggests that delivery of vitamin D to the placenta may be important. This exploratory study identifies placental amino acid transporters which may be altered in response to modifiable maternal factors and provides a basis for further studies.

  15. Thermodynamic model for the solubility of BaSeO4(cr) in the aqueous Ba2+-SeO42--Na+-H+-OH--H2O system: Extending to high selenate concentrations

    SciTech Connect

    Rai, Dhanpat; Felmy, Andrew R.; Moore, Dean A.; Kitamura, Akira; Yoshikawa, Hideki; Doi, Reisuke; Yoshida, Yasushi

    2014-09-15

    The solubility of Ba(SeO4, SO4) precipitates was determined as a function of the BaSeO4 mole fractions, ranging from 0.0015 to 0.3830, and time with an equilibration period extending to as long as 302 days. Equilibrium/steady state conditions in this system are reached in ≤ 65 days. Pitzer’s ion interaction model was used to calculate solid and aqueous phase activity coefficients. Thermodynamic analyses showed that the data do not satisfy Gibbs-Duhem equation, thereby demonstrating that a single-solid solution phase does not control both the selenate and sulfate concentrations. Our extensive data with log10 [Ba]) ranging from -3.6 to -5.9 mol.kg-1, log10 [SeO4]) ranging from -3.6 to -5.2 mol.kg-1, and log10 [SO4] ranging from -4.0 to -5.3 mol.kg-1 can be explained with the formation of an ideal BaSeO4 solid solution phase that controls the selenium concentrations and a slightly disordered/less-crystalline BaSO4(s) (log10 K0sp = -9.5 instead of -10.05 for barite) that controls the sulfate concentrations. In these experiments the BaSO4 component of the solid solution phase never reaches thermodynamic equilibrium with the aqueous phase. Thermodynamic interpretations of the data show that both the ideal BaSeO4 solid solution phase and less-crystalline BaSO4(s) phase are in equilibrium with each other in the entire range of BaSeO4 mole fractions investigated in this study.

  16. Proteins wriggle.

    PubMed Central

    Cahill, Michael; Cahill, Sean; Cahill, Kevin

    2002-01-01

    We propose an algorithmic strategy for improving the efficiency of Monte Carlo searches for the low-energy states of proteins. Our strategy is motivated by a model of how proteins alter their shapes. In our model, when proteins fold under physiological conditions, their backbone dihedral angles change synchronously in groups of four or more to avoid steric clashes and respect the kinematic conservation laws. They wriggle; they do not thrash. We describe a simple algorithm that can be used to incorporate wriggling in Monte Carlo simulations of protein folding. We have tested this wriggling algorithm against a code in which the dihedral angles are varied independently (thrashing). Our standard of success is the average root-mean-square distance (rmsd) between the alpha-carbons of the folding protein and those of its native structure. After 100,000 Monte Carlo sweeps, the relative decrease in the mean rmsd, as one switches from thrashing to wriggling, rises from 11% for the protein 3LZM with 164 amino acids (aa) to 40% for the protein 1A1S with 313 aa and 47% for the protein 16PK with 415 aa. These results suggest that wriggling is useful and that its utility increases with the size of the protein. One may implement wriggling on a parallel computer or a computer farm. PMID:11964253

  17. A single amino acid change resulting in loss of fluorescence of eGFP in a viral fusion protein confers fitness and growth advantage to the recombinant vesicular stomatitis virus

    SciTech Connect

    Dinh, Phat X.; Panda, Debasis; Das, Phani B.; Das, Subash C.; Das, Anshuman; Pattnaik, Asit K.

    2012-10-25

    Using a recombinant vesicular stomatitis virus encoding eGFP fused in-frame with an essential viral replication protein, the phosphoprotein P, we show that during passage in culture, the virus mutates the nucleotide C289 within eGFP of the fusion protein PeGFP to A or T, resulting in R97S/C amino acid substitution and loss of fluorescence. The resultant non-fluorescent virus exhibits increased fitness and growth advantage over its fluorescent counterpart. The growth advantage of the non-fluorescent virus appears to be due to increased transcription and replication activities of the PeGFP protein carrying the R97S/C substitution. Further, our results show that the R97S/C mutation occurs prior to accumulation of mutations that can result in loss of expression of the gene inserted at the G-L gene junction. These results suggest that fitness gain is more important for the recombinant virus than elimination of expression of the heterologous gene.

  18. The combination of oligo- and polysaccharides and reticulated protein for the control of symptoms in patients with irritable bowel syndrome: Results of a randomised, placebo-controlled, double-blind, parallel group, multicentre clinical trial

    PubMed Central

    Alexea, Octavian; Bacarea, Vlad

    2015-01-01

    Background A medical device containing the film-forming agent reticulated protein and a prebiotic mixture of vegetable oligo- and polysaccharides has been developed, recently receiving European approval as MED class III for the treatment of chronic/functional or recidivant diarrhoea due to different causes including irritable bowel syndrome (IBS). In the present paper, we evaluate a protein preparation containing these components in comparison with placebo in adult patients with diarrhoea-predominant IBS. Methods In a randomised, placebo-controlled, double-blind, parallel group, multicentre clinical trial, patients were randomly assigned to receive the combination of oligo- and polysaccharides and reticulated protein and placebo (four oral tablets/day for 56 days). Demographic, clinical and quality of life characteristics and presence and intensity of abdominal pain and flatulence (seven-point Likert scale) were assessed at three study visits (baseline and at 28 and 56 days). Stool emissions were recorded on the diary card using the seven-point Bristol Stool Scale. Results A total of 128 patients were randomised to receive either tablets containing the combination (n = 63) or placebo (n = 65). Treatment with oligo- and polysaccharides and reticulated protein was safe and well tolerated. A significant improvement in symptoms across the study was observed in patients treated with oligo- and polysaccharides and reticulated protein between visit 2 and visit 3 in abdominal pain (p = 0.0167) and flatulence (p = 0.0373). We also detected a statistically significant increase in the quality of life of patients receiving the active treatment from baseline to visit 3 (p < 0.0001). Conclusions Treatment with oligo- and polysaccharides and reticulated protein is safe, improving IBS symptoms and quality of life of patients with diarrhoea-predominant IBS. PMID:27403313

  19. Vaccination with a Fusion Protein That Introduces HIV-1 Gag Antigen into a Multitrimer CD40L Construct Results in Enhanced CD8+ T Cell Responses and Protection from Viral Challenge by Vaccinia-Gag

    PubMed Central

    Gupta, Sachin; Termini, James M.; Raffa, Francesca N.; Williams, Cindi-Ann; Kornbluth, Richard S.

    2014-01-01

    CD40 ligand (CD40L, CD154) is a membrane protein that is important for the activation of dendritic cells (DCs) and DC-induced CD8+ T cell responses. To be active, CD40L must cluster CD40 receptors on responding cells. To produce a soluble form of CD40L that clusters CD40 receptors necessitates the use of a multitrimer construct. With this in mind, a tripartite fusion protein was made from surfactant protein D (SPD), HIV-1 Gag as a test antigen, and CD40L, where SPD serves as a scaffold for the multitrimer protein complex. This SPD-Gag-CD40L protein activated CD40-bearing cells and bone marrow-derived DCs in vitro. Compared to a plasmid for Gag antigen alone (pGag), DNA vaccination of mice with pSPD-Gag-CD40L induced an increased number of Gag-specific CD8+ T cells with increased avidity for major histocompatibility complex class I-restricted Gag peptide and improved vaccine-induced protection from challenge by vaccinia-Gag virus. The importance of the multitrimeric nature of the complex was shown using a plasmid lacking the N terminus of SPD that produced a single trimer fusion protein. This plasmid, pTrimer-Gag-CD40L, was only weakly active on CD40-bearing cells and did not elicit strong CD8+ T cell responses or improve protection from vaccinia-Gag challenge. An adenovirus 5 (Ad5) vaccine incorporating SPD-Gag-CD40L was much stronger than Ad5 expressing Gag alone (Ad5-Gag) and induced complete protection (i.e., sterilizing immunity) from vaccinia-Gag challenge. Overall, these results show the potential of a new vaccine design in which antigen is introduced into a construct that expresses a multitrimer soluble form of CD40L, leading to strongly protective CD8+ T cell responses. PMID:24227853

  20. Activation of the Ras/Mitogen-Activated Protein Kinase Pathway by Kinase-Defective Epidermal Growth Factor Receptors Results in Cell Survival but Not Proliferation

    PubMed Central

    Walker, Francesca; Kato, Akiko; Gonez, L. Jorge; Hibbs, Margaret L.; Pouliot, Normand; Levitzki, Alexander; Burgess, Antony W.

    1998-01-01

    Signalling by the epidermal growth factor (EGF) receptor (EGFR) has been studied intensively, but for most cell types the analysis is complicated by the fact that EGFR not only homodimerizes but can also form heterodimers with other EGFR family members. Heterodimerization is a particular problem in the study of EGFR mutants, where the true phenotype of the mutants is confounded by the contribution of the heterodimer partner to signal transduction. We have made use of the murine hemopoietic cell line BaF/3, which does not express EGFR family members, to express wild-type (WT) EGFR, three kinase-defective EGFR mutants (V741G, Y740F, and K721R), or a C-terminally truncated EGFR (CT957) and have measured their responses to EGF. We found that under the appropriate conditions EGF can stimulate cell proliferation of BaF/3 cells expressing WT or CT957 EGFRs but not that of cells expressing the kinase-defective mutants. However, EGF promotes the survival of BaF/3 cells expressing either of the kinase-defective receptors (V741G and Y740F), indicating that these receptors can still transmit a survival signal. Analysis of the early signalling events by the WT, V741G, and Y740F mutant EGF receptors indicated that EGF stimulates comparable levels of Shc phosphorylation, Shc–GRB-2 association, and activation of Ras, B-Raf, and Erk-1. Blocking the mitogen-activated protein kinase (MAPK) signalling pathway with the specific inhibitor PD98059 abrogates completely the EGF-dependent survival of cells expressing the kinase-defective EGFR mutants but has no effect on the EGF-dependent proliferation mediated by WT and CT957 EGFRs. Similarly, the Src family kinase inhibitor PP1 abrogates EGF-dependent survival without affecting proliferation. However blocking phosphatidylinositol-3-kinase or JAK-2 kinase with specific inhibitors does arrest growth factor-dependent cell proliferation. Thus, EGFR-mediated mitogenic signalling in BaF/3 cells requires an intact EGFR tyrosine kinase activity

  1. Hyperproduction of alpha-toxin by Staphylococcus aureus results in paradoxically reduced virulence in experimental endocarditis: a host defense role for platelet microbicidal proteins.

    PubMed Central

    Bayer, A S; Ramos, M D; Menzies, B E; Yeaman, M R; Shen, A J; Cheung, A L

    1997-01-01

    Staphylococcal alpha-toxin targets several cell types which are important components of cardiac vegetations in endocarditis, including platelets, erythrocytes, and endothelial cells. We evaluated the in vivo role of Staphylococcus aureus alpha-toxin in experimental endocarditis by using isogenic strains differing in the capacity to produce functional alpha-toxin, including 8325-4 (wild-type strain), DU-1090 (a mutant strain with allelic replacement of the alpha-toxin gene [hla]), DU1090(pH35L) (a mutant strain producing a target cell-binding but nonlytic toxin), DU1090(pDU1212) (a variant of DU1090 carrying the cloned hla gene on a multicopy plasmid), and DU1090(pCL84::hla) (a variant of DU1090 with a single copy of the hla gene cloned into the chromosomal lipase locus). In vitro, wild-type alpha-toxin (from parental strain 8325-4) extensively lysed both erythrocytes and platelets. In contrast, mutant alpha-toxin [from strain DU1090(pH35L)] lysed neither cell type. Following exposure to the wild-type alpha-toxin, platelet lysates were found to contain microbicidal activity against Bacillus subtilis (but not against Micrococcus luteus), as well as against the parental and alpha-toxin variant S. aureus strains noted above. Furthermore, lysate microbicidal activity was heat stable, neutralized by polyanionic filters or compounds, and recoverable from anionic filter membranes by hypertonic saline elution. These characteristics are consistent with those of cationic platelet microbicidal proteins (PMPs). Reverse-phase high-pressure liquid chromatography and polyacrylamide gel electrophoresis confirmed the presence of three distinct PMPs (1, 2, and 3) in platelet lysates. In experimental endocarditis, the two variant staphylococcal strains producing either minimal alpha-toxin or nonlytic alpha-toxin in vitro [strains DU1090 and DU1090(pH35L), respectively] exhibited significantly lower virulence in vivo than the parental strain (decreased intravegetation staphylococcal

  2. A Marine Protein-based Dietary Supplement for Subclinical Hair Thinning/Loss: Results of a Multisite, Double-blind, Placebo-controlled Clinical Trial

    PubMed Central

    Rizer, Ronald L; Stephens, Thomas J; Herndon, James H; Sperber, Brian R; Murphy, James; Ablon, Glynis R

    2015-01-01

    Introduction: Since skin and hair quality are potent vitality signals, and hair growth deficiency can cause significant psychological morbidity. In addition to clearly-defined hair loss disorders, milder forms of hair thinning or hair loss appear to be increasingly common, with a suggestion that sub-optimal diets and stressful lifestyles may be involved. Methods: Here we assess the value of a dietary marine-extract based dietary supplement in premenopausal women with subclinical hair thinning or hair loss conditions. This multi-site, randomized double-blind, placebo-controlled clinical trial was conducted with impact on hair shedding rate and hair fiber diameter (assessed by phototrichogram) as primary end points upon consumption of the oral supplement compared to a placebo. A total of 96 eligible female subjects were enrolled aged 21–55 years of age from Asian, Caucasian, and Hispanic ethnic backgrounds. Results: This study showed that hair shedding was significantly reduced in the first 3–6 months of daily consumption of the oral supplement. Moreover, phototrichogram image analysis revealed a statistically significant increase in the mean vellus-like hair diameter after 6 months of supplement consumption, when compared to the mean vellus-like hair diameters measured at baseline. Discussion: These results support the view that a nutritional supplement approach may be useful for women in this age group to deal with subclinical hair thinning or hair loss conditions, and those components of this marine extract-based oral supplement may be a useful adjunct. PMID:26903744

  3. Impact of adjuvants on CD4(+) T cell and B cell responses to a protein antigen vaccine: Results from a phase II, randomized, multicenter trial.

    PubMed

    Leroux-Roels, Geert; Marchant, Arnaud; Levy, Jack; Van Damme, Pierre; Schwarz, Tino F; Horsmans, Yves; Jilg, Wolfgang; Kremsner, Peter G; Haelterman, Edwige; Clément, Frédéric; Gabor, Julian J; Esen, Meral; Hens, Annick; Carletti, Isabelle; Fissette, Laurence; Tavares Da Silva, Fernanda; Burny, Wivine; Janssens, Michel; Moris, Philippe; Didierlaurent, Arnaud M; Van Der Most, Robbert; Garçon, Nathalie; Van Belle, Pascale; Van Mechelen, Marcelle

    2016-08-01

    Immunogenicity and safety of different adjuvants combined with a model antigen (HBsAg) were compared. Healthy HBV-naïve adults were randomized to receive HBs adjuvanted with alum or Adjuvant Systems AS01B, AS01E, AS03A or AS04 at Days 0 and 30. Different frequencies of HBs-specific CD4+ T cells 14days post dose 2 but similar polyfunctionality profiles were induced by the different adjuvants with frequencies significantly higher in the AS01B and AS01E groups than in the other groups. Antibody concentrations 30days post-dose 2 were significantly higher in AS01B, AS01E and AS03A than in other groups. Limited correlations were observed between HBs-specific CD4+ T cell and antibody responses. Injection site pain was the most common solicited local symptom and was more frequent in AS groups than in alum group. Different adjuvants formulated with the same antigen induced different adaptive immune responses and reactogenicity patterns in healthy naïve adults. The results summary for this study (GSK study number 112115 - NCT# NCT00805389) is available on the GSK Clinical Study Register and can be accessed at www.gsk-clinicalstudyregister.com.

  4. Structure−Activity Relationships in Peptide Modulators of β-Amyloid Protein Aggregation: Variation in α,α-Disubstitution Results in Altered Aggregate Size and Morphology

    PubMed Central

    2010-01-01

    Neuronal cytotoxicity observed in Alzheimer’s disease (AD) is linked to the aggregation of β-amyloid peptide (Aβ) into toxic forms. Increasing evidence points to oligomeric materials as the neurotoxic species, not Aβ fibrils; disruption or inhibition of Aβ self-assembly into oligomeric or fibrillar forms remains a viable therapeutic strategy to reduce Aβ neurotoxicity. We describe the synthesis and characterization of amyloid aggregation mitigating peptides (AAMPs) whose structure is based on the Aβ “hydrophobic core” Aβ17−20, with α,α-disubstituted amino acids (ααAAs) added into this core as potential disrupting agents of fibril self-assembly. The number, positional distribution, and side-chain functionality of ααAAs incorporated into the AAMP sequence were found to influence the resultant aggregate morphology as indicated by ex situ experiments using atomic force microscopy (AFM) and transmission electron microscopy (TEM). For instance, AAMP-5, incorporating a sterically hindered ααAA with a diisobutyl side chain in the core sequence, disrupted Aβ1−40 fibril formation. However, AAMP-6, with a less sterically hindered ααAA with a dipropyl side chain, altered fibril morphology, producing shorter and larger sized fibrils (compared with those of Aβ1−40). Remarkably, ααAA-AAMPs caused disassembly of existing Aβ fibrils to produce either spherical aggregates or protofibrillar structures, suggesting the existence of equilibrium between fibrils and prefibrillar structures. PMID:22778850

  5. Impact of adjuvants on CD4(+) T cell and B cell responses to a protein antigen vaccine: Results from a phase II, randomized, multicenter trial.

    PubMed

    Leroux-Roels, Geert; Marchant, Arnaud; Levy, Jack; Van Damme, Pierre; Schwarz, Tino F; Horsmans, Yves; Jilg, Wolfgang; Kremsner, Peter G; Haelterman, Edwige; Clément, Frédéric; Gabor, Julian J; Esen, Meral; Hens, Annick; Carletti, Isabelle; Fissette, Laurence; Tavares Da Silva, Fernanda; Burny, Wivine; Janssens, Michel; Moris, Philippe; Didierlaurent, Arnaud M; Van Der Most, Robbert; Garçon, Nathalie; Van Belle, Pascale; Van Mechelen, Marcelle

    2016-08-01

    Immunogenicity and safety of different adjuvants combined with a model antigen (HBsAg) were compared. Healthy HBV-naïve adults were randomized to receive HBs adjuvanted with alum or Adjuvant Systems AS01B, AS01E, AS03A or AS04 at Days 0 and 30. Different frequencies of HBs-specific CD4+ T cells 14days post dose 2 but similar polyfunctionality profiles were induced by the different adjuvants with frequencies significantly higher in the AS01B and AS01E groups than in the other groups. Antibody concentrations 30days post-dose 2 were significantly higher in AS01B, AS01E and AS03A than in other groups. Limited correlations were observed between HBs-specific CD4+ T cell and antibody responses. Injection site pain was the most common solicited local symptom and was more frequent in AS groups than in alum group. Different adjuvants formulated with the same antigen induced different adaptive immune responses and reactogenicity patterns in healthy naïve adults. The results summary for this study (GSK study number 112115 - NCT# NCT00805389) is available on the GSK Clinical Study Register and can be accessed at www.gsk-clinicalstudyregister.com. PMID:27236001

  6. Reduced β-lactoglobulin IgE binding upon in vitro digestion as a result of the interaction of the protein with casein glycomacropeptide.

    PubMed

    Martinez, María J; Martos, Gustavo; Molina, Elena; Pilosof, Ana M R

    2016-02-01

    The aim of this work was to evaluate the effect of the presence of casein glycomacropeptide (CMP) on the in vitro digestibility and potential allergenicity of β-lactoglobulin (β-lg)-CMP mixtures. The digestion products were analyzed by RP-HPLC and RP-HPLC-ESI-MS/MS. The potential allergenicity of the digestion products was studied by human IgE binding by inhibition ELISA with serum samples from children with clinical allergic symptoms to β-lg. No differences were observed by HPLC in the mixtures hydrolysates due to CMP-β-lg interactions. RP-HPLC-ESI-MS/MS results showed different peptides occurring in the mixtures hydrolysates. Additionally, it was observed a significant reduction of β-lg IgE binding in the presence of CMP. The disappearance of epitopes in the digested mixtures could explain the lower IgE binding observed in these systems compared to β-lg. It can be concluded that the presence of CMP in products containing β-lg may modify the digestion products that may reduce the potential allergenicity of β-lg.

  7. A Prototype Recombinant-Protein Based Chlamydia pecorum Vaccine Results in Reduced Chlamydial Burden and Less Clinical Disease in Free-Ranging Koalas (Phascolarctos cinereus)

    PubMed Central

    Waugh, Courtney; Khan, Shahneaz Ali; Carver, Scott; Hanger, Jonathan; Loader, Joanne; Polkinghorne, Adam; Beagley, Kenneth; Timms, Peter

    2016-01-01

    Diseases associated with Chlamydia pecorum infection are a major cause of decline in koala populations in Australia. While koalas in care can generally be treated, a vaccine is considered the only option to effectively reduce the threat of infection and disease at the population level. In the current study, we vaccinated 30 free-ranging koalas with a prototype Chlamydia pecorum vaccine consisting of a recombinant chlamydial MOMP adjuvanted with an immune stimulating complex. An additional cohort of 30 animals did not receive any vaccine and acted as comparison controls. Animals accepted into this study were either uninfected (Chlamydia PCR negative) at time of initial vaccination, or infected (C. pecorum positive) at either urogenital (UGT) and/or ocular sites (Oc), but with no clinical signs of chlamydial disease. All koalas were vaccinated / sampled and then re-released into their natural habitat before re-capturing and re-sampling at 6 and 12 months. All vaccinated koalas produced a strong immune response to the vaccine, as indicated by high titres of specific plasma antibodies. The incidence of new infections in vaccinated koalas over the 12-month period post-vaccination was slightly less than koalas in the control group, however, this was not statistically significant. Importantly though, the vaccine was able to significantly reduce the infectious load in animals that were Chlamydia positive at the time of vaccination. This effect was evident at both the Oc and UGT sites and was stronger at 6 months than at 12 months post-vaccination. Finally, the vaccine was also able to reduce the number of animals that progressed to disease during the 12-month period. While the sample sizes were small (statistically speaking), results were nonetheless striking. This study highlights the potential for successful development of a Chlamydia vaccine for koalas in a wild setting. PMID:26756624

  8. A Prototype Recombinant-Protein Based Chlamydia pecorum Vaccine Results in Reduced Chlamydial Burden and Less Clinical Disease in Free-Ranging Koalas (Phascolarctos cinereus).

    PubMed

    Waugh, Courtney; Khan, Shahneaz Ali; Carver, Scott; Hanger, Jonathan; Loader, Joanne; Polkinghorne, Adam; Beagley, Kenneth; Timms, Peter

    2016-01-01

    Diseases associated with Chlamydia pecorum infection are a major cause of decline in koala populations in Australia. While koalas in care can generally be treated, a vaccine is considered the only option to effectively reduce the threat of infection and disease at the population level. In the current study, we vaccinated 30 free-ranging koalas with a prototype Chlamydia pecorum vaccine consisting of a recombinant chlamydial MOMP adjuvanted with an immune stimulating complex. An additional cohort of 30 animals did not receive any vaccine and acted as comparison controls. Animals accepted into this study were either uninfected (Chlamydia PCR negative) at time of initial vaccination, or infected (C. pecorum positive) at either urogenital (UGT) and/or ocular sites (Oc), but with no clinical signs of chlamydial disease. All koalas were vaccinated/sampled and then re-released into their natural habitat before re-capturing and re-sampling at 6 and 12 months. All vaccinated koalas produced a strong immune response to the vaccine, as indicated by high titres of specific plasma antibodies. The incidence of new infections in vaccinated koalas over the 12-month period post-vaccination was slightly less than koalas in the control group, however, this was not statistically significant. Importantly though, the vaccine was able to significantly reduce the infectious load in animals that were Chlamydia positive at the time of vaccination. This effect was evident at both the Oc and UGT sites and was stronger at 6 months than at 12 months post-vaccination. Finally, the vaccine was also able to reduce the number of animals that progressed to disease during the 12-month period. While the sample sizes were small (statistically speaking), results were nonetheless striking. This study highlights the potential for successful development of a Chlamydia vaccine for koalas in a wild setting. PMID:26756624

  9. A Prototype Recombinant-Protein Based Chlamydia pecorum Vaccine Results in Reduced Chlamydial Burden and Less Clinical Disease in Free-Ranging Koalas (Phascolarctos cinereus).

    PubMed

    Waugh, Courtney; Khan, Shahneaz Ali; Carver, Scott; Hanger, Jonathan; Loader, Joanne; Polkinghorne, Adam; Beagley, Kenneth; Timms, Peter

    2016-01-01

    Diseases associated with Chlamydia pecorum infection are a major cause of decline in koala populations in Australia. While koalas in care can generally be treated, a vaccine is considered the only option to effectively reduce the threat of infection and disease at the population level. In the current study, we vaccinated 30 free-ranging koalas with a prototype Chlamydia pecorum vaccine consisting of a recombinant chlamydial MOMP adjuvanted with an immune stimulating complex. An additional cohort of 30 animals did not receive any vaccine and acted as comparison controls. Animals accepted into this study were either uninfected (Chlamydia PCR negative) at time of initial vaccination, or infected (C. pecorum positive) at either urogenital (UGT) and/or ocular sites (Oc), but with no clinical signs of chlamydial disease. All koalas were vaccinated/sampled and then re-released into their natural habitat before re-capturing and re-sampling at 6 and 12 months. All vaccinated koalas produced a strong immune response to the vaccine, as indicated by high titres of specific plasma antibodies. The incidence of new infections in vaccinated koalas over the 12-month period post-vaccination was slightly less than koalas in the control group, however, this was not statistically significant. Importantly though, the vaccine was able to significantly reduce the infectious load in animals that were Chlamydia positive at the time of vaccination. This effect was evident at both the Oc and UGT sites and was stronger at 6 months than at 12 months post-vaccination. Finally, the vaccine was also able to reduce the number of animals that progressed to disease during the 12-month period. While the sample sizes were small (statistically speaking), results were nonetheless striking. This study highlights the potential for successful development of a Chlamydia vaccine for koalas in a wild setting.

  10. A single Alal 39-to-Glu substitution in the Renibacterium salmoninarum virulence-associated protein p57 results in antigenic variation and is associated with enhanced p57 binding to Chinook salmon leukocytes

    USGS Publications Warehouse

    Wiens, Gregory D.; Pascho, Ron; Winton, James R.

    2002-01-01

    The gram-positive bacterium Renibacterium salmoninarum produces relatively large amounts of a 57-kDa protein (p57) implicated in the pathogenesis of salmonid bacterial kidney disease. Antigenic variation in p57 was identified by using monoclonal antibody 4C11, which exhibited severely decreased binding to R. salmoninarum strain 684 p57 and bound robustly to the p57 proteins of seven other R. salmoninarum strains. This difference in binding was not due to alterations in p57 synthesis, secretion, or bacterial cell association. The molecular basis of the 4C11 epitope loss was determined by amplifying and sequencing the two identical genes encoding p57, msa1 and msa2. The 5′ and coding sequences of the 684 msa1 and msa2 genes were identical to those of the ATCC 33209 msa1and msa2 genes except for a single C-to-A nucleotide mutation. This mutation was identified in both the msa1 and msa2 genes of strain 684 and resulted in an Ala139-to-Glu substitution in the amino-terminal region of p57. We examined whether this mutation in p57 altered salmonid leukocyte and rabbit erythrocyte binding activities. R. salmoninarum strain 684 extracellular protein exhibited a twofold increase in agglutinating activity for chinook salmon leukocytes and rabbit erythrocytes compared to the activity of the ATCC 33209 extracellular protein. A specific and quantitative p57 binding assay confirmed the increased binding activity of 684 p57. Monoclonal antibody 4C11 blocked the agglutinating activity of the ATCC 33209 extracellular protein but not the agglutinating activity of the 684 extracellular protein. These results indicate that the Ala139-to-Glu substitution altered immune recognition and was associated with enhanced biological activity of R. salmoninarum 684 p57.

  11. Increasing the metabolizable protein supply enhanced growth performance and led to variable results on innate and humoral immune response of preconditioning beef steers.

    PubMed

    Moriel, P; Artioli, L F A; Poore, M H; Confer, A W; Marques, R S; Cooke, R F

    2015-09-01

    . 100% and 115% MP steers (3.1, 2.4, and 2.5 ± 0.21 log, respectively). Preconditioning MP supply did not affect ( ≥ 0.26) ubsequent finishing growth performance and carcass characteristics. Thus, increasing MP supply from 85% to 115% of daily requirement of preconditioning beef steers had variable results on innate and humoral immune response and enhanced growth performance during a 42-d preconditioning period without affecting carcass characteristics at slaughter. PMID:26440347

  12. Cytotoxicity and Genotoxicity Assessment of Sandalwood Essential Oil in Human Breast Cell Lines MCF-7 and MCF-10A.

    PubMed

    Ortiz, Carmen; Morales, Luisa; Sastre, Miguel; Haskins, William E; Matta, Jaime

    2016-01-01

    Sandalwood essential oil (SEO) is extracted from Santalum trees. Although α-santalol, a main constituent of SEO, has been studied as a chemopreventive agent, the genotoxic activity of the whole oil in human breast cell lines is still unknown. The main objective of this study was to assess the cytotoxic and genotoxic effects of SEO in breast adenocarcinoma (MCF-7) and nontumorigenic breast epithelial (MCF-10A) cells. Proteins associated with SEO genotoxicity were identified using a proteomics approach. Commercially available, high-purity, GC/MS characterized SEO was used to perform the experiments. The main constituents reported in the oil were (Z)-α-santalol (25.34%), (Z)-nuciferol (18.34%), (E)-β-santalol (10.97%), and (E)-nuciferol (10.46%). Upon exposure to SEO (2-8 μg/mL) for 24 hours, cell proliferation was determined by the MTT assay. Alkaline and neutral comet assays were used to assess genotoxicity. SEO exposure induced single- and double-strand breaks selectively in the DNA of MCF-7 cells. Quantitative LC/MS-based proteomics allowed identification of candidate proteins involved in this response: Ku70 (p = 1.37E - 2), Ku80 (p = 5.8E - 3), EPHX1 (p = 3.3E - 3), and 14-3-3ζ (p = 4.0E - 4). These results provide the first evidence that SEO is genotoxic and capable of inducing DNA single- and double-strand breaks in MCF-7 cells. PMID:27293457

  13. Cytotoxicity and Genotoxicity Assessment of Sandalwood Essential Oil in Human Breast Cell Lines MCF-7 and MCF-10A

    PubMed Central

    Ortiz, Carmen; Morales, Luisa; Sastre, Miguel; Haskins, William E.; Matta, Jaime

    2016-01-01

    Sandalwood essential oil (SEO) is extracted from Santalum trees. Although α-santalol, a main constituent of SEO, has been studied as a chemopreventive agent, the genotoxic activity of the whole oil in human breast cell lines is still unknown. The main objective of this study was to assess the cytotoxic and genotoxic effects of SEO in breast adenocarcinoma (MCF-7) and nontumorigenic breast epithelial (MCF-10A) cells. Proteins associated with SEO genotoxicity were identified using a proteomics approach. Commercially available, high-purity, GC/MS characterized SEO was used to perform the experiments. The main constituents reported in the oil were (Z)-α-santalol (25.34%), (Z)-nuciferol (18.34%), (E)-β-santalol (10.97%), and (E)-nuciferol (10.46%). Upon exposure to SEO (2–8 μg/mL) for 24 hours, cell proliferation was determined by the MTT assay. Alkaline and neutral comet assays were used to assess genotoxicity. SEO exposure induced single- and double-strand breaks selectively in the DNA of MCF-7 cells. Quantitative LC/MS-based proteomics allowed identification of candidate proteins involved in this response: Ku70 (p = 1.37E − 2), Ku80 (p = 5.8E − 3), EPHX1 (p = 3.3E − 3), and 14-3-3ζ (p = 4.0E − 4). These results provide the first evidence that SEO is genotoxic and capable of inducing DNA single- and double-strand breaks in MCF-7 cells. PMID:27293457

  14. Toxicity of selenium (Na sub 2 SeO sub 3 ) and mercury (HgCl sub 2 ) on the planarian Dugesia gonocephala

    SciTech Connect

    Congiu, A.M.; Casu, S.; Ugazio, G. )

    1989-10-01

    The toxicity of selenium (Na{sub 2}SeO{sub 3}) and mercury (HgCl{sub 2}) was determined by using a freshwater planarian which is particularly sensitive to pollution, and belongs to a fissiparous breed of Dugesia gonocephala. The mortality and fissiparity frequency of the subjects were studied. They were exposed to intense treatments (48 hours) or for medium to long periods of time (21 days) to either the single compounds or a combination of both, and were fed or fasting. The lethal effect of sodium selenite is correlated to the food intake, whereas the toxicity of mercurous chloride is probably the result of a fixative effect which does not depend on feeding. The 21-day treatment with the first compound has a non-negligible lethal effect which is probably due to an accumulation phenomenon. At doses where an antioxidant effect prevails, fissiparity is stimulated. On the other hand, the second compound reduces reproduction frequency to half the base values. Compared to the Paracentrotus lividus, the Dugesia gonocephala offers various advantages concerning toxicological experiments; besides being easier to handle in the laboratory, it is available all year round and is not subject to seasonal cycles. It is also more susceptible to the toxic effect of mercury, which is a common and highly toxic pollutant, than the sea urchin.

  15. Rhizobium selenitireducens proteins involved in the reduction of selenite to elemental selenium

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Microbial based bioremediation barriers can remove the metalloid selenite (SeO3–2) from flowing groundwater. The organisms associated with the process include microorganisms from within the bacterial and archaeal domains that can reduce soluble SeO3–2 to the insoluble and reddish-colored elemental ...

  16. Constitutive or seed-specific overexpression of Arabidopsis G-protein γ subunit 3 (AGG3) results in increased seed and oil production and improved stress tolerance in Camelina sativa.

    PubMed

    Roy Choudhury, Swarup; Riesselman, Adam J; Pandey, Sona

    2014-01-01

    Heterotrimeric G-proteins consisting of Gα, Gβ and Gγ subunits play an integral role in mediating multiple signalling pathways in plants. A novel, recently identified plant-specific Gγ protein, AGG3, has been proposed to be an important regulator of organ size and mediator of stress responses in Arabidopsis, whereas its potential homologs in rice are major quantitative trait loci for seed size and panicle branching. To evaluate the role of AGG3 towards seed and oil yield improvement, the gene was overexpressed in Camelina sativa, an oilseed crop of the Brassicaceae family. Analysis of multiple homozygous T4 transgenic Camelina lines showed that constitutive overexpression of AGG3 resulted in faster vegetative as well as reproductive growth accompanied by an increase in photosynthetic efficiency. Moreover, when expressed constitutively or specifically in seed tissue, AGG3 was found to increase seed size, seed mass and seed number per plant by 15%-40%, effectively resulting in significantly higher oil yield per plant. AGG3 overexpressing Camelina plants also exhibited improved stress tolerance. These observations draw a strong link between the roles of AGG3 in regulating two critical yield parameters, seed traits and plant stress responses, and reveal an effective biotechnological tool to dramatically increase yield in agricultural crops. PMID:24102738

  17. Nanotechnologies in protein microarrays.

    PubMed

    Krizkova, Sona; Heger, Zbynek; Zalewska, Marta; Moulick, Amitava; Adam, Vojtech; Kizek, Rene

    2015-01-01

    Protein microarray technology became an important research tool for study and detection of proteins, protein-protein interactions and a number of other applications. The utilization of nanoparticle-based materials and nanotechnology-based techniques for immobilization allows us not only to extend the surface for biomolecule immobilization resulting in enhanced substrate binding properties, decreased background signals and enhanced reporter systems for more sensitive assays. Generally in contemporarily developed microarray systems, multiple nanotechnology-based techniques are combined. In this review, applications of nanoparticles and nanotechnologies in creating protein microarrays, proteins immobilization and detection are summarized. We anticipate that advanced nanotechnologies can be exploited to expand promising fields of proteins identification, monitoring of protein-protein or drug-protein interactions, or proteins structures. PMID:26039143

  18. Effects of lactulose and lactitol on protein digestion and metabolism in conventional and germ free animal models: relevance of the results to their use in the treatment of portosystemic encephalopathy.

    PubMed Central

    Bird, S P; Hewitt, D; Ratcliffe, B; Gurr, M I

    1990-01-01

    Protein digestion and metabolism have been studied in laboratory rats and miniature pigs to investigate the mechanisms of action of lactulose and lactitol when used in the treatment of patients with portosystemic encephalopathy. Lactulose (beta-D-galactopyranosyl-(1----4)-beta-D-fructofuranose) and lactitol (beta-D-galactopyranosyl-(1----4)-D-glucitol) increased the excretion of nitrogenous material in the faeces and decreased nitrogen excretion in the urine in a similar degree to that reported for human patients. In studies with germ free rats given lactulose no such effect was observed, suggesting that, for lactulose at least, these effects are mediated by the gut flora. Measurement of the alpha-, epsilon-diaminopimelic acid content of the faeces confirmed that the enhancement of faecal nitrogen was due to an increased contribution from bacteria. The similarity in the results for lactulose and lactitol suggests that, from the perspective of protein metabolism, lactitol acts in a similar way to lactulose in the treatment of portosystemic encephalopathy. PMID:2265782

  19. Deletion of 43 amino acids in the NH2-terminal half of the large tumor antigen of simian virus 40 results in a non-karyophilic protein capable of transforming established cells.

    PubMed Central

    Fischer-Fantuzzi, L; Vesco, C

    1985-01-01

    We have characterized a simian virus 40 (SV40) mutant, derived from the viral DNA insertion present in simian cell transformants, which carries a deletion affecting the NH2-terminal region of the SV40 large tumor antigen. This mutant protein is 6% smaller than normal, has lost the typical nuclear localization of the SV40 large tumor antigen, and accumulates in the cytoplasm. The deletion begins at nucleotide position 4490 of the SV40 DNA and ends in-frame at nucleotide position 4362. The missing 43 amino acids begin with proline-110 and end with serine-152 of the predicted sequence; they include a cluster of basic residues, presumably important for the viral origin-DNA binding, and most of the phosphorylation sites present in the NH2-terminal half of the molecule. The protein can still be phosphorylated considerably in vivo. This mutant viral genome is replication-defective but has conserved the competence to transform established cells, such as NIH/3T3 cells. Transfection of cloned mutant DNA into such cells resulted in the production of full transformants. Full transformants were not produced in similar transfections carried out in primary rat embryo fibroblasts, although some primary transfectants expressing the non-karyophilic large tumor antigen might be considered minimally transformed. Images PMID:2984671

  20. Growth hormone deficiency in 'little' mice results in aberrant body composition, reduced insulin-like growth factor-I and insulin-like growth factor-binding protein-3 (IGFBP-3), but does not affect IGFBP-2, -1 or -4.

    PubMed

    Donahue, L R; Beamer, W G

    1993-01-01

    Although GH is known to regulate somatic growth during development, its role in regulating adult body composition is less well defined. The effects of GH on individual body compartments--water, fat, protein and mineral--are achieved both by the action of GH and by a GH-induced hormone, insulin-like growth factor-I (IGF-I). We used a genetic model of GH deficiency, the 'little' (gene symbol lit) mouse, to determine the GH regulation of IGF-I and its insulin-like growth factor-binding proteins (IGFBPs) and to define the interaction between these hormones and each body compartment in adults. Our results showed that GH-deficient lit/lit mice had reduced levels of serum IGF-I (range 38-130 micrograms/l) compared with normal lit/+ littermates (range 432-567 micrograms/l) between 2 and 52 weeks of age. The lit/lit mice did not experience the fivefold increase in IGF-I between 2 and 4 weeks of age that was seen in lit/+ mice. In lit/lit serum, overall binding of 125I-labelled IGF-I to the four IGFBPs was reduced, solely in response to a reduced amount of IGFBP-3. No overall differences were found between lit/lit and lit/+ mice in the binding of 125I-labelled IGF-I to IGFBP-2, -1 or -4. Age-related declines in IGF-I and IGFBPs were seen in lit/lit mice. However, adult levels of IGF-I were maintained in lit/+ mice to at least 52 weeks of age, as were levels of IGFBP-1 and -4, while IGFBP-3 and -2 declined with age. With respect to body composition, comparison of lit/lit with lit/+ mice showed that the lit/lit mice were characterized by abnormally large adipose tissue stores and reduced body water, protein and mineral from 2 weeks onward. These changes occurred despite normal energy intake in lit/lit mice up to 52 weeks of age, indicating that neither undernutrition nor hyperphagia is characteristic of this GH-induced model of obesity. Furthermore, lit/lit males accrued more body fat beginning at an earlier age than lit/lit females. With advancing age, the per cent body fat

  1. Protein Crystallization

    NASA Technical Reports Server (NTRS)

    Chernov, Alexander A.

    2005-01-01

    Nucleation, growth and perfection of protein crystals will be overviewed along with crystal mechanical properties. The knowledge is based on experiments using optical and force crystals behave similar to inorganic crystals, though with a difference in orders of magnitude in growing parameters. For example, the low incorporation rate of large biomolecules requires up to 100 times larger supersaturation to grow protein, rather than inorganic crystals. Nucleation is often poorly reproducible, partly because of turbulence accompanying the mixing of precipitant with protein solution. Light scattering reveals fluctuations of molecular cluster size, its growth, surface energies and increased clustering as protein ages. Growth most often occurs layer-by-layer resulting in faceted crystals. New molecular layer on crystal face is terminated by a step where molecular incorporation occurs. Quantitative data on the incorporation rate will be discussed. Rounded crystals with molecularly disordered interfaces will be explained. Defects in crystals compromise the x-ray diffraction resolution crucially needed to find the 3D atomic structure of biomolecules. The defects are immobile so that birth defects stay forever. All lattice defects known for inorganics are revealed in protein crystals. Contribution of molecular conformations to lattice disorder is important, but not studied. This contribution may be enhanced by stress field from other defects. Homologous impurities (e.g., dimers, acetylated molecules) are trapped more willingly by a growing crystal than foreign protein impurities. The trapped impurities induce internal stress eliminated in crystals exceeding a critical size (part of mni for ferritin, lysozyme). Lesser impurities are trapped from stagnant, as compared to the flowing, solution. Freezing may induce much more defects unless quickly amorphysizing intracrystalline water.

  2. Overexpression of the Candida albicans ALA1 Gene in Saccharomyces cerevisiae Results in Aggregation following Attachment of Yeast Cells to Extracellular Matrix Proteins, Adherence Properties Similar to Those of Candida albicans

    PubMed Central

    Gaur, Nand K.; Klotz, Stephen A.; Henderson, Ramona L.

    1999-01-01

    Candida albicans maintains a commensal relationship with human hosts, probably by adhering to mucosal tissue in a variety of physiological conditions. We show that adherence due to the C. albicans gene ALA1 when transformed into Saccharomyces cerevisiae, is comprised of two sequential steps. Initially, C. albicans rapidly attaches to extracellular matrix (ECM) protein-coated magnetic beads in small numbers (the attachment phase). This is followed by a relatively slower step in which cell-to-cell interactions predominate (the aggregation phase). Neither of these phases is observed in S. cerevisiae. However, expression of the C. albicans ALA1 gene from a low-copy vector causes S. cerevisiae transformants to attach to ECM-coated magnetic beads without appreciable aggregation. Expression of ALA1 from a high-copy vector results in both attachment and aggregation. Moreover, transcriptional fusion of ALA1 with the galactose-inducible promoters GALS, GALL, and GAL1, allowing for low, moderate, and high levels of inducible transcription, respectively, causes attachment and aggregation that correlates with the strength of the GAL promoter. The adherence of C. albicans and S. cerevisiae overexpressing ALA1 to a number of protein ligands occurs over a broad pH range, is resistant to shear forces generated by vortexing, and is unaffected by the presence of sugars, high salt levels, free ligands, or detergents. Adherence is, however, inhibited by agents that disrupt hydrogen bonds. The similarities in the adherence and aggregation properties of C. albicans and S. cerevisiae overexpressing ALA1 suggest a role in adherence and aggregation for ALA1 and ALA1-like genes in C. albicans. PMID:10531265

  3. Bacteriophage protein-protein interactions.

    PubMed

    Häuser, Roman; Blasche, Sonja; Dokland, Terje; Haggård-Ljungquist, Elisabeth; von Brunn, Albrecht; Salas, Margarita; Casjens, Sherwood; Molineux, Ian; Uetz, Peter

    2012-01-01

    Bacteriophages T7, λ, P22, and P2/P4 (from Escherichia coli), as well as ϕ29 (from Bacillus subtilis), are among the best-studied bacterial viruses. This chapter summarizes published protein interaction data of intraviral protein interactions, as well as known phage-host protein interactions of these phages retrieved from the literature. We also review the published results of comprehensive protein interaction analyses of Pneumococcus phages Dp-1 and Cp-1, as well as coliphages λ and T7. For example, the ≈55 proteins encoded by the T7 genome are connected by ≈43 interactions with another ≈15 between the phage and its host. The chapter compiles published interactions for the well-studied phages λ (33 intra-phage/22 phage-host), P22 (38/9), P2/P4 (14/3), and ϕ29 (20/2). We discuss whether different interaction patterns reflect different phage lifestyles or whether they may be artifacts of sampling. Phages that infect the same host can interact with different host target proteins, as exemplified by E. coli phage λ and T7. Despite decades of intensive investigation, only a fraction of these phage interactomes are known. Technical limitations and a lack of depth in many studies explain the gaps in our knowledge. Strategies to complete current interactome maps are described. Although limited space precludes detailed overviews of phage molecular biology, this compilation will allow future studies to put interaction data into the context of phage biology. PMID:22748812

  4. Vaccination with a Live Attenuated Cytomegalovirus Devoid of a Protein Kinase R Inhibitory Gene Results in Reduced Maternal Viremia and Improved Pregnancy Outcome in a Guinea Pig Congenital Infection Model

    PubMed Central

    Bierle, Craig J.; Swanson, Elizabeth C.; McVoy, Michael A.; Wang, Jian Ben; Al-Mahdi, Zainab; Geballe, Adam P.

    2015-01-01

    ABSTRACT Development of a vaccine to prevent congenital cytomegalovirus infection is a major public health priority. Live vaccines attenuated through mutations targeting viral mechanisms responsible for evasion of host defense may be both safe and efficacious. Safety and vaccine efficacy were evaluated using a guinea pig cytomegalovirus (GPCMV) model. Recombinant GPCMV with a targeted deletion of gp145 (designated Δ145), a viral protein kinase R (PKR) inhibitor, was generated. Attenuation was evaluated following inoculation of 107 PFU of Δ145 or parental virus into guinea pigs immunosuppressed with cyclophosphamide. Efficacy was evaluated by immunizing GPCMV-naive guinea pigs twice with either 105 or 106 PFU of Δ145, establishing pregnancy, and challenging the guinea pigs with salivary gland-adapted GPCMV. The immune response, maternal viral load, pup mortality, and congenital infection rates in the vaccine and control groups were compared. Δ145 was substantially attenuated for replication in immunocompromised guinea pigs. Vaccination with Δ145 induced enzyme-linked immunosorbent assay (ELISA) and neutralizing antibody levels comparable to those achieved in natural infection. In the higher- and lower-dose vaccine groups, pup mortality was reduced to 1/24 (4%) and 4/29 (14%) pups, respectively, whereas it was 26/31 (81%) in unvaccinated control pups (P < 0.0001 for both groups versus the control group). Congenital infection occurred in 20/31 (65%) control pups but only 8/24 (33%) pups in the group vaccinated with 106 PFU (P < 0.05). Significant reductions in the magnitude of maternal DNAemia and pup viral load were noted in the vaccine groups compared to those in the controls. Deletion of a GPCMV genome-encoded PKR inhibitor results in a highly attenuated virus that is immunogenic and protective as a vaccine against transplacental infection. IMPORTANCE Previous attempts to develop successful immunization against cytomegalovirus have largely centered on subunit

  5. Aromatic interactions impact ligand binding and function at serotonin 5-HT2C G protein-coupled receptors: receptor homology modelling, ligand docking, and molecular dynamics results validated by experimental studies

    NASA Astrophysics Data System (ADS)

    Córdova-Sintjago, Tania; Villa, Nancy; Fang, Lijuan; Booth, Raymond G.

    2014-02-01

    The serotonin (5-hydroxytryptamine, 5-HT) 5-HT2 G protein-coupled receptor (GPCR) family consists of types 2A, 2B, and 2C that share ∼75% transmembrane (TM) sequence identity. Agonists for 5-HT2C receptors are under development for psychoses; whereas, at 5-HT2A receptors, antipsychotic effects are associated with antagonists - in fact, 5-HT2A agonists can cause hallucinations and 5-HT2B agonists cause cardiotoxicity. It is known that 5-HT2A TM6 residues W6.48, F6.51, and F6.52 impact ligand binding and function; however, ligand interactions with these residues at the 5-HT2C receptor have not been reported. To predict and validate molecular determinants for 5-HT2C-specific activation, results from receptor homology modelling, ligand docking, and molecular dynamics simulation studies were compared with experimental results for ligand binding and function at wild type and W6.48A, F6.51A, and F6.52A point-mutated 5-HT2C receptors.

  6. Protein Condensation

    NASA Astrophysics Data System (ADS)

    Gunton, James D.; Shiryayev, Andrey; Pagan, Daniel L.

    2007-09-01

    Preface; 1. Introduction; 2. Globular protein structure; 3. Experimental methods; 4. Thermodynamics and statistical mechanics; 5. Protein-protein interactions; 6. Theoretical studies of equilibrium; 7. Nucleation theory; 8. Experimental studies of nucleation; 9. Lysozyme; 10. Some other globular proteins; 11. Membrane proteins; 12. Crystallins and cataracts; 13. Sickle hemoglobin and sickle cell anemia; 14, Alzheimer's disease; Index.

  7. Protein Condensation

    NASA Astrophysics Data System (ADS)

    Gunton, James D.; Shiryayev, Andrey; Pagan, Daniel L.

    2014-07-01

    Preface; 1. Introduction; 2. Globular protein structure; 3. Experimental methods; 4. Thermodynamics and statistical mechanics; 5. Protein-protein interactions; 6. Theoretical studies of equilibrium; 7. Nucleation theory; 8. Experimental studies of nucleation; 9. Lysozyme; 10. Some other globular proteins; 11. Membrane proteins; 12. Crystallins and cataracts; 13. Sickle hemoglobin and sickle cell anemia; 14, Alzheimer's disease; Index.

  8. Inferring Protein Associations Using Protein Pulldown Assays

    SciTech Connect

    Sharp, Julia L.; Anderson, Kevin K.; Daly, Don S.; Auberry, Deanna L.; Borkowski, John J.; Cannon, William R.

    2007-02-01

    Background: One method to infer protein-protein associations is through a “bait-prey pulldown” assay using a protein affinity agent and an LC-MS (liquid chromatography-mass spectrometry)-based protein identification method. False positive and negative protein identifications are not uncommon, however, leading to incorrect inferences. Methods: A pulldown experiment generates a protein association matrix wherein each column represents a sample from one bait protein, each row represents one prey protein and each cell contains a presence/absence association indicator. Our method evaluates the presence/absence pattern across a prey protein (row) with a Likelihood Ratio Test (LRT), computing its p-value with simulated LRT test statistic distributions after a check with simulated binomial random variates disqualified the large sample 2 test. A pulldown experiment often involves hundreds of tests so we apply the false discovery rate method to control the false positive rate. Based on the p-value, each prey protein is assigned a category (specific association, non-specific association, or not associated) and appraised with respect to the pulldown experiment’s goal and design. The method is illustrated using a pulldown experiment investigating the protein complexes of Shewanella oneidensis MR-1. Results: The Monte Carlo simulated LRT p-values objectively reveal specific and ubiquitous prey, as well as potential systematic errors. The example analysis shows the results to be biologically sensible and more realistic than the ad hoc screening methods previously utilized. Conclusions: The method presented appears to be informative for screening for protein-protein associations.

  9. [Influence of gravity discharge on the content of isatin-binding proteins in mice: results of ground-based and space research under the program Bion-M №1].

    PubMed

    Ivanov, A S; Medvedev, A E; Buneeva, O A; Gnedenko, O V; Ershov, P V; Mezencev, Y V; Yablokov, E O; Kaluzhsky, L A; Florinskaya, A V; Moskaleva, N E; Zgoda, V G

    2015-01-01

    Isatin-binding activity of mice liver proteins has been investigated in the samples from the control and flight groups by using the methods of biosensor and proteomic analysis. It was found the higher isatin-binding activity in mice of flight group. The content of a number of individual isatin-binding proteins in the samples of the flight groups differ slightly from the ground control. However, in samples from animals which have weekly post-flight adaptation, the level of certain proteins was significantly increased. The latter allows us to assume that the main events in the proteome of mice (at least in subproteome of isatin-binding proteins), occurs in early post-flight period. PMID:26539872

  10. [Influence of gravity discharge on the content of isatin-binding proteins in mice: results of ground-based and space research under the program Bion-M №1].

    PubMed

    Ivanov, A S; Medvedev, A E; Buneeva, O A; Gnedenko, O V; Ershov, P V; Mezencev, Y V; Yablokov, E O; Kaluzhsky, L A; Florinskaya, A V; Moskaleva, N E; Zgoda, V G

    2015-01-01

    Isatin-binding activity of mice liver proteins has been investigated in the samples from the control and flight groups by using the methods of biosensor and proteomic analysis. It was found the higher isatin-binding activity in mice of flight group. The content of a number of individual isatin-binding proteins in the samples of the flight groups differ slightly from the ground control. However, in samples from animals which have weekly post-flight adaptation, the level of certain proteins was significantly increased. The latter allows us to assume that the main events in the proteome of mice (at least in subproteome of isatin-binding proteins), occurs in early post-flight period.

  11. Molecular interactions of agonist and inverse agonist ligands at serotonin 5-HT2C G protein-coupled receptors: computational ligand docking and molecular dynamics studies validated by experimental mutagenesis results

    NASA Astrophysics Data System (ADS)

    Córdova-Sintjago, Tania C.; Liu, Yue; Booth, Raymond G.

    2015-02-01

    To understand molecular determinants for ligand activation of the serotonin 5-HT2C G protein-coupled receptor (GPCR), a drug target for obesity and neuropsychiatric disorders, a 5-HT2C homology model was built according to an adrenergic β2 GPCR (β2AR) structure and validated using a 5-HT2B GPCR crystal structure. The models were equilibrated in a simulated phosphatidyl choline membrane for ligand docking and molecular dynamics studies. Ligands included (2S, 4R)-(-)-trans-4-(3'-bromo- and trifluoro-phenyl)-N,N-dimethyl-1,2,3,4-tetrahydronaphthalene-2-amine (3'-Br-PAT and 3'-CF3-PAT), a 5-HT2C agonist and inverse agonist, respectively. Distinct interactions of 3'-Br-PAT and 3'-CF3-PAT at the wild-type (WT) 5-HT2C receptor model were observed and experimental 5-HT2C receptor mutagenesis studies were undertaken to validate the modelling results. For example, the inverse agonist 3'-CF3-PAT docked deeper in the WT 5-HT2C binding pocket and altered the orientation of transmembrane helices (TM) 6 in comparison to the agonist 3'-Br-PAT, suggesting that changes in TM orientation that result from ligand binding impact function. For both PATs, mutation of 5-HT2C residues S3.36, T3.37, and F5.47 to alanine resulted in significantly decreased affinity, as predicted from modelling results. It was concluded that upon PAT binding, 5-HT2C residues T3.37 and F5.47 in TMs 3 and 5, respectively, engage in inter-helical interactions with TMs 4 and 6, respectively. The movement of TMs 5 and 6 upon agonist and inverse agonist ligand binding observed in the 5-HT2C receptor modelling studies was similar to movements reported for the activation and deactivation of the β2AR, suggesting common mechanisms among aminergic neurotransmitter GPCRs.

  12. [Protein-losing enteropathy].

    PubMed

    Amiot, A

    2015-07-01

    Protein-losing enteropathy is a rare syndrome of gastrointestinal protein loss. The primary causes can be classified into lymphatic leakage due to increased interstitial pressure and increased leakage of protein-rich fluids due to erosive or non-erosive gastrointestinal disorders. The diagnosis of protein-losing enteropathy should be considered in patients with chronic diarrhea and peripheral oedema. The diagnosis of protein-losing enteropathy is most commonly based on the determination of fecal alpha-1 antitrypsin clearance. Most protein-losing enteropathy cases are the result of either lymphatic obstruction or a variety of gastrointestinal disorders and cardiac diseases, while primary intestinal lymphangiectasia (Waldmann's disease) is less common. Treatment of protein-losing enteropathy targets the underlying disease but also includes dietary modification, such as high-protein and low-fat diet along with medium-chain triglyceride supplementation. PMID:25618488

  13. Oral Debio1143 (AT406), an antagonist of inhibitor of apoptosis proteins, in combination with daunorubicin and cytarabine in patients with poor-risk acute myeloid leukemia - results of a phase I dose escalation study

    PubMed Central

    Erba, Harry P.; Larson, Richard A.; Luger, Selina M.; Tallman, Martin S.; Brill, Jeffrey M.; Vuagniaux, Gregoire; Rouits, Elisabeth; Sorensen, J. Mel; Zanna, Claudio

    2016-01-01

    Background Treatment of acute myeloid leukemia (AML) remains difficult due to the development of treatment resistance which might be overcome through antagonists of inhibitors of apoptosis proteins (IAPs). Patients and methods This multi-center, open-label, dose escalation study aimed to evaluate tolerability, pharmacokinetics (PK), pharmacodynamics (PD), and efficacy of Debio1143 (formerly AT-406), a new IAP antagonist, when given along with a standard "7 plus 3 regimen" of daunorubicin and cytarabine to poor-risk patients with AML during the induction cycle. Consecutive patient cohorts received once daily 100, 200, 300, or 400 mg of oral Debio1143 on treatment days 1–5. Blood samples were collected regularly until hematological recovery or response; bone marrow samples on day 0, 14, and 29; and PK and PD samples on days 1, 3, 5, 8, 10 and 1, 2, 8, respectively. Results Of 29 enrolled patients, 23 completed the study. Most common adverse events of any grade deemed related to treatment were nausea (31% of patients), diarrhea (14%), and febrile neutropenia (14%). Exposure exceeded dose-proportionality, without accumulation over 5 days. Inhibition of cIAP-1 was detectable in CD34/CD117+ cells and blasts. A total of 11 (38%) patients achieved complete remission, the majority in the 100 mg dose cohort. Of these, 6 (56%) relapsed still within the study period. Responders more frequently showed plasma increases of TNFα and IL-8 post-first dose of Debio1143. Conclusion Debio1143 up to 400 mg/day showed good tolerability in combination with daunorubicin and cytarabine; further studies in subsets of patients with AML are warranted. PMID:25842225

  14. Circulating concentrations of insulin-like growth factor-I, insulin-like growth factor binding protein-3, genetic polymorphisms and mammographic density in premenopausal Mexican women: results from the ESMaestras cohort

    PubMed Central

    Rinaldi, S.; Biessy, C.; Hernandez, M; Lesueur, F.; dos-Santos-Silva, I.; Rice, M. S.; Lajous, M.; Lopez-Ridaura, R.; Torres-Mejía, G.; Romieu, I.

    2015-01-01

    The insulin-like growth factor (IGF) axis plays an essential role in the development of the mammary gland. High circulating levels of IGF-I and of its major binding protein IGFBP3 have been related with increased mammographic density in Caucasian premenopausal women. Some common single nucleotide polymorphisms (SNPs) in genes of the IGF pathway have also been suggested to play a role in mammographic density. We conducted a cross-sectional study nested within the large Mexican ESMaestras cohort, to investigate the relation between circulating levels of IGF-I, IGFBP-3, the IGF-I/IGFBP-3 ratio, five common SNPs in the IGF-1, IGFBP-3 and IGF-1R genes, and mammographic density in 593 premenopausal Mexican women. Mean age at mammogram was 43.1 (standard deviation–SD=3.7) years, and average body mass index (BMI) at recruitment was 28.5 kg/m2. Mean percent mammographic density was 36.5% (SD: 17.1), with mean dense tissue area of 48.3 (SD: 33.3) cm2. Mean IGF-I and IGFBP-3 concentrations were 15.33 (SD: 5.52) nmol/l and 114.96 (SD: 21.34) nmol/l, respectively. No significant associations were seen between percent density and biomarker concentrations but women with higher IGF-I and IGF-I/IGFBP-3 concentrations had lower absolute dense (ptrend =0.03 and 0.09, respectively) and non-dense tissue areas (ptrend <0.001 for both parameters). However, these associations were null after adjustment by BMI. SNPs in specific genes were associated with circulating levels of growth factors, but not with mammographic density features. These results do not support the hypothesis of a strong association between circulating levels of growth hormones and mammographic density in Mexican premenopausal women. PMID:24037648

  15. Circulating concentrations of insulin-like growth factor-I, insulin-like growth factor-binding protein-3, genetic polymorphisms and mammographic density in premenopausal Mexican women: results from the ESMaestras cohort.

    PubMed

    Rinaldi, S; Biessy, C; Hernandez, M; Lesueur, F; dos-Santos-Silva, I; Rice, M S; Lajous, M; Lopez-Ridaura, R; Torres-Mejía, G; Romieu, I

    2014-03-15

    The insulin-like growth factor (IGF) axis plays an essential role in the development of the mammary gland. High circulating levels of IGF-I and of its major binding protein IGFBP3 have been related with increased mammographic density in Caucasian premenopausal women. Some common single nucleotide polymorphisms (SNPs) in genes of the IGF pathway have also been suggested to play a role in mammographic density. We conducted a cross-sectional study nested within the large Mexican ESMaestras cohort to investigate the relation between circulating levels of IGF-I, IGFBP-3, the IGF-I/IGFBP-3 ratio, five common SNPs in the IGF-1, IGFBP-3 and IGF-1R genes and mammographic density in 593 premenopausal Mexican women. Mean age at mammogram was 43.1 (standard deviation, SD = 3.7) years, and average body mass index (BMI) at recruitment was 28.5 kg/m(2). Mean percent mammographic density was 36.5% (SD: 17.1), with mean dense tissue area of 48.3 (SD: 33.3) cm(2) . Mean IGF-I and IGFBP-3 concentrations were 15.33 (SD: 5.52) nmol/l and 114.96 (SD: 21.34) nmol/l, respectively. No significant associations were seen between percent density and biomarker concentrations, but women with higher IGF-I and IGF-I/IGFBP-3 concentrations had lower absolute dense (p(trend) = 0.03 and 0.09, respectively) and nondense tissue areas (p(trend) < 0.001 for both parameters). However, these associations were null after adjustment by BMI. SNPs in specific genes were associated with circulating levels of growth factors, but not with mammographic density features. These results do not support the hypothesis of a strong association between circulating levels of growth hormones and mammographic density in Mexican premenopausal women.

  16. Impact of Protein Supplementation and Care and Support on Body Composition and CD4 Count among HIV-Infected Women Living in Rural India: Results from a Randomized Pilot Clinical Trial

    PubMed Central

    Nyamathi, Adeline; Sinha, Sanjeev; Ganguly, Kalyan K; Ramakrishna, Padma; Suresh, P.; Carpenter, Catherine L

    2013-01-01

    Body composition in HIV-infected individuals is subject to many influences. We conducted a pilot six-month randomized trial of 68 WLA (women living with AIDS) from rural India. High protein intervention combined with education and supportive care delivered by HIV-trained village women (Asha [Activated Social Health Activist] Life [AL]) was compared to standard protein with usual care delivered by village community assistants (Usual Care [UC]). Measurements included CD4 counts, ART adherence, socio-demographics, disease characteristics (questionnaires); and anthropometry (bioimpedance analyzer). Repeated measures analysis of variance modeled associations. AL significantly gained in BMI, muscle mass, fat mass, ART adherence, and CD4 counts compared to UC, with higher weight and muscle mass gains among ART adherent (≥ 66%) participants who had healthier immunity (CD4 ≥ 450). BMI of WLA improved through high protein supplementation combined with education and supportive care. Future research is needed to determine which intervention aspect was most responsible. PMID:23370835

  17. Protein and older adults.

    PubMed

    Chernoff, Ronni

    2004-12-01

    Body composition changes as people get older. One of the noteworthy alterations is the reduction in total body protein. A decrease in skeletal muscle is the most noticeable manifestation of this change but there is also a reduction in other physiologic proteins such as organ tissue, blood components, and immune bodies as well as declines in total body potassium and water. This contributes to impaired wound healing, loss of skin elasticity, and an inability to fight infection. The recommended dietary allowance (RDA) for adults for protein is 0.8 grams of protein per kilogram of body weight. Protein tissue accounts for 30% of whole-body protein turnover but that rate declines to 20% or less by age 70. The result of this phenomenon is that older adults require more protein/kilogram body weight than do younger adults. Recently, it has become clear that the requirement for exogenous protein is at least 1.0 gram/kilogram body weight. Adequate dietary intake of protein may be more difficult for older adults to obtain. Dietary animal protein is the primary source of high biological value protein, iron, vitamin B(12), folic acid, biotin and other essential nutrients. In fact, egg protein is the standard against which all other proteins are compared. Compared to other high-quality protein sources like meat, poultry and seafood, eggs are the least expensive. The importance of dietary protein cannot be underestimated in the diets of older adults; inadequate protein intake contributes to a decrease in reserve capacity, increased skin fragility, decreased immune function, poorer healing, and longer recuperation from illness.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background: It has been suggested that perturbations in branched-chain amino acid (BCAA) catabolism are associated with insulin resistance and contribute to elevated systemic BCAAs. Evidence in rodents suggests dietary protein rich in BCAAs can increase BCAA catabolism, but there is limited evidence...

  19. Solute exclusion by polymer and protein-dominated water: correlation with results of nuclear magnetic resonance (NMR) and calorimetric studies and their significance for the understanding of the physical state of water in living cells.

    PubMed

    Ling, G N

    1988-06-01

    According to the polarized multilayer (PM) theory of cell water proteins with their backbones fully extended and their NHCO groups directly exposed to bulk water, polarize water in multilayers. Experimental testing of the theory led to a new understanding of the uniqueness of gelatin, due to its permanently maintained fully extended conformation and its ability to polarize the bulk phase water in multilayers with reduced solubilities for solutes in a size dependent manner ("size rule"). Other models which behave like gelatin are urea-denatured proteins, synthetic polymers like polyethylene oxide (PEO), and polyvinylpyrrolidine (PVP), but not native proteins. NMR studies showed that the majority of water molecules dominated by these polymers does indeed suffer rotational (and translational) motional restriction as predicted by the PM theory. In conjunction with ultra-high frequency dielectric studies but particularly quasielastic neutron scattering of both model systems (e.g., PEO) and living cells (i.e., brine shrimp cysts and frog muscle), this finding offers confirmation of the PM theory of living cell water and model systems. Studies of the freezing point depression showed that the presence of as much as 50% of native proteins had no effect on the freezing point of water while inclusion of gelatin, PEO, etc., caused concentration-dependent lowering of the freezing temperature. These findings demonstrate the key role of polarized water in the phenomena of freezing point depression and the unusual ice forms seen in living cells. PMID:3041574

  20. Protein solubility modeling

    NASA Technical Reports Server (NTRS)

    Agena, S. M.; Pusey, M. L.; Bogle, I. D.

    1999-01-01

    A thermodynamic framework (UNIQUAC model with temperature dependent parameters) is applied to model the salt-induced protein crystallization equilibrium, i.e., protein solubility. The framework introduces a term for the solubility product describing protein transfer between the liquid and solid phase and a term for the solution behavior describing deviation from ideal solution. Protein solubility is modeled as a function of salt concentration and temperature for a four-component system consisting of a protein, pseudo solvent (water and buffer), cation, and anion (salt). Two different systems, lysozyme with sodium chloride and concanavalin A with ammonium sulfate, are investigated. Comparison of the modeled and experimental protein solubility data results in an average root mean square deviation of 5.8%, demonstrating that the model closely follows the experimental behavior. Model calculations and model parameters are reviewed to examine the model and protein crystallization process. Copyright 1999 John Wiley & Sons, Inc.

  1. Racemic protein crystallography.

    PubMed

    Yeates, Todd O; Kent, Stephen B H

    2012-01-01

    Although natural proteins are chiral and are all of one "handedness," their mirror image forms can be prepared by chemical synthesis. This opens up new opportunities for protein crystallography. A racemic mixture of the enantiomeric forms of a protein molecule can crystallize in ways that natural proteins cannot. Recent experimental data support a theoretical prediction that this should make racemic protein mixtures highly amenable to crystallization. Crystals obtained from racemic mixtures also offer advantages in structure determination strategies. The relevance of these potential advantages is heightened by advances in synthetic methods, which are extending the size limit for proteins that can be prepared by chemical synthesis. Recent ideas and results in the area of racemic protein crystallography are reviewed.

  2. Smoking interacts with HLA-DRB1 shared epitope in the development of anti-citrullinated protein antibody-positive rheumatoid arthritis: results from the Malaysian Epidemiological Investigation of Rheumatoid Arthritis (MyEIRA)

    PubMed Central

    2012-01-01

    Introduction Rheumatoid arthritis (RA) is a multifactorial autoimmune disease in which genetic and environmental factors interact in the etiology. In this study, we investigated whether smoking and HLA-DRB1 shared-epitope (SE) alleles interact differently in the development of the two major subgroups of rheumatoid arthritis (RA), anti-citrullinated proteins antibody (ACPA)-positive and ACPA-negative disease, in a multiethnic population of Asian descent. Methods A case-control study comprising early diagnosed RA cases was carried out in Malaysia between 2005 and 2009. In total, 1,076 cases and 1,612 matched controls participated in the study. High-resolution HLA-DRB1 genotyping was performed for shared-epitope (SE) alleles. All participants answered a questionnaire on a broad range of issues, including smoking habits. The odds ratio (OR) of developing ACPA-positive and ACPA-negative disease was calculated for smoking and the presence of any SE alleles separately. Potential interaction between smoking history (defined as "ever" and "never" smoking) and HLA-DRB1 SE alleles also was calculated. Results In our multiethnic study, both the SE alleles and smoking were associated with an increased risk of developing ACPA-positive RA (OR SE alleles, 4.7; 95% confidence interval (CI), 3.6 to 6.2; OR smoking, 4.1; 95% CI, 1.9 to 9.2). SE-positive smokers had an odds ratio of ACPA-positive RA of 25.6 (95% CI, 10.4 to 63.4), compared with SE-negative never-smokers. The interaction between smoking and SE alleles was significant (attributable proportion due to interaction (AP) was 0.7 (95% CI, 0.5 to 1.0)). The HLA-DRB1*04:05 SE allele, which is common in Asian populations, but not among Caucasians, was associated with an increased risk of ACPA-positive RA, and this allele also showed signs of interaction with smoking (AP, 0.4; 95% CI, -0.1 to 0.9). Neither smoking nor SE alleles nor their combination was associated with an increased risk of ACPA-negative RA. Conclusions The risk

  3. Exposure to the three structurally different PCB congeners (PCB 118, 153, and 126) results in decreased protein expression and altered steroidogenesis in the human adrenocortical carcinoma cell line H295R.

    PubMed

    Tremoen, Nina Hårdnes; Fowler, Paul A; Ropstad, Erik; Verhaegen, Steven; Krogenæs, Anette

    2014-01-01

    Polychlorinated biphenyls (PCB), synthetic, persistent organic pollutants (POP), are detected ubiquitously, in water, soil, air, and sediments, as well as in animals and humans. PCB are associated with range of adverse health effects, such as interference with the immune system and nervous system, reproductive abnormalities, fetotoxicity, carcinogenicity, and endocrine disruption. Our objective was to determine the effects of three structurally different PCB congeners, PCB118, PCB 126, and PCB 153, each at two concentrations, on the steroidogenic capacity and proteome of human adrenocortical carcinoma cell line cultures (H295R) . After 48 h of exposure, cell viability was monitored and estradiol, testosterone, cortisol and progesterone secretion measured to quantify steroidogenic capacity of the cells. Two-dimensional (2D) gel-based proteomics was used to screen for proteome alterations in H295R cells in response to the PCB. Exposure to PCB 118 increased estradiol and cortisol secretion, while exposure to PCB 153 elevated estradiol secretion. PCB 126 was the most potent congener, increasing estradiol, cortisol, and progesterone secretion in exposed H295R cells. Seventy-three of the 711 spots analyzed showed a significant difference in normalized spot volumes between controls (vehicle only) and at least one exposure group. Fourteen of these protein spots were identified by liquid chromatography with mass spectroscopy (LC-MS/MS). Exposure to three PCB congeners with different chemical structure perturbed steroidogenesis and protein expression in the H295R in vitro model. This study represents an initial analysis of the effects on proteins and hormones in the H295R cell model, and additional studies are required in order to obtain a more complete understanding of the pathways disturbed by PCB congeners in H295R cells. Overall, alterations in protein regulation and steroid hormone synthesis suggest that exposure to PCB disturbs several cellular processes, including

  4. Total protein

    MedlinePlus

    ... page: //medlineplus.gov/ency/article/003483.htm Total protein To use the sharing features on this page, please enable JavaScript. The total protein test measures the total amount of two classes ...

  5. Forces stabilizing proteins.

    PubMed

    Nick Pace, C; Scholtz, J Martin; Grimsley, Gerald R

    2014-06-27

    The goal of this article is to summarize what has been learned about the major forces stabilizing proteins since the late 1980s when site-directed mutagenesis became possible. The following conclusions are derived from experimental studies of hydrophobic and hydrogen bonding variants. (1) Based on studies of 138 hydrophobic interaction variants in 11 proteins, burying a -CH2- group on folding contributes 1.1±0.5 kcal/mol to protein stability. (2) The burial of non-polar side chains contributes to protein stability in two ways: first, a term that depends on the removal of the side chains from water and, more importantly, the enhanced London dispersion forces that result from the tight packing in the protein interior. (3) Based on studies of 151 hydrogen bonding variants in 15 proteins, forming a hydrogen bond on folding contributes 1.1±0.8 kcal/mol to protein stability. (4) The contribution of hydrogen bonds to protein stability is strongly context dependent. (5) Hydrogen bonds by side chains and peptide groups make similar contributions to protein stability. (6) Polar group burial can make a favorable contribution to protein stability even if the polar group is not hydrogen bonded. (7) Hydrophobic interactions and hydrogen bonds both make large contributions to protein stability.

  6. Forces Stabilizing Proteins

    PubMed Central

    Pace, C. Nick; Scholtz, J. Martin; Grimsley, Gerald R.

    2014-01-01

    The goal of this article is to summarize what has been learned about the major forces stabilizing proteins since the late 1980s when site-directed mutagenesis became possible. The following conclusions are derived from experimental studies of hydrophobic and hydrogen bonding variants. 1. Based on studies of 138 hydrophobic interaction variants in 11 proteins, burying a –CH2– group on folding contributes 1.1 ± 0.5 kcal/mol to protein stability. 2. The burial of nonpolar side chains contributes to protein stability in two ways: first, a term that depends on the removal of the side chains from water and, more importantly, the enhanced London dispersion forces that result from the tight packing in the protein interior. 3. Based on studies of 151 hydrogen bonding variants in 15 proteins, forming a hydrogen bond on folding contributes 1.1 ± 0.8 kcal/mol to protein stability. 4. The contribution of hydrogen bonds to protein stability is strongly context dependent. 5. Hydrogen bonds by side chains and peptide groups make similar contributions to protein stability. 6. Polar group burial can make a favorable contribution to protein stability even if the polar group is not hydrogen bonded. 7. Hydrophobic interactions and hydrogen bonds both make large contributions to protein stability. PMID:24846139

  7. Protein Complexes in Bacteria

    PubMed Central

    Caufield, J. Harry; Abreu, Marco; Wimble, Christopher; Uetz, Peter

    2015-01-01

    Large-scale analyses of protein complexes have recently become available for Escherichia coli and Mycoplasma pneumoniae, yielding 443 and 116 heteromultimeric soluble protein complexes, respectively. We have coupled the results of these mass spectrometry-characterized protein complexes with the 285 “gold standard” protein complexes identified by EcoCyc. A comparison with databases of gene orthology, conservation, and essentiality identified proteins conserved or lost in complexes of other species. For instance, of 285 “gold standard” protein complexes in E. coli, less than 10% are fully conserved among a set of 7 distantly-related bacterial “model” species. Complex conservation follows one of three models: well-conserved complexes, complexes with a conserved core, and complexes with partial conservation but no conserved core. Expanding the comparison to 894 distinct bacterial genomes illustrates fractional conservation and the limits of co-conservation among components of protein complexes: just 14 out of 285 model protein complexes are perfectly conserved across 95% of the genomes used, yet we predict more than 180 may be partially conserved across at least half of the genomes. No clear relationship between gene essentiality and protein complex conservation is observed, as even poorly conserved complexes contain a significant number of essential proteins. Finally, we identify 183 complexes containing well-conserved components and uncharacterized proteins which will be interesting targets for future experimental studies. PMID:25723151

  8. Protein Microarrays

    NASA Astrophysics Data System (ADS)

    Ricard-Blum, S.

    Proteins are key actors in the life of the cell, involved in many physiological and pathological processes. Since variations in the expression of messenger RNA are not systematically correlated with variations in the protein levels, the latter better reflect the way a cell functions. Protein microarrays thus supply complementary information to DNA chips. They are used in particular to analyse protein expression profiles, to detect proteins within complex biological media, and to study protein-protein interactions, which give information about the functions of those proteins [3-9]. They have the same advantages as DNA microarrays for high-throughput analysis, miniaturisation, and the possibility of automation. Section 18.1 gives a brief overview of proteins. Following this, Sect. 18.2 describes how protein microarrays can be made on flat supports, explaining how proteins can be produced and immobilised on a solid support, and discussing the different kinds of substrate and detection method. Section 18.3 discusses the particular format of protein microarrays in suspension. The diversity of protein microarrays and their applications are then reported in Sect. 18.4, with applications to therapeutics (protein-drug interactions) and diagnostics. The prospects for future developments of protein microarrays are then outlined in the conclusion. The bibliography provides an extensive list of reviews and detailed references for those readers who wish to go further in this area. Indeed, the aim of the present chapter is not to give an exhaustive or detailed analysis of the state of the art, but rather to provide the reader with the basic elements needed to understand how proteins are designed and used.

  9. Dietary Proteins

    MedlinePlus

    ... meat, dairy products, nuts, and certain grains and beans. Proteins from meat and other animal products are complete proteins. This means they supply all of the amino acids the body can't make on its own. Most plant proteins are incomplete. You should eat different types ...

  10. Protein Structure

    ERIC Educational Resources Information Center

    Asmus, Elaine Garbarino

    2007-01-01

    Individual students model specific amino acids and then, through dehydration synthesis, a class of students models a protein. The students clearly learn amino acid structure, primary, secondary, tertiary, and quaternary structure in proteins and the nature of the bonds maintaining a protein's shape. This activity is fun, concrete, inexpensive and…

  11. Environments of the four tryptophans in the extracellular domain of human tissue factor: comparison of results from absorption and fluorescence difference spectra of tryptophan replacement mutants with the crystal structure of the wild-type protein.

    PubMed Central

    Hasselbacher, C A; Rusinova, E; Waxman, E; Rusinova, R; Kohanski, R A; Lam, W; Guha, A; Du, J; Lin, T C; Polikarpov, I

    1995-01-01

    The local environments of the four tryptophan residues of the extracellular domain of human tissue factor (sTF) were assessed from difference absorption and fluorescence spectra. The difference spectra were derived by subtracting spectra from single Trp-to-Phe or Trp-to-Tyr replacement mutants from the corresponding spectrum of the wild-type protein. Each of the mutants was capable of enhancing the proteolytic activity of factor VIIa showing that the mutations did not introduce major structural changes, although the mutants were more susceptible to denaturation by guanidinium chloride. The difference spectra indicate that the Trp residues are buried to different extents within the protein matrix. This evaluation was compared with the x-ray crystal structure of sTF. There is excellent agreement between predictions from the difference spectra and the environments of the Trp residues observed in the x-ray crystal structure, demonstrating that difference absorption and particularly fluorescence spectra derived from functional single-Trp replacement mutants can be used to obtain information about the local environments of individual Trp residues in multi-tryptophan proteins. Images FIGURE 7 FIGURE 8 PMID:7669897

  12. Protein- protein interaction detection system using fluorescent protein microdomains

    DOEpatents

    Waldo, Geoffrey S.; Cabantous, Stephanie

    2010-02-23

    The invention provides a protein labeling and interaction detection system based on engineered fragments of fluorescent and chromophoric proteins that require fused interacting polypeptides to drive the association of the fragments, and further are soluble and stable, and do not change the solubility of polypeptides to which they are fused. In one embodiment, a test protein X is fused to a sixteen amino acid fragment of GFP (.beta.-strand 10, amino acids 198-214), engineered to not perturb fusion protein solubility. A second test protein Y is fused to a sixteen amino acid fragment of GFP (.beta.-strand 11, amino acids 215-230), engineered to not perturb fusion protein solubility. When X and Y interact, they bring the GFP strands into proximity, and are detected by complementation with a third GFP fragment consisting of GFP amino acids 1-198 (strands 1-9). When GFP strands 10 and 11 are held together by interaction of protein X and Y, they spontaneous association with GFP strands 1-9, resulting in structural complementation, folding, and concomitant GFP fluorescence.

  13. Use of protein-protein interactions in affinity chromatography.

    PubMed

    Muronetz, V I; Sholukh, M; Korpela, T

    2001-10-30

    Biospecific recognition between proteins is a phenomenon that can be exploited for designing affinity-chromatographic purification systems for proteins. In principle, the approach is straightforward, and there are usually many alternative ways, since a protein can be always found which binds specifically enough to the desired protein. Routine immunoaffinity chromatography utilizes the recognition of antigenic epitopes by antibodies. However, forces involved in protein-protein interactions as well the forces keeping the three-dimensional structures of proteins intact are complicated, and proteins are easily unfolded by various factors with unpredictable results. Because of this and because of the generally high association strength between proteins, the correct adjustment of binding forces between an immobilized protein and the protein to be purified as well as the release of bound proteins in biologically active form from affinity complexes are the main problem. Affinity systems involving interactions like enzyme-enzyme, subunit-oligomer, protein-antibody, protein-chaperone and the specific features involved in each case are presented as examples. This article also aims to sketch prospects for further development of the use of protein-protein interactions for the purification of proteins. PMID:11694271

  14. Protein sulfhydration.

    PubMed

    Paul, Bindu D; Snyder, Solomon H

    2015-01-01

    Hydrogen sulfide (H2S) is one of the gasotransmitters that modulates various biological processes and participates in multiple signaling pathways. H2S signals by a process termed sulfhydration. Sulfhydration has recently been recognized as a posttranslational modification similar to nitrosylation. Sulfhydration occurs at reactive cysteine residues in proteins and results in the conversion of an -SH group of cysteine to an -SSH or a persulfide group. Sulfhydration is highly prevalent in vivo, and aberrant sulfhydration patterns have been observed under several pathological conditions ranging from heart disease to neurodegenerative diseases such as Parkinson's disease. The biotin switch assay, originally developed to detect nitrosylation, has been modified to detect sulfhydration. In this chapter, we discuss the physiological roles of sulfhydration and the methodologies used to detect this modification.

  15. Protein oxidation and peroxidation.

    PubMed

    Davies, Michael J

    2016-04-01

    Proteins are major targets for radicals and two-electron oxidants in biological systems due to their abundance and high rate constants for reaction. With highly reactive radicals damage occurs at multiple side-chain and backbone sites. Less reactive species show greater selectivity with regard to the residues targeted and their spatial location. Modification can result in increased side-chain hydrophilicity, side-chain and backbone fragmentation, aggregation via covalent cross-linking or hydrophobic interactions, protein unfolding and altered conformation, altered interactions with biological partners and modified turnover. In the presence of O2, high yields of peroxyl radicals and peroxides (protein peroxidation) are formed; the latter account for up to 70% of the initial oxidant flux. Protein peroxides can oxidize both proteins and other targets. One-electron reduction results in additional radicals and chain reactions with alcohols and carbonyls as major products; the latter are commonly used markers of protein damage. Direct oxidation of cysteine (and less commonly) methionine residues is a major reaction; this is typically faster than with H2O2, and results in altered protein activity and function. Unlike H2O2, which is rapidly removed by protective enzymes, protein peroxides are only slowly removed, and catabolism is a major fate. Although turnover of modified proteins by proteasomal and lysosomal enzymes, and other proteases (e.g. mitochondrial Lon), can be efficient, protein hydroperoxides inhibit these pathways and this may contribute to the accumulation of modified proteins in cells. Available evidence supports an association between protein oxidation and multiple human pathologies, but whether this link is causal remains to be established.

  16. Protein oxidation and peroxidation

    PubMed Central

    Davies, Michael J.

    2016-01-01

    Proteins are major targets for radicals and two-electron oxidants in biological systems due to their abundance and high rate constants for reaction. With highly reactive radicals damage occurs at multiple side-chain and backbone sites. Less reactive species show greater selectivity with regard to the residues targeted and their spatial location. Modification can result in increased side-chain hydrophilicity, side-chain and backbone fragmentation, aggregation via covalent cross-linking or hydrophobic interactions, protein unfolding and altered conformation, altered interactions with biological partners and modified turnover. In the presence of O2, high yields of peroxyl radicals and peroxides (protein peroxidation) are formed; the latter account for up to 70% of the initial oxidant flux. Protein peroxides can oxidize both proteins and other targets. One-electron reduction results in additional radicals and chain reactions with alcohols and carbonyls as major products; the latter are commonly used markers of protein damage. Direct oxidation of cysteine (and less commonly) methionine residues is a major reaction; this is typically faster than with H2O2, and results in altered protein activity and function. Unlike H2O2, which is rapidly removed by protective enzymes, protein peroxides are only slowly removed, and catabolism is a major fate. Although turnover of modified proteins by proteasomal and lysosomal enzymes, and other proteases (e.g. mitochondrial Lon), can be efficient, protein hydroperoxides inhibit these pathways and this may contribute to the accumulation of modified proteins in cells. Available evidence supports an association between protein oxidation and multiple human pathologies, but whether this link is causal remains to be established. PMID:27026395

  17. Physics of protein motility and motor proteins

    NASA Astrophysics Data System (ADS)

    Kolomeisky, Anatoly B.

    2013-09-01

    Motor proteins are enzymatic molecules that transform chemical energy into mechanical motion and work. They are critically important for supporting various cellular activities and functions. In the last 15 years significant progress in understanding the functioning of motor proteins has been achieved due to revolutionary breakthroughs in single-molecule experimental techniques and strong advances in theoretical modelling. However, microscopic mechanisms of protein motility are still not well explained, and the collective efforts of many scientists are needed in order to solve these complex problems. In this special section the reader will find the latest advances on the difficult road to mapping motor proteins dynamics in various systems. Recent experimental developments have allowed researchers to monitor and to influence the activity of single motor proteins with a high spatial and temporal resolution. It has stimulated significant theoretical efforts to understand the non-equilibrium nature of protein motility phenomena. The latest results from all these advances are presented and discussed in this special section. We would like to thank the scientists from all over the world who have reported their latest research results for this special section. We are also grateful to the staff and editors of Journal of Physics: Condensed Matter for their invaluable help in handling all the administrative and refereeing activities. The field of motor proteins and protein motility is fast moving, and we hope that this collection of articles will be a useful source of information in this highly interdisciplinary area. Physics of protein motility and motor proteins contents Physics of protein motility and motor proteinsAnatoly B Kolomeisky Identification of unique interactions between the flexible linker and the RecA-like domains of DEAD-box helicase Mss116 Yuan Zhang, Mirkó Palla, Andrew Sun and Jung-Chi Liao The load dependence of the physical properties of a molecular motor

  18. Protopia: a protein-protein interaction tool

    PubMed Central

    Real-Chicharro, Alejandro; Ruiz-Mostazo, Iván; Navas-Delgado, Ismael; Kerzazi, Amine; Chniber, Othmane; Sánchez-Jiménez, Francisca; Medina, Miguel Ángel; Aldana-Montes, José F

    2009-01-01

    Background Protein-protein interactions can be considered the basic skeleton for living organism self-organization and homeostasis. Impressive quantities of experimental data are being obtained and computational tools are essential to integrate and to organize this information. This paper presents Protopia, a biological tool that offers a way of searching for proteins and their interactions in different Protein Interaction Web Databases, as a part of a multidisciplinary initiative of our institution for the integration of biological data . Results The tool accesses the different Databases (at present, the free version of Transfac, DIP, Hprd, Int-Act and iHop), and results are expressed with biological protein names or databases codes and can be depicted as a vector or a matrix. They can be represented and handled interactively as an organic graph. Comparison among databases is carried out using the Uniprot codes annotated for each protein. Conclusion The tool locates and integrates the current information stored in the aforementioned databases, and redundancies among them are detected. Results are compatible with the most important network analysers, so that they can be compared and analysed by other world-wide known tools and platforms. The visualization possibilities help to attain this goal and they are especially interesting for handling multiple-step or complex networks. PMID:19828077

  19. Regulators of G-protein-signaling proteins: negative modulators of G-protein-coupled receptor signaling.

    PubMed

    Woodard, Geoffrey E; Jardín, Isaac; Berna-Erro, A; Salido, Gines M; Rosado, Juan A

    2015-01-01

    Regulators of G-protein-signaling (RGS) proteins are a category of intracellular proteins that have an inhibitory effect on the intracellular signaling produced by G-protein-coupled receptors (GPCRs). RGS along with RGS-like proteins switch on through direct contact G-alpha subunits providing a variety of intracellular functions through intracellular signaling. RGS proteins have a common RGS domain that binds to G alpha. RGS proteins accelerate GTPase and thus enhance guanosine triphosphate hydrolysis through the alpha subunit of heterotrimeric G proteins. As a result, they inactivate the G protein and quickly turn off GPCR signaling thus terminating the resulting downstream signals. Activity and subcellular localization of RGS proteins can be changed through covalent molecular changes to the enzyme, differential gene splicing, and processing of the protein. Other roles of RGS proteins have shown them to not be solely committed to being inhibitors but behave more as modulators and integrators of signaling. RGS proteins modulate the duration and kinetics of slow calcium oscillations and rapid phototransduction and ion signaling events. In other cases, RGS proteins integrate G proteins with signaling pathways linked to such diverse cellular responses as cell growth and differentiation, cell motility, and intracellular trafficking. Human and animal studies have revealed that RGS proteins play a vital role in physiology and can be ideal targets for diseases such as those related to addiction where receptor signaling seems continuously switched on.

  20. The 3;21 translocation in myelodysplasia results in a fusion transcript between the AML1 gene and the gene for EAP, a highly conserved protein associated with the Epstein-Barr virus small RNA EBER 1.

    PubMed Central

    Nucifora, G; Begy, C R; Erickson, P; Drabkin, H A; Rowley, J D

    1993-01-01

    In the 8;21 translocation, the AML1 gene, located at chromosome band 21q22, is translocated to chromosome 8 (q22), where it is fused to the ETO gene and transcribed as a chimeric gene. AML1 is the human homolog of the recently cloned mouse gene pebp2 alpha B, homologous to the DNA binding alpha subunit of the polyoma enhancer factor pebp2. AML1 is also involved in a translocation with chromosome 3 that is seen in patients with therapy-related acute myeloid leukemia and myelodysplastic syndrome and in chronic myelogenous leukemia in blast crisis. We have isolated a fusion cDNA clone from a t(3;21) library derived from a patient with therapy-related myelodysplastic syndrome; this clone contains sequences from AML1 and from EAP, which we have now localized to band 3q26. EAP has previously been characterized as a highly expressed small nuclear protein of 128 residues (EBER 1) associated with Epstein-Barr virus small RNA. The fusion clone contains the DNA binding 5' part of AML1 that is fused to ETO in the t(8;21) and, in addition, at least one other exon. The translocation replaces the last nine codons of AML1 with the last 96 codons of EAP. The fusion does not maintain the correct reading frame of EAP and may not lead to a functional chimeric protein. Images Fig. 1 Fig. 2 Fig. 4 Fig. 5 Fig. 6 PMID:8395054

  1. Transport proteins.

    PubMed

    Thatcher, Jack D

    2013-04-16

    This Teaching Resource provides and describes two animated lessons that illustrate general properties of transport proteins. The lesson called "transport protein classes" depicts major classes and subclasses of transport proteins. The "transporters, mechanism of action" lesson explains how transporters and P class ATPase (adenosine triphosphatase) pumps function. These animations serve as valuable resources for any collegiate-level course that describes these important factors. Courses that might use them include introductory biology, biochemistry, cell biology, physiology, and biophysics.

  2. Discovery of binding proteins for a protein target using protein-protein docking-based virtual screening.

    PubMed

    Zhang, Changsheng; Tang, Bo; Wang, Qian; Lai, Luhua

    2014-10-01

    Target structure-based virtual screening, which employs protein-small molecule docking to identify potential ligands, has been widely used in small-molecule drug discovery. In the present study, we used a protein-protein docking program to identify proteins that bind to a specific target protein. In the testing phase, an all-to-all protein-protein docking run on a large dataset was performed. The three-dimensional rigid docking program SDOCK was used to examine protein-protein docking on all protein pairs in the dataset. Both the binding affinity and features of the binding energy landscape were considered in the scoring function in order to distinguish positive binding pairs from negative binding pairs. Thus, the lowest docking score, the average Z-score, and convergency of the low-score solutions were incorporated in the analysis. The hybrid scoring function was optimized in the all-to-all docking test. The docking method and the hybrid scoring function were then used to screen for proteins that bind to tumor necrosis factor-α (TNFα), which is a well-known therapeutic target for rheumatoid arthritis and other autoimmune diseases. A protein library containing 677 proteins was used for the screen. Proteins with scores among the top 20% were further examined. Sixteen proteins from the top-ranking 67 proteins were selected for experimental study. Two of these proteins showed significant binding to TNFα in an in vitro binding study. The results of the present study demonstrate the power and potential application of protein-protein docking for the discovery of novel binding proteins for specific protein targets.

  3. Protein-Losing Gastroenteropathy

    PubMed Central

    Pops, Martin A.

    1966-01-01

    In the past 10 years with the development of improved methods, particularly radioisotope techniques, it has been demonstrated that a number of patients with gastrointestinal disease and depletion of plasma proteins become hypoproteinemic because of actual leakage of albumin and other plasma proteins into the lumen of the gastrointestinal tract. The site of protein leakage is variable depending on the underlying pathological state but the loss of protein-containing lymph through the gastrointestinal lymphatic channels seems to be the major mechanism for hypoproteinemia. It has become apparent that there exists a normal mechanism for secretion of plasma proteins into the gastrointestinal tract as part of the overall metabolism of the plasma proteins. When the process is exaggerated so that resynthesis of plasma protein cannot keep pace with its degradation, sometimes severe hypoproteinemia is the result. Such a pathological process has now been described in approximately 40 disease states. A review of all the techniques which can demonstrate gastroenteric protein loss reveals that there are no widely available quantitative tests but that accurate quantitation is not necessary for the diagnosis of protein losing gastroenteropathy. PMID:18730025

  4. The Halophile protein database.

    PubMed

    Sharma, Naveen; Farooqi, Mohammad Samir; Chaturvedi, Krishna Kumar; Lal, Shashi Bhushan; Grover, Monendra; Rai, Anil; Pandey, Pankaj

    2014-01-01

    Halophilic archaea/bacteria adapt to different salt concentration, namely extreme, moderate and low. These type of adaptations may occur as a result of modification of protein structure and other changes in different cell organelles. Thus proteins may play an important role in the adaptation of halophilic archaea/bacteria to saline conditions. The Halophile protein database (HProtDB) is a systematic attempt to document the biochemical and biophysical properties of proteins from halophilic archaea/bacteria which may be involved in adaptation of these organisms to saline conditions. In this database, various physicochemical properties such as molecular weight, theoretical pI, amino acid composition, atomic composition, estimated half-life, instability index, aliphatic index and grand average of hydropathicity (Gravy) have been listed. These physicochemical properties play an important role in identifying the protein structure, bonding pattern and function of the specific proteins. This database is comprehensive, manually curated, non-redundant catalogue of proteins. The database currently contains 59 897 proteins properties extracted from 21 different strains of halophilic archaea/bacteria. The database can be accessed through link. Database URL: http://webapp.cabgrid.res.in/protein/

  5. Human Plasma Protein C

    PubMed Central

    Kisiel, Walter

    1979-01-01

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

  6. Molecular simulations of lipid-mediated protein-protein interactions.

    PubMed

    de Meyer, Frédérick Jean-Marie; Venturoli, Maddalena; Smit, Berend

    2008-08-01

    Recent experimental results revealed that lipid-mediated interactions due to hydrophobic forces may be important in determining the protein topology after insertion in the membrane, in regulating the protein activity, in protein aggregation and in signal transduction. To gain insight into the lipid-mediated interactions between two intrinsic membrane proteins, we developed a mesoscopic model of a lipid bilayer with embedded proteins, which we studied with dissipative particle dynamics. Our calculations of the potential of mean force between transmembrane proteins show that hydrophobic forces drive long-range protein-protein interactions and that the nature of these interactions depends on the length of the protein hydrophobic segment, on the three-dimensional structure of the protein and on the properties of the lipid bilayer. To understand the nature of the computed potentials of mean force, the concept of hydrophilic shielding is introduced. The observed protein interactions are interpreted as resulting from the dynamic reorganization of the system to maintain an optimal hydrophilic shielding of the protein and lipid hydrophobic parts, within the constraint of the flexibility of the components. Our results could lead to a better understanding of several membrane processes in which protein interactions are involved. PMID:18487292

  7. Protein-protein interactions: methods for detection and analysis.

    PubMed Central

    Phizicky, E M; Fields, S

    1995-01-01

    The function and activity of a protein are often modulated by other proteins with which it interacts. This review is intended as a practical guide to the analysis of such protein-protein interactions. We discuss biochemical methods such as protein affinity chromatography, affinity blotting, coimmunoprecipitation, and cross-linking; molecular biological methods such as protein probing, the two-hybrid system, and phage display: and genetic methods such as the isolation of extragenic suppressors, synthetic mutants, and unlinked noncomplementing mutants. We next describe how binding affinities can be evaluated by techniques including protein affinity chromatography, sedimentation, gel filtration, fluorescence methods, solid-phase sampling of equilibrium solutions, and surface plasmon resonance. Finally, three examples of well-characterized domains involved in multiple protein-protein interactions are examined. The emphasis of the discussion is on variations in the approaches, concerns in evaluating the results, and advantages and disadvantages of the techniques. PMID:7708014

  8. The t(10;11)(p13;q14) in the U937 cell line results in the fusion of the AF10 gene and CALM, encoding a new member of the AP-3 clathrin assembly protein family.

    PubMed Central

    Dreyling, M H; Martinez-Climent, J A; Zheng, M; Mao, J; Rowley, J D; Bohlander, S K

    1996-01-01

    The translocation t(10;11)(p13;q14) is a recurring chromosomal abnormality that has been observed in patients with acute lymphoblastic leukemia as well as acute myeloid leukemia. We have recently reported that the monocytic cell line U937 has a t(10;11)(p13;q14) translocation. Using a combination of positional cloning and candidate gene approach, we cloned the breakpoint and were able to show that AF10 is fused to a novel gene that we named CALM (Clathrin Assembly Lymphoid Myeloid leukemia gene) located at 11q14. AF10, a putative transcription factor, had recently been cloned as one of the fusion partners of MLL. CALM has a very high homology in its N-terminal third to the murine ap-3 gene which is one of the clathrin assembly proteins. The N-terminal region of ap-3 has been shown to bind to clathrin and to have a high-affinity binding site for phosphoinositols. The identification of the CALM/AF10 fusion gene in the widely used U937 cell line will contribute to our understanding of the malignant phenotype of this line. Images Fig. 1 Fig. 3 PMID:8643484

  9. Correlation of C-reactive protein haplotypes with serum C-reactive protein level and response to anti-tumor necrosis factor therapy in UK rheumatoid arthritis patients: results from the Biologics in Rheumatoid Arthritis Genetics and Genomics Study Syndicate cohort

    PubMed Central

    2012-01-01

    Introduction In many European countries, restrictions exist around the prescription of anti-tumor necrosis factor (anti-TNF) treatments for rheumatoid arthritis (RA). Eligibility and response to treatment is assessed by using the disease activity score 28 (DAS28) algorithm, which incorporates one of two inflammatory markers, erythrocyte sedimentation rate (ESR) or C-reactive protein (CRP). Although DAS28-CRP provides a more reliable measure of disease activity, functional variants exist within the CRP gene that affect basal CRP production. Therefore, we aimed to determine the relation between functional genetic variants at the CRP gene locus and levels of serum CRP in RA patients, and whether these variants, alone or in combination, are correlated with DAS28-CRP and change in DAS28-CRP after anti-TNF treatment. Methods DNA samples from the Biologics in Rheumatoid Arthritis Genetics and Genomics Study Syndicate (BRAGGSS) were genotyped for rs1205, rs1800947, and rs3091244 by using either TaqMan or the Sequenom MassARRAY iPLEX system. Estimated haplotypes were constructed for each sample by using the expectation maximization algorithm implemented in the haplo.stats package within the R statistical program. CRP values were log transformed, and the association between single nucleotide polymorphisms (SNPs), haplotypes of SNPs and baseline CRP, baseline DAS28-CRP, and change in DAS28-CRP were evaluated by using linear regression in STATA v.10. Results Baseline CRP measurements were available for 599 samples with 442 also having data 6 months after treatment with an anti-TNF. For these 442 samples, the study had > 80% power to detect a clinically meaningful difference of 0.6 DAS28 Units for an allele frequency of 5%. Estimated haplotype frequencies corresponded with previous frequencies reported in the literature. Overall, no significant association was observed between any of the markers investigated and baseline CRP levels. Further, CRP haplotypes did not correlate

  10. Protein Unfolding and Alzheimer's

    NASA Astrophysics Data System (ADS)

    Cheng, Kelvin

    2012-10-01

    Early interaction events of beta-amyloid (Aβ) proteins with neurons have been associated with the pathogenesis of Alzheimer's disease. Knowledge pertaining to the role of lipid molecules, particularly cholesterol, in modulating the single Aβ interactions with neurons at the atomic length and picosecond time resolutions, remains unclear. In our research, we have used atomistic molecular dynamics simulations to explore early molecular events including protein insertion kinetics, protein unfolding, and protein-induced membrane disruption of Aβ in lipid domains that mimic the nanoscopic raft and non-raft regions of the neural membrane. In this talk, I will summarize our current work on investigating the role of cholesterol in regulating the Aβ interaction events with membranes at the molecular level. I will also explain how our results will provide new insights into understanding the pathogenesis of Alzheimer's disease associated with the Aβ proteins.

  11. Protein disulfide engineering.

    PubMed

    Dombkowski, Alan A; Sultana, Kazi Zakia; Craig, Douglas B

    2014-01-21

    Improving the stability of proteins is an important goal in many biomedical and industrial applications. A logical approach is to emulate stabilizing molecular interactions found in nature. Disulfide bonds are covalent interactions that provide substantial stability to many proteins and conform to well-defined geometric conformations, thus making them appealing candidates in protein engineering efforts. Disulfide engineering is the directed design of novel disulfide bonds into target proteins. This important biotechnological tool has achieved considerable success in a wide range of applications, yet the rules that govern the stabilizing effects of disulfide bonds are not fully characterized. Contrary to expectations, many designed disulfide bonds have resulted in decreased stability of the modified protein. We review progress in disulfide engineering, with an emphasis on the issue of stability and computational methods that facilitate engineering efforts.

  12. Protein crystallization in microgravity.

    PubMed

    Aibara, S; Shibata, K; Morita, Y

    1997-12-01

    A space experiment involving protein crystallization was conducted in a microgravity environment using the space shuttle "Endeavour" of STS-47, on a 9-day mission from September 12th to 20th in 1992. The crystallization was carried out according to a batch method, and 5 proteins were selected as flight samples for crystallization. Two of these proteins: hen egg-white lysozyme and co-amino acid: pyruvate aminotransferase from Pseudomonas sp. F-126, were obtained as single crystals of good diffraction quality. Since 1992 we have carried out several space experiments for protein crystallization aboard space shuttles and the space station MIR. Our experimental results obtained mainly from hen egg-white lysozyme are described below, focusing on the effects of microgravity on protein crystal growth.

  13. Delayed formation of zero-valent selenium nanoparticles by Bacillus mycoides SeITE01 as a consequence of selenite reduction under aerobic conditions

    PubMed Central

    2014-01-01

    Background Selenite (SeO32−) oxyanion shows severe toxicity to biota. Different bacterial strains exist that are capable of reducing SeO32− to non-toxic elemental selenium (Se0), with the formation of Se nanoparticles (SeNPs). These SeNPs might be exploited for technological applications due to their physico-chemical and biological characteristics. The present paper discusses the reduction of selenite to SeNPs by a strain of Bacillus sp., SeITE01, isolated from the rhizosphere of the Se-hyperaccumulator legume Astragalus bisulcatus. Results Use of 16S rRNA and GyrB gene sequence analysis positioned SeITE01 phylogenetically close to B. mycoides. On agarized medium, this strain showed rhizoid growth whilst, in liquid cultures, it was capable of reducing 0.5 and 2.0 mM SeO32− within 12 and 24 hours, respectively. The resultant Se0 aggregated to form nanoparticles and the amount of Se0 measured was equivalent to the amount of selenium originally added as selenite to the growth medium. A delay of more than 24 hours was observed between the depletion of SeO32 and the detection of SeNPs. Nearly spherical-shaped SeNPs were mostly found in the extracellular environment whilst rarely in the cytoplasmic compartment. Size of SeNPs ranged from 50 to 400 nm in diameter, with dimensions greatly influenced by the incubation times. Different SeITE01 protein fractions were assayed for SeO32− reductase capability, revealing that enzymatic activity was mainly associated with the membrane fraction. Reduction of SeO32− was also detected in the supernatant of bacterial cultures upon NADH addition. Conclusions The selenite reducing bacterial strain SeITE01 was attributed to the species Bacillus mycoides on the basis of phenotypic and molecular traits. Under aerobic conditions, the formation of SeNPs were observed both extracellularly or intracellullarly. Possible mechanisms of Se0 precipitation and SeNPs assembly are suggested. SeO32− is proposed to be enzimatically reduced to

  14. EDITORIAL: Precision proteins Precision proteins

    NASA Astrophysics Data System (ADS)

    Demming, Anna

    2010-06-01

    Since the birth of modern day medicine, during the times of Hippocrates in ancient Greece, the profession has developed from the rudimentary classification of disease into a rigorous science with an inspiring capability to treat and cure. Scientific methodology has distilled clinical diagnostic tools from the early arts of prognosis, which used to rely as much on revelation and prophecy, as intuition and judgement [1]. Over the past decade, research into the interactions between proteins and nanosystems has provided some ingenious and apt techniques for delving into the intricacies of anatomical systems. In vivo biosensing has emerged as a vibrant field of research, as much of medical diagnosis relies on the detection of substances or an imbalance in the chemicals in the body. The inherent properties of nanoscale structures, such as cantilevers, make them well suited to biosensing applications that demand the detection of molecules at very low concentrations. Measurable deflections in cantilevers functionalised with antibodies provide quantitative indicators of the presence of specific antigens when the two react. Such developments have roused mounting interest in the interactions of proteins with nanostructures, such as carbon nanotubes [3], which have demonstrated great potential as generic biomarkers. Plasmonic properties are also being exploited in sensing applications, such as the molecular sentinel recently devised by researchers in the US. The device uses the plasmonic properties of a silver nanoparticle linked to a Raman labelled hairpin DNA probe to signal changes in the probe geometry resulting from interactions with substances in the environment. Success stories so far include the detection of two specific genes associated with breast cancer [4]. A greater understanding of how RNA interference regulates gene expression has highlighted the potential of using this natural process as another agent for combating disease in personalized medicine. However, the

  15. EDITORIAL: Precision proteins Precision proteins

    NASA Astrophysics Data System (ADS)

    Demming, Anna

    2010-06-01

    Since the birth of modern day medicine, during the times of Hippocrates in ancient Greece, the profession has developed from the rudimentary classification of disease into a rigorous science with an inspiring capability to treat and cure. Scientific methodology has distilled clinical diagnostic tools from the early arts of prognosis, which used to rely as much on revelation and prophecy, as intuition and judgement [1]. Over the past decade, research into the interactions between proteins and nanosystems has provided some ingenious and apt techniques for delving into the intricacies of anatomical systems. In vivo biosensing has emerged as a vibrant field of research, as much of medical diagnosis relies on the detection of substances or an imbalance in the chemicals in the body. The inherent properties of nanoscale structures, such as cantilevers, make them well suited to biosensing applications that demand the detection of molecules at very low concentrations. Measurable deflections in cantilevers functionalised with antibodies provide quantitative indicators of the presence of specific antigens when the two react. Such developments have roused mounting interest in the interactions of proteins with nanostructures, such as carbon nanotubes [3], which have demonstrated great potential as generic biomarkers. Plasmonic properties are also being exploited in sensing applications, such as the molecular sentinel recently devised by researchers in the US. The device uses the plasmonic properties of a silver nanoparticle linked to a Raman labelled hairpin DNA probe to signal changes in the probe geometry resulting from interactions with substances in the environment. Success stories so far include the detection of two specific genes associated with breast cancer [4]. A greater understanding of how RNA interference regulates gene expression has highlighted the potential of using this natural process as another agent for combating disease in personalized medicine. However, the

  16. Protein based Block Copolymers

    PubMed Central

    Rabotyagova, Olena S.; Cebe, Peggy; Kaplan, David L.

    2011-01-01

    Advances in genetic engineering have led to the synthesis of protein-based block copolymers with control of chemistry and molecular weight, resulting in unique physical and biological properties. The benefits from incorporating peptide blocks into copolymer designs arise from the fundamental properties of proteins to adopt ordered conformations and to undergo self-assembly, providing control over structure formation at various length scales when compared to conventional block copolymers. This review covers the synthesis, structure, assembly, properties, and applications of protein-based block copolymers. PMID:21235251

  17. Amino acids and proteins.

    PubMed

    van Goudoever, Johannes B; Vlaardingerbroek, Hester; van den Akker, Chris H; de Groof, Femke; van der Schoor, Sophie R D

    2014-01-01

    Amino acids and protein are key factors for growth. The neonatal period requires the highest intake in life to meet the demands. Those demands include amino acids for growth, but proteins and amino acids also function as signalling molecules and function as neurotransmitters. Often the nutritional requirements are not met, resulting in a postnatal growth restriction. However, current knowledge on adequate levels of both amino acid as well as protein intake can avoid under nutrition in the direct postnatal phase, avoid the need for subsequent catch-up growth and improve later outcome.

  18. Protein-protein interaction databases: keeping up with growing interactomes

    PubMed Central

    2009-01-01

    Over the past few years, the number of known protein-protein interactions has increased substantially. To make this information more readily available, a number of publicly available databases have set out to collect and store protein-protein interaction data. Protein-protein interactions have been retrieved from six major databases, integrated and the results compared. The six databases (the Biological General Repository for Interaction Datasets [BioGRID], the Molecular INTeraction database [MINT], the Biomolecular Interaction Network Database [BIND], the Database of Interacting Proteins [DIP], the IntAct molecular interaction database [IntAct] and the Human Protein Reference Database [HPRD]) differ in scope and content; integration of all datasets is non-trivial owing to differences in data annotation. With respect to human protein-protein interaction data, HPRD seems to be the most comprehensive. To obtain a complete dataset, however, interactions from all six databases have to be combined. To overcome this limitation, meta-databases such as the Agile Protein Interaction Database (APID) offer access to integrated protein-protein interaction datasets, although these also currently have certain restrictions. PMID:19403463

  19. Ontology integration to identify protein complex in protein interaction networks

    PubMed Central

    2011-01-01

    Background Protein complexes can be identified from the protein interaction networks derived from experimental data sets. However, these analyses are challenging because of the presence of unreliable interactions and the complex connectivity of the network. The integration of protein-protein interactions with the data from other sources can be leveraged for improving the effectiveness of protein complexes detection algorithms. Methods We have developed novel semantic similarity method, which use Gene Ontology (GO) annotations to measure the reliability of protein-protein interactions. The protein interaction networks can be converted into a weighted graph representation by assigning the reliability values to each interaction as a weight. Following the approach of that of the previously proposed clustering algorithm IPCA which expands clusters starting from seeded vertices, we present a clustering algorithm OIIP based on the new weighted Protein-Protein interaction networks for identifying protein complexes. Results The algorithm OIIP is applied to the protein interaction network of Sacchromyces cerevisiae and identifies many well known complexes. Experimental results show that the algorithm OIIP has higher F-measure and accuracy compared to other competing approaches. PMID:22165991

  20. EIGER characterization results

    NASA Astrophysics Data System (ADS)

    Dinapoli, Roberto; Bergamaschi, Anna; Greiffenberg, Dominic; Henrich, Beat; Horisberger, Roland; Johnson, Ian; Mozzanica, Aldo; Radicci, Valeria; Schmitt, Bernd; Shi, Xintian; Tinti, Gemma

    2013-12-01

    Characterization and performance measurements have been done on several EIGER detector systems, produced with chips coming from two different lots, both with a lab X-ray source and at the Swiss Light Source (SLS). Results on the detector calibration, electronic noise, threshold dispersion, minimum achievable energy threshold, maximum detectable incoming photon flux and maximum frame rate are presented. An EIGER module is constructed from a ∼4×8 cm2 monolithic silicon sensor bump-bonded to 2 ×4 readout chips and contains 0.5 Mpixel. The first EIGER 500 K systems have been produced and images taken with these detectors are shown. Modules can be tiled together to form large area detectors; both a 9 Mpixel and a 16 Mpixel systems are at present under development for the coherent small angle X-ray scattering and protein crystallography beamlines of the SLS.

  1. Tetramer formation in Arabidopsis MADS domain proteins: analysis of a protein-protein interaction network

    PubMed Central

    2014-01-01

    Background MADS domain proteins are transcription factors that coordinate several important developmental processes in plants. These proteins interact with other MADS domain proteins to form dimers, and it has been proposed that they are able to associate as tetrameric complexes that regulate transcription of target genes. Whether the formation of functional tetramers is a widespread property of plant MADS domain proteins, or it is specific to few of these transcriptional regulators remains unclear. Results We analyzed the structure of the network of physical interactions among MADS domain proteins in Arabidopsis thaliana. We determined the abundance of subgraphs that represent the connection pattern expected for a MADS domain protein heterotetramer. These subgraphs were significantly more abundant in the MADS domain protein interaction network than in randomized analogous networks. Importantly, these subgraphs are not significantly frequent in a protein interaction network of TCP plant transcription factors, when compared to expectation by chance. In addition, we found that MADS domain proteins in tetramer-like subgraphs are more likely to be expressed jointly than proteins in other subgraphs. This effect is mainly due to proteins in the monophyletic MIKC clade, as there is no association between tetramer-like subgraphs and co-expression for proteins outside this clade. Conclusions Our results support that the tendency to form functional tetramers is widespread in the MADS domain protein-protein interaction network. Our observations also suggest that this trend is prevalent, or perhaps exclusive, for proteins in the MIKC clade. Because it is possible to retrodict several experimental results from our analyses, our work can be an important aid to make new predictions and facilitates experimental research on plant MADS domain proteins. PMID:24468197

  2. Fullerene sorting proteins.

    PubMed

    Calvaresi, Matteo; Zerbetto, Francesco

    2011-07-01

    Proteins bind fullerenes. Hydrophobic pockets can accommodate a carbon cage either in full or in part. However, the identification of proteins able to discriminate between different cages is an open issue. Prediction of candidates able to perform this function is desirable and is achieved with an inverse docking procedure that accurately accounts for (i) van der Waals interactions between the cage and the protein surface, (ii) desolvation free energy, (iii) shape complementarity, and (iv) minimization of the number of steric clashes through conformational variations. A set of more than 1000 protein structures is divided into four categories that either select C(60) or C(70) (p-C(60) or p-C(70)) and either accommodate the cages in the same pocket (homosaccic proteins, from σακκoζ meaning pocket) or in different pockets (heterosaccic proteins). In agreement with the experiments, the KcsA Potassium Channel is predicted to have one of the best performances for both cages. Possible ways to exploit the results and efficiently separate the two cages with proteins are also discussed.

  3. Protein-protein interaction network-based detection of functionally similar proteins within species.

    PubMed

    Song, Baoxing; Wang, Fen; Guo, Yang; Sang, Qing; Liu, Min; Li, Dengyun; Fang, Wei; Zhang, Deli

    2012-07-01

    Although functionally similar proteins across species have been widely studied, functionally similar proteins within species showing low sequence similarity have not been examined in detail. Identification of these proteins is of significant importance for understanding biological functions, evolution of protein families, progression of co-evolution, and convergent evolution and others which cannot be obtained by detection of functionally similar proteins across species. Here, we explored a method of detecting functionally similar proteins within species based on graph theory. After denoting protein-protein interaction networks using graphs, we split the graphs into subgraphs using the 1-hop method. Proteins with functional similarities in a species were detected using a method of modified shortest path to compare these subgraphs and to find the eligible optimal results. Using seven protein-protein interaction networks and this method, some functionally similar proteins with low sequence similarity that cannot detected by sequence alignment were identified. By analyzing the results, we found that, sometimes, it is difficult to separate homologous from convergent evolution. Evaluation of the performance of our method by gene ontology term overlap showed that the precision of our method was excellent.

  4. Protein enriched pasta: structure and digestibility of its protein network.

    PubMed

    Laleg, Karima; Barron, Cécile; Santé-Lhoutellier, Véronique; Walrand, Stéphane; Micard, Valérie

    2016-02-01

    Wheat (W) pasta was enriched in 6% gluten (G), 35% faba (F) or 5% egg (E) to increase its protein content (13% to 17%). The impact of the enrichment on the multiscale structure of the pasta and on in vitro protein digestibility was studied. Increasing the protein content (W- vs. G-pasta) strengthened pasta structure at molecular and macroscopic scales but reduced its protein digestibility by 3% by forming a higher covalently linked protein network. Greater changes in the macroscopic and molecular structure of the pasta were obtained by varying the nature of protein used for enrichment. Proteins in G- and E-pasta were highly covalently linked (28-32%) resulting in a strong pasta structure. Conversely, F-protein (98% SDS-soluble) altered the pasta structure by diluting gluten and formed a weak protein network (18% covalent link). As a result, protein digestibility in F-pasta was significantly higher (46%) than in E- (44%) and G-pasta (39%). The effect of low (55 °C, LT) vs. very high temperature (90 °C, VHT) drying on the protein network structure and digestibility was shown to cause greater molecular changes than pasta formulation. Whatever the pasta, a general strengthening of its structure, a 33% to 47% increase in covalently linked proteins and a higher β-sheet structure were observed. However, these structural differences were evened out after the pasta was cooked, resulting in identical protein digestibility in LT and VHT pasta. Even after VHT drying, F-pasta had the best amino acid profile with the highest protein digestibility, proof of its nutritional interest.

  5. Protein enriched pasta: structure and digestibility of its protein network.

    PubMed

    Laleg, Karima; Barron, Cécile; Santé-Lhoutellier, Véronique; Walrand, Stéphane; Micard, Valérie

    2016-02-01

    Wheat (W) pasta was enriched in 6% gluten (G), 35% faba (F) or 5% egg (E) to increase its protein content (13% to 17%). The impact of the enrichment on the multiscale structure of the pasta and on in vitro protein digestibility was studied. Increasing the protein content (W- vs. G-pasta) strengthened pasta structure at molecular and macroscopic scales but reduced its protein digestibility by 3% by forming a higher covalently linked protein network. Greater changes in the macroscopic and molecular structure of the pasta were obtained by varying the nature of protein used for enrichment. Proteins in G- and E-pasta were highly covalently linked (28-32%) resulting in a strong pasta structure. Conversely, F-protein (98% SDS-soluble) altered the pasta structure by diluting gluten and formed a weak protein network (18% covalent link). As a result, protein digestibility in F-pasta was significantly higher (46%) than in E- (44%) and G-pasta (39%). The effect of low (55 °C, LT) vs. very high temperature (90 °C, VHT) drying on the protein network structure and digestibility was shown to cause greater molecular changes than pasta formulation. Whatever the pasta, a general strengthening of its structure, a 33% to 47% increase in covalently linked proteins and a higher β-sheet structure were observed. However, these structural differences were evened out after the pasta was cooked, resulting in identical protein digestibility in LT and VHT pasta. Even after VHT drying, F-pasta had the best amino acid profile with the highest protein digestibility, proof of its nutritional interest. PMID:26829164

  6. Proteins interacting with cloning scars: a source of false positive protein-protein interactions.

    PubMed

    Banks, Charles A S; Boanca, Gina; Lee, Zachary T; Florens, Laurence; Washburn, Michael P

    2015-02-23

    A common approach for exploring the interactome, the network of protein-protein interactions in cells, uses a commercially available ORF library to express affinity tagged bait proteins; these can be expressed in cells and endogenous cellular proteins that copurify with the bait can be identified as putative interacting proteins using mass spectrometry. Control experiments can be used to limit false-positive results, but in many cases, there are still a surprising number of prey proteins that appear to copurify specifically with the bait. Here, we have identified one source of false-positive interactions in such studies. We have found that a combination of: 1) the variable sequence of the C-terminus of the bait with 2) a C-terminal valine "cloning scar" present in a commercially available ORF library, can in some cases create a peptide motif that results in the aberrant co-purification of endogenous cellular proteins. Control experiments may not identify false positives resulting from such artificial motifs, as aberrant binding depends on sequences that vary from one bait to another. It is possible that such cryptic protein binding might occur in other systems using affinity tagged proteins; this study highlights the importance of conducting careful follow-up studies where novel protein-protein interactions are suspected.

  7. Fc epsilon RI-mediated association of 6-micron beads with RBL-2H3 mast cells results in exclusion of signaling proteins from the forming phagosome and abrogation of normal downstream signaling

    PubMed Central

    1996-01-01

    Cells of the mucosal mast cell line, RBL-2H3, are normally stimulated to degranulate after aggregation of high affinity receptors for IgE (Fc epsilon RI) by soluble cross-linking ligands. This cellular degranulation process requires sustained elevation of cytoplasmic Ca2+. In this study, we investigated the response of RBL-2H3 cells to 6- micron beads coated with IgE-specific ligands. These ligand-coated beads cause only small, transient Ca2+ responses, even though the same ligands added in soluble form cause larger, more sustained Ca2+ responses. The ligand-coated 6-micron beads also fail to stimulate significant degranulation of RBL-2H3 cells, whereas much larger ligand- coated Sepharose beads stimulate ample degranulation. Confocal fluorescence microscopy shows that the 6-micron beads (but not the Sepharose beads) are phagocytosed by RBL-2H3 cells and that, beginning with the initial stages of bead engulfment, there is exclusion of many plasma membrane components from the 6-micron bead/cell interface, including p53/56lyn and several other markers for detergent-resistant membrane domains, as well as an integrin and unliganded IgE-Fc epsilon RI. The fluorescent lipid probe DiIC16 is a marker for the membrane domains that is excluded from the cell/bead interface, whereas a structural analogue, fast DiI, which differs from DiIC16 by the presence of unsaturated acyl chains, is not substantially excluded from the interface. None of these components are excluded from the interface of RBL-2H3 cells and the large Sepharose beads. Additional confocal microscopy analysis indicates that microfilaments are involved in the exclusion of plasma membrane components from the cell/bead interface. These results suggest that initiation of phagocytosis diverts normal signaling pathways in a cytoskeleton-driven membrane clearance process that alters the physiological response of the cells. PMID:8830772

  8. Effects of dabigatran on the cellular and protein phase of coagulation in patients with coronary artery disease on dual antiplatelet therapy with aspirin and clopidogrel. Results from a prospective, randomised, double-blind, placebo-controlled study.

    PubMed

    Franchi, Francesco; Rollini, Fabiana; Cho, Jung Rae; King, Rhodri; Phoenix, Fladia; Bhatti, Mona; DeGroat, Christopher; Tello-Montoliu, Antonio; Zenni, Martin M; Guzman, Luis A; Bass, Theodore A; Ajjan, Ramzi A; Angiolillo, Dominick J

    2016-03-01

    There is growing interest in understanding the effects of adding an oral anticoagulant in patients on dual antiplatelet therapy (DAPT). Vitamin K antagonists (VKAs) and clopidogrel represent the most broadly utilised oral anticoagulant and P2Y12 receptor inhibitor, respectively. However, VKAs can interfere with clopidogrel metabolism via the cytochrome P450 (CYP) system which in turn may result in an increase in platelet reactivity. Dabigatran is a direct acting (anti-II) oral anticoagulant which does not interfere with CYP and has favourable safety and efficacy profiles compared with VKAs. The pharmacodynamic (PD) effects on platelet reactivity and clot kinetic of adjunctive dabigatran therapy in patients on DAPT are poorly explored. In this prospective, randomised, double-blind, placebo-controlled PD study, patients (n=30) on maintenance DAPT with aspirin and clopidogrel were randomised to either dabigatran 150 mg bid or placebo for seven days. PD testing was performed before and after treatment using four different assays exploring multiple pathways of platelet aggregation and fibrin clot kinetics: light transmittance aggregometry (LTA), multiple electrode aggregometry (MEA), kaolin-activated thromboelastography (TEG) and turbidimetric assays. There were no differences in multiple measures of platelet reactivity investigating purinergic and non-purinergic signaling pathways assessed by LTA, MEA and TEG platelet mapping. Dabigatran significantly increased parameters related to thrombin activity and thrombus generation, and delayed fibrin clot formation, without affecting clot structure or fibrinolysis. In conclusion, in patients on DAPT with aspirin and clopidogrel, adjunctive dabigatran therapy is not associated with modulation of profiles of platelet reactivity as determined by several assays assessing multiple platelet signalling pathways. However, dabigatran significantly interferes with parameters related to thrombin activity and delays fibrin clot formation

  9. Prophylaxis vs. on-demand treatment with BAY 81-8973, a full-length plasma protein-free recombinant factor VIII product: results from a randomized trial (LEOPOLD II)

    PubMed Central

    Kavakli, K; Yang, R; Rusen, L; Beckmann, H; Tseneklidou-Stoeter, D; Maas Enriquez, M

    2015-01-01

    Background BAY 81-8973 is a new full-length human recombinant factor VIII product manufactured with technologies to improve consistency in glycosylation and expression to optimize clinical performance. Objectives To demonstrate superiority of prophylaxis vs. on-demand therapy with BAY 81-8973 in patients with severe hemophilia A. Patients/Methods In this multinational, randomized, open-label crossover study (LEOPOLD II; ClinicalTrials.gov identifier: NCT01233258), males aged 12–65 years with severe hemophilia A were randomized to twice-weekly prophylaxis (20–30 IU kg−1), 3-times-weekly prophylaxis (30–40 IU kg−1), or on-demand treatment with BAY 81-8973. Potency labeling for BAY 81-8973 was based on the chromogenic substrate assay or adjusted to the one-stage assay. Primary efficacy endpoint was annualized number of all bleeds (ABR). Adverse events (AEs) and immunogenicity were also assessed. Results Eighty patients (on demand, n = 21; twice-weekly prophylaxis, n = 28; 3-times-weekly prophylaxis, n = 31) were treated and analyzed. Mean ± SD ABR was significantly lower with prophylaxis (twice-weekly, 5.7 ± 7.2; 3-times-weekly, 4.3 ± 6.5; combined, 4.9 ± 6.8) vs. on-demand treatment (57.7 ± 24.6; P < 0.0001, anova). Median ABR was reduced by 97% with prophylaxis (twice-weekly, 4.0; 3-times-weekly, 2.0; combined, 2.0) vs. on-demand treatment (60.0). Median ABR was higher with twice-weekly vs. 3-times-weekly prophylaxis during the first 6-month treatment period (4.1 vs. 2.0) but was comparable in the second 6-month period (1.1 vs. 2.0). Few patients reported treatment-related AEs (4%); no treatment-related serious AEs or inhibitors were reported. Conclusions Twice-weekly or 3-times-weekly prophylaxis with BAY 81-8973 reduced median ABR by 97% compared with on-demand therapy, confirming the superiority of prophylaxis. Treatment with BAY 81-8973 was well tolerated. PMID:25546368

  10. Recombinant protein production technology

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Recombinant protein production is an important technology for antibody production, biochemical activity study, and structural determination during the post-genomic era. Limiting factors in recombinant protein production include low-level protein expression, protein precipitation, and loss of protein...

  11. Protein crystal growth in microgravity

    NASA Technical Reports Server (NTRS)

    Rosenblum, William M.; Delucas, Lawrence J.; Wilson, William W.

    1989-01-01

    Major advances have been made in several of the experimental aspects of protein crystallography, leaving protein crystallization as one of the few remaining bottlenecks. As a result, it has become important that the science of protein crystal growth is better understood and that improved methods for protein crystallization are developed. Preliminary experiments with both small molecules and proteins indicate that microgravity may beneficially affect crystal growth. For this reason, a series of protein crystal growth experiments using the Space Shuttle was initiated. The preliminary space experiments were used to evolve prototype hardware that will form the basis for a more advanced system that can be used to evaluate effects of gravity on protein crystal growth. Various optical techniques are being utilized to monitor the crystal growth process from the incipient or nucleation stage and throughout the growth phase. The eventual goal of these studies is to develop a system which utilizes optical monitoring for dynamic control of the crystallization process.

  12. Highly specific protein-protein interactions, evolution and negative design.

    PubMed

    Sear, Richard P

    2004-12-01

    We consider highly specific protein-protein interactions in proteomes of simple model proteins. We are inspired by the work of Zarrinpar et al (2003 Nature 426 676). They took a binding domain in a signalling pathway in yeast and replaced it with domains of the same class but from different organisms. They found that the probability of a protein binding to a protein from the proteome of a different organism is rather high, around one half. We calculate the probability of a model protein from one proteome binding to the protein of a different proteome. These proteomes are obtained by sampling the space of functional proteomes uniformly. In agreement with Zarrinpar et al we find that the probability of a protein binding a protein from another proteome is rather high, of order one tenth. Our results, together with those of Zarrinpar et al, suggest that designing, say, a peptide to block or reconstitute a single signalling pathway, without affecting any other pathways, requires knowledge of all the partners of the class of binding domains the peptide is designed to mimic. This knowledge is required to use negative design to explicitly design out interactions of the peptide with proteins other than its target. We also found that patches that are required to bind with high specificity evolve more slowly than those that are required only to not bind to any other patch. This is consistent with some analysis of sequence data for proteins engaged in highly specific interactions.

  13. Cotton and Protein Interactions

    SciTech Connect

    Goheen, Steven C.; Edwards, J. V.; Rayburn, Alfred R.; Gaither, Kari A.; Castro, Nathan J.

    2006-06-30

    The adsorbent properties of important wound fluid proteins and cotton cellulose are reviewed. This review focuses on the adsorption of albumin to cotton-based wound dressings and some chemically modified derivatives targeted for chronic wounds. Adsorption of elastase in the presence of albumin was examined as a model to understand the interactive properties of these wound fluid components with cotton fibers. In the chronic non-healing wound, elastase appears to be over-expressed, and it digests tissue and growth factors, interfering with the normal healing process. Albumin is the most prevalent protein in wound fluid, and in highly to moderately exudative wounds, it may bind significantly to the fibers of wound dressings. Thus, the relative binding properties of both elastase and albumin to wound dressing fibers are of interest in the design of more effective wound dressings. The present work examines the binding of albumin to two different derivatives of cotton, and quantifies the elastase binding to the same derivatives following exposure of albumin to the fiber surface. An HPLC adsorption technique was employed coupled with a colorimetric enzyme assay to quantify the relative binding properties of albumin and elastase to cotton. The results of wound protein binding are discussed in relation to the porosity and surface chemistry interactions of cotton and wound proteins. Studies are directed to understanding the implications of protein adsorption phenomena in terms of fiber-protein models that have implications for rationally designing dressings for chronic wounds.

  14. Protein Adsorption in Three Dimensions

    PubMed Central

    Vogler, Erwin A.

    2011-01-01

    initially-adsorbed protein. Interphase protein concentration CI increases as VI decreases, resulting in slow reduction in interfacial energetics. Steady-state is governed by a net partition coefficient P=(/CBCI). In the process of occupying space within the interphase, adsorbing protein molecules must displace an equivalent volume of interphase water. Interphase water is itself associated with surface-bound water through a network of transient hydrogen bonds. Displacement of interphase water thus requires an amount of energy that depends on the adsorbent surface chemistry/energy. This “adsorption-dehydration” step is the significant free-energy cost of adsorption that controls the maximum amount of protein that can be adsorbed at steady state to a unit adsorbent-surface area (the adsorbent capacity). As adsorbent hydrophilicity increases, protein adsorption monotonically decreases because the energetic cost of surface dehydration increases, ultimately leading to no protein adsorption near an adsorbent water wettability (surface energy) characterized by a water contact angle θ → 65°. Consequently, protein does not adsorb (accumulate at interphase concentrations greater than bulk solution) to more hydrophilic adsorbents exhibiting θ < 65° . For adsorbents bearing strong Lewis acid/base chemistry such as ion-exchange resins, protein/surface interactions can be highly favorable, causing protein to adsorb in multilayers in a relatively thick interphase. A straightforward, three-component free energy relationship captures salient features of protein adsorption to all surfaces predicting that the overall free energy of protein adsorption ΔGadso is a relatively small multiple of thermal energy for any surface chemistry (except perhaps for bioengineered surfaces bearing specific ligands for adsorbing protein) because a surface chemistry that interacts chemically with proteins must also interact with water through hydrogen bonding. In this way, water moderates protein

  15. Protein-protein interaction network analysis of cirrhosis liver disease

    PubMed Central

    Safaei, Akram; Rezaei Tavirani, Mostafa; Arefi Oskouei, Afsaneh; Zamanian Azodi, Mona; Mohebbi, Seyed Reza; Nikzamir, Abdol Rahim

    2016-01-01

    Aim: Evaluation of biological characteristics of 13 identified proteins of patients with cirrhotic liver disease is the main aim of this research. Background: In clinical usage, liver biopsy remains the gold standard for diagnosis of hepatic fibrosis. Evaluation and confirmation of liver fibrosis stages and severity of chronic diseases require a precise and noninvasive biomarkers. Since the early detection of cirrhosis is a clinical problem, achieving a sensitive, specific and predictive novel method based on biomarkers is an important task. Methods: Essential analysis, such as gene ontology (GO) enrichment and protein-protein interactions (PPI) was undergone EXPASy, STRING Database and DAVID Bioinformatics Resources query. Results: Based on GO analysis, most of proteins are located in the endoplasmic reticulum lumen, intracellular organelle lumen, membrane-enclosed lumen, and extracellular region. The relevant molecular functions are actin binding, metal ion binding, cation binding and ion binding. Cell adhesion, biological adhesion, cellular amino acid derivative, metabolic process and homeostatic process are the related processes. Protein-protein interaction network analysis introduced five proteins (fibroblast growth factor receptor 4, tropomyosin 4, tropomyosin 2 (beta), lectin, Lectin galactoside-binding soluble 3 binding protein and apolipoprotein A-I) as hub and bottleneck proteins. Conclusion: Our result indicates that regulation of lipid metabolism and cell survival are important biological processes involved in cirrhosis disease. More investigation of above mentioned proteins will provide a better understanding of cirrhosis disease. PMID:27099671

  16. Protein structure modeling with MODELLER.

    PubMed

    Webb, Benjamin; Sali, Andrej

    2014-01-01

    Genome sequencing projects have resulted in a rapid increase in the number of known protein sequences. In contrast, only about one-hundredth of these sequences have been characterized at atomic resolution using experimental structure determination methods. Computational protein structure modeling techniques have the potential to bridge this sequence-structure gap. In this chapter, we present an example that illustrates the use of MODELLER to construct a comparative model for a protein with unknown structure. Automation of a similar protocol has resulted in models of useful accuracy for domains in more than half of all known protein sequences.

  17. Comparative effects of selenite and selenate on nitrate assimilation in barley seedlings

    NASA Technical Reports Server (NTRS)

    Aslam, M.; Harbit, K. B.; Huffaker, R. C.

    1990-01-01

    The effect of SeO3= and SeO4= on NO3- assimilation in 8-d-old barley (Hordeum vulgare L.) seedlings was studied over a 24-h period. Selenite at 0.1 mol m-3 in the uptake solutions severely inhibited the induction of NO3- uptake and active nitrate reductases. Selenate, at 1.0 mol m-3 in the nutrient solution, had little effect on induction of activities of these systems until after 12 h; however, when the seedlings were pretreated with 1.0 mol m-3 SeO4= for 24 h, subsequent NO3- uptake from SeO4(=) -free solutions was inhibited about 60%. Sulphate partially alleviated the inhibitory effect of SeO3= when supplied together in the ambient solutions, but had no effect in seedlings pretreated with SeO3=. By contrast, SO4= partially alleviated the inhibitory effect of SeO4= even in seedlings pretreated with SeO4=. Since uptake of NO3- by intact seedlings was also inhibited by SO3=, the percentage of the absorbed NO3- that was reduced was not affected. By contrast, SeO4=, which affected NO3- uptake much less, inhibited the percentage reduced of that absorbed. However, when supplied to detached leaves, both SeO3= and SeO4= inhibited the in vivo reduction of NO3- as well as induction of nitrate reductase and nitrite reductase activities. Selenite was more inhibitory than SeO4= ; approximately a five to 10 times higher concentration of SeO4= than SeO3= was required to achieve similar inhibition. In detached leaves, the inhibitory effect of both SeO3= and SeO4= on in vivo NO3- reduction as well as on the induction of nitrate reductase activity was partially alleviated by SO4=. The inhibitory effects of Se salts on the induction of the nitrite reductase were, however, completely alleviated by SO4=. The results show that in barley seedlings SeO3= is more toxic than SeO4=. The reduction of SeO4= to SeO3= may be a rate limiting step in causing Se toxicity.

  18. Prediction of thermodynamic instabilities of protein solutions from simple protein-protein interactions

    NASA Astrophysics Data System (ADS)

    D'Agostino, Tommaso; Solana, José Ramón; Emanuele, Antonio

    2013-10-01

    Statistical thermodynamics of protein solutions is often studied in terms of simple, microscopic models of particles interacting via pairwise potentials. Such modelling can reproduce the short range structure of protein solutions at equilibrium and predict thermodynamics instabilities of these systems. We introduce a square well model of effective protein-protein interaction that embeds the solvent’s action. We modify an existing model [45] by considering a well depth having an explicit dependence on temperature, i.e. an explicit free energy character, thus encompassing the statistically relevant configurations of solvent molecules around proteins. We choose protein solutions exhibiting demixing upon temperature decrease (lysozyme, enthalpy driven) and upon temperature increase (haemoglobin, entropy driven). We obtain satisfactory fits of spinodal curves for both the two proteins without adding any mean field term, thus extending the validity of the original model. Our results underline the solvent role in modulating or stretching the interaction potential.

  19. Young proteins experience more variable selection pressures than old proteins.

    PubMed

    Vishnoi, Anchal; Kryazhimskiy, Sergey; Bazykin, Georgii A; Hannenhalli, Sridhar; Plotkin, Joshua B

    2010-11-01

    It is well known that young proteins tend to experience weaker purifying selection and evolve more quickly than old proteins. Here, we show that, in addition, young proteins tend to experience more variable selection pressures over time than old proteins. We demonstrate this pattern in three independent taxonomic groups: yeast, Drosophila, and mammals. The increased variability of selection pressures on young proteins is highly significant even after controlling for the fact that young proteins are typically shorter and experience weaker purifying selection than old proteins. The majority of our results are consistent with the hypothesis that the function of a young gene tends to change over time more readily than that of an old gene. At the same time, our results may be caused in part by young genes that serve constant functions over time, but nevertheless appear to evolve under changing selection pressures due to depletion of adaptive mutations. In either case, our results imply that the evolution of a protein-coding sequence is partly determined by its age and origin, and not only by the phenotypic properties of the encoded protein. We discuss, via specific examples, the consequences of these findings for understanding of the sources of evolutionary novelty.

  20. A new protein structure representation for efficient protein function prediction.

    PubMed

    Maghawry, Huda A; Mostafa, Mostafa G M; Gharib, Tarek F

    2014-12-01

    One of the challenging problems in bioinformatics is the prediction of protein function. Protein function is the main key that can be used to classify different proteins. Protein function can be inferred experimentally with very small throughput or computationally with very high throughput. Computational methods are sequence based or structure based. Structure-based methods produce more accurate protein function prediction. In this article, we propose a new protein structure representation for efficient protein function prediction. The representation is based on three-dimensional patterns of protein residues. In the analysis, we used protein function based on enzyme activity through six mechanistically diverse enzyme superfamilies: amidohydrolase, crotonase, haloacid dehalogenase, isoprenoid synthase type I, and vicinal oxygen chelate. We applied three different classification methods, naïve Bayes, k-nearest neighbors, and random forest, to predict the enzyme superfamily of a given protein. The prediction accuracy using the proposed representation outperforms a recently introduced representation method that is based only on the distance patterns. The results show that the proposed representation achieved prediction accuracy up to 98%, with improvement of about 10% on average.

  1. Converting a marginally hydrophobic soluble protein into a membrane protein.

    PubMed

    Nørholm, Morten H H; Cunningham, Fiona; Deber, Charles M; von Heijne, Gunnar

    2011-03-18

    δ-Helices are marginally hydrophobic α-helical segments in soluble proteins that exhibit certain sequence characteristics of transmembrane (TM) helices [Cunningham, F., Rath, A., Johnson, R. M. & Deber, C. M. (2009). Distinctions between hydrophobic helices in globular proteins and TM segments as factors in protein sorting. J. Biol. Chem., 284, 5395-402]. In order to better understand the difference between δ-helices and TM helices, we have studied the insertion of five TM-like δ-helices into dog pancreas microsomal membranes. Using model constructs in which an isolated δ-helix is engineered into a bona fide membrane protein, we find that, for two δ-helices originating from secreted proteins, at least three single-nucleotide mutations are necessary to obtain efficient membrane insertion, whereas one mutation is sufficient in a δ-helix from the cytosolic protein P450BM-3. We further find that only when the entire upstream region of the mutated δ-helix in the intact cytochrome P450BM-3 is deleted does a small fraction of the truncated protein insert into microsomes. Our results suggest that upstream portions of the polypeptide, as well as embedded charged residues, protect δ-helices in globular proteins from being recognized by the signal recognition particle-Sec61 endoplasmic-reticulum-targeting machinery and that δ-helices in secreted proteins are mutationally more distant from TM helices than δ-helices in cytosolic proteins.

  2. Cholesterol testing and results

    MedlinePlus

    ... VLDL cholesterol) Lipoproteins are made of fat and protein. They carry cholesterol, triglycerides, and other fats, called ... Pencina MJ, Navar-Boggan AM, D'Agostino RB Sr, Williams K, Neely B, Sniderman AD, Peterson ED. ...

  3. Development of a Split SNAP-CLIP Double Labeling System for Tracking Proteins Following Dissociation from Protein-Protein Complexes in Living Cells.

    PubMed

    Mie, Masayasu; Naoki, Tatsuhiko; Kobatake, Eiry

    2016-08-16

    The split SNAP-tag protein-fragment complementation assay (PCA) is a useful tool for imaging protein-protein interactions (PPIs) in living cells. In contrast to conventional methods employed for imaging PPIs, the split SNAP-tag PCA enables tracking of proteins following dissociation from protein-protein complexes. A limitation of this system, however, is that it only allows for labeling and tracking of one of the proteins forming the protein-protein complex. To track both proteins forming a protein-protein complex, each protein needs to be appropriately labeled. In this study, a split SNAP-CLIP double labeling system is developed and applied for tracking of each protein forming a protein-protein complex. As a proof-of concept, FM protein for PPIs and protein kinase C alpha (PKCα) for translocation are introduced to a split SNAP-CLIP double labeling system. The results show a split SNAP-CLIP double labeling system enables labeling of both proteins in a protein-protein complex and subsequent tracking of each of the proteins following dissociation from the protein-protein complexes in living cells.

  4. Protein electrophoresis - serum

    MedlinePlus

    ... of protein and fat, called lipoproteins (such as LDL cholesterol). ... globulin proteins may indicate: Abnormally low level of LDL cholesterol Malnutrition Increased gamma globulin proteins may indicate: Bone ...

  5. Protein energy malnutrition.

    PubMed

    Grover, Zubin; Ee, Looi C

    2009-10-01

    Protein energy malnutrition (PEM) is a common problem worldwide and occurs in both developing and industrialized nations. In the developing world, it is frequently a result of socioeconomic, political, or environmental factors. In contrast, protein energy malnutrition in the developed world usually occurs in the context of chronic disease. There remains much variation in the criteria used to define malnutrition, with each method having its own limitations. Early recognition, prompt management, and robust follow up are critical for best outcomes in preventing and treating PEM.

  6. Modeling Mercury in Proteins

    SciTech Connect

    Smith, Jeremy C; Parks, Jerry M

    2016-01-01

    Mercury (Hg) is a naturally occurring element that is released into the biosphere both by natural processes and anthropogenic activities. Although its reduced, elemental form Hg(0) is relatively non-toxic, other forms such as Hg2+ and, in particular, its methylated form, methylmercury, are toxic, with deleterious effects on both ecosystems and humans. Microorganisms play important roles in the transformation of mercury in the environment. Inorganic Hg2+ can be methylated by certain bacteria and archaea to form methylmercury. Conversely, bacteria also demethylate methylmercury and reduce Hg2+ to relatively inert Hg(0). Transformations and toxicity occur as a result of mercury interacting with various proteins. Clearly, then, understanding the toxic effects of mercury and its cycling in the environment requires characterization of these interactions. Computational approaches are ideally suited to studies of mercury in proteins because they can provide a detailed picture and circumvent issues associated with toxicity. Here we describe computational methods for investigating and characterizing how mercury binds to proteins, how inter- and intra-protein transfer of mercury is orchestrated in biological systems, and how chemical reactions in proteins transform the metal. We describe quantum chemical analyses of aqueous Hg(II), which reveal critical factors that determine ligand binding propensities. We then provide a perspective on how we used chemical reasoning to discover how microorganisms methylate mercury. We also highlight our combined computational and experimental studies of the proteins and enzymes of the mer operon, a suite of genes that confers mercury resistance in many bacteria. Lastly, we place work on mercury in proteins in the context of what is needed for a comprehensive multi-scale model of environmental mercury cycling.

  7. The Hedgehog protein family.

    PubMed

    Bürglin, Thomas R

    2008-01-01

    The Hedgehog (Hh) pathway is one of the fundamental signal transduction pathways in animal development and is also involved in stem-cell maintenance and carcinogenesis. The hedgehog (hh) gene was first discovered in Drosophila, and members of the family have since been found in most metazoa. Hh proteins are composed of two domains, an amino-terminal domain HhN, which has the biological signal activity, and a carboxy-terminal autocatalytic domain HhC, which cleaves Hh into two parts in an intramolecular reaction and adds a cholesterol moiety to HhN. HhC has sequence similarity to the self-splicing inteins, and the shared region is termed Hint. New classes of proteins containing the Hint domain have been discovered recently in bacteria and eukaryotes, and the Hog class, of which Hh proteins comprise one family, is widespread throughout eukaryotes. The non-Hh Hog proteins have carboxy-terminal domains (the Hog domain) highly similar to HhC, although they lack the HhN domain, and instead have other amino-terminal domains. Hog proteins are found in many protists, but the Hh family emerged only in early metazoan evolution. HhN is modified by cholesterol at its carboxyl terminus and by palmitate at its amino terminus in both flies and mammals. The modified HhN is released from the cell and travels through the extracellular space. On binding its receptor Patched, it relieves the inhibition that Patched exerts on Smoothened, a G-protein-coupled receptor. The resulting signaling cascade converges on the transcription factor Cubitus interruptus (Ci), or its mammalian counterparts, the Gli proteins, which activate or repress target genes.

  8. Protein-water dynamics in antifreeze protein III activity

    NASA Astrophysics Data System (ADS)

    Xu, Yao; Bäumer, Alexander; Meister, Konrad; Bischak, Connor G.; DeVries, Arthur L.; Leitner, David M.; Havenith, Martina

    2016-03-01

    We combine Terahertz absorption spectroscopy (THz) and molecular dynamics (MD) simulations to investigate the underlying molecular mechanism for the antifreeze activity of one class of antifreeze protein, antifreeze protein type III (AFP-III) with a focus on the collective water hydrogen bond dynamics near the protein. After summarizing our previous work on AFPs, we present a new investigation of the effects of cosolutes on protein antifreeze activity by adding sodium citrate to the protein solution of AFP-III. Our results reveal that for AFP-III, unlike some other AFPs, the addition of the osmolyte sodium citrate does not affect the hydrogen bond dynamics at the protein surface significantly, as indicated by concentration dependent THz measurements. The present data, in combination with our previous THz measurements and molecular simulations, confirm that while long-range solvent perturbation is a necessary condition for the antifreeze activity of AFP-III, the local binding affinity determines the size of the hysteresis.

  9. Protein Adsorption in Three Dimensions

    PubMed Central

    Vogler, Erwin A.

    2011-01-01

    initially-adsorbed protein. Interphase protein concentration CI increases as VI decreases, resulting in slow reduction in interfacial energetics. Steady-state is governed by a net partition coefficient P=(/CBCI). In the process of occupying space within the interphase, adsorbing protein molecules must displace an equivalent volume of interphase water. Interphase water is itself associated with surface-bound water through a network of transient hydrogen bonds. Displacement of interphase water thus requires an amount of energy that depends on the adsorbent surface chemistry/energy. This “adsorption-dehydration” step is the significant free-energy cost of adsorption that controls the maximum amount of protein that can be adsorbed at steady state to a unit adsorbent-surface area (the adsorbent capacity). As adsorbent hydrophilicity increases, protein adsorption monotonically decreases because the energetic cost of surface dehydration increases, ultimately leading to no protein adsorption near an adsorbent water wettability (surface energy) characterized by a water contact angle θ → 65°. Consequently, protein does not adsorb (accumulate at interphase concentrations greater than bulk solution) to more hydrophilic adsorbents exhibiting θ < 65° . For adsorbents bearing strong Lewis acid/base chemistry such as ion-exchange resins, protein/surface interactions can be highly favorable, causing protein to adsorb in multilayers in a relatively thick interphase. A straightforward, three-component free energy relationship captures salient features of protein adsorption to all surfaces predicting that the overall free energy of protein adsorption ΔGadso is a relatively small multiple of thermal energy for any surface chemistry (except perhaps for bioengineered surfaces bearing specific ligands for adsorbing protein) because a surface chemistry that interacts chemically with proteins must also interact with water through hydrogen bonding. In this way, water moderates protein

  10. Protein farnesyltransferase and protein prenylation in Plasmodium falciparum.

    PubMed

    Chakrabarti, Debopam; Da Silva, Thiago; Barger, Jennifer; Paquette, Steve; Patel, Hetal; Patterson, Shelley; Allen, Charles M

    2002-11-01

    Comparison of the malaria parasite and mammalian protein prenyltransferases and their cellular substrates is important for establishing this enzyme as a target for developing antimalarial agents. Nineteen heptapeptides differing only in their carboxyl-terminal amino acid were tested as alternative substrates of partially purified Plasmodium falciparum protein farnesyltransferase. Only NRSCAIM and NRSCAIQ serve as substrates, with NRSCAIM being the best. Peptidomimetics, FTI-276 and GGTI-287, inhibit the transferase with IC(50) values of 1 and 32 nm, respectively. Incubation of P. falciparum-infected erythrocytes with [(3)H]farnesol labels 50- and 22-28-kDa proteins, whereas [(3)H]geranylgeraniol labels only 22-28-kDa proteins. The 50-kDa protein is shown to be farnesylated, whereas the 22-28-kDa proteins are geranylgeranylated, irrespective of the labeling prenol. Protein labeling is inhibited more than 50% by either 5 microm FTI-277 or GGTI-298. The same concentration of inhibitors also inhibits parasite growth from the ring stage by 50%, decreases expression of prenylated proteins as measured with prenyl-specific antibody, and inhibits parasite differentiation beyond the trophozoite stage. Furthermore, differentiation specific prenylation of P. falciparum proteins is demonstrated. Protein labeling is detected predominantly during the trophozoite to schizont and schizont to ring transitions. These results demonstrate unique properties of protein prenylation in P. falciparum: a limited specificity of the farnesyltransferase for peptide substrates compared with mammalian enzymes, the ability to use farnesol to label both farnesyl and geranylgeranyl moieties on proteins, differentiation specific protein prenylation, and the ability of peptidomimetic prenyltransferase inhibitors to block parasite differentiation.

  11. Proteins aggregation and human diseases

    NASA Astrophysics Data System (ADS)

    Hu, Chin-Kun

    2015-04-01

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

  12. Phylogenomics of Prokaryotic Ribosomal Proteins

    PubMed Central

    Yutin, Natalya; Puigbò, Pere; Koonin, Eugene V.; Wolf, Yuri I.

    2012-01-01

    Archaeal and bacterial ribosomes contain more than 50 proteins, including 34 that are universally conserved in the three domains of cellular life (bacteria, archaea, and eukaryotes). Despite the high sequence conservation, annotation of ribosomal (r-) protein genes is often difficult because of their short lengths and biased sequence composition. We developed an automated computational pipeline for identification of r-protein genes and applied it to 995 completely sequenced bacterial and 87 archaeal genomes available in the RefSeq database. The pipeline employs curated seed alignments of r-proteins to run position-specific scoring matrix (PSSM)-based BLAST searches against six-frame genome translations, mitigating possible gene annotation errors. As a result of this analysis, we performed a census of prokaryotic r-protein complements, enumerated missing and paralogous r-proteins, and analyzed the distributions of ribosomal protein genes among chromosomal partitions. Phyletic patterns of bacterial and archaeal r-protein genes were mapped to phylogenetic trees reconstructed from concatenated alignments of r-proteins to reveal the history of likely multiple independent gains and losses. These alignments, available for download, can be used as search profiles to improve genome annotation of r-proteins and for further comparative genomics studies. PMID:22615861

  13. Exosome engineering for efficient intracellular delivery of soluble proteins using optically reversible protein-protein interaction module.

    PubMed

    Yim, Nambin; Ryu, Seung-Wook; Choi, Kyungsun; Lee, Kwang Ryeol; Lee, Seunghee; Choi, Hojun; Kim, Jeongjin; Shaker, Mohammed R; Sun, Woong; Park, Ji-Ho; Kim, Daesoo; Heo, Won Do; Choi, Chulhee

    2016-01-01

    Nanoparticle-mediated delivery of functional macromolecules is a promising method for treating a variety of human diseases. Among nanoparticles, cell-derived exosomes have recently been highlighted as a new therapeutic strategy for the in vivo delivery of nucleotides and chemical drugs. Here we describe a new tool for intracellular delivery of target proteins, named 'exosomes for protein loading via optically reversible protein-protein interactions' (EXPLORs). By integrating a reversible protein-protein interaction module controlled by blue light with the endogenous process of exosome biogenesis, we are able to successfully load cargo proteins into newly generated exosomes. Treatment with protein-loaded EXPLORs is shown to significantly increase intracellular levels of cargo proteins and their function in recipient cells in vitro and in vivo. These results clearly indicate the potential of EXPLORs as a mechanism for the efficient intracellular transfer of protein-based therapeutics into recipient cells and tissues. PMID:27447450

  14. Benchtop Detection of Proteins

    NASA Technical Reports Server (NTRS)

    Scardelletti, Maximilian C.; Varaljay, Vanessa

    2007-01-01

    A process, and a benchtop-scale apparatus for implementing the process, have been developed to detect proteins associated with specific microbes in water. The process and apparatus may also be useful for detection of proteins in other, more complex liquids. There may be numerous potential applications, including monitoring lakes and streams for contamination, testing of blood and other bodily fluids in medical laboratories, and testing for microbial contamination of liquids in restaurants and industrial food-processing facilities. A sample can be prepared and analyzed by use of this process and apparatus within minutes, whereas an equivalent analysis performed by use of other processes and equipment can often take hours to days. The process begins with the conjugation of near-infrared-fluorescent dyes to antibodies that are specific to a particular protein. Initially, the research has focused on using near-infrared dyes to detect antigens or associated proteins in solution, which has proven successful vs. microbial cells, and streamlining the technique in use for surface protein detection on microbes would theoretically render similar results. However, it is noted that additional work is needed to transition protein-based techniques to microbial cell detection. Consequently, multiple such dye/antibody pairs could be prepared to enable detection of multiple selected microbial species, using a different dye for each species. When excited by near-infrared light of a suitable wavelength, each dye fluoresces at a unique longer wavelength that differs from those of the other dyes, enabling discrimination among the various species. In initial tests, the dye/antibody pairs are mixed into a solution suspected of containing the selected proteins, causing the binding of the dye/antibody pairs to such suspect proteins that may be present. The solution is then run through a microcentrifuge that includes a membrane that acts as a filter in that it retains the dye/antibody/protein

  15. Recombinant protein polymers in biomaterials.

    PubMed

    Kim, Wookhyun

    2013-01-01

    Naturally occurring protein-based materials have been found that function as critical components in biomechanical response, fibers and adhesives. A relatively small but growing number of recombinant protein-based materials that mimic the desired features of their natural sources, such as collagens, elastins and silks, are considered as an alternative to conventional synthetic polymers. Advances in genetic engineering have facilitated the synthesis of repetitive protein polymers with precise control of molecular weights which are designed by using synthetic genes encoding tandem repeats of oligopeptide originating from a modular domain of natural proteins. Many repeat sequences as protein polymer building blocks adopt a well-defined secondary structure and undergo self-assembly to result in physically cross-linked networks or with chemical cross-linking so that further form three-dimensional architectures similar to natural counterparts. In this review, recombinant protein polymers currently developed will be presented that have emerged as promising class of next generation biomaterials. PMID:23276922

  16. NOXclass: prediction of protein-protein interaction types

    PubMed Central

    Zhu, Hongbo; Domingues, Francisco S; Sommer, lngolf; Lengauer, Thomas

    2006-01-01

    Background Structural models determined by X-ray crystallography play a central role in understanding protein-protein interactions at the molecular level. Interpretation of these models requires the distinction between non-specific crystal packing contacts and biologically relevant interactions. This has been investigated previously and classification approaches have been proposed. However, less attention has been devoted to distinguishing different types of biological interactions. These interactions are classified as obligate and non-obligate according to the effect of the complex formation on the stability of the protomers. So far no automatic classification methods for distinguishing obligate, non-obligate and crystal packing interactions have been made available. Results Six interface properties have been investigated on a dataset of 243 protein interactions. The six properties have been combined using a support vector machine algorithm, resulting in NOXclass, a classifier for distinguishing obligate, non-obligate and crystal packing interactions. We achieve an accuracy of 91.8% for the classification of these three types of interactions using a leave-one-out cross-validation procedure. Conclusion NOXclass allows the interpretation and analysis of protein quaternary structures. In particular, it generates testable hypotheses regarding the nature of protein-protein interactions, when experimental results are not available. We expect this server will benefit the users of protein structural models, as well as protein crystallographers and NMR spectroscopists. A web server based on the method and the datasets used in this study are available at . PMID:16423290

  17. Protein function prediction using neighbor relativity in protein-protein interaction network.

    PubMed

    Moosavi, Sobhan; Rahgozar, Masoud; Rahimi, Amir

    2013-04-01

    There is a large gap between the number of discovered proteins and the number of functionally annotated ones. Due to the high cost of determining protein function by wet-lab research, function prediction has become a major task for computational biology and bioinformatics. Some researches utilize the proteins interaction information to predict function for un-annotated proteins. In this paper, we propose a novel approach called "Neighbor Relativity Coefficient" (NRC) based on interaction network topology which estimates the functional similarity between two proteins. NRC is calculated for each pair of proteins based on their graph-based features including distance, common neighbors and the number of paths between them. In order to ascribe function to an un-annotated protein, NRC estimates a weight for each neighbor to transfer its annotation to the unknown protein. Finally, the unknown protein will be annotated by the top score transferred functions. We also investigate the effect of using different coefficients for various types of functions. The proposed method has been evaluated on Saccharomyces cerevisiae and Homo sapiens interaction networks. The performance analysis demonstrates that NRC yields better results in comparison with previous protein function prediction approaches that utilize interaction network.

  18. Novel computational methods to design protein-protein interactions

    NASA Astrophysics Data System (ADS)

    Zhou, Alice Qinhua; O'Hern, Corey; Regan, Lynne

    2014-03-01

    Despite the abundance of structural data, we still cannot accurately predict the structural and energetic changes resulting from mutations at protein interfaces. The inadequacy of current computational approaches to the analysis and design of protein-protein interactions has hampered the development of novel therapeutic and diagnostic agents. In this work, we apply a simple physical model that includes only a minimal set of geometrical constraints, excluded volume, and attractive van der Waals interactions to 1) rank the binding affinity of mutants of tetratricopeptide repeat proteins with their cognate peptides, 2) rank the energetics of binding of small designed proteins to the hydrophobic stem region of the influenza hemagglutinin protein, and 3) predict the stability of T4 lysozyme and staphylococcal nuclease mutants. This work will not only lead to a fundamental understanding of protein-protein interactions, but also to the development of efficient computational methods to rationally design protein interfaces with tunable specificity and affinity, and numerous applications in biomedicine. NSF DMR-1006537, PHY-1019147, Raymond and Beverly Sackler Institute for Biological, Physical and Engineering Sciences, and Howard Hughes Medical Institute.

  19. Membrane Protein Solubilization and Composition of Protein Detergent Complexes.

    PubMed

    Duquesne, Katia; Prima, Valérie; Sturgis, James N

    2016-01-01

    Membrane proteins are typically expressed in heterologous systems with a view to in vitro characterization. A critical step in the preparation of membrane proteins after expression in any system is the solubilization of the protein in aqueous solution, typically using detergents and lipids, to obtain the protein in a form suitable for purification, structural or functional analysis. This process is particularly difficult as the objective is to prepare the protein in an unnatural environment, a protein detergent complex, separating it from its natural lipid partners while causing the minimum destabilization or modification of the structure. Although the process is difficult, and relatively hard to master, an increasing number of membrane proteins have been successfully isolated after expression in a wide variety of systems. In this chapter we give a general protocol for preparing protein detergent complexes that is aimed at guiding the reader through the different critical steps. In the second part of the chapter we illustrate how to analyze the composition of protein detergent complexes; this analysis is important as it has been found that compositional variation often causes irreproducible results. PMID:27485340

  20. Exploring the repeat protein universe through computational protein design.

    PubMed

    Brunette, T J; Parmeggiani, Fabio; Huang, Po-Ssu; Bhabha, Gira; Ekiert, Damian C; Tsutakawa, Susan E; Hura, Greg L; Tainer, John A; Baker, David

    2015-12-24

    A central question in protein evolution is the extent to which naturally occurring proteins sample the space of folded structures accessible to the polypeptide chain. Repeat proteins composed of multiple tandem copies of a modular structure unit are widespread in nature and have critical roles in molecular recognition, signalling, and other essential biological processes. Naturally occurring repeat proteins have been re-engineered for molecular recognition and modular scaffolding applications. Here we use computational protein design to investigate the space of folded structures that can be generated by tandem repeating a simple helix-loop-helix-loop structural motif. Eighty-three designs with sequences unrelated to known repeat proteins were experimentally characterized. Of these, 53 are monomeric and stable at 95 °C, and 43 have solution X-ray scattering spectra consistent with the design models. Crystal structures of 15 designs spanning a broad range of curvatures are in close agreement with the design models with root mean square deviations ranging from 0.7 to 2.5 Å. Our results show that existing repeat proteins occupy only a small fraction of the possible repeat protein sequence and structure space and that it is possible to design novel repeat proteins with precisely specified geometries, opening up a wide array of new possibilities for biomolecular engineering.

  1. Water-protein interactions from high-resolution protein crystallography.

    PubMed Central

    Nakasako, Masayoshi

    2004-01-01

    To understand the role of water in life at molecular and atomic levels, structures and interactions at the protein-water interface have been investigated by cryogenic X-ray crystallography. The method enabled a much clearer visualization of definite hydration sites on the protein surface than at ambient temperature. Using the structural models of proteins, including several hydration water molecules, the characteristics in hydration structures were systematically analysed for the amount, the interaction geometries between water molecules and proteins, and the local and global distribution of water molecules on the surface of proteins. The tetrahedral hydrogen-bond geometry of water molecules in bulk solvent was retained at the interface and enabled the extension of a three-dimensional chain connection of a hydrogen-bond network among hydration water molecules and polar protein atoms over the entire surface of proteins. Networks of hydrogen bonds were quite flexible to accommodate and/or to regulate the conformational changes of proteins such as domain motions. The present experimental results may have profound implications in the understanding of the physico-chemical principles governing the dynamics of proteins in an aqueous environment and a discussion of why water is essential to life at a molecular level. PMID:15306376

  2. Modular protein switches derived from antibody mimetic proteins.

    PubMed

    Nicholes, N; Date, A; Beaujean, P; Hauk, P; Kanwar, M; Ostermeier, M

    2016-02-01

    Protein switches have potential applications as biosensors and selective protein therapeutics. Protein switches built by fusion of proteins with the prerequisite input and output functions are currently developed using an ad hoc process. A modular switch platform in which existing switches could be readily adapted to respond to any ligand would be advantageous. We investigated the feasibility of a modular protein switch platform based on fusions of the enzyme TEM-1 β-lactamase (BLA) with two different antibody mimetic proteins: designed ankyrin repeat proteins (DARPins) and monobodies. We created libraries of random insertions of the gene encoding BLA into genes encoding a DARPin or a monobody designed to bind maltose-binding protein (MBP). From these libraries, we used a genetic selection system for β-lactamase activity to identify genes that conferred MBP-dependent ampicillin resistance to Escherichia coli. Some of these selected genes encoded switch proteins whose enzymatic activity increased up to 14-fold in the presence of MBP. We next introduced mutations into the antibody mimetic domain of these switches that were known to cause binding to different ligands. To different degrees, introduction of the mutations resulted in switches with the desired specificity, illustrating the potential modularity of these platforms.

  3. Phosphorylation of protein phosphatase inhibitor-1 by protein kinase C.

    PubMed

    Sahin, Bogachan; Shu, Hongjun; Fernandez, Joseph; El-Armouche, Ali; Molkentin, Jeffery D; Nairn, Angus C; Bibb, James A

    2006-08-25

    Inhibitor-1 becomes a potent inhibitor of protein phosphatase 1 when phosphorylated by cAMP-dependent protein kinase at Thr(35). Moreover, Ser(67) of inhibitor-1 serves as a substrate for cyclin-dependent kinase 5 in the brain. Here, we report that dephosphoinhibitor-1 but not phospho-Ser(67) inhibitor-1 was efficiently phosphorylated by protein kinase C at Ser(65) in vitro. In contrast, Ser(67) phosphorylation by cyclin-dependent kinase 5 was unaffected by phospho-Ser(65). Protein kinase C activation in striatal tissue resulted in the concomitant phosphorylation of inhibitor-1 at Ser(65) and Ser(67), but not Ser(65) alone. Selective pharmacological inhibition of protein phosphatase activity suggested that phospho-Ser(65) inhibitor-1 is dephosphorylated by protein phosphatase 1 in the striatum. In vitro studies confirmed these findings and suggested that phospho-Ser(67) protects phospho-Ser(65) inhibitor-1 from dephosphorylation by protein phosphatase 1 in vivo. Activation of group I metabotropic glutamate receptors resulted in the up-regulation of diphospho-Ser(65)/Ser(67) inhibitor-1 in this tissue. In contrast, the activation of N-methyl-d-aspartate-type ionotropic glutamate receptors opposed increases in striatal diphospho-Ser(65)/Ser(67) inhibitor-1 levels. Phosphomimetic mutation of Ser(65) and/or Ser(67) did not convert inhibitor-1 into a protein phosphatase 1 inhibitor. On the other hand, in vitro and in vivo studies suggested that diphospho-Ser(65)/Ser(67) inhibitor-1 is a poor substrate for cAMP-dependent protein kinase. These observations extend earlier studies regarding the function of phospho-Ser(67) and underscore the possibility that phosphorylation in this region of inhibitor-1 by multiple protein kinases may serve as an integrative signaling mechanism that governs the responsiveness of inhibitor-1 to cAMP-dependent protein kinase activation.

  4. Are protein-protein interfaces special regions on a protein's surface?

    NASA Astrophysics Data System (ADS)

    Tonddast-Navaei, Sam; Skolnick, Jeffrey

    2015-12-01

    Protein-protein interactions (PPIs) are involved in many cellular processes. Experimentally obtained protein quaternary structures provide the location of protein-protein interfaces, the surface region of a given protein that interacts with another. These regions are termed half-interfaces (HIs). Canonical HIs cover roughly one third of a protein's surface and were found to have more hydrophobic residues than the non-interface surface region. In addition, the classical view of protein HIs was that there are a few (if not one) HIs per protein that are structurally and chemically unique. However, on average, a given protein interacts with at least a dozen others. This raises the question of whether they use the same or other HIs. By copying HIs from monomers with the same folds in solved quaternary structures, we introduce the concept of geometric HIs (HIs whose geometry has a significant match to other known interfaces) and show that on average they cover three quarters of a protein's surface. We then demonstrate that in some cases, these geometric HI could result in real physical interactions (which may or may not be biologically relevant). The composition of the new HIs is on average more charged compared to most known ones, suggesting that the current protein interface database is biased towards more hydrophobic, possibly more obligate, complexes. Finally, our results provide evidence for interface fuzziness and PPI promiscuity. Thus, the classical view of unique, well defined HIs needs to be revisited as HIs are another example of coarse-graining that is used by nature.

  5. Modeling Mercury in Proteins.

    PubMed

    Parks, J M; Smith, J C

    2016-01-01

    Mercury (Hg) is a naturally occurring element that is released into the biosphere both by natural processes and anthropogenic activities. Although its reduced, elemental form Hg(0) is relatively nontoxic, other forms such as Hg(2+) and, in particular, its methylated form, methylmercury, are toxic, with deleterious effects on both ecosystems and humans. Microorganisms play important roles in the transformation of mercury in the environment. Inorganic Hg(2+) can be methylated by certain bacteria and archaea to form methylmercury. Conversely, bacteria also demethylate methylmercury and reduce Hg(2+) to relatively inert Hg(0). Transformations and toxicity occur as a result of mercury interacting with various proteins. Clearly, then, understanding the toxic effects of mercury and its cycling in the environment requires characterization of these interactions. Computational approaches are ideally suited to studies of mercury in proteins because they can provide a detailed molecular picture and circumvent issues associated with toxicity. Here, we describe computational methods for investigating and characterizing how mercury binds to proteins, how inter- and intraprotein transfer of mercury is orchestrated in biological systems, and how chemical reactions in proteins transform the metal. We describe quantum chemical analyses of aqueous Hg(II), which reveal critical factors that determine ligand-binding propensities. We then provide a perspective on how we used chemical reasoning to discover how microorganisms methylate mercury. We also highlight our combined computational and experimental studies of the proteins and enzymes of the mer operon, a suite of genes that confer mercury resistance in many bacteria. Lastly, we place work on mercury in proteins in the context of what is needed for a comprehensive multiscale model of environmental mercury cycling.

  6. Modeling Mercury in Proteins.

    PubMed

    Parks, J M; Smith, J C

    2016-01-01

    Mercury (Hg) is a naturally occurring element that is released into the biosphere both by natural processes and anthropogenic activities. Although its reduced, elemental form Hg(0) is relatively nontoxic, other forms such as Hg(2+) and, in particular, its methylated form, methylmercury, are toxic, with deleterious effects on both ecosystems and humans. Microorganisms play important roles in the transformation of mercury in the environment. Inorganic Hg(2+) can be methylated by certain bacteria and archaea to form methylmercury. Conversely, bacteria also demethylate methylmercury and reduce Hg(2+) to relatively inert Hg(0). Transformations and toxicity occur as a result of mercury interacting with various proteins. Clearly, then, understanding the toxic effects of mercury and its cycling in the environment requires characterization of these interactions. Computational approaches are ideally suited to studies of mercury in proteins because they can provide a detailed molecular picture and circumvent issues associated with toxicity. Here, we describe computational methods for investigating and characterizing how mercury binds to proteins, how inter- and intraprotein transfer of mercury is orchestrated in biological systems, and how chemical reactions in proteins transform the metal. We describe quantum chemical analyses of aqueous Hg(II), which reveal critical factors that determine ligand-binding propensities. We then provide a perspective on how we used chemical reasoning to discover how microorganisms methylate mercury. We also highlight our combined computational and experimental studies of the proteins and enzymes of the mer operon, a suite of genes that confer mercury resistance in many bacteria. Lastly, we place work on mercury in proteins in the context of what is needed for a comprehensive multiscale model of environmental mercury cycling. PMID:27497164

  7. [The photometric determination of total bile protein].

    PubMed

    Miroshnichenko, V P; Savel'ev, V G

    1989-01-01

    Studies of bilirubin absorption spectra by protein measurements with the use of the biuret test and Lowry's method have shown that bilirubin influences much the protein absorption spectrum, provoking higher results in examinations of the bile. To eliminate bilirubin effects, the authors recommend bilirubin extraction with ethyl-acetone mixture in a 1:1 ratio after protein sedimentation with trichloroacetic acid. The biuret test with bilirubin-free protein yields results compatible with those obtained by nonphotometric techniques not involving bilirubin effects.

  8. Molecular dynamics of membrane proteins.

    SciTech Connect

    Woolf, Thomas B.; Crozier, Paul Stewart; Stevens, Mark Jackson

    2004-10-01

    Understanding the dynamics of the membrane protein rhodopsin will have broad implications for other membrane proteins and cellular signaling processes. Rhodopsin (Rho) is a light activated G-protein coupled receptor (GPCR). When activated by ligands, GPCRs bind and activate G-proteins residing within the cell and begin a signaling cascade that results in the cell's response to external stimuli. More than 50% of all current drugs are targeted toward G-proteins. Rho is the prototypical member of the class A GPCR superfamily. Understanding the activation of Rho and its interaction with its Gprotein can therefore lead to a wider understanding of the mechanisms of GPCR activation and G-protein activation. Understanding the dark to light transition of Rho is fully analogous to the general ligand binding and activation problem for GPCRs. This transition is dependent on the lipid environment. The effect of lipids on membrane protein activity in general has had little attention, but evidence is beginning to show a significant role for lipids in membrane protein activity. Using the LAMMPS program and simulation methods benchmarked under the IBIG program, we perform a variety of allatom molecular dynamics simulations of membrane proteins.

  9. Engineering Protein Farnesyltransferase for Enzymatic Protein Labeling Applications

    PubMed Central

    2015-01-01

    Creating covalent protein conjugates is an active area of research due to the wide range of uses for protein conjugates spanning everything from biological studies to protein therapeutics. Protein Farnesyltransferase (PFTase) has been used for the creation of site-specific protein conjugates, and a number of PFTase substrates have been developed to facilitate that work. PFTase is an effective catalyst for protein modification because it transfers Farnesyl diphosphate (FPP) analogues to protein substrates on a cysteine four residues from the C-terminus. While much work has been done to synthesize various FPP analogues, there are few reports investigating how mutations in PFTase alter the kinetics with these unnatural analogues. Herein we examined how different mutations within the PFTase active site alter the kinetics of the PFTase reaction with a series of large FPP analogues. We found that mutating either a single tryptophan or tyrosine residue to alanine results in greatly improved catalytic parameters, particularly in kcat. Mutation of tryptophan 102β to alanine caused a 4-fold increase in kcat and a 10-fold decrease in KM for a benzaldehyde-containing FPP analogue resulting in an overall 40-fold increase in catalytic efficiency. Similarly, mutation of tyrosine 205β to alanine caused a 25-fold increase in kcat and a 10-fold decrease in KM for a coumarin-containing analogue leading to a 300-fold increase in catalytic efficiency. Smaller but significant changes in catalytic parameters were also obtained for cyclo-octene- and NBD-containing FPP analogues. The latter compound was used to create a fluorescently labeled form of Ciliary Neurotrophic Factor (CNTF), a protein of therapeutic importance. Additionally, computational modeling was performed to study how the large non-natural isoprenoid analogues can fit into the active sites enlarged via mutagenesis. Overall, these results demonstrate that PFTase can be improved via mutagenesis in ways that will be useful

  10. Mathematics of protein pathological misfolding.

    PubMed

    Armah, Ebenezer O

    2007-07-01

    "Protein folding is defined as a process by which a polypeptide chain performs a search in conformational space with the objective of achieving the so-called native conformation to global free-energy minimum under a given set of physiochemical conditions of the medium." Misfolding then, is the process by which this objective is not achieved. Protein Folding Quality Assessment (PFQA), is characterized by a three-parameter distribution function Phi(T) referred to as the PFQA function. It uses results of protein folding processes to assess the output quality of protein folding. Protein misfolding is implicated in the initial cause of many conformational diseases. Folding of cytosolic protein can be regarded as the performance of the protein after it is produced or manufactured by the synthesis processes. Protein folding through different mechanisms and pathways has been extensively covered in [J.D. Bryngelson, P.G. Wolynes, Spin glass and statistical mechanics of protein folding, Proc. Natl. Acad. Sci. USA 84 (1987) 7524; J. Wang, Statistics, pathways and dynamics of single molecule folding, J. Chem. Phys. 118 (2) (2003) 953; N.D. Socci, J.N. Onuchic, P.G. Wolynes, Diffusive dynamics of the reaction coordinates for protein folding funnels, J. Chem. Phys. 104 (14) (1996); D. Thirumalai, From minimal models to real proteins, time scales for protein folding kinetics, J. Phys. I France 5 (1995) 1457]. The model is based on growth models of Ratkowsky, Richards, etc. [D.A. Ratkowski, T.J. Reeds, Choosing near-linear parameters logistic model for radio-ligand and related assays, Biometrics 42 (1986) 575] for a three-parameters model to handle the quality assessment of the folding process. Thus a complete distribution can be found, thanks to the scale, location and shape parameters.

  11. TGF-beta signaling proteins and the Protein Ontology

    PubMed Central

    Arighi, Cecilia N; Liu, Hongfang; Natale, Darren A; Barker, Winona C; Drabkin, Harold; Blake, Judith A; Smith, Barry; Wu, Cathy H

    2009-01-01

    Background The Protein Ontology (PRO) is designed as a formal and principled Open Biomedical Ontologies (OBO) Foundry ontology for proteins. The components of PRO extend from a classification of proteins on the basis of evolutionary relationships at the homeomorphic level to the representation of the multiple protein forms of a gene, including those resulting from alternative splicing, cleavage and/or post-translational modifications. Focusing specifically on the TGF-beta signaling proteins, we describe the building, curation, usage and dissemination of PRO. Results PRO is manually curated on the basis of PrePRO, an automatically generated file with content derived from standard protein data sources. Manual curation ensures that the treatment of the protein classes and the internal and external relationships conform to the PRO framework. The current release of PRO is based upon experimental data from mouse and human proteins wherein equivalent protein forms are represented by single terms. In addition to the PRO ontology, the annotation of PRO terms is released as a separate PRO association file, which contains, for each given PRO term, an annotation from the experimentally characterized sub-types as well as the corresponding database identifiers and sequence coordinates. The annotations are added in the form of relationship to other ontologies. Whenever possible, equivalent forms in other species are listed to facilitate cross-species comparison. Splice and allelic variants, gene fusion products and modified protein forms are all represented as entities in the ontology. Therefore, PRO provides for the representation of protein entities and a resource for describing the associated data. This makes PRO useful both for proteomics studies where isoforms and modified forms must be differentiated, and for studies of biological pathways, where representations need to take account of the different ways in which the cascade of events may depend on specific protein

  12. Proteomic Analysis of Membrane Proteins of Vero Cells: Exploration of Potential Proteins Responsible for Virus Entry

    PubMed Central

    Guo, Donghua; Zhu, Qinghe; Zhang, Hong

    2014-01-01

    Vero cells are highly susceptible to many viruses in humans and animals, and its membrane proteins (MPs) are responsible for virus entry. In our study, the MP proteome of the Vero cells was investigated using a shotgun LC-MS/MS approach. Six hundred twenty-seven proteins, including a total of 1839 peptides, were identified in MP samples of the Vero cells. In 627 proteins, 307 proteins (48.96%) were annotated in terms of biological process of gene ontology (GO) categories; 356 proteins (56.78%) were annotated in terms of molecular function of GO categories; 414 proteins (66.03%) were annotated in terms of cellular components of GO categories. Of 627 identified proteins, seventeen proteins had been revealed to be virus receptor proteins. The resulting protein lists and highlighted proteins may provide valuable information to increase understanding of virus infection of Vero cells. PMID:24286161

  13. Disentangling protein-silica interactions.

    PubMed

    Giussani, Lara; Tabacchi, Gloria; Gianotti, Enrica; Coluccia, Salvatore; Fois, Ettore

    2012-03-28

    We present the results of modelling studies aimed at the understanding of the interaction of a 7 nm sized water droplet containing a negatively charged globular protein with flat silica surfaces. We show how the droplet interaction with the surface depends on the electrostatic surface charge, and that adhesion of the droplet occurs when the surface is negatively charged as well. The key role of water and of the charge-balancing counter ions in mediating the surface-protein adhesion is highlighted. The relevance of the present results with respect to the production of bioinorganic hybrids via encapsulation of proteins inside mesoporous silica materials is discussed.

  14. Fusion-protein-assisted protein crystallization.

    PubMed

    Kobe, Bostjan; Ve, Thomas; Williams, Simon J

    2015-07-01

    Fusion proteins can be used directly in protein crystallization to assist crystallization in at least two different ways. In one approach, the `heterologous fusion-protein approach', the fusion partner can provide additional surface area to promote crystal contact formation. In another approach, the `fusion of interacting proteins approach', protein assemblies can be stabilized by covalently linking the interacting partners. The linker connecting the proteins plays different roles in the two applications: in the first approach a rigid linker is required to reduce conformational heterogeneity; in the second, conversely, a flexible linker is required that allows the native interaction between the fused proteins. The two approaches can also be combined. The recent applications of fusion-protein technology in protein crystallization from the work of our own and other laboratories are briefly reviewed.

  15. Protein-protein interaction networks (PPI) and complex diseases

    PubMed Central

    Safari-Alighiarloo, Nahid; Taghizadeh, Mohammad; Rezaei-Tavirani, Mostafa; Goliaei, Bahram

    2014-01-01

    The physical interaction of proteins which lead to compiling them into large densely connected networks is a noticeable subject to investigation. Protein interaction networks are useful because of making basic scientific abstraction and improving biological and biomedical applications. Based on principle roles of proteins in biological function, their interactions determine molecular and cellular mechanisms, which control healthy and diseased states in organisms. Therefore, such networks facilitate the understanding of pathogenic (and physiologic) mechanisms that trigger the onset and progression of diseases. Consequently, this knowledge can be translated into effective diagnostic and therapeutic strategies. Furthermore, the results of several studies have proved that the structure and dynamics of protein networks are disturbed in complex diseases such as cancer and autoimmune disorders. Based on such relationship, a novel paradigm is suggested in order to confirm that the protein interaction networks can be the target of therapy for treatment of complex multi-genic diseases rather than individual molecules with disrespect the network. PMID:25436094

  16. Dynamics of protein conformations

    NASA Astrophysics Data System (ADS)

    Stepanova, Maria

    2010-10-01

    A novel theoretical methodology is introduced to identify dynamic structural domains and analyze local flexibility in proteins. The methodology employs a multiscale approach combining identification of essential collective coordinates based on the covariance analysis of molecular dynamics trajectories, construction of the Mori projection operator with these essential coordinates, and analysis of the corresponding generalized Langevin equations [M.Stepanova, Phys.Rev.E 76(2007)051918]. Because the approach employs a rigorous theory, the outcomes are physically transparent: the dynamic domains are associated with regions of relative rigidity in the protein, whereas off-domain regions are relatively soft. This also allows scoring the flexibility in the macromolecule with atomic-level resolution [N.Blinov, M.Berjanskii, D.S.Wishart, and M.Stepanova, Biochemistry, 48(2009)1488]. The applications include the domain coarse-graining and characterization of conformational stability in protein G and prion proteins. The results are compared with published NMR experiments. Potential applications for structural biology, bioinformatics, and drug design are discussed.

  17. [ALR, the multifunctional protein].

    PubMed

    Balogh, Tibor; Szarka, András

    2015-03-29

    ALR is a mystic protein. It has a so called "long" 22 kDa and a "short" 15 kDa forms. It has been described after partial hepatectomy and it has just been considered as a key protein of liver regeneration. At the beginning of the 21st century it has been revealed that the "long" form is localized in the mitochondrial intermembrane space and it is an element of the mitochondrial protein import and disulphide relay system. Several proteins of the substrates of the mitochondrial disulphide relay system are necessary for the proper function of the mitochondria, thus any mutation of the ALR gene leads to mitochondrial diseases. The "short" form of ALR functions as a secreted extracellular growth factor and it promotes the protection, regeneration and proliferation of hepatocytes. The results gained on the recently generated conditional ALR mutant mice suggest that ALR can play an important role in the pathogenesis of alcoholic and non-alcoholic steatosis. Since the serum level of ALR is modified in several liver diseases it can be a promising marker molecule in laboratory diagnostics. PMID:25796277

  18. Protein stability in ice.

    PubMed

    Strambini, Giovanni B; Gonnelli, Margherita

    2007-03-15

    This study presents an experimental approach, based on the change of Trp fluorescence between native and denatured states of proteins, which permits to monitor unfolding equilibria and the thermodynamic stability (DeltaG degrees ) of these macromolecules in frozen aqueous solutions. The results obtained by guanidinium chloride denaturation of the azurin mutant C112S from Pseudomonas aeruginosa, in the temperature range from -8 to -16 degrees C, demonstrate that the stability of the native fold may be significantly perturbed in ice depending mainly on the size of the liquid water pool (V(L)) in equilibrium with the solid phase. The data establish a threshold, around V(L)=1.5%, below which in ice DeltaG degrees decreases progressively relative to liquid state, up to 3 kcal/mole for V(L)=0.285%. The sharp dependence of DeltaG degrees on V(L) is consistent with a mechanism based on adsorption of the protein to the ice surface. The reduction in DeltaG degrees is accompanied by a corresponding decrease in m-value indicating that protein-ice interactions increase the solvent accessible surface area of the native fold or reduce that of the denatured state, or both. The method opens the possibility for examining in a more quantitative fashion the influence of various experimental conditions on the ice perturbation and in particular to test the effectiveness of numerous additives used in formulations to preserve labile pharmaco proteins.

  19. Protein Cross-Linking Capillary Electrophoresis for Protein-Protein Interaction Analysis.

    PubMed

    Ouimet, Claire M; Shao, Hao; Rauch, Jennifer N; Dawod, Mohamed; Nordhues, Bryce; Dickey, Chad A; Gestwicki, Jason E; Kennedy, Robert T

    2016-08-16

    Capillary electrophoresis (CE) has been identified as a useful platform for detecting, quantifying, and screening for modulators of protein-protein interactions (PPIs). In this method, one protein binding partner is labeled with a fluorophore, the protein binding partners are mixed, and then, the complex is separated from free protein to allow direct determination of bound to free ratios. Although it possesses many advantages for PPI studies, the method is limited by the need to have separation conditions that both prevent protein adsorption to capillary and maintain protein interactions during the separation. In this work, we use protein cross-linking capillary electrophoresis (PXCE) to overcome this limitation. In PXCE, the proteins are cross-linked under binding conditions and then separated. This approach eliminates the need to maintain noncovalent interactions during electrophoresis and facilitates method development. We report PXCE methods for an antibody-antigen interaction and heterodimer and homodimer heat shock protein complexes. Complexes are cross-linked by short treatments with formaldehyde after reaching binding equilibrium. Cross-linked complexes are separated by electrophoretic mobility using free solution CE or by size using sieving electrophoresis of SDS complexes. The method gives good quantitative results; e.g., a lysozyme-antibody interaction was found to have Kd = 24 ± 3 nM by PXCE and Kd = 17 ± 2 nM using isothermal calorimetry (ITC). Heat shock protein 70 (Hsp70) in complex with bcl2 associated athanogene 3 (Bag3) was found to have Kd = 25 ± 5 nM by PXCE which agrees with Kd values reported without cross-linking. Hsp70-Bag3 binding site mutants and small molecule inhibitors of Hsp70-Bag3 were characterized by PXCE with good agreement to inhibitory constants and IC50 values obtained by a bead-based flow cytometry protein interaction assay (FCPIA). PXCE allows rapid method development for quantitative analysis of PPIs. PMID:27434096

  20. Effect of phosphorus levels on the protein profiles of secreted protein and root surface protein of rice.

    PubMed

    Shinano, Takuro; Yoshimura, Tomoko; Watanabe, Toshihiro; Unno, Yusuke; Osaki, Mitsuru; Nanjo, Yohei; Komatsu, Setsuko

    2013-11-01

    Plant roots are complicated organs that absorb water and nutrients from the soil. Roots also play an essential role in protecting plants from attack by soil pathogens and develop a beneficial role with some soil microorganisms. Plant-derived rhizosphere proteins (e.g., root secretory proteins and root surface binding proteins) are considered to play important roles in developing mutual relationships in the rhizosphere. In the rhizosphere, where plant roots meet the surrounding environment, it has been suggested that root secretory protein and root surface binding protein are important factors. Furthermore, it is not known how the physiological status of the plant affects the profile of these proteins. In this study, rice plants were grown aseptically, with or without phosphorus nutrition, and proteins were obtained from root bathing solution (designated as root secretory proteins) and obtained using 0.2 M CaCl2 solution (designated as root surface binding proteins). The total number of identified proteins in the root bathing solution was 458, and the number of root surface binding proteins was 256. More than half of the proteins were observed in both fractions. Most of the proteins were categorized as either having signal peptides or no membrane transport helix sites. The functional categorization suggested that most of the proteins seemed to have secretory pathways and were involved in defense/disease-related functions. These characteristics seem to be unique to rhizosphere proteins, and the latter might be part of the plants strategy to defeat pathogens in the soil. The low phosphorus treatment significantly increased the number of pathogenesis-related proteins in the root secretory proteins, whereas the change was small in the case of the root surface binding proteins. The results suggested that the roots are actively and selectively secreting protein into the rhizosphere. PMID:24083427

  1. Theory of Acoustic Raman Modes in Proteins

    NASA Astrophysics Data System (ADS)

    DeWolf, Timothy; Gordon, Reuven

    2016-09-01

    We present a theoretical analysis that associates the resonances of extraordinary acoustic Raman (EAR) spectroscopy [Wheaton et al., Nat. Photonics 9, 68 (2015)] with the collective modes of proteins. The theory uses the anisotropic elastic network model to find the protein acoustic modes, and calculates Raman intensity by treating the protein as a polarizable ellipsoid. Reasonable agreement is found between EAR spectra and our theory. Protein acoustic modes have been extensively studied theoretically to assess the role they play in protein function; this result suggests EAR spectroscopy as a new experimental tool for studies of protein acoustic modes.

  2. Protein-Polymer Functionalized Nanopatterned Surfaces

    NASA Astrophysics Data System (ADS)

    Wang, Haoyu; Akcora, Pinar

    2015-03-01

    Understanding and controlling the protein interactions with surfaces for biosensors and biomedical implants is a fundamental problem for biocompatible nanomaterial design. Proteins attached in ordered nanopores can exhibit superior biological activities compared to smooth microstructured surfaces. We developed heterogeneous and nanopatterned surfaces decorated with polymer brushes and proteins to control protein fates through elasticity. The heterogeneity of surfaces is controlled with well-defined chemistry, pattern size and geometry, stiffness of polymers and protein types. We will present our recent nanoindentation results on nanopatterned and biofunctionalized flat surfaces and discuss the pattern size effect on protein activity, hence conformation.

  3. Quantitative Analysis of Spatial Protein-protein Proximity in Fluorescence Confocal Microscopy

    NASA Astrophysics Data System (ADS)

    Wu, Yong; Liu, Yi-Kuang; Eghbali, Mansoureh; Stefani, Enrico

    2009-02-01

    To quantify spatial protein-protein proximity (colocalization) in fluorescence microscopic images, cross-correlation and autocorrelation functions were decomposed into fast and slowly decaying components. The fast component results from clusters of proteins specifically labeled and the slow one from background/image heterogeneity. We show that the calculation of the protein-protein proximity index and the correlation coefficient are more reliably determined by extracting the fast-decaying component.

  4. Analysis of Stable and Transient Protein-Protein Interactions

    PubMed Central

    Byrum, Stephanie; Smart, Sherri K.; Larson, Signe; Tackett, Alan J.

    2012-01-01

    The assembly of proteins into defined complexes drives a plethora of cellular activities. These protein complexes often have a set of more stably interacting proteins as well as more unstable or transient interactions. Studying the in vivo components of these protein complexes is challenging as many of the techniques used for isolation result in the purification of only the most stable components and the transient interactions are lost. A technology called transient isotopic differentiation of interactions as random or targeted (transient I-DIRT) has been developed to identify these transiently interacting proteins as well as the stable interactions. Described here are the detailed methodological approaches used for a transient I-DIRT analysis of a multi-subunit complex, NuA3, that acetylates histone H3 and functions to activate gene transcription. Transcription is known to involve a concert of protein assemblies performing different activities on the chromatin/gene template, thus understanding the less stable or transient protein interactions with NuA3 will shed light onto the protein complexes that function synergistically, or antagonistically, to regulate gene transcription and chromatin remodeling. PMID:22183593

  5. Modularity in protein structures: study on all-alpha proteins.

    PubMed

    Khan, Taushif; Ghosh, Indira

    2015-01-01

    Modularity is known as one of the most important features of protein's robust and efficient design. The architecture and topology of proteins play a vital role by providing necessary robust scaffolds to support organism's growth and survival in constant evolutionary pressure. These complex biomolecules can be represented by several layers of modular architecture, but it is pivotal to understand and explore the smallest biologically relevant structural component. In the present study, we have developed a component-based method, using protein's secondary structures and their arrangements (i.e. patterns) in order to investigate its structural space. Our result on all-alpha protein shows that the known structural space is highly populated with limited set of structural patterns. We have also noticed that these frequently observed structural patterns are present as modules or "building blocks" in large proteins (i.e. higher secondary structure content). From structural descriptor analysis, observed patterns are found to be within similar deviation; however, frequent patterns are found to be distinctly occurring in diverse functions e.g. in enzymatic classes and reactions. In this study, we are introducing a simple approach to explore protein structural space using combinatorial- and graph-based geometry methods, which can be used to describe modularity in protein structures. Moreover, analysis indicates that protein function seems to be the driving force that shapes the known structure space.

  6. Intersurf: dynamic interface between proteins.

    PubMed

    Ray, Nicolas; Cavin, Xavier; Paul, Jean-Claude; Maigret, Bernard

    2005-01-01

    Protein docking is a fundamental biological process that links two proteins. This link is typically defined by an interaction between two large zones of the protein boundaries. Visualizing such an interface is useful to understand the process thanks to 3D protein structures, to estimate the quality of docking simulation results, and to classify interactions in order to predict docking affinity between classes of interacting zones. Since the interface may be defined by a surface that separates the two proteins, it is possible to create a map of interaction that allows comparisons to be performed in 2D. This paper presents a very fast algorithm that extracts an interface surface and creates a valid and low-distorted interaction map. Another benefit of our approach is that a pre-computed part of the algorithm enables the surface to be updated in real-time while residues are moved. PMID:15670955

  7. The protein composition of reconstituted 30S ribosomal subunits: the effects of single protein omission.

    PubMed

    Buck, M A; Olah, T V; Perrault, A R; Cooperman, B S

    1991-06-01

    Using reverse phase HPLC, we have been able to quantify the protein compositions of reconstituted 30S ribosomal subunits, formed either with the full complement of 30S proteins in the reconstitution mix or with a single protein omitted. We denote particles formed in the latter case as SPORE (single protein omission reconstitution) particles. An important goal in 30S reconstitution studies is the formation of reconstituted subunits having uniform protein composition, preferably corresponding to one copy of each protein per reconstituted particle. Here we describe procedures involving variation of the protein:rRNA ratio that approach this goal. In SPORE particles the omission of one protein often results in the partial loss in uptake of other proteins. We also describe procedures to increase the uptake of such proteins into SPORE particles, thus enhancing the utility of the SPORE approach in defining the role of specific proteins in 30S structure and function. The losses of proteins other than the omitted protein provide a measure of protein:protein interaction within the 30S subunit. Most of these losses are predictable on the basis of other such measures. However, we do find evidence for several long-range protein:protein interactions (S6:S3, S6:S12, S10:S16, and S6:S4) that have not been described previously.

  8. Text Mining for Protein Docking

    PubMed Central

    Badal, Varsha D.; Kundrotas, Petras J.; Vakser, Ilya A.

    2015-01-01

    The rapidly growing amount of publicly available information from biomedical research is readily accessible on the Internet, providing a powerful resource for predictive biomolecular modeling. The accumulated data on experimentally determined structures transformed structure prediction of proteins and protein complexes. Instead of exploring the enormous search space, predictive tools can simply proceed to the solution based on similarity to the existing, previously determined structures. A similar major paradigm shift is emerging due to the rapidly expanding amount of information, other than experimentally determined structures, which still can be used as constraints in biomolecular structure prediction. Automated text mining has been widely used in recreating protein interaction networks, as well as in detecting small ligand binding sites on protein structures. Combining and expanding these two well-developed areas of research, we applied the text mining to structural modeling of protein-protein complexes (protein docking). Protein docking can be significantly improved when constraints on the docking mode are available. We developed a procedure that retrieves published abstracts on a specific protein-protein interaction and extracts information relevant to docking. The procedure was assessed on protein complexes from Dockground (http://dockground.compbio.ku.edu). The results show that correct information on binding residues can be extracted for about half of the complexes. The amount of irrelevant information was reduced by conceptual analysis of a subset of the retrieved abstracts, based on the bag-of-words (features) approach. Support Vector Machine models were trained and validated on the subset. The remaining abstracts were filtered by the best-performing models, which decreased the irrelevant information for ~ 25% complexes in the dataset. The extracted constraints were incorporated in the docking protocol and tested on the Dockground unbound benchmark set

  9. Protein Crystal Based Nanomaterials

    NASA Technical Reports Server (NTRS)

    Bell, Jeffrey A.; VanRoey, Patrick

    2001-01-01

    This is the final report on a NASA Grant. It concerns a description of work done, which includes: (1) Protein crystals cross-linked to form fibers; (2) Engineering of protein to favor crystallization; (3) Better knowledge-based potentials for protein-protein contacts; (4) Simulation of protein crystallization.

  10. Shotgun protein sequencing.

    SciTech Connect

    Faulon, Jean-Loup Michel; Heffelfinger, Grant S.

    2009-06-01

    A novel experimental and computational technique based on multiple enzymatic digestion of a protein or protein mixture that reconstructs protein sequences from sequences of overlapping peptides is described in this SAND report. This approach, analogous to shotgun sequencing of DNA, is to be used to sequence alternative spliced proteins, to identify post-translational modifications, and to sequence genetically engineered proteins.

  11. Effects of influenza A virus NS1 protein on protein expression: the NS1 protein enhances translation and is not required for shutoff of host protein synthesis.

    PubMed

    Salvatore, Mirella; Basler, Christopher F; Parisien, Jean-Patrick; Horvath, Curt M; Bourmakina, Svetlana; Zheng, Hongyong; Muster, Thomas; Palese, Peter; García-Sastre, Adolfo

    2002-02-01

    The influenza A virus NS1 protein, a virus-encoded alpha/beta interferon (IFN-alpha/beta) antagonist, appears to be a key regulator of protein expression in infected cells. We now show that NS1 protein expression results in enhancement of reporter gene activity from transfected plasmids. This effect appears to be mediated at the translational level, and it is reminiscent of the activity of the adenoviral virus-associated I (VAI) RNA, a known inhibitor of the antiviral, IFN-induced, PKR protein. To study the effects of the NS1 protein on viral and cellular protein synthesis during influenza A virus infection, we used recombinant influenza viruses lacking the NS1 gene (delNS1) or expressing truncated NS1 proteins. Our results demonstrate that the NS1 protein is required for efficient viral protein synthesis in COS-7 cells. This activity maps to the amino-terminal domain of the NS1 protein, since cells infected with wild-type virus or with a mutant virus expressing a truncated NS1 protein-lacking approximately half of its carboxy-terminal end-showed similar kinetics of viral and cellular protein expression. Interestingly, no major differences in host cell protein synthesis shutoff or in viral protein expression were found among NS1 mutant viruses in Vero cells. Thus, another viral component(s) different from the NS1 protein is responsible for the inhibition of host protein synthesis during viral infection. In contrast to the earlier proposal suggesting that the NS1 protein regulates the levels of spliced M2 mRNA, no effects on M2 protein accumulation were seen in Vero cells infected with delNS1 virus.

  12. Mercury-binding proteins of Mytilus edulis

    SciTech Connect

    Roesijadi, G.; Morris, J. E.; Calabrese, A.

    1981-11-01

    Mytilus edulis possesses low molecular weight, mercury-binding proteins. The predominant protein isolated from gill tissue is enriched in cysteinyl residues (8%) and possesses an amino acid composition similar to cadmium-binding proteins of mussels and oysters. Continuous exposure of mussels to 5 ..mu..g/l mercury results in spillover of mercury from these proteins to high molecular weight proteins. Antibodies to these proteins have been isolated, and development of immunoassays is presently underway. Preliminary studies to determine whether exposure of adult mussels to mercury will result in induction of mercury-binding proteins in offspring suggest that such proteins occur in larvae although additional studies are indicated for a conclusive demonstration.

  13. Quantitative study of protein-protein interactions by quartz nanopipettes.

    PubMed

    Tiwari, Purushottam Babu; Astudillo, Luisana; Miksovska, Jaroslava; Wang, Xuewen; Li, Wenzhi; Darici, Yesim; He, Jin

    2014-09-01

    In this report, protein-modified quartz nanopipettes were used to quantitatively study protein-protein interactions in attoliter sensing volumes. As shown by numerical simulations, the ionic current through the conical-shaped nanopipette is very sensitive to the surface charge variation near the pore mouth. With the appropriate modification of negatively charged human neuroglobin (hNgb) onto the inner surface of a nanopipette, we were able to detect concentration-dependent current change when the hNgb-modified nanopipette tip was exposed to positively charged cytochrome c (Cyt c) with a series of concentrations in the bath solution. Such current change is due to the adsorption of Cyt c to the inner surface of the nanopipette through specific interactions with hNgb. In contrast, a smaller current change with weak concentration dependence was observed when Cyt c was replaced with lysozyme, which does not specifically bind to hNgb. The equilibrium dissociation constant (KD) for the Cyt c-hNgb complex formation was derived and the value matched very well with the result from surface plasmon resonance measurement. This is the first quantitative study of protein-protein interactions by a conical-shaped nanopore based on charge sensing. Our results demonstrate that nanopipettes can potentially be used as a label-free analytical tool to quantitatively characterize protein-protein interactions.

  14. Dual targeting of peroxisomal proteins

    PubMed Central

    Ast, Julia; Stiebler, Alina C.; Freitag, Johannes; Bölker, Michael

    2013-01-01

    Cellular compartmentalization into organelles serves to separate biological processes within the environment of a single cell. While some metabolic reactions are specific to a single organelle, others occur in more than one cellular compartment. Specific targeting of proteins to compartments inside of eukaryotic cells is mediated by defined sequence motifs. To achieve multiple targeting to different compartments cells use a variety of strategies. Here, we focus on mechanisms leading to dual targeting of peroxisomal proteins. In many instances, isoforms of peroxisomal proteins with distinct intracellular localization are encoded by separate genes. But also single genes can give rise to differentially localized proteins. Different isoforms can be generated by use of alternative transcriptional start sites, by differential splicing or ribosomal read-through of stop codons. In all these cases different peptide variants are produced, of which only one carries a peroxisomal targeting signal. Alternatively, peroxisomal proteins contain additional signals that compete for intracellular targeting. Dual localization of proteins residing in both the cytoplasm and in peroxisomes may also result from use of inefficient targeting signals. The recent observation that some bona fide cytoplasmic enzymes were also found in peroxisomes indicates that dual targeting of proteins to both the cytoplasm and the peroxisome might be more widespread. Although current knowledge of proteins exhibiting only partial peroxisomal targeting is far from being complete, we speculate that the metabolic capacity of peroxisomes might be larger than previously assumed. PMID:24151469

  15. Nanobiomechanics of proteins and biomembrane.

    PubMed

    Ikai, Atsushi

    2008-06-27

    A review of the work done in the Laboratory of Biodynamics of Tokyo Institute of Technology in the last decade has been summarized in this article in relation to the results reported from other laboratories. The emphasis here is the application of nanomechanics based on the force mode of atomic force microscopy (AFM) to proteins and protein-based biological structures. Globular proteins were stretched in various ways to detect the localized rigidity inside of the molecule. When studied by this method, bovine carbonic anhydrase II (BCA II), calmodulin and OspA protein all showed the presence of localized rigid structures inside the molecules. Protein compression experiments were done on BCA II to obtain an estimate of the Young modulus and its change in the process of denaturation. Then, the AFM probe method was turned on to cell membranes and cytoplasmic components. Force curves accompanying the extraction process of membrane proteins from intact cells were analysed in relation to their interaction with the cytoskeletal components. By pushing the AFM probe further into the cytoplasm, mRNAs were recovered from a live cell with minimal damage, and multiplied using PCR technology for their identification. Altogether, the work introduced here forms the basis of nanomechanics of protein and protein-based biostructures and application of the nanomechanical technology to cell biology.

  16. Dual targeting of peroxisomal proteins.

    PubMed

    Ast, Julia; Stiebler, Alina C; Freitag, Johannes; Bölker, Michael

    2013-10-18

    Cellular compartmentalization into organelles serves to separate biological processes within the environment of a single cell. While some metabolic reactions are specific to a single organelle, others occur in more than one cellular compartment. Specific targeting of proteins to compartments inside of eukaryotic cells is mediated by defined sequence motifs. To achieve multiple targeting to different compartments cells use a variety of strategies. Here, we focus on mechanisms leading to dual targeting of peroxisomal proteins. In many instances, isoforms of peroxisomal proteins with distinct intracellular localization are encoded by separate genes. But also single genes can give rise to differentially localized proteins. Different isoforms can be generated by use of alternative transcriptional start sites, by differential splicing or ribosomal read-through of stop codons. In all these cases different peptide variants are produced, of which only one carries a peroxisomal targeting signal. Alternatively, peroxisomal proteins contain additional signals that compete for intracellular targeting. Dual localization of proteins residing in both the cytoplasm and in peroxisomes may also result from use of inefficient targeting signals. The recent observation that some bona fide cytoplasmic enzymes were also found in peroxisomes indicates that dual targeting of proteins to both the cytoplasm and the peroxisome might be more widespread. Although current knowledge of proteins exhibiting only partial peroxisomal targeting is far from being complete, we speculate that the metabolic capacity of peroxisomes might be larger than previously assumed.

  17. Protein knot server: detection of knots in protein structures.

    PubMed

    Kolesov, Grigory; Virnau, Peter; Kardar, Mehran; Mirny, Leonid A

    2007-07-01

    KNOTS (http://knots.mit.edu) is a web server that detects knots in protein structures. Several protein structures have been reported to contain intricate knots. The physiological role of knots and their effect on folding and evolution is an area of active research. The user submits a PDB id or uploads a 3D protein structure in PDB or mmCIF format. The current implementation of the server uses the Alexander polynomial to detect knots. The results of the analysis that are presented to the user are the location of the knot in the structure, the type of the knot and an interactive visualization of the knot. The results can also be downloaded and viewed offline. The server also maintains a regularly updated list of known knots in protein structures.

  18. Measurements of Protein Crystal Face Growth Rates

    NASA Technical Reports Server (NTRS)

    Gorti, S.

    2014-01-01

    Protein crystal growth rates will be determined for several hyperthermophile proteins.; The growth rates will be assessed using available theoretical models, including kinetic roughening.; If/when kinetic roughening supersaturations are established, determinations of protein crystal quality over a range of supersaturations will also be assessed.; The results of our ground based effort may well address the existence of a correlation between fundamental growth mechanisms and protein crystal quality.

  19. Protein immobilization strategies for protein biochips.

    PubMed

    Rusmini, Federica; Zhong, Zhiyuan; Feijen, Jan

    2007-06-01

    In the past few years, protein biochips have emerged as promising proteomic and diagnostic tools for obtaining information about protein functions and interactions. Important technological innovations have been made. However, considerable development is still required, especially regarding protein immobilization, in order to fully realize the potential of protein biochips. In fact, protein immobilization is the key to the success of microarray technology. Proteins need to be immobilized onto surfaces with high density in order to allow the usage of small amount of sample solution. Nonspecific protein adsorption needs to be avoided or at least minimized in order to improve detection performances. Moreover, full retention of protein conformation and activity is a challenging task to be accomplished. Although a large number of review papers on protein biochips have been published in recent years, few have focused on protein immobilization technology. In this review, current protein immobilization strategies, including physical, covalent, and bioaffinity immobilization for the fabrication of protein biochips, are described. Particular consideration has been given to oriented immobilization, also referred to as site-specific immobilization, which is believed will improve homogeneous surface covering and accessibility of the active site.

  20. Immunogenicity evaluation of protein hydrolysates for hypoallergenic infant formulae.

    PubMed

    Cordle, C T; Mahmoud, M I; Moore, V

    1991-10-01

    Casein and soy protein were enzymatically hydrolyzed for potential use in a hypoallergenic infant formula. To assess the relative immunoreactivity of the hydrolysates, rabbits were immunized with either the intact proteins or the protein hydrolysates using a vigorous immunization protocol. Serum samples were tested using ELISA methods that quantitated IgG antibody specific for the immunizing protein hydrolysates and the corresponding intact proteins. The results showed that the protein hydrolysates had substantially lower immunogenicity than the parent proteins. Also, antibody specific for the parent protein showed very low cross-reactivity with the hydrolysates. Both of the protein hydrolysates seem to be promising candidates for use in hypoallergenic infant feeding systems.

  1. Protein-losing enteropathy

    MedlinePlus

    ... this page: //medlineplus.gov/ency/article/007338.htm Protein-losing enteropathy To use the sharing features on this page, please enable JavaScript. Protein-losing enteropathy is an abnormal loss of protein ...

  2. Protein in diet

    MedlinePlus

    ... basic structure of protein is a chain of amino acids. You need protein in your diet to help ... Protein foods are broken down into parts called amino acids during digestion. The human body needs a number ...

  3. Identifying the singleplex and multiplex proteins based on transductive learning for protein subcellular localization prediction.

    PubMed

    Cao, Junzhe; Liu, Wenqi; He, Jianjun; Gu, Hong

    2013-07-01

    A new method is proposed to identify whether a query protein is singleplex or multiplex for improving the quality of protein subcellular localization prediction. Based on the transductive learning technique, this approach utilizes the information from the both query proteins and known proteins to estimate the subcellular location number of every query protein so that the singleplex and multiplex proteins can be recognized and distinguished. Each query protein is then dealt with by a targeted single-label or multi-label predictor to achieve a high-accuracy prediction result. We assess the performance of the proposed approach by applying it to three groups of protein sequences datasets. Simulation experiments show that the proposed approach can effectively identify the singleplex and multiplex proteins. Through a comparison, the reliably of this method for enhancing the power of predicting protein subcellular localization can also be verified.

  4. Evolutionary optimization of protein folding.

    PubMed

    Debès, Cédric; Wang, Minglei; Caetano-Anollés, Gustavo; Gräter, Frauke

    2013-01-01

    Nature has shaped the make up of proteins since their appearance, [Formula: see text]3.8 billion years ago. However, the fundamental drivers of structural change responsible for the extraordinary diversity of proteins have yet to be elucidated. Here we explore if protein evolution affects folding speed. We estimated folding times for the present-day catalog of protein domains directly from their size-modified contact order. These values were mapped onto an evolutionary timeline of domain appearance derived from a phylogenomic analysis of protein domains in 989 fully-sequenced genomes. Our results show a clear overall increase of folding speed during evolution, with known ultra-fast downhill folders appearing rather late in the timeline. Remarkably, folding optimization depends on secondary structure. While alpha-folds showed a tendency to fold faster throughout evolution, beta-folds exhibited a trend of folding time increase during the last [Formula: see text]1.5 billion years that began during the "big bang" of domain combinations. As a consequence, these domain structures are on average slow folders today. Our results suggest that fast and efficient folding of domains shaped the universe of protein structure. This finding supports the hypothesis that optimization of the kinetic and thermodynamic accessibility of the native fold reduces protein aggregation propensities that hamper cellular functions. PMID:23341762

  5. FUNCTIONAL INTERACTOMICS: DETERMINING THE ROLES PLAYED BY MEMBERS OF THE POPULAR BIOMASS PROTEIN-PROTEIN INTERACTOME

    SciTech Connect

    Beers, Eric; Brunner, Amy; Helm, Richard

    2015-07-31

    Proteins are molecular machines that are required for nearly all biological functions based on interactions with other molecules such as carbohydrates, lipids, other low molecular weight molecules, nucleic acids and other proteins. Here we map protein-protein interactions relevant to biomass production by focusing on proteins coexpressed in poplar xylem, the site of the majority of lignocellulose synthesis and hence biomass accumulation in poplar. Work proposed here will yield novel biological and bioinformatic resources that can benefit a variety of ongoing and future projects focusing on plant biomass/cell wall biology. The protein-protein interaction map that results from these studies will comprise an advanced view of protein-protein interactions in a model biomass tissue. Results will be made available to the biomass research community to serve as tools for developing new strategies for altering biomass quality and quantity.

  6. Genome-wide protein-protein interaction screening by protein-fragment complementation assay (PCA) in living cells.

    PubMed

    Rochette, Samuel; Diss, Guillaume; Filteau, Marie; Leducq, Jean-Baptiste; Dubé, Alexandre K; Landry, Christian R

    2015-01-01

    Proteins are the building blocks, effectors and signal mediators of cellular processes. A protein's function, regulation and localization often depend on its interactions with other proteins. Here, we describe a protocol for the yeast protein-fragment complementation assay (PCA), a powerful method to detect direct and proximal associations between proteins in living cells. The interaction between two proteins, each fused to a dihydrofolate reductase (DHFR) protein fragment, translates into growth of yeast strains in presence of the drug methotrexate (MTX). Differential fitness, resulting from different amounts of reconstituted DHFR enzyme, can be quantified on high-density colony arrays, allowing to differentiate interacting from non-interacting bait-prey pairs. The high-throughput protocol presented here is performed using a robotic platform that parallelizes mating of bait and prey strains carrying complementary DHFR-fragment fusion proteins and the survival assay on MTX. This protocol allows to systematically test for thousands of protein-protein interactions (PPIs) involving bait proteins of interest and offers several advantages over other PPI detection assays, including the study of proteins expressed from their endogenous promoters without the need for modifying protein localization and for the assembly of complex reporter constructs.

  7. Protein crystallization in hydrogel beads.

    PubMed

    Willaert, Ronnie; Zegers, Ingrid; Wyns, Lode; Sleutel, Mike

    2005-09-01

    The use of hydrogel beads for the crystallization of proteins is explored in this contribution. The dynamic behaviour of the internal precipitant, protein concentration and relative supersaturation in a gel bead upon submerging the bead in a precipitant solution is characterized theoretically using a transient diffusion model. Agarose and calcium alginate beads have been used for the crystallization of a low-molecular-weight (14.4 kDa, hen egg-white lysozyme) and a high-molecular-weight (636.0 kDa, alcohol oxidase) protein. Entrapment of the protein in the agarose-gel matrix was accomplished using two methods. In the first method, a protein solution is mixed with the agarose sol solution. Gel beads are produced by immersing drops of the protein-agarose sol mixture in a cold paraffin solution. In the second method (which was used to produce calcium alginate and agarose beads), empty gel beads are first produced and subsequently filled with protein by diffusion from a bulk solution into the bead. This latter method has the advantage that a supplementary purification step is introduced (for protein aggregates and large impurities) owing to the diffusion process in the gel matrix. Increasing the precipitant, gel concentration and protein loading resulted in a larger number of crystals of smaller size. Consequently, agarose as well as alginate gels act as nucleation promoters. The supersaturation in a gel bead can be dynamically controlled by changing the precipitant and/or the protein concentration in the bulk solution. Manipulation of the supersaturation allowed the nucleation rate to be varied and led to the production of large crystals which were homogeneously distributed in the gel bead.

  8. The Geobiochemistry of Methanogen Proteins

    NASA Astrophysics Data System (ADS)

    Prasad, A.; Shock, E.

    2013-12-01

    A principle of geobiochemistry is that adaptation over evolutionary time includes a thermodynamic drive to minimize costs of making biomolecules like proteins and lipids. If so, then biomolecule abundances will reflect, at least in part, their relative stabilities at the conditions imposed by external environments. We tested this hypothesis by comparing relative stabilities of 138 orthologous proteins between a representative lake-sediment methanogen (Methanoculleus marisnigri) and a representative rumen methanogen (Methanospirillum hungatei) at the compositional constraints of their respective environments. Chemical affinities of the proteins were calculated based on pH, temperature, and concentrations of dissolved hydrogen, bicarbonate, ammonia, and hydrogen sulfide, together with standard Gibbs energies of formation of proteins from the elements predicted with a group additivity algorithm for unfolded proteins [1]. Methanogens were chosen as they are chemoautotrophs and their metabolism proceeds at relatively small affinities. Also, they are found in a variety of compositionally varying habitats like rumen, sediments, hydrothermal systems and sewage. The methanogens selected belong to the same order of taxonomy and are closely related. Preliminary results show that a majority of the proteins belonging to the rumen methanogen (66%) are more stable in the rumen environment, while a majority of the proteins belonging to the lake-sediment methanogen (58%) are more stable at sediment conditions. In a separate observation, it was noted that while the complete protein ';proteasome subunit alpha' of another rumen methanogen (Methanobrevibacter smithii) was less stable in its more reducing habitat as compared to a sewage methanogen (Methanothermobacter thermoautotophicus), its first 26 amino acid residues (N terminal) were in fact more stable in its own environment. These 26 residues are reported to be unique as compared to other proteasome proteins and are suggested to

  9. Prediction of lipid-binding regions in cytoplasmic and extracellular loops of membrane proteins as exemplified by protein translocation membrane proteins.

    PubMed

    Keller, Rob C A

    2013-01-01

    The presence of possible lipid-binding regions in the cytoplasmic or extracellular loops of membrane proteins with an emphasis on protein translocation membrane proteins was investigated in this study using bioinformatics. Recent developments in approaches recognizing lipid-binding regions in proteins were found to be promising. In this study a total bioinformatics approach specialized in identifying lipid-binding helical regions in proteins was explored. Two features of the protein translocation membrane proteins, the position of the transmembrane regions and the identification of additional lipid-binding regions, were analyzed. A number of well-studied protein translocation membrane protein structures were checked in order to demonstrate the predictive value of the bioinformatics approach. Furthermore, the results demonstrated that lipid-binding regions in the cytoplasmic and extracellular loops in protein translocation membrane proteins can be predicted, and it is proposed that the interaction of these regions with phospholipids is important for proper functioning during protein translocation. PMID:22961045

  10. Isolation of proteins and protein complexes by immunoprecipitation.

    PubMed

    Kaboord, Barbara; Perr, Maria

    2008-01-01

    Immunoprecipitation (IP) uses the specificity of antibodies to isolate target proteins (antigens) out of complex sample mixtures. Three different approaches for performing IP will be discussed; traditional (classical) method, oriented affinity method and direct affinity method. The traditional method of incubating the IP antibody with the sample and sequentially binding to Protein A or G agarose beads (resin) facilitates the most efficient target antigen recovery. However, this approach results in the target protein becoming contaminated with the IP antibody that can interfere with downstream analyses. The orientated affinity method uses Protein A or G beads to serve as an anchor to which the IP antibody is crosslinked thereby preventing the antibody from co-eluting with the target protein. Similarly, the direct affinity method also immobilizes the IP antibody except in this case it is directly attached to a chemically activated support. Both methods prevent co-elution of the IP antibody enabling reuse of the immunomatrix. All three approaches have unique advantages and can also be used for co-immunoprecipitation to study protein:protein interactions and investigate the functional proteome.

  11. Protein domain architectures.

    PubMed

    Mulder, Nicola J

    2010-01-01

    Proteins are composed of functional units, or domains, that can be found alone or in combination with other domains. Analysis of protein domain architectures and the movement of protein domains within and across different genomes provide clues about the evolution of protein function. The classification of proteins into families and domains is provided through publicly available tools and databases that use known protein domains to predict other members in new proteins sequences. Currently at least 80% of the main protein sequence databases can be classified using these tools, thus providing a large data set to work from for analyzing protein domain architectures. Each of the protein domain databases provide intuitive web interfaces for viewing and analyzing their domain classifications and provide their data freely for downloading. Some of the main protein family and domain databases are described here, along with their Web-based tools for analyzing domain architectures.

  12. The N and C Termini of ZO-1 Are Surrounded by Distinct Proteins and Functional Protein Networks*

    PubMed Central

    Van Itallie, Christina M.; Aponte, Angel; Tietgens, Amber Jean; Gucek, Marjan; Fredriksson, Karin; Anderson, James Melvin

    2013-01-01

    The proteins and functional protein networks of the tight junction remain incompletely defined. Among the currently known proteins are barrier-forming proteins like occludin and the claudin family; scaffolding proteins like ZO-1; and some cytoskeletal, signaling, and cell polarity proteins. To define a more complete list of proteins and infer their functional implications, we identified the proteins that are within molecular dimensions of ZO-1 by fusing biotin ligase to either its N or C terminus, expressing these fusion proteins in Madin-Darby canine kidney epithelial cells, and purifying and identifying the resulting biotinylated proteins by mass spectrometry. Of a predicted proteome of ∼9000, we identified more than 400 proteins tagged by biotin ligase fused to ZO-1, with both identical and distinct proteins near the N- and C-terminal ends. Those proximal to the N terminus were enriched in transmembrane tight junction proteins, and those proximal to the C terminus were enriched in cytoskeletal proteins. We also identified many unexpected but easily rationalized proteins and verified partial colocalization of three of these proteins with ZO-1 as examples. In addition, functional networks of interacting proteins were tagged, such as the basolateral but not apical polarity network. These results provide a rich inventory of proteins and potential novel insights into functions and protein networks that should catalyze further understanding of tight junction biology. Unexpectedly, the technique demonstrates high spatial resolution, which could be generally applied to defining other subcellular protein compartmentalization. PMID:23553632

  13. Detection and identification of protein interactions of S100 proteins by ProteinChip technology.

    PubMed

    Lehmann, Roland; Melle, Christian; Escher, Niko; von Eggeling, Ferdinand

    2005-01-01

    The aim of this work was to establish an approach for identification of protein interactions. This assay used an anti-S100A8 antibody coupled on beads and incubated with cell extract. The bead eluates were analyzed using ProteinChip technology and subsequently subjected to an appropriate digestion. Molecular masses of digestion fragments were determined by SELDI-MS, and database analysis revealed S100A10 as interacting protein. This result was confirmed by co-immunoprecipitation and immunocapturing. Using S100A10 as new bait, a specific interaction with S100A7 was detectable. PMID:16212425

  14. Protein imprinting in polyacrylamide-based gels

    PubMed Central

    Zayats, Maya; Brenner, Andrew J.; Searson, Peter C.

    2015-01-01

    Protein imprinting in hydrogels is a method to produce materials capable of selective recognition and capture of a target protein. Here we report on the imprinting of fluorescently-labeled maltose binding protein (MBP) in acrylamide (AAm)/N-isopropylacrylamide (NIPAm) hydrogels. The targeting efficiency and selectivity of protein recognition is usually characterized by the imprinting factor, which in the simplest case is the ratio of protein uptake in an imprinted film divided by the uptake by the corresponding non-imprinted film. Our objective in this work is to study the dynamics of protein binding and elution in imprinted and non-imprinted films to elucidate the processes that control protein recognition. Protein elution from imprinted and non-imprinted films suggests that imprinting results in sites with a distribution of binding energies, and that only a relatively small fraction of these sites exhibit strong binding. PMID:25034963

  15. Protein imprinting in polyacrylamide-based gels.

    PubMed

    Zayats, Maya; Brenner, Andrew J; Searson, Peter C

    2014-10-01

    Protein imprinting in hydrogels is a method to produce materials capable of selective recognition and capture of a target protein. Here we report on the imprinting of fluorescently-labeled maltose binding protein (MBP) in acrylamide (AAm)/N-isopropylacrylamide (NIPAm) hydrogels. The targeting efficiency and selectivity of protein recognition is usually characterized by the imprinting factor, which in the simplest case is the ratio of protein uptake in an imprinted film divided by the uptake by the corresponding non-imprinted film. Our objective in this work is to study the dynamics of protein binding and elution in imprinted and non-imprinted films to elucidate the processes that control protein recognition. Protein elution from imprinted and non-imprinted films suggests that imprinting results in sites with a distribution of binding energies, and that only a relatively small fraction of these sites exhibit strong binding.

  16. Protein imprinting in polyacrylamide-based gels.

    PubMed

    Zayats, Maya; Brenner, Andrew J; Searson, Peter C

    2014-10-01

    Protein imprinting in hydrogels is a method to produce materials capable of selective recognition and capture of a target protein. Here we report on the imprinting of fluorescently-labeled maltose binding protein (MBP) in acrylamide (AAm)/N-isopropylacrylamide (NIPAm) hydrogels. The targeting efficiency and selectivity of protein recognition is usually characterized by the imprinting factor, which in the simplest case is the ratio of protein uptake in an imprinted film divided by the uptake by the corresponding non-imprinted film. Our objective in this work is to study the dynamics of protein binding and elution in imprinted and non-imprinted films to elucidate the processes that control protein recognition. Protein elution from imprinted and non-imprinted films suggests that imprinting results in sites with a distribution of binding energies, and that only a relatively small fraction of these sites exhibit strong binding. PMID:25034963

  17. Distinguishing proteins from arbitrary amino acid sequences.

    PubMed

    Yau, Stephen S-T; Mao, Wei-Guang; Benson, Max; He, Rong Lucy

    2015-01-01

    What kinds of amino acid sequences could possibly be protein sequences? From all existing databases that we can find, known proteins are only a small fraction of all possible combinations of amino acids. Beginning with Sanger's first detailed determination of a protein sequence in 1952, previous studies have focused on describing the structure of existing protein sequences in order to construct the protein universe. No one, however, has developed a criteria for determining whether an arbitrary amino acid sequence can be a protein. Here we show that when the collection of arbitrary amino acid sequences is viewed in an appropriate geometric context, the protein sequences cluster together. This leads to a new computational test, described here, that has proved to be remarkably accurate at determining whether an arbitrary amino acid sequence can be a protein. Even more, if the results of this test indicate that the sequence can be a protein, and it is indeed a protein sequence, then its identity as a protein sequence is uniquely defined. We anticipate our computational test will be useful for those who are attempting to complete the job of discovering all proteins, or constructing the protein universe. PMID:25609314

  18. A Protein Complex Map of Trypanosoma brucei

    PubMed Central

    Mehta, Vaibhav; Najafabadi, Hamed S.; Moshiri, Houtan; Jardim, Armando; Salavati, Reza

    2016-01-01

    The functions of the majority of trypanosomatid-specific proteins are unknown, hindering our understanding of the biology and pathogenesis of Trypanosomatida. While protein-protein interactions are highly informative about protein function, a global map of protein interactions and complexes is still lacking for these important human parasites. Here, benefiting from in-depth biochemical fractionation, we systematically interrogated the co-complex interactions of more than 3354 protein groups in procyclic life stage of Trypanosoma brucei, the protozoan parasite responsible for human African trypanosomiasis. Using a rigorous methodology, our analysis led to identification of 128 high-confidence complexes encompassing 716 protein groups, including 635 protein groups that lacked experimental annotation. These complexes correlate well with known pathways as well as for proteins co-expressed across the T. brucei life cycle, and provide potential functions for a large number of previously uncharacterized proteins. We validated the functions of several novel proteins associated with the RNA-editing machinery, identifying a candidate potentially involved in the mitochondrial post-transcriptional regulation of T. brucei. Our data provide an unprecedented view of the protein complex map of T. brucei, and serve as a reliable resource for further characterization of trypanosomatid proteins. The presented results in this study are available at: www.TrypsNetDB.org. PMID:26991453

  19. Proteins Among the Polysaccharides

    PubMed Central

    Wen, Fushi; Curlango-Rivera, Gilberto

    2007-01-01

    Charles Darwin recognized the power of the root cap as a model for plant signalling and behavior, and used it to explore the ways plants sense and respond to diverse stimuli. Over ensuing decades, various groups have reported tantalizing clues regarding the role of a complex extracellular matrix that ensheaths the tip region housing the apical and root cap meristems. In the course of characterizing root tip resistance to infection and injury and the role border cells play in this phenomenon, we confirmed and extended early- and mid-20th century studies reporting enzyme activities secreted from the root cap. Multidimensional protein analysis revealed, in fact, that >100 proteins are actively synthesized and secreted from the root cap and border cells. This ‘root cap secretome’ appears to be a critical component of root tip resistance to infection. We have developed a microscopic assay to quantify the protein-based extracellular response to dynamic changes in environmental conditions including hydroponic culture, and present the results here. This tool provides a simple, direct measure that can be used to explore the ways border cells may function in the manner of white blood cells to trap, immobilize and neutralize threats to the growing root tip. PMID:19704617

  20. Infrared Protein Crystallography

    SciTech Connect

    J Sage; Y Zhang; J McGeehan; R Ravelli; M Weik; J van Thor

    2011-12-31

    We consider the application of infrared spectroscopy to protein crystals, with particular emphasis on exploiting molecular orientation through polarization measurements on oriented single crystals. Infrared microscopes enable transmission measurements on individual crystals using either thermal or nonthermal sources, and can accommodate flow cells, used to measure spectral changes induced by exposure to soluble ligands, and cryostreams, used for measurements of flash-cooled crystals. Comparison of unpolarized infrared measurements on crystals and solutions probes the effects of crystallization and can enhance the value of the structural models refined from X-ray diffraction data by establishing solution conditions under which they are most relevant. Results on several proteins are consistent with similar equilibrium conformational distributions in crystal and solutions. However, the rates of conformational change are often perturbed. Infrared measurements also detect products generated by X-ray exposure, including CO{sub 2}. Crystals with favorable symmetry exhibit infrared dichroism that enhances the synergy with X-ray crystallography. Polarized infrared measurements on crystals can distinguish spectral contributions from chemically similar sites, identify hydrogen bonding partners, and, in opportune situations, determine three-dimensional orientations of molecular groups. This article is part of a Special Issue entitled: Protein Structure and Function in the Crystalline State.

  1. PREFACE: Protein protein interactions: principles and predictions

    NASA Astrophysics Data System (ADS)

    Nussinov, Ruth; Tsai, Chung-Jung

    2005-06-01

    Proteins are the `workhorses' of the cell. Their roles span functions as diverse as being molecular machines and signalling. They carry out catalytic reactions, transport, form viral capsids, traverse membranes and form regulated channels, transmit information from DNA to RNA, making possible the synthesis of new proteins, and they are responsible for the degradation of unnecessary proteins and nucleic acids. They are the vehicles of the immune response and are responsible for viral entry into the cell. Given their importance, considerable effort has been centered on the prediction of protein function. A prime way to do this is through identification of binding partners. If the function of at least one of the components with which the protein interacts is known, that should let us assign its function(s) and the pathway(s) in which it plays a role. This holds since the vast majority of their chores in the living cell involve protein-protein interactions. Hence, through the intricate network of these interactions we can map cellular pathways, their interconnectivities and their dynamic regulation. Their identification is at the heart of functional genomics; their prediction is crucial for drug discovery. Knowledge of the pathway, its topology, length, and dynamics may provide useful information for forecasting side effects. The goal of predicting protein-protein interactions is daunting. Some associations are obligatory, others are continuously forming and dissociating. In principle, from the physical standpoint, any two proteins can interact, but under what conditions and at which strength? The principles of protein-protein interactions are general: the non-covalent interactions of two proteins are largely the outcome of the hydrophobic effect, which drives the interactions. In addition, hydrogen bonds and electrostatic interactions play important roles. Thus, many of the interactions observed in vitro are the outcome of experimental overexpression. Protein disorder

  2. Leveraging Genomics Software to Improve Proteomics Results

    SciTech Connect

    Fodor, I K; Nelson, D O

    2005-09-06

    Rigorous data analysis techniques are essential in quantifying the differential expression of proteins in biological samples of interest. Statistical methods from the microarray literature were applied to the analysis of two-dimensional difference gel electrophoresis (2-D DIGE) proteomics experiments, in the context of technical variability studies involving human plasma. Protein expression measurements were corrected to account for observed intensity-dependent biases within gels, and normalized to mitigate observed gel to gel variations. The methods improved upon the results achieved using the best currently available 2-D DIGE proteomics software. The spot-wise protein variance was reduced by 10% and the number of apparently differentially expressed proteins was reduced by over 50%.

  3. Protein sequence comparison and protein evolution

    SciTech Connect

    Pearson, W.R.

    1995-12-31

    This tutorial was one of eight tutorials selected to be presented at the Third International Conference on Intelligent Systems for Molecular Biology which was held in the United Kingdom from July 16 to 19, 1995. This tutorial examines how the information conserved during the evolution of a protein molecule can be used to infer reliably homology, and thus a shared proteinfold and possibly a shared active site or function. The authors start by reviewing a geological/evolutionary time scale. Next they look at the evolution of several protein families. During the tutorial, these families will be used to demonstrate that homologous protein ancestry can be inferred with confidence. They also examine different modes of protein evolution and consider some hypotheses that have been presented to explain the very earliest events in protein evolution. The next part of the tutorial will examine the technical aspects of protein sequence comparison. Both optimal and heuristic algorithms and their associated parameters that are used to characterize protein sequence similarities are discussed. Perhaps more importantly, they survey the statistics of local similarity scores, and how these statistics can both be used to improve the selectivity of a search and to evaluate the significance of a match. They them examine distantly related members of three protein families, the serine proteases, the glutathione transferases, and the G-protein-coupled receptors (GCRs). Finally, the discuss how sequence similarity can be used to examine internal repeated or mosaic structures in proteins.

  4. Protein-protein binding site identification by enumerating the configurations

    PubMed Central

    2012-01-01

    Background The ability to predict protein-protein binding sites has a wide range of applications, including signal transduction studies, de novo drug design, structure identification and comparison of functional sites. The interface in a complex involves two structurally matched protein subunits, and the binding sites can be predicted by identifying structural matches at protein surfaces. Results We propose a method which enumerates “all” the configurations (or poses) between two proteins (3D coordinates of the two subunits in a complex) and evaluates each configuration by the interaction between its components using the Atomic Contact Energy function. The enumeration is achieved efficiently by exploring a set of rigid transformations. Our approach incorporates a surface identification technique and a method for avoiding clashes of two subunits when computing rigid transformations. When the optimal transformations according to the Atomic Contact Energy function are identified, the corresponding binding sites are given as predictions. Our results show that this approach consistently performs better than other methods in binding site identification. Conclusions Our method achieved a success rate higher than other methods, with the prediction quality improved in terms of both accuracy and coverage. Moreover, our method is being able to predict the configurations of two binding proteins, where most of other methods predict only the binding sites. The software package is available at http://sites.google.com/site/guofeics/dobi for non-commercial use. PMID:22768846

  5. Protein designs in HP models

    NASA Astrophysics Data System (ADS)

    Gupta, Arvind; Khodabakhshi, Alireza Hadj; Maňuch, Ján; Rafiey, Arash; Stacho, Ladislav

    2009-07-01

    The inverse protein folding problem is that of designing an amino acid sequence which folds into a prescribed shape. This problem arises in drug design where a particular structure is necessary to ensure proper protein-protein interactions and could have applications in nanotechnology. A major challenge in designing proteins with native folds that attain a specific shape is to avoid proteins that have multiple native folds (unstable proteins). In this technical note we present our results on protein designs in the variant of Hydrophobic-Polar (HP) model introduced by Dill [6] on 2D square lattice. The HP model distinguishes only polar and hydrophobic monomers and only counts the number of hydrophobic contacts in the energy function. To achieve better stability of our designs we use the Hydrophobic-Polar-Cysteine (HPC) model which distinguishes the third type of monomers called "cysteines" and incorporates also the disulfid bridges (SS-bridges) into the energy function. We present stable designs in 2D square lattice and 3D hexagonal prism lattice in the HPC model.

  6. Protein intake and ovulatory infertility

    PubMed Central

    Chavarro, Jorge E.; Rich-Edwards, Janet W.; Rosner, Bernard A.; Willett, Walter C.

    2011-01-01

    Objective To evaluate whether intake of protein from animal and vegetable origin is associated with ovulatory infertility. Study Design 18,555 married women without a history of infertility were followed as they attempted a pregnancy or became pregnant during an eight year period. Dietary assessments were related to the incidence of ovulatory infertility. Results During follow-up, 438 women reported ovulatory infertility. The multivariate-adjusted relative risk [RR] (95% CI; P, trend) of ovulatory infertility comparing the highest to the lowest quintile of animal protein intake was 1.39 (1.01 – 1.90; 0.03). The corresponding RR (95% CI; P, trend) for vegetable protein intake was 0.78 (0.54 – 1.12; 0.07). Further, consuming 5% of total energy intake as vegetable protein rather than as animal protein was associated with a more than 50% lower risk of ovulatory infertility (P = 0.007). Conclusions Replacing animal sources of protein with vegetable sources of protein may reduce ovulatory infertility risk. PMID:18226626

  7. Succination of proteins in diabetes.

    PubMed

    Frizzell, Norma; Lima, Maria; Baynes, John W

    2011-01-01

    Cysteine is arguably the most reactive amino acid in protein. A wide range of cysteine derivatives is formed in vivo, resulting from oxidation, nitrosation, alkylation and acylation reactions. This review describes succination of proteins, an irreversible chemical modification of cysteine by the Krebs cycle intermediate, fumarate, yielding S-(2-succinyl)cysteine (2SC). Intracellular fumarate concentration and succination of proteins are increased by hyperpolarization of the inner mitochondrial membrane and develop in concert with mitochondrial and oxidative stress in diabetes. Increased succination of glyceraldehyde-3-phosphate dehydrogenase explains the loss in specific activity of this enzyme in muscle of streptozotocin-diabetic rats and increased succination of adiponectin may explain the decreased secretion of adiponectin from adipose tissue in type 2 diabetes. In addition to GAPDH and adiponectin, other succinated proteins identified in adipocytes include cytoskeletal proteins (tubulin, actin) and chaperone proteins in the endoplasmic reticulum. Succination of adipocyte protein in vitro is inhibited by uncouplers of oxidative phosphorylation and by inhibitors of ER stress. 2SC serves as a biomarker of mitochondrial stress and recent studies suggest that succination is the mechanistic link between mitochondrial and ER stress in diabetes.

  8. Protein surface-distribution and protein-protein interactions in the binding of peripheral proteins to charged lipid membranes.

    PubMed Central

    Heimburg, T; Marsh, D

    1995-01-01

    The binding of native cytochrome c to negatively charged lipid dispersions of dioleoyl phosphatidylglycerol has been studied over a wide range of ionic strengths. Not only is the strength of protein binding found to decrease rapidly with increasing ionic strength, but also the binding curves reach an apparent saturation level that decreases rapidly with increasing ionic strength. Analysis of the binding isotherms with a general statistical thermodynamic model that takes into account not only the free energy of the electrostatic double layer, but also the free energy of the surface distribution of the protein, demonstrates that the apparent saturation effects could arise from a competition between the out-of-plane binding reaction and the lateral in-plane interactions between proteins at the surface. It is found that association with nonlocalized sites results in binding isotherms that display the apparent saturation effect to a much more pronounced extent than does the Langmuir adsorption isotherm for binding to localized sites. With the model for nonlocalized sites, the binding isotherms of native cytochrome c can be described adequately by taking into account only the entropy of the surface distribution of the protein, without appreciable enthalpic interactions between the bound proteins. The binding of cytochrome c to dioleoyl phosphatidylglycerol dispersions at a temperature at which the bound protein is denatured on the lipid surface, but is nondenatured when free in solution, has also been studied. The binding curves for the surface-denatured protein differ from those for the native protein in that the apparent saturation at high ionic strength is less pronounced. This indicates the tendency of the denatured protein to aggregate on the lipid surface, and can be described by the binding isotherms for nonlocalized sites only if attractive interactions between the surface-bound proteins are included in addition to the distributional entropic terms. Additionally

  9. Protein structure alignment beyond spatial proximity.

    PubMed

    Wang, Sheng; Ma, Jianzhu; Peng, Jian; Xu, Jinbo

    2013-01-01

    Protein structure alignment is a fundamental problem in computational structure biology. Many programs have been developed for automatic protein structure alignment, but most of them align two protein structures purely based upon geometric similarity without considering evolutionary and functional relationship. As such, these programs may generate structure alignments which are not very biologically meaningful from the evolutionary perspective. This paper presents a novel method DeepAlign for automatic pairwise protein structure alignment. DeepAlign aligns two protein structures using not only spatial proximity of equivalent residues (after rigid-body superposition), but also evolutionary relationship and hydrogen-bonding similarity. Experimental results show that DeepAlign can generate structure alignments much more consistent with manually-curated alignments than other automatic tools especially when proteins under consideration are remote homologs. These results imply that in addition to geometric similarity, evolutionary information and hydrogen-bonding similarity are essential to aligning two protein structures.

  10. Dietary proteins in obesity and in diabetes.

    PubMed

    Keller, Ulrich

    2011-03-01

    Dietary proteins influence body weight by affecting four targets for body weight regulation: satiety, thermogenesis, energy efficiency, and body composition. Protein ingestion results in higher ratings of satiety than equicaloric amounts of carbohydrates or fat. Their effect on satiety is mainly due to oxidation of amino acids fed in excess; this effect is higher with ingestion of specific "incomplete" proteins (vegetal) than with animal proteins. Diet-induced thermogenesis is higher for proteins than for other macronutrients. The increase in energy expenditure is caused by protein and urea synthesis and by gluconeogenesis. This effect is higher with animal proteins containing larger amounts of essential amino acids than with vegetable proteins. Specifically, diet-induced thermogenesis increases after protein ingestion by 20 - 30 %, but by only 5 - 10 % after carbohydrates and 0 - 5 % after ingestion of fat. Consumption of higher amounts of protein during dietary treatment of obesity resulted in greater weight loss than with lower amounts of protein in dietary studies lasting up to one year. During weight loss and decreased caloric intake, a relatively increased protein content of the diet maintained fat-free mass (i. e. muscle mass) and increased calcium balance, resulting in preservation of bone mineral content. This is of particular importance during weight loss after bariatric surgery because these patients are at risk for protein malnutrition. Adequate dietary protein intake in diabetes type 2 is of specific importance since proteins are relatively neutral with regard to glucose and lipid metabolism, and they preserve muscle and bone mass, which may be decreased in subjects with poorly controlled diabetes. Ingestion of dietary proteins in diabetes type 1 exerts a delayed postprandial increase in blood glucose levels due to protein-induced stimulation of pancreatic glucagon secretion. Higher than minimal amounts of protein in the diet needed for nitrogen

  11. Mirror image proteins.

    PubMed

    Zhao, Le; Lu, Wuyuan

    2014-10-01

    Proteins composed entirely of unnatural d-amino acids and the achiral amino acid glycine are mirror image forms of their native l-protein counterparts. Recent advances in chemical protein synthesis afford unique and facile synthetic access to domain-sized mirror image d-proteins, enabling protein research to be conducted through 'the looking glass' and in a way previously unattainable. d-Proteins can facilitate structure determination of their native l-forms that are difficult to crystallize (racemic X-ray crystallography); d-proteins can serve as the bait for library screening to ultimately yield pharmacologically superior d-peptide/d-protein therapeutics (mirror-image phage display); d-proteins can also be used as a powerful mechanistic tool for probing molecular events in biology. This review examines recent progress in the application of mirror image proteins to structural biology, drug discovery, and immunology.

  12. High-throughput and multiplexed protein array technology: protein-DNA and protein-protein interactions.

    PubMed

    Sakanyan, Vehary

    2005-02-01

    Miniaturized protein arrays address protein interactions with various types of molecules in a high-throughput and multiplexed fashion. This review focuses on achievements in the analysis of protein-DNA and protein-protein interactions. The technological feasibility of protein arrays depends on the different factors that enable the arrayed proteins to recognize molecular partners and on the specificity of the interactions involved. Proteome-scale studies of molecular interactions require high-throughput approaches for both the production and purification of functionally active proteins. Various solutions have been proposed to avoid non-specific protein interactions on array supports and to monitor low-abundance molecules. The data accumulated indicate that this emerging technology is perfectly suited to resolve networks of protein interactions involved in complex physiological and pathological phenomena in different organisms and to develop sensitive tools for biomedical applications.

  13. Multiple protein-protein interactions converging on the Prp38 protein during activation of the human spliceosome.

    PubMed

    Schütze, Tonio; Ulrich, Alexander K C; Apelt, Luise; Will, Cindy L; Bartlick, Natascha; Seeger, Martin; Weber, Gert; Lührmann, Reinhard; Stelzl, Ulrich; Wahl, Markus C

    2016-02-01

    Spliceosomal Prp38 proteins contain a conserved amino-terminal domain, but only higher eukaryotic orthologs also harbor a carboxy-terminal RS domain, a hallmark of splicing regulatory SR proteins. We show by crystal structure analysis that the amino-terminal domain of human Prp38 is organized around three pairs of antiparallel α-helices and lacks similarities to RNA-binding domains found in canonical SR proteins. Instead, yeast two-hybrid analyses suggest that the amino-terminal domain is a versatile protein-protein interaction hub that possibly binds 12 other spliceosomal proteins, most of which are recruited at the same stage as Prp38. By quantitative, alanine surface-scanning two-hybrid screens and biochemical analyses we delineated four distinct interfaces on the Prp38 amino-terminal domain. In vitro interaction assays using recombinant proteins showed that Prp38 can bind at least two proteins simultaneously via two different interfaces. Addition of excess Prp38 amino-terminal domain to in vitro splicing assays, but not of an interaction-deficient mutant, stalled splicing at a precatalytic stage. Our results show that human Prp38 is an unusual SR protein, whose amino-terminal domain is a multi-interface protein-protein interaction platform that might organize the relative positioning of other proteins during splicing. PMID:26673105

  14. An ontology-based search engine for protein-protein interactions

    PubMed Central

    2010-01-01

    Background Keyword matching or ID matching is the most common searching method in a large database of protein-protein interactions. They are purely syntactic methods, and retrieve the records in the database that contain a keyword or ID specified in a query. Such syntactic search methods often retrieve too few search results or no results despite many potential matches present in the database. Results We have developed a new method for representing protein-protein interactions and the Gene Ontology (GO) using modified Gödel numbers. This representation is hidden from users but enables a search engine using the representation to efficiently search protein-protein interactions in a biologically meaningful way. Given a query protein with optional search conditions expressed in one or more GO terms, the search engine finds all the interaction partners of the query protein by unique prime factorization of the modified Gödel numbers representing the query protein and the search conditions. Conclusion Representing the biological relations of proteins and their GO annotations by modified Gödel numbers makes a search engine efficiently find all protein-protein interactions by prime factorization of the numbers. Keyword matching or ID matching search methods often miss the interactions involving a protein that has no explicit annotations matching the search condition, but our search engine retrieves such interactions as well if they satisfy the search condition with a more specific term in the ontology. PMID:20122195

  15. Detergent-mediated protein aggregation.

    PubMed

    Neale, Chris; Ghanei, Hamed; Holyoake, John; Bishop, Russell E; Privé, Gilbert G; Pomès, Régis

    2013-04-01

    Because detergents are commonly used to solvate membrane proteins for structural evaluation, much attention has been devoted to assessing the conformational bias imparted by detergent micelles in comparison to the native environment of the lipid bilayer. Here, we conduct six 500-ns simulations of a system with >600,000 atoms to investigate the spontaneous self assembly of dodecylphosphocholine detergent around multiple molecules of the integral membrane protein PagP. This detergent formed equatorial micelles in which acyl chains surround the protein's hydrophobic belt, confirming existing models of the detergent solvation of membrane proteins. In addition, unexpectedly, the extracellular and periplasmic apical surfaces of PagP interacted with the headgroups of detergents in other micelles 85 and 60% of the time, respectively, forming complexes that were stable for hundreds of nanoseconds. In some cases, an apical surface of one molecule of PagP interacted with an equatorial micelle surrounding another molecule of PagP. In other cases, the apical surfaces of two molecules of PagP simultaneously bound a neat detergent micelle. In these ways, detergents mediated the non-specific aggregation of folded PagP. These simulation results are consistent with dynamic light scattering experiments, which show that, at detergent concentrations ≥600 mM, PagP induces the formation of large scattering species that are likely to contain many copies of the PagP protein. Together, these simulation and experimental results point to a potentially generic mechanism of detergent-mediated protein aggregation.

  16. Protein interfacial structure and nanotoxicology

    NASA Astrophysics Data System (ADS)

    White, John W.; Perriman, Adam W.; McGillivray, Duncan J.; Lin, Jhih-Min

    2009-02-01

    Here we briefly recapitulate the use of X-ray and neutron reflectometry at the air-water interface to find protein structures and thermodynamics at interfaces and test a possibility for understanding those interactions between nanoparticles and proteins which lead to nanoparticle toxicology through entry into living cells. Stable monomolecular protein films have been made at the air-water interface and, with a specially designed vessel, the substrate changed from that which the air-water interfacial film was deposited. This procedure allows interactions, both chemical and physical, between introduced species and the monomolecular film to be studied by reflectometry. The method is briefly illustrated here with some new results on protein-protein interaction between β-casein and κ-casein at the air-water interface using X-rays. These two proteins are an essential component of the structure of milk. In the experiments reported, specific and directional interactions appear to cause different interfacial structures if first, a β-casein monolayer is attacked by a κ-casein solution compared to the reverse. The additional contrast associated with neutrons will be an advantage here. We then show the first results of experiments on the interaction of a β-casein monolayer with a nanoparticle titanium oxide sol, foreshadowing the study of the nanoparticle "corona" thought to be important for nanoparticle-cell wall penetration.

  17. A β-hairpin-binding protein for three different disease-related amyloidogenic proteins.

    PubMed

    Shaykhalishahi, Hamed; Mirecka, Ewa A; Gauhar, Aziz; Grüning, Clara S R; Willbold, Dieter; Härd, Torleif; Stoldt, Matthias; Hoyer, Wolfgang

    2015-02-01

    Amyloidogenic proteins share a propensity to convert to the β-structure-rich amyloid state that is associated with the progression of several protein-misfolding disorders. Here we show that a single engineered β-hairpin-binding protein, the β-wrapin AS10, binds monomers of three different amyloidogenic proteins, that is, amyloid-β peptide, α-synuclein, and islet amyloid polypeptide, with sub-micromolar affinity. AS10 binding inhibits the aggregation and toxicity of all three proteins. The results demonstrate common conformational preferences and related binding sites in a subset of the amyloidogenic proteins. These commonalities enable the generation of multispecific monomer-binding agents.

  18. Hyperglycemic effect of low protein cassava diet.

    PubMed

    Sreeja, V G; Leelamma, S

    1998-03-01

    Hyperglycemic effect of cassava diet in presence of varying amounts of protein has been carried out. The rats fed a low protein high cyanide diet showed an increase in the blood glucose and a decrease in the liver glycogen. The activity of glycogen phosphorylase, glucose 6-phosphatase and phosphoglucomutase showed higher levels in the liver of low protein high cyanide group compared to the control group. Also, the activity of hexokinase, and isocitrate dehydrogenase activity in the liver of high cyanide low protein were significantly low. The results suggests that cassava diet with the low protein can induce hyperglycemia. PMID:9754064

  19. When is protein binding important?

    PubMed

    Heuberger, Jules; Schmidt, Stephan; Derendorf, Hartmut

    2013-09-01

    The present paper is an ode to a classic citation by Benet and Hoener (2002. Clin Pharm Ther 71(3):115-121). The now classic paper had a huge impact on drug development and the way the issue of protein binding is perceived and interpreted. Although the authors very clearly pointed out the limitations and underlying assumptions for their delineations, these are too often overlooked and the classic paper's message is misinterpreted by broadening to cases that were not intended. Some members of the scientific community concluded from the paper that protein binding is not important. This was clearly not intended by the authors, as they finished their paper with a paragraph entitled: "When is protein binding important?" Misinterpretation of the underlying assumptions in the classic work can result in major pitfalls in drug development. Therefore, we revisit the topic of protein binding with the intention of clarifying when clinically relevant changes should be considered during drug development.

  20. Superantigenicity of streptococcal M protein

    PubMed Central

    1990-01-01

    M proteins that define the serotypes of group A streptococci are powerful blastogens for human T lymphocytes. The mechanism by which they activate T cells was investigated and compared with the conventional T cell mitogen phytohemagglutinin, and the known superantigen staphylococcal enterotoxin B. Although major histocompatibility complex (MHC) class II molecules are required for presentation, there is no MHC restriction, since allogeneic class II molecules presented the bacterial protein to human T cells. Type 5 M protein appears to bind class II molecules on the antigen-presenting cells and stimulate T cells bearing V beta 8 sequences. Our results indicate that this streptococcal M protein is a superantigen and suggest a possible mechanism of its role in the pathogenesis of the postinfectious autoimmune sequelae. PMID:2358781

  1. De novo protein design: how do we expand into the universe of possible protein structures?

    PubMed

    Woolfson, Derek N; Bartlett, Gail J; Burton, Antony J; Heal, Jack W; Niitsu, Ai; Thomson, Andrew R; Wood, Christopher W

    2015-08-01

    Protein scientists are paving the way to a new phase in protein design and engineering. Approaches and methods are being developed that could allow the design of proteins beyond the confines of natural protein structures. This possibility of designing entirely new proteins opens new questions: What do we build? How do we build into protein-structure space where there are few, if any, natural structures to guide us? To what uses can the resulting proteins be put? And, what, if anything, does this pursuit tell us about how natural proteins fold, function and evolve? We describe the origins of this emerging area of fully de novo protein design, how it could be developed, where it might lead, and what challenges lie ahead.

  2. A general approach to visualize protein binding and DNA conformation without protein labelling.

    PubMed

    Song, Dan; Graham, Thomas G W; Loparo, Joseph J

    2016-03-08

    Single-molecule manipulation methods, such as magnetic tweezers and flow stretching, generally use the measurement of changes in DNA extension as a proxy for examining interactions between a DNA-binding protein and its substrate. These approaches are unable to directly measure protein-DNA association without fluorescently labelling the protein, which can be challenging. Here we address this limitation by developing a new approach that visualizes unlabelled protein binding on DNA with changes in DNA conformation in a relatively high-throughput manner. Protein binding to DNA molecules sparsely labelled with Cy3 results in an increase in fluorescence intensity due to protein-induced fluorescence enhancement (PIFE), whereas DNA length is monitored under flow of buffer through a microfluidic flow cell. Given that our assay uses unlabelled protein, it is not limited to the low protein concentrations normally required for single-molecule fluorescence imaging and should be broadly applicable to studying protein-DNA interactions.

  3. Efficient isolation and elution of cellular proteins using aptamer-mediated protein precipitation assay.

    PubMed

    Kim, Kiseok; Lee, SeungJin; Ryu, Sungho; Han, Dongil

    2014-05-23

    Protein precipitation is one of the most widely used methods for antigen detection and purification in biological research. We developed a reproducible aptamer-mediated magnetic protein precipitation method that is able to efficiently capture, purify and isolate the target proteins. We discovered DNA aptamers having individually high affinity and specificity against human epidermal growth factor receptor (EGFR) and human insulin receptor (INSR). Using aptamers and magnetic beads, we showed it is highly efficient technique to enrich endogenous proteins complex and is applicable to identify physiologically relevant protein-protein interactions with minimized nonspecific binding of proteins. The results presented here indicate that aptamers would be applicable as a useful and cost-effective tool to identify the presence of the particular target protein with their specific protein partners.

  4. Computational Prediction of Protein–Protein Interaction Networks: Algo-rithms and Resources

    PubMed Central

    Zahiri, Javad; Bozorgmehr, Joseph Hannon; Masoudi-Nejad, Ali

    2013-01-01

    Protein interactions play an important role in the discovery of protein functions and pathways in biological processes. This is especially true in case of the diseases caused by the loss of specific protein-protein interactions in the organism. The accuracy of experimental results in finding protein-protein interactions, however, is rather dubious and high throughput experimental results have shown both high false positive beside false negative information for protein interaction. Computational methods have attracted tremendous attention among biologists because of the ability to predict protein-protein interactions and validate the obtained experimental results. In this study, we have reviewed several computational methods for protein-protein interaction prediction as well as describing major databases, which store both predicted and detected protein-protein interactions, and the tools used for analyzing protein interaction networks and improving protein-protein interaction reliability. PMID:24396273

  5. [Protein expression and purification].

    PubMed

    Růčková, E; Müller, P; Vojtěšek, B

    2014-01-01

    Production of recombinant proteins is essential for many applications in both basic research and also in medicine, where recombinant proteins are used as pharmaceuticals. This review summarizes procedures involved in recombinant protein expression and purification, including molecular cloning of target genes into expression vectors, selection of the appropriate expression system, and protein purification techniques. Recombinant DNA technology allows protein engineering to modify protein stability, activity and function or to facilitate protein purification by affinity tag fusions. A wide range of cloning systems enabling fast and effective design of expression vectors is currently available. A first choice of protein expression system is usually the bacteria Escherichia coli. The main advantages of this prokaryotic expression system are low cost and simplicity; on the other hand this system is often unsuitable for production of complex mammalian proteins. Protein expression mediated by eukaryotic cells (yeast, insect and mammalian cells) usually produces properly folded and posttranslationally modified proteins. How-ever, cultivation of insect and, especially, mammalian cells is time consuming and expensive. Affinity tagged recombinant proteins are purified efficiently using affinity chromatography. An affinity tag is a protein or peptide that mediates specific binding to a chromatography column, unbound proteins are removed during a washing step and pure protein is subsequently eluted. PMID:24945544

  6. Urine Protein and Urine Protein to Creatinine Ratio

    MedlinePlus

    ... limited. Home Visit Global Sites Search Help? Urine Protein and Urine Protein to Creatinine Ratio Share this page: Was this page helpful? Also known as: 24-Hour Urine Protein; Urine Total Protein; Urine Protein to Creatinine Ratio; ...

  7. Biofortification of Carrot (Daucus carota L.) with Iodine and Selenium in a Field Experiment.

    PubMed

    Smoleń, Sylwester; Skoczylas, Łukasz; Ledwożyw-Smoleń, Iwona; Rakoczy, Roksana; Kopeć, Aneta; Piątkowska, Ewa; Bieżanowska-Kopeć, Renata; Koronowicz, Aneta; Kapusta-Duch, Joanna

    2016-01-01

    The low content of iodine (I) and selenium (Se) forms available to plants in soil is one of the main causes of their insufficient transfer in the soil-plant-consumer system. Their deficiency occurs in food in the majority of human and farm animal populations around the world. Both elements are classified as beneficial elements. However, plant response to simultaneous fertilization with I and Se has not been investigated in depth. The study (conducted in 2012-2014) included soil fertilization of carrot cv. "Kazan F1" in the following combinations: (1) Control; (2) KI; (3) KIO3; (4) Na2SeO4; (5) Na2SeO3; (6) KI+Na2SeO4; (7) KIO3+Na2SeO4; (8) KI+Na2SeO3; (9) KIO3+Na2SeO3. I and Se were applied twice: before sowing and as top-dressing in a total dose of 5 kg I⋅ha(-1) and 1 kg Se⋅ha(-1). No negative effects of I and Se fertilization were noted with respect to carrot yield. Higher accumulation and the uptake by leaves and storage roots of I and Se were obtained after the application of KI than KIO3, as well as of Na2SeO4 than Na2SeO3, respectively. Transfer factor values for leaves and roots were about a dozen times higher for Se than for I. Selenomethionine content in carrot was higher after fertilization with Na2SeO4 than with Na2SeO3. However, it was the application of Na2SeO3, KI+Na2SeO3 and KIO3+Na2SeO3 that resulted in greater evenness within the years and a higher share of Se from selenomethionine in total Se in carrot plants. Consumption of 100 g f.w. of carrots fertilized with KI+Na2SeO3 and KIO3+Na2SeO3 can supply approximately or slightly exceed 100% of the Recommended Daily Allowance for I and Se. Moreover, the molar ratio of I and Se content in carrot fertilized with KI+Na2SeO3 and KIO3+Na2SeO3 was the best among the research plots. PMID:27303423

  8. Biofortification of Carrot (Daucus carota L.) with Iodine and Selenium in a Field Experiment.

    PubMed

    Smoleń, Sylwester; Skoczylas, Łukasz; Ledwożyw-Smoleń, Iwona; Rakoczy, Roksana; Kopeć, Aneta; Piątkowska, Ewa; Bieżanowska-Kopeć, Renata; Koronowicz, Aneta; Kapusta-Duch, Joanna

    2016-01-01

    The low content of iodine (I) and selenium (Se) forms available to plants in soil is one of the main causes of their insufficient transfer in the soil-plant-consumer system. Their deficiency occurs in food in the majority of human and farm animal populations around the world. Both elements are classified as beneficial elements. However, plant response to simultaneous fertilization with I and Se has not been investigated in depth. The study (conducted in 2012-2014) included soil fertilization of carrot cv. "Kazan F1" in the following combinations: (1) Control; (2) KI; (3) KIO3; (4) Na2SeO4; (5) Na2SeO3; (6) KI+Na2SeO4; (7) KIO3+Na2SeO4; (8) KI+Na2SeO3; (9) KIO3+Na2SeO3. I and Se were applied twice: before sowing and as top-dressing in a total dose of 5 kg I⋅ha(-1) and 1 kg Se⋅ha(-1). No negative effects of I and Se fertilization were noted with respect to carrot yield. Higher accumulation and the uptake by leaves and storage roots of I and Se were obtained after the application of KI than KIO3, as well as of Na2SeO4 than Na2SeO3, respectively. Transfer factor values for leaves and roots were about a dozen times higher for Se than for I. Selenomethionine content in carrot was higher after fertilization with Na2SeO4 than with Na2SeO3. However, it was the application of Na2SeO3, KI+Na2SeO3 and KIO3+Na2SeO3 that resulted in greater evenness within the years and a higher share of Se from selenomethionine in total Se in carrot plants. Consumption of 100 g f.w. of carrots fertilized with KI+Na2SeO3 and KIO3+Na2SeO3 can supply approximately or slightly exceed 100% of the Recommended Daily Allowance for I and Se. Moreover, the molar ratio of I and Se content in carrot fertilized with KI+Na2SeO3 and KIO3+Na2SeO3 was the best among the research plots.

  9. Biofortification of Carrot (Daucus carota L.) with Iodine and Selenium in a Field Experiment

    PubMed Central

    Smoleń, Sylwester; Skoczylas, Łukasz; Ledwożyw-Smoleń, Iwona; Rakoczy, Roksana; Kopeć, Aneta; Piątkowska, Ewa; Bieżanowska-Kopeć, Renata; Koronowicz, Aneta; Kapusta-Duch, Joanna

    2016-01-01

    The low content of iodine (I) and selenium (Se) forms available to plants in soil is one of the main causes of their insufficient transfer in the soil-plant-consumer system. Their deficiency occurs in food in the majority of human and farm animal populations around the world. Both elements are classified as beneficial elements. However, plant response to simultaneous fertilization with I and Se has not been investigated in depth. The study (conducted in 2012–2014) included soil fertilization of carrot cv. “Kazan F1” in the following combinations: (1) Control; (2) KI; (3) KIO3; (4) Na2SeO4; (5) Na2SeO3; (6) KI+Na2SeO4; (7) KIO3+Na2SeO4; (8) KI+Na2SeO3; (9) KIO3+Na2SeO3. I and Se were applied twice: before sowing and as top-dressing in a total dose of 5 kg I⋅ha-1 and 1 kg Se⋅ha-1. No negative effects of I and Se fertilization were noted with respect to carrot yield. Higher accumulation and the uptake by leaves and storage roots of I and Se were obtained after the application of KI than KIO3, as well as of Na2SeO4 than Na2SeO3, respectively. Transfer factor values for leaves and roots were about a dozen times higher for Se than for I. Selenomethionine content in carrot was higher after fertilization with Na2SeO4 than with Na2SeO3. However, it was the application of Na2SeO3, KI+Na2SeO3 and KIO3+Na2SeO3 that resulted in greater evenness within the years and a higher share of Se from selenomethionine in total Se in carrot plants. Consumption of 100 g f.w. of carrots fertilized with KI+Na2SeO3 and KIO3+Na2SeO3 can supply approximately or slightly exceed 100% of the Recommended Daily Allowance for I and Se. Moreover, the molar ratio of I and Se content in carrot fertilized with KI+Na2SeO3 and KIO3+Na2SeO3 was the best among the research plots. PMID:27303423

  10. Autonomous Soaring Flight Results

    NASA Technical Reports Server (NTRS)

    Allen, Michael J.

    2006-01-01

    A viewgraph presentation on autonomous soaring flight results for Unmanned Aerial Vehicles (UAV)'s is shown. The topics include: 1) Background; 2) Thermal Soaring Flight Results; 3) Autonomous Dolphin Soaring; and 4) Future Plans.

  11. Origins of Myc Proteins – Using Intrinsic Protein Disorder to Trace Distant Relatives

    PubMed Central

    Mahani, Amir; Henriksson, Johan; Wright, Anthony P. H.

    2013-01-01

    Mammalian Myc proteins are important determinants of cell proliferation as well as the undifferentiated state of stem cells and their activity is frequently deregulated in cancer. Based mainly on conservation in the C-terminal DNA-binding and dimerization domain, Myc-like proteins have been reported in many simpler organisms within and outside the Metazoa but they have not been found in fungi or plants. Several important signature motifs defining mammalian Myc proteins are found in the N-terminal domain but the extent to which these are found in the Myc-like proteins from simpler organisms is not well established. The extent of N-terminal signature sequence conservation would give important insights about the evolution of Myc proteins and their current function in mammalian physiology and disease. In a systematic study of Myc-like proteins we show that N-terminal signature motifs are not readily detectable in individual Myc-like proteins from invertebrates but that weak similarities to Myc boxes 1 and 2 can be found in the N-termini of the simplest Metazoa as well as the unicellular choanoflagellate, Monosiga brevicollis, using multiple protein alignments. Phylogenetic support for the connections of these proteins to established Myc proteins is however poor. We show that the pattern of predicted protein disorder along the length of Myc proteins can be used as a complementary approach to making dendrograms of Myc proteins that aids the classification of Myc proteins. This suggests that the pattern of disorder within Myc proteins is more conserved through evolution than their amino acid sequence. In the disorder-based dendrograms the Myc-like proteins from simpler organisms, including M. brevicollis, are connected to established Myc proteins with a higher degree of certainty. Our results suggest that protein disorder based dendrograms may be of general significance for studying distant relationships between proteins, such as transcription factors, that have high

  12. Predicting the protein-protein interactions using primary structures with predicted protein surface

    PubMed Central

    2010-01-01

    Background Many biological functions involve various protein-protein interactions (PPIs). Elucidating such interactions is crucial for understanding general principles of cellular systems. Previous studies have shown the potential of predicting PPIs based on only sequence information. Compared to approaches that require other auxiliary information, these sequence-based approaches can be applied to a broader range of applications. Results This study presents a novel sequence-based method based on the assumption that protein-protein interactions are more related to amino acids at the surface than those at the core. The present method considers surface information and maintains the advantage of relying on only sequence data by including an accessible surface area (ASA) predictor recently proposed by the authors. This study also reports the experiments conducted to evaluate a) the performance of PPI prediction achieved by including the predicted surface and b) the quality of the predicted surface in comparison with the surface obtained from structures. The experimental results show that surface information helps to predict interacting protein pairs. Furthermore, the prediction performance achieved by using the surface estimated with the ASA predictor is close to that using the surface obtained from protein structures. Conclusion This work presents a sequence-based method that takes into account surface information for predicting PPIs. The proposed procedure of surface identification improves the prediction performance with an F-measure of 5.1%. The extracted surfaces are also valuable in other biomedical applications that require similar information. PMID:20122202

  13. Measuring protein-protein and protein-nucleic Acid interactions by biolayer interferometry.

    PubMed

    Sultana, Azmiri; Lee, Jeffrey E

    2015-01-01

    Biolayer interferometry (BLI) is a simple, optical dip-and-read system useful for measuring interactions between proteins, peptides, nucleic acids, small molecules, and/or lipids in real time. In BLI, a biomolecular bait is immobilized on a matrix at the tip of a fiber-optic sensor. The binding between the immobilized ligand and another molecule in an analyte solution produces a change in optical thickness at the tip and results in a wavelength shift proportional to binding. BLI provides direct binding affinities and rates of association and dissociation. This unit describes an efficient approach using streptavidin-based BLI to analyze DNA-protein and protein-protein interactions. A quantitative set of equilibrium binding affinities (K(d)) and rates of association and dissociation (k(a)/k(d)) can be measured in minutes using nanomole quantities of sample.

  14. Origins of Protein Functions in Cells

    NASA Technical Reports Server (NTRS)

    Seelig, Burchard; Pohorille, Andrzej

    2011-01-01

    . Recently it was found that, as in the previous case, the proteins have a structure unknown among modern enzymes. In this case, in vitro evolution started from a small, non-enzymatic protein. A similar selection process initiated from a library of random polypeptides is in progress. These results not only allow for estimating the occurrence of function in random protein assemblies but also provide evidence for the possibility of alternative protein worlds. Extant proteins might simply represent a frozen accident in the world of possible proteins. Alternative collections of proteins, even with similar functions, could originate alternative evolutionary paths.

  15. Designing Fluorinated Proteins.

    PubMed

    Marsh, E N G

    2016-01-01

    As methods to incorporate noncanonical amino acid residues into proteins have become more powerful, interest in their use to modify the physical and biological properties of proteins and enzymes has increased. This chapter discusses the use of highly fluorinated analogs of hydrophobic amino acids, for example, hexafluoroleucine, in protein design. In particular, fluorinated residues have proven to be generally effective in increasing the thermodynamic stability of proteins. The chapter provides an overview of the different fluorinated amino acids that have been used in protein design and the various methods available for producing fluorinated proteins. It discusses model proteins systems into which highly fluorinated amino acids have been introduced and the reasons why fluorinated residues are generally stabilizing, with particular reference to thermodynamic and structural studies from our laboratory. Lastly, details of the methodology we have developed to measure the thermodynamic stability of oligomeric fluorinated proteins are presented, as this may be generally applicable to many proteins. PMID:27586337

  16. A method for investigating protein-protein interactions related to Salmonella typhimurium pathogenesis

    SciTech Connect

    Chowdhury, Saiful M.; Shi, Liang; Yoon, Hyunjin; Ansong, Charles; Rommereim, Leah M.; Norbeck, Angela D.; Auberry, Kenneth J.; Moore, R. J.; Adkins, Joshua N.; Heffron, Fred; Smith, Richard D.

    2009-02-10

    We successfully modified an existing method to investigate protein-protein interactions in the pathogenic bacterium Salmonella typhimurium (STM). This method includes i) addition of a histidine-biotin-histidine tag to the bait proteins via recombinant DNA techniques; ii) in vivo cross-linking with formaldehyde; iii) tandem affinity purification of bait proteins under fully denaturing conditions; and iv) identification of the proteins cross-linked to the bait proteins by liquid-chromatography in conjunction with tandem mass-spectrometry. In vivo cross-linking stabilized protein interactions permitted the subsequent two-step purification step conducted under denaturing conditions. The two-step purification greatly reduced nonspecific binding of non-cross-linked proteins to bait proteins. Two different negative controls were employed to reduce false-positive identification. In an initial demonstration of this approach, we tagged three selected STM proteins- HimD, PduB and PhoP- with known binding partners that ranged from stable (e.g., HimD) to transient (i.e., PhoP). Distinct sets of interacting proteins were identified with each bait protein, including the known binding partners such as HimA for HimD, as well as anticipated and unexpected binding partners. Our results suggest that novel protein-protein interactions may be critical to pathogenesis by Salmonella typhimurium. .

  17. Microstructural Changes in High-Protein Nutrition Bars Formulated with Extruded or Toasted Milk Protein Concentrate.

    PubMed

    Banach, J C; Clark, S; Lamsal, B P

    2016-02-01

    Milk protein concentrates with more than 80% protein (that is, MPC80) are underutilized as the primary protein source in high-protein nutrition bars as they impart crumbliness and cause hardening during storage. High-protein nutrition bar texture changes are often associated with internal protein aggregations and macronutrient phase separation. These changes were investigated in model high-protein nutrition bars formulated with MPC80 and physically modified MPC80s. High-protein nutrition bars formulated with extruded MPC80s hardened slower than those formulated with toasted or unmodified MPC80. Extruded MPC80 had reduced free sulfhydryl group exposure, whereas measurable increases were seen in the toasted MPC80. High-protein nutrition bar textural performance may be related to the number of exposed free sulfhydryl groups in MPC80. Protein aggregations resulting from ingredient modification and high-protein nutrition bar storage were studied with sodium dodecyl sulfate polyacrylamide gel electrophoresis. Disulfide-based protein aggregations and changes in free sulfhydryl concentration were not consistently relatable to high-protein nutrition bar texture change. However, the high-protein nutrition bars formulated with extruded MPC80 were less prone to phase separations, as depicted by confocal laser scanning microscopy, and underwent less texture change during storage than those formulated with toasted or unmodified MPC80.

  18. Post-translational control of protein function with light using a LOV-intein fusion protein.

    PubMed

    Jones, D C; Mistry, I N; Tavassoli, A

    2016-04-01

    Methods for the post-translational control of protein function with light hold much value as tools in cell biology. To this end, we report a fusion protein that consists of DnaE split-inteins, flanking the light sensitive LOV2 domain of Avena sativa. The resulting chimera combines the activities of these two unrelated proteins to enable controlled formation of a functional protein via upregulation of intein splicing with blue light in bacterial and human cells. PMID:26940144

  19. PPLook: an automated data mining tool for protein-protein interaction

    PubMed Central

    2010-01-01

    Background Extracting and visualizing of protein-protein interaction (PPI) from text literatures are a meaningful topic in protein science. It assists the identification of interactions among proteins. There is a lack of tools to extract PPI, visualize and classify the results. Results We developed a PPI search system, termed PPLook, which automatically extracts and visualizes protein-protein interaction (PPI) from text. Given a query protein name, PPLook can search a dataset for other proteins interacting with it by using a keywords dictionary pattern-matching algorithm, and display the topological parameters, such as the number of nodes, edges, and connected components. The visualization component of PPLook enables us to view the interaction relationship among the proteins in a three-dimensional space based on the OpenGL graphics interface technology. PPLook can also provide the functions of selecting protein semantic class, counting the number of semantic class proteins which interact with query protein, counting the literature number of articles appearing the interaction relationship about the query protein. Moreover, PPLook provides heterogeneous search and a user-friendly graphical interface. Conclusions PPLook is an effective tool for biologists and biosystem developers who need to access PPI information from the literature. PPLook is freely available for non-commercial users at http://meta.usc.edu/softs/PPLook. PMID:20550717

  20. Identification of Essential Proteins Based on a New Combination of Local Interaction Density and Protein Complexes

    PubMed Central

    Luo, Jiawei; Qi, Yi

    2015-01-01

    Background Computational approaches aided by computer science have been used to predict essential proteins and are faster than expensive, time-consuming, laborious experimental approaches. However, the performance of such approaches is still poor, making practical applications of computational approaches difficult in some fields. Hence, the development of more suitable and efficient computing methods is necessary for identification of essential proteins. Method In this paper, we propose a new method for predicting essential proteins in a protein interaction network, local interaction density combined with protein complexes (LIDC), based on statistical analyses of essential proteins and protein complexes. First, we introduce a new local topological centrality, local interaction density (LID), of the yeast PPI network; second, we discuss a new integration strategy for multiple bioinformatics. The LIDC method was then developed through a combination of LID and protein complex information based on our new integration strategy. The purpose of LIDC is discovery of important features of essential proteins with their neighbors in real protein complexes, thereby improving the efficiency of identification. Results Experimental results based on three different PPI(protein-protein interaction) networks of Saccharomyces cerevisiae and Escherichia coli showed that LIDC outperformed classical topological centrality measures and some recent combinational methods. Moreover, when predicting MIPS datasets, the better improvement of performance obtained by LIDC is over all nine reference methods (i.e., DC, BC, NC, LID, PeC, CoEWC, WDC, ION, and UC). Conclusions LIDC is more effective for the prediction of essential proteins than other recently developed methods. PMID:26125187

  1. Dephosphorylation of chicken cardiac myofibril C-protein by protein phosphatases 1 and 2A

    SciTech Connect

    Thysseril, T.J.; Hegazy, M.G.; Schlender, K.K.

    1987-05-01

    C-Protein, which is a regulatory component of cardiac muscle myofibrils, is phosphorylated in response to US -adrenergic agonists by a cAMP-dependent mechanism and dephosphorylated in response to cholinergic agonists. It is believed that the cAMP-dependent phosphorylation is due to cAMP-dependent protein kinase. The protein phosphatase(s) involved in the dephosphorylation of C-protein has not been determined. In this study, chicken cardiac C-protein was phosphorylated with the cAMP-dependent protein kinase to about 3 mol phosphate/mol C-protein. Incubation of (TSP)C-protein with the catalytic subunit of protein phosphatase 1 or 2A rapidly removed 30-40% of TS(P). Phosphopeptide maps and phosphoamino acid analysis revealed that the major site(s) dephosphorylated by either phosphatase was a phosphothreonine residue(s) located on the same tryptic peptide and on the same CNBr fragment. Increasing the incubation period or the phosphatase concentration did not result in any further dephosphorylation of C-protein by phosphatase 1, but phosphatase 2A completely dephosphorylated C-protein. Preliminary studies showed that the major protein phosphatase associated with the myofibril was phosphatase 2A. These results indicate the phosphatase 2A may be important in the regulation of the phosphorylation state of C-protein.

  2. Stabilization of a protein nanocage through the plugging of a protein-protein interfacial water pocket.