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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

    Plissier, Hlne 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

    2014-06-01

    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:24905822

  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. PMID:25763691

  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. Uncertain role of MtSEO-F3 in assembly of Medicago truncatula forisomes

    PubMed Central

    Groscurth, Sira; Mller, Boje; Visser, Franziska; Blob, Bernhard; Menzel, Matthias; Rping, Boris A; Twyman, Richard M; Prfer, Dirk; Noll, Gundula A

    2014-01-01

    Forisomes are specialized multimeric protein complexes found only in the papilionoid legumes. They undergo a reversible conformational change in response to phloem injury to enable the occlusion of sieve tubes, thus preventing the loss of photoassimilates. The individual subunits are designated by the letters SEO-F (sieve element occlusion by forisomes) and are part of the larger SEO protein family, which also includes the typical P-proteins found in most dicots and some monocots. When specific SEO-F subunits from different species are expressed in a heterologous background, they self-assemble into fully-functional artificial forisomes. However, with the exception of basal species such as Dipteryx panamensis, the geometry of these artificial forisomes differs from that of their native counterparts. Studies involving SEO-F proteins from the model legume Medicago truncatula have shown that a combination of 3 of the 4 subunits can fine-tune the geometry of artificial forisomes. However, MtSEO-F3 was excluded from these studies because it was not incorporated into either the native or artificial forisomes in our original experiments. In this addendum, we present further data concerning the interactive properties of the SEO-F proteins and confirm that all 4 MtSEO-F proteins interact in all possible pairwise combinations. These data indicate that the exclusion of MtSEO-F3 from the compact forisome may reflect the steric hindrance of binding sites rather than an inability to interact with other forisome subunits. PMID:25763696

  6. SEOS frame camera applications study

    NASA Technical Reports Server (NTRS)

    1974-01-01

    A research and development satellite is discussed which will provide opportunities for observation of transient phenomena that fall within the fixed viewing circle of the spacecraft. The evaluation of possible applications for frame cameras, for SEOS, are studied. The computed lens characteristics for each camera are listed.

  7. 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.

  8. Incommensurate Phase in ?-Irradiated RbH3(SeO3)2 and RbD3(SeO3)2

    NASA Astrophysics Data System (ADS)

    Yamamoto, Kenichi; Fukui, Minoru; Abe, Ryuji; Sakai, Akira; Yagi, Toshirou

    1984-01-01

    ESR spectra and dielectric constant of ?-irradiated RbH3(SeO3)2 and RbD3(SeO3)2 were measured simultaneously around the phase transition temperature (TC). It has been confirmed that in these substances with the doze of about 5 Mard, an incommensurate phase appears in the temperature range of about 46 K above TC and the range broadens slightly with the increase of doze. From the analysis of temperature dependence of A tensor for SeO2- radical, it is clarified that SeO3(II) in RbD3(SeO3)2 rotates in a quite similar way with that in RbH3(SeO3)2 below TC. It was found for the first time that in RbD3(SeO3)2 the dielectric constant peak along the a-axis at TC is much larger than that along the b-axis. On these results some discussion is presented.

  9. Pb4V6O16(SeO3)3(H2O), Pb2VO2(SeO3)2Cl, and PbVO2(SeO3)F: new lead(II)-vanadium(V) mixed-metal selenites featuring novel anionic skeletons.

    PubMed

    Cao, Xue-Li; Kong, Fang; Hu, Chun-Li; Xu, Xiang; Mao, Jiang-Gao

    2014-08-18

    Hydrothermal reactions of PbCO3 (or PbCl2), V2O5, and SeO2 in KOH solution or HF solution resulted in three new lead(II)-vanadium(V) mixed-metal selenites, namely, Pb4V6O16(SeO3)3(H2O) (1), Pb2VO2(SeO3)2Cl (2), and PbVO2(SeO3)F (3). Compounds 1 and 2 are polar (space group P21), whereas compound 3 is centrosymmetric (space group Pbca). Compound 1 displays an unusual [V6O16(SeO3)3](8-) anionic chain, which is composed by a 1D [V4O12](2-) anionic chain that is further decorated by dimeric [V2O6(SeO3)3](8-) units via corner-sharing. Compound 2 features two types of 1D chains, a cationic [Pb2Cl](3+) chain and a [VO2(SeO3)2](3-) anionic chain, whereas compound 3 contains dimeric [V2O4(SeO3)2F2](2-) units. The powder second-harmonic-generating (SHG) measurements indicate that compound 1 shows a weak SHG response of about 0.2 KDP (KH2PO4) under 1400 nm laser radiation. Thermal stability and optical properties as well as theoretical calculations based on density functional theory methods were also performed. PMID:25102013

  10. 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.

  11. Structure-Directing Effect of Alkali Metal Cations in New Molybdenum Selenites, Na2Mo2O5(SeO3)2, K2Mo2O5(SeO3)2, and Rb2Mo3O7(SeO3)3.

    PubMed

    Bang, Seong-eun; Ok, Kang Min

    2015-09-01

    Both single crystals and pure polycrystalline samples of three new quaternary alkali metal molybdenum selenites, Na2Mo2O5(SeO3)2, K2Mo2O5(SeO3)2, and Rb2Mo3O7(SeO3)3, have been synthesized through hydrothermal and solid-state reactions using A2CO3 (A = Na, K, and Rb), MoO3, and SeO2 as reagents. The frameworks of all three materials consist of both families of second-order Jahn-Teller distortive cations, i.e., the d(0) cation (Mo(6+)) and the lone pair cation (Se(4+)). Although the extent of framework distortions and the resulting occupation sites of alkali metal cations are dissimilar, Na2Mo2O5(SeO3)2 and K2Mo2O5(SeO3)2 exhibit similar three-dimensional networks that are composed of highly asymmetric Mo2O11 dimers and SeO3 polyhedra. Rb2Mo3O7(SeO3)3 reveals a two-dimensional structure that is built with Mo3O15 trimers and SeO3 intralayer linkers. Close structural examinations suggest that the structure-directing effect of alkali metal cations is significant in determining the framework distortions and the dimensions of the molybdenum selenites. UV-vis diffuse reflectance and infrared spectroscopy, thermogravimetric analyses, and ion-exchange reactions are reported, as are out-of-center distortion and dipole moment calculations. PMID:26307853

  12. 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.

  13. Rapid, room-temperature synthesis of amorphous selenium/protein composites using Capsicum annuum L extract

    NASA Astrophysics Data System (ADS)

    Li, Shikuo; Shen, Yuhua; Xie, Anjian; Yu, Xuerong; Zhang, Xiuzhen; Yang, Liangbao; Li, Chuanhao

    2007-10-01

    We describe the formation of amorphous selenium (?-Se)/protein composites using Capsicum annuum L extract to reduce selenium ions (SeO32-) at room temperature. The reaction occurs rapidly and the process is simple and easy to handle. A protein with a molecular weight of 30 kDa extracted from Capsicum annuum L not only reduces the SeO32- ions to Se0, but also controls the nucleation and growth of Se0, and even participates in the formation of ?-Se/protein composites. The size and shell thickness of the ?-Se/protein composites increases with high Capsicum annuum L extract concentration, and decreases with low reaction solution pH. The results suggest that this eco-friendly, biogenic synthesis strategy could be widely used for preparing inorganic/organic biocomposites. In addition, we also discuss the possible mechanism of the reduction of SeO32- ions by Capsicum annuum L extract.

  14. 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).

  15. LiMn3(SeO3)2(HSeO3)6.

    PubMed

    Johnston, Magnus G; Harrison, William T A

    2007-04-01

    The title compound, lithium trimanganese bis[trioxoselenate(IV)] hexakis[hydrogentrioxoselenate(IV)], is built up from a vertex-sharing network of distorted Mn(III)O(6) octahedra, SeO(3) and HSeO(3) pyramids and unusual Li(OH)(6) octahedra, resulting in a dense three-dimensional structure. Mn, Li and one Se atom have site symmetries of -1, -3, and 3, respectively. An O-H...O hydrogen bond helps to establish the crystal packing. PMID:17413211

  16. Investigation of local symmetry in LiH3(SeO3)2 single crystals by 1H and 7Li nuclear magnetic resonance

    NASA Astrophysics Data System (ADS)

    Lim, Ae Ran

    2013-10-01

    The local environments of 1H and 7Li nuclei in LiH3(SeO3)2 crystals were investigated using FT NMR. The 7Li spectrum does changes from three resonance lines to one resonance line near Tm (=383 K). The variation in the splitting of the 7Li resonance lines with temperature indicates that the EFG at the Li sites produced by the (SeO3)2- groups varies with temperature. The changes in the temperature dependence of the intensity, line width, and spin-lattice relaxation time T1 near Tm for the 1H and 7Li nuclei coincide with the distortion of the structural framework surrounding each 1H and 7Li ion. Finally, the NMR results obtained here are compared to MH3(SeO3)2 (M = Na, K, and Cs) crystals previously reported.

  17. Structure and properties of a non-traditional glass containing TeO2, SeO2 and MoO3

    NASA Astrophysics Data System (ADS)

    Bachvarova-Nedelcheva, A.; Iordanova, R.; Kostov, K. L.; Yordanov, St.; Ganev, V.

    2012-09-01

    A glass containing SeO2, TeO2, MoO3 and La2O3 was obtained at high oxygen pressure (P = 36 MPa) using pure oxides as precursors. The real bulk chemical composition of the glass according to LA-ICP-MS analysis is 17SeO250TeO232MoO31La2O3 (wt.%). The glass was characterized by X-ray diffraction, scanning electron microscopy (SEM), differential thermal analysis (DTA), UV-Vis, XPS, IR and EPR spectroscopy. According to DTA the glass transition temperature (Tg) is below 300 C. By IR and X-ray photoelectron spectroscopy was determined the main building units (TeO3, TeO4, SeO3, Mo2O8) and the existing of mixed bridging bonds only, which build up the amorphous network. It was established by UV-Vis that the glass is transparent above 490 nm. As a result of a lengthy heat treatment, crystallization took place and crystals rich in SeO2 and TeO2 were found incorporated into the amorphous part containing all components.

  18. Syntheses, structures, and properties of Ag 4(Mo 2O 5)(SeO 4) 2(SeO 3) and Ag 2(MoO 3) 3SeO 3

    NASA Astrophysics Data System (ADS)

    Ling, Jie; Albrecht-Schmitt, Thomas E.

    2007-05-01

    Ag 4(Mo 2O 5)(SeO 4) 2(SeO 3) has been synthesized by reacting AgNO 3, MoO 3, and selenic acid under mild hydrothermal conditions. The structure of this compound consists of cis-MoO 22+ molybdenyl units that are bridged to neighboring molybdenyl moieties by selenate anions and by a bridging oxo anion. These dimeric units are joined by selenite anions to yield zigzag one-dimensional chains that extended down the c-axis. Individual chains are polar with the C2 distortion of the Mo(VI) octahedra aligning on one side of each chain. However, the overall structure is centrosymmetric because neighboring chains have opposite alignment of the C2 distortion. Upon heating Ag 4(Mo 2O 5)(SeO 4) 2(SeO 3) looses SeO 2 in two distinct steps to yield Ag 2MoO 4. Crystallographic data: (193 K; Mo K?, ?=0.71073 ): orthorhombic, space group Pbcm, a=5.6557(3), b=15.8904(7), c=15.7938(7) , V=1419.41(12), Z=4, R( F)=2.72% for 121 parameters with 1829 reflections with I>2 ?( I). Ag 2(MoO 3) 3SeO 3 was synthesized by reacting AgNO 3 with MoO 3, SeO 2, and HF under hydrothermal conditions. The structure of Ag 2(MoO 3) 3SeO 3 consists of three crystallographically unique Mo(VI) centers that are in 2+2+2 coordination environments with two long, two intermediate, and two short bonds. These MoO 6 units are connected to form a molybdenyl ribbon that extends along the c-axis. These ribbons are further connected together through tridentate selenite anions to form two-dimensional layers in the [ bc] plane. Crystallographic data: (193 K; Mo K?, ?=0.71073 ): monoclinic, space group P2 1/n, a=7.7034(5), b=11.1485(8), c=12.7500(9) , ?=105.018(1) V=1002.7(2), Z=4, R( F)=3.45% for 164 parameters with 2454 reflections with I>2 ?( I). Ag 2(MoO 3) 3SeO 3 decomposes to Ag 2Mo 3O 10 on heating above 550 C.

  19. Thallium(III) selenite, Tl2(SeO3)3.

    PubMed

    Harrison, William T A

    2005-07-01

    The structure of Tl2(SeO3)3 [dithallium(III) triselenium(IV) nonaoxide] is monoclinic (P21/n symmetry), with all atoms in general positions. It is built up from TlO6 octahedra, distorted TlO7 pentagonal bipyramids and (SeO3)2- pyramids sharing vertices and edges to form corrugated (001) layers. The Se lone pairs of electrons are accommodated in the interlayer regions. PMID:15997051

  20. Crystal chemistry of selenates with mineral-like structures: VII. The structure of (H3O)[(UO2)(SeO4)(SeO2OH)] and some structural features of selenite-selenates

    NASA Astrophysics Data System (ADS)

    Krivovichev, S. V.

    2009-12-01

    The crystal structure of a new compound, (H3O)[(UO2)(SeO4)(SeO2OH)] (monoclinic, P21/ n, a = 8.6682(19), b = 10.6545(16), c = 9.846(2) , ? = 97.881(17), V = 900.7(3) 3), was solved by direct methods and refined to R 1 = 0.050. The structure contains two symmetrically different Se atoms. The Se1 site is coordinated by three O atoms as is characteristic of Se4+ cations. The Se2 site is coordinated by four O atoms and forms selenate anion SeO{4/2-}. The structure is based on selenite-selenate sheets [(UO2)(SeO4)(SeO2OH)]- linked by the interlayer H3O- ions. The sheets are parallel to (101). The structure is compared to that of schmiederite, Pb2Cu2(SeO3)(SeO4)(OH)4.

  1. 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.

  2. SeO II addition on PVA-based photopolymer for improving photostorage stabilities and diffraction efficiencies

    NASA Astrophysics Data System (ADS)

    Kim, Daeheum; Nam, Seungwoong; Yeo, Seungbyung; Lim, Jiyun

    2006-08-01

    Polyvinyl alcohol/Acrylamide(PVA/AA)based photopolymer systems modified with SeO II crystals were prepared and photostorage characteristics mainly including diffraction efficiencies were examined and compared with pure PVA/AA films using green laser light (532nm). The photosensitive films were composed of polymeric film-forming binder (PVA), monomer (acrylamide, AA), photoinitiator (triethanol amine, TEA), photosensitizer (Eosin YR), and SeO II crystals. The best optical recording characteristics were observed at the composition of: polymer binder (PVA) : AA : TEA : SeO II : Eosin Y = 1.0 : 0.3 : 0.225 : 0.1 : 0.0015. Diffraction efficiencies as high as 85% with energetic sensitivity of 0.5 mW/cm2 have been obtained in the photopolymer film, and the photopolymer film with SeO II showed higher diffraction efficiencies and lower initial sensitivity than the photopolymer film without SeO II. The morphology of SeO II was expected to be nano crystals since they didn't scatter optical lights and didn't show any peaks in X-ray diffraction spectra.

  3. Emulating exhalative chemistry: synthesis and structural characterization of ilinskite, Na[Cu5O2](SeO3)2Cl3, and its K-analogue

    NASA Astrophysics Data System (ADS)

    Kovrugin, Vadim M.; Siidra, Oleg I.; Colmont, Marie; Mentré, Olivier; Krivovichev, Sergey V.

    2015-08-01

    The K- and Na-synthetic analogues of the fumarolic mineral ilinskite have been synthesized by the chemical vapor transport (CVT) reactions method. The A[Cu5O2](SeO3)2Cl3 ( A + = K+, Na+) compounds crystallize in the orthorhombic space group Pnma: a = 18.1691(6) Å, b = 6.4483(2) Å, c = 10.5684(4) Å, V = 1238.19(7) Å3, R 1 = 0.018 for 1957 unique reflections with F > 4σ F for K[Cu5O2](SeO3)2Cl3 ( KI), and a = 17.7489(18) Å, b = 6.4412(6) Å, c = 10.4880(12) Å, V = 1199.0(2) Å3, R 1 = 0.049 for 1300 unique reflections with F > 4σ F for Na[Cu5O2](SeO3)2Cl3 ( NaI). The crystal structures of KI and NaI are based upon the [O2Cu5]6+ sheets consisting of corner-sharing (OCu4)6+ tetrahedra. The Na-for-K substitution results in the significant expansion of the interlayer space and changes in local coordination of some of the Cu2+ cations. The A + cation coordination changes from fivefold (for Na+) to ninefold (for K+). The CVT reactions method provides a unique opportunity to model physicochemical conditions existing in fumarolic environments and may be used not only to model exhalative processes, but also to predict possible mineral phases that may form in fumaroles. In particular, the K analogue of ilinskite is not known in nature, whereas it may well form from volcanic gases in a K-rich local geochemical environment.

  4. New bismuth selenium oxides: syntheses, structures, and characterizations of centrosymmetric Bi2(SeO3)2(SeO4) and Bi2(TeO3)2(SeO4) and noncentrosymmetric Bi(SeO3)(HSeO3).

    PubMed

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

    2013-04-01

    Three new mixed metal selenium oxides materials, Bi2(SeO3)2(SeO4), Bi2(TeO3)2(SeO4), and Bi(SeO3)(HSeO3), have been synthesized by hydrothermal and solid-state reactions using Bi(NO3)35H2O, SeO2 (or TeO2), H2SeO4, and Bi2O3 as reagents. The reported materials have been structurally characterized by single crystal X-ray diffraction. While Bi2(SeO3)2(SeO4) and Bi2(TeO3)2(SeO4) are crystallographically centrosymmetric (CS), Bi(SeO3)(HSeO3) crystallizes in a noncentrosymmetric (NCS) space group. The isostructural Bi2(SeO3)2(SeO4) and Bi2(TeO3)2(SeO4) exhibit three-dimensional framework structures that are composed of BiO6, Se(4+)O3 (or Te(4+)O3), and Se(6+)O4 polyhedra. However, Bi(SeO3)(HSeO3) exhibits corrugated layers that are composed of BiO5, Se(4+)O3, and Se(4+)O2(OH) polyhedra. All three materials contain local asymmetric coordination environments attributable to the lone pairs on the Bi(3+), Se(4+), and/or Te(4+) cations. Powder second-harmonic generation (SHG) measurements on NCS Bi(SeO3)(HSeO3) using 1064 nm radiation indicate that the material has a SHG efficiency of approximately 20 times that of ?-SiO2 and is not phase-matchable (type 1). The origin and magnitude of the SHG efficiency of Bi(SeO3)(HSeO3) is explained by determining the net direction of the polarizations arising from individual asymmetric polyhedra. Infrared spectroscopy, thermal analysis, elemental analysis, and dipole moment calculations for the reported materials are also presented. PMID:23506341

  5. 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

  6. A new phase in the MnII-SeIV-MoVI-O system, Mn(MoO3)(SeO3)(H2O): Hydrothermal synthesis, crystal structure and properties

    NASA Astrophysics Data System (ADS)

    Zhen, Yanzhong; Wang, Danjun; Liu, Bin; Fu, Feng; Xue, Ganglin

    2013-11-01

    A new phase in the MnII-SeIV-MoVI-O system, Mn(MoO3)(SeO3)(H2O) (1), has been hydrothermally synthesized with a high yield (82%), and characterized by IR, TG-DSC, magnetism measurement and single crystal X-ray diffraction. The structure of Mn(MoO3)(SeO3)(H2O) features a complicated 3D network composed of the 1D molybdenum(VI) oxide chains and the 1D manganese(II) selenite chains interconnected via Se-O-Mo and Mn-O-Mo bridges. It is stable up to approximately 340 C, and losses water molecule at 340 C, then release SeO2 at about 420 C. The result of magnetic property measurements has indicated that there exist antiferromagnetic interactions between Mn(II) centers. Photocatalysis experimental result illustrates that the compound exhibits good photocatalytic performance for degradation of RhB under visible light irradiation.

  7. Cs(TaO2)3(SeO3)2 and Cs(TiOF)3(SeO3)2: structural and second harmonic generation changes induced by the different d(0)-TM coordination octahedra.

    PubMed

    Cao, Xue-Li; Hu, Chun-Li; Kong, Fang; Mao, Jiang-Gao

    2015-04-20

    Two new cesium selenites containing TaO6 or TiO4F2 octahedra, namely, Cs(TaO2)3(SeO3)2 (1) and Cs(TiOF)3(SeO3)2 (2), have been prepared using standard high temperature solid-state method and hydrothermal reaction, respectively. Compound 1 crystallizes in P3?m1 and features an unusual [(TaO2)3(SeO3)2](-) sandwich-like double layer in which two [Ta(1)O3(SeO3)](3-) layers are bridged by central Ta(2)O6 octahedra via corner-sharing, whereas Cs(TiOF)3(SeO3)2 with a polar space group P63mc features an interesting hexagonal tungsten oxide (HTO) layered topology and presents a strong second harmonic generation (SHG) of about 5 KDP (KH2PO4), which is much larger than those of A(VO2)3(QO3)2 (A = K, Tl, Rb, Cs, or NH4; Q = Se, Te) with a similar HTO layered structure. Cs(TiOF)3(SeO3)2 is also type-I phase matching. The SHG of above-mentioned HTO materials can be enhanced greatly with the replacement of VO6 octahedra by TiO4F2 octahedra. Furthermore, thermal stabilities, UV-vis diffuse reflectance spectra, infrared spectra, relationship between crystal structure and SHG, and theoretical calculations were also reported. PMID:25835387

  8. TiO2-SEO Block Copolymer Nanocomposites as Solid-State Electrolytes for Lithium Metal Batteries

    NASA Astrophysics Data System (ADS)

    Gurevitch, Inna; Buonsanti, Raffaella; Teran, Alexander; Cabana, Jordi; Balsara, Nitash

    2013-03-01

    Replacing the liquid electrolyte in lithium batteries by a solid has been a long-standing goal of the battery industry due to the promise of better safety and the potential to produce batteries with higher energy densities. Recently, symmetric polystyrene-block-poly(ethylene oxide) (SEO) copolymers/LiX salt mixtures with high ionic conductivity and high shear modulus were developed as solid electrolytes. For an enhancement in mechanical properties and its effect on the dendrite growth from lithium metal electrodes, we study the effect of adding TiO2 nanoparticles to the SEO/LiX mixtures. We find that TiO2/SEO/LiX nanocomposite electrolytes have stable performance against the lithium metal electrodes. There appears to be a correlation between the stability of the electrolytes, morphology, and mechanical properties.

  9. Insertion of Mutant Proteolipid Protein Results in Missorting of Myelin Proteins

    PubMed Central

    Vaurs-Barriere, Catherine; Wong, Kondi; Weibel, Thais D.; Abu-Asab, Mones; Weiss, Michael D.; Kaneski, Christine R.; Mixon, Tong-Hui; Bonavita, Simona; Creveaux, Isabelle; Heiss, John D.; Tsokos, Maria; Goldin, Ehud; Quarles, Richard H.; Boespflug-Tanguy, Odile; Schiffmann, Raphael

    2014-01-01

    Two brothers with a leukodystrophy, progressive spastic diplegia, and peripheral neuropathy were found to have proteinaceous aggregates in the peripheral nerve myelin sheath. The patients mother had only subclinical peripheral neuropathy, but the maternal grandmother had adult-onset leukodystrophy. Sequencing of the proteolipid protein (PLP) gene showed a point mutation IVS4 + 1 G?A within the donor splice site of intron 4. We identified one transcript with a deletion of exon 4 (?ex4, 169bp) encoding for PLP and DM20 proteins and lacking two transmembrane domains, and a second transcript with exon 4 + 10bp encoding three transmembrane domains. Immunohistochemistry showed abnormal aggregation in the myelin sheath of MBP and P0. Myelin-associated glycoprotein was present in the SchmidtLanterman clefts but significantly reduced in the periaxonal region. Using immunogold electron microscopy, we demonstrated the presence of mutated PLP/DM20 and the absence of the intact protein in the patient peripheral myelin sheath. We conclude that insertion of mutant PLP/DM20 with resulting aberrant distribution of other myelin proteins in peripheral nerve may constitute an important mechanism of dysmyelination in disorders associated with PLP mutations. PMID:14681886

  10. Crystal structure and magnetic properties of two new cobalt selenite halides: Co 5(SeO 3) 4X2 ( X=Cl, Br)

    NASA Astrophysics Data System (ADS)

    Becker, Richard; Prester, Mladen; Berger, Helmuth; Hui Lin, Ping; Johnsson, Mats; Drobac, Djuro; Zivkovic, Ivica

    2007-03-01

    Two new isostructural cobalt selenite halides Co 5(SeO 3) 4Cl 2 and Co 5(SeO 3) 4Br 2 have been synthesized. They crystallize in the triclinic system space group P-1 with the following lattice parameters for Co 5(SeO 3) 4Cl 2: a=6.4935(8) , b=7.7288(8) , c=7.7443(10) , ?=66.051(11), ?=73.610(11), ?=81.268(9), and Z=1. The crystal structures were solved from single-crystal X-ray data, R1=3.73 and 4.03 for Co 5(SeO 3) 4Cl 2 and Co 5(SeO 3) 4Br 2, respectively. The new compounds are isostructural to Ni 5(SeO 3) 4Br 2. Magnetic susceptibility measurements on oriented single-crystalline samples show anisotropic response in a broad temperature range. The anisotropic susceptibility is quantitatively interpreted within the zero-field splitting schemes for Co 2+ and Ni 2+ ions. Sharp low-temperature susceptibility features, at TN=18 and 20 K for Co 5(SeO 3) 4Cl 2 and Co 5(SeO 3) 4Br 2, respectively, are ascribed to antiferromagnetic ordering in a minority magnetic subsystem. In isostructural Ni 5(SeO 3) 4Br 2 magnetically ordered subsystem represents a majority fraction ( TN=46 K). Nevertheless, anisotropic susceptibility of Ni 5(SeO 3) 4Br 2 is dominated at low temperatures by a minority fraction, subject to single-ion anisotropy effects and increasing population of S z=0 (singlet) ground state of octahedrally coordinated Ni 2+.

  11. Formation of a selenium-substituted rhodanese by reaction with selenite and glutathione: possible role of a protein perselenide in a selenium delivery system.

    PubMed

    Ogasawara, Y; Lacourciere, G; Stadtman, T C

    2001-08-14

    Selenophosphate is the active selenium-donor compound required by bacteria and mammals for the specific synthesis of Secys-tRNA, the precursor of selenocysteine in selenoenzymes. Although free selenide can be used in vitro for the synthesis of selenophosphate, the actual physiological selenium substrate has not been identified. Rhodanese (EC ) normally occurs as a persulfide of a critical cysteine residue and is believed to function as a sulfur-delivery protein. Also, it has been demonstrated that a selenium-substituted rhodanese (E-Se form) can exist in vitro. In this study, we have prepared and characterized an E-Se rhodanese. Persulfide-free bovine-liver rhodanese (E form) did not react with SeO(3)(2-) directly, but in the presence of reduced glutathione (GSH) and SeO(3)(2-) E-Se rhodanese was generated. These results indicate that the intermediates produced from the reaction of GSH with SeO(3)(2-) are required for the formation of a selenium-substituted rhodanese. E-Se rhodanese was stable in the presence of excess GSH at neutral pH at 37 degrees C. E-Se rhodanese could effectively replace the high concentrations of selenide normally used in the selenophosphate synthetase in vitro assay in which the selenium-dependent hydrolysis of ATP is measured. These results show that a selenium-bound rhodanese could be used as the selenium donor in the in vitro selenophosphate synthetase assay. PMID:11493708

  12. High-pressure single-crystal X-ray diffraction of Tl 2SeO 4

    NASA Astrophysics Data System (ADS)

    Grzechnik, Andrzej; Breczewski, Tomasz; Friese, Karen

    2008-11-01

    The effect of pressure on the crystal structure of thallium selenate (Tl 2SeO 4) ( Pmcn, Z=4), containing the Tl + cations with electron lone pairs, has been studied with single-crystal X-ray diffraction in a diamond anvil cell up to 3.64 GPa at room temperature. No phase transition has been observed. The compressibility data are fitted by a Murnaghan equation of state with the zero-pressure bulk modulus B0=29(1) GPa and the unit-cell volume at ambient pressure V0=529.6(8) 3 ( B'=4.00). Tl 2SeO 4 is the least compressible in the c direction, while the pressure-induced changes of the a and b lattice parameters are quite similar. These observations can be explained by different pressure effects on the nine- and 11-fold coordination polyhedra around the two non-equivalent Tl atoms. The SeO 42- tetrahedra are not rigid units and become more distorted. Their contribution to the compressibility is small. The effect of pressure on the isotypical oxide materials A 2TO 4 with the ?-K 2SO 4 structure is discussed. It appears that the presence of electron lone pairs on the Tl + cation does not seem to influence the compressibility of Tl 2SeO 4.

  13. Success in Mathematics within a Challenged Minority: The Case of Students of Ethiopian Origin in Israel (SEO)

    ERIC Educational Resources Information Center

    Mulat, Tiruwork; Arcavi, Abraham

    2009-01-01

    Many studies have reported on the economical, social, and educational difficulties encountered by Ethiopian Jews since their immigration to Israel. Furthermore, the overall academic underachievement and poor representation of students of Ethiopian origin (SEO) in the advanced mathematics and science classes were highlighted and described. Yet,

  14. 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

  15. Theoretical study of potential energy curves, spectroscopic constants, and radiative lifetimes of low-lying states in an SeO molecule

    NASA Astrophysics Data System (ADS)

    Li, Rui; Lian, Ke-Yan; Li, Qi-Nan; Miao, Feng-Juan; Yan, Bing; Jin, Ming-Xing

    2012-12-01

    The low-lying potential energy curves of the SeO molecule are computed by means of an ab initio multireference configuration interaction technique, taking into account relativistic (scalar plus spinorbit coupling) effects. The spectroscopic constants of ? states for X3?-, a1?, b1?+, A3?, A'3?, and A? 3?+ states are obtained, and they are in good accordance with available experimental values. The FranckCondon factors and transition dipole moments to the ground state are computed, and the natural radiative lifetimes of low-lying ? states are theoretically obtained. Comparisons of the natural lifetimes of ? states with previous experimental results and those of isovalent TeO molecule are made.

  16. 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

    2015-10-01

    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 (13)C? 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

  17. 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

  18. 31 CFR 30.3 - Q-3: How are the SEOs and most highly compensated employees identified for purposes of compliance...

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 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...

  19. 31 CFR 30.3 - Q-3: How are the SEOs and most highly compensated employees identified for purposes of compliance...

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 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...

  20. 31 CFR 30.3 - Q-3: How are the SEOs and most highly compensated employees identified for purposes of compliance...

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 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...

  1. 31 CFR 30.3 - Q-3: How are the SEOs and most highly compensated employees identified for purposes of compliance...

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 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...

  2. 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.

  3. Bi6(SeO3)3O5Br2: A new bismuth oxo-selenite bromide

    NASA Astrophysics Data System (ADS)

    Berdonosov, Peter S.; Olenev, Andrei V.; Kirsanova, Maria A.; Lebed, Julia B.; Dolgikh, Valery A.

    2012-12-01

    A new bismuth oxo-selenite bromide Bi6(SeO3)3O5Br2 was synthesized and structurally characterized. The crystal structure belongs to the triclinic system (space group P1, Z=2, a=7.1253(7) , b=10.972(1) , c=12.117(1) , ?=67.765(7), ?=82.188(8), ?=78.445(7)) and is unrelated to those of other known oxo-selenite halides. It can be considered as an open framework composed of BiOx or BiOyBrz polyhedrons forming channels running along [1 0 0] direction which contain the selenium atoms in pyramidal shape oxygen coordination (SeO3E). The spectroscopic properties and thermal stability were studied. The new compound is stable up to 400 C.

  4. Infrared evidence for multiple structural transitions in single crystal Cu3(SeO3)2 Cl

    NASA Astrophysics Data System (ADS)

    Miller, Kevin H.; Berger, Helmuth; Tanner, David B.

    2013-03-01

    Infrared reflection and transmission over a broad temperature range (10-300 K) have been measured on the anisotropic single-crystal Cu3(SeO3)2 Cl. Two distinct space groups have previously been reported for Cu3(SeO3)2 Cl at 300 K (monoclinic C2/m and triclinic P1bar). Comparing the number of observed infrared active phonons with group theoretical predictions points towards the existence of the triclinic structure at 300 K; however, an impurity-rich monoclinic structure cannot be ruled out. New phonon modes are observed upon cooling below 90 K, and again upon cooling below 40 K. The latter temperature range corresponds to the onset of long range magnetic order in the material. The structural and magnetic properties of Cu3(SeO3)2 Cl will be discussed in terms of our infrared spectra, group theoretical predictions, and comparisons to related compounds. Supported by the US DOE through contract DE-FG02-02ER45984 at UF.

  5. Silencing of Soybean Seed Storage Proteins Results in a Rebalanced Protein Composition Preserving Seed Protein Content without Major Collateral Changes in the Metabolome and Transcriptome[W][OA

    PubMed Central

    Schmidt, Monica A.; Barbazuk, W. Brad; Sandford, Michael; May, Greg; Song, Zhihong; Zhou, Wenxu; Nikolau, Basil J.; Herman, Eliot M.

    2011-01-01

    The ontogeny of seed structure and the accumulation of seed storage substances is the result of a determinant genetic program. Using RNA interference, the synthesis of soybean (Glycine max) glycinin and conglycinin storage proteins has been suppressed. The storage protein knockdown (SP−) seeds are overtly identical to the wild type, maturing to similar size and weight, and in developmental ontogeny. The SP− seeds rebalance the proteome, maintaining wild-type levels of protein and storage triglycerides. The SP− soybeans were evaluated with systems biology techniques of proteomics, metabolomics, and transcriptomics using both microarray and next-generation sequencing transcript sequencing (RNA-Seq). Proteomic analysis shows that rebalancing of protein content largely results from the selective increase in the accumulation of only a few proteins. The rebalancing of protein composition occurs with small alterations to the seed’s transcriptome and metabolome. The selectivity of the rebalancing was further tested by introgressing into the SP− line a green fluorescent protein (GFP) glycinin allele mimic and quantifying the resulting accumulation of GFP. The GFP accumulation was similar to the parental GFP-expressing line, showing that the GFP glycinin gene mimic does not participate in proteome rebalancing. The results show that soybeans make large adjustments to the proteome during seed filling and compensate for the shortage of major proteins with the increased selective accumulation of other proteins that maintains a normal protein content. PMID:21398260

  6. Evolutionary Rate Covariation in Meiotic Proteins Results from Fluctuating Evolutionary Pressure in Yeasts and Mammals

    PubMed Central

    Clark, Nathan L.; Alani, Eric; Aquadro, Charles F.

    2013-01-01

    Evolutionary rates of functionally related proteins tend to change in parallel over evolutionary time. Such evolutionary rate covariation (ERC) is a sequence-based signature of coevolution and a potentially useful signature to infer functional relationships between proteins. One major hypothesis to explain ERC is that fluctuations in evolutionary pressure acting on entire pathways cause parallel rate changes for functionally related proteins. To explore this hypothesis we analyzed ERC within DNA mismatch repair (MMR) and meiosis proteins over phylogenies of 18 yeast species and 22 mammalian species. We identified a strong signature of ERC between eight yeast proteins involved in meiotic crossing over, which seems to have resulted from relaxation of constraint specifically in Candida glabrata. These and other meiotic proteins in C. glabrata showed marked rate acceleration, likely due to its apparently clonal reproductive strategy and the resulting infrequent use of meiotic proteins. This correlation between change of reproductive mode and change in constraint supports an evolutionary pressure origin for ERC. Moreover, we present evidence for similar relaxations of constraint in additional pathogenic yeast species. Mammalian MMR and meiosis proteins also showed statistically significant ERC; however, there was not strong ERC between crossover proteins, as observed in yeasts. Rather, mammals exhibited ERC in different pathways, such as piRNA-mediated defense against transposable elements. Overall, if fluctuation in evolutionary pressure is responsible for ERC, it could reveal functional relationships within entire protein pathways, regardless of whether they physically interact or not, so long as there was variation in constraint on that pathway. PMID:23183665

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

    NASA Astrophysics Data System (ADS)

    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 (p H 7 ) 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.

  8. A single amino acid substitution results in a retinoblastoma protein defective in phosphorylation and oncoprotein binding

    SciTech Connect

    Kaye, F.J.; Gerster, J.L. Uniformed Services Univ. of Health Sciences, Bethesda, MD ); Kratzke, R.A. ); Horowitz, J.M. )

    1990-09-01

    The authors have previously identified a small-cell lung cancer cell line (NCI-H209) that expresses an aberrant, underphosphorylated form of the retinoblastoma protein RB1. Molecular analysis of RB1 mRNA from this cell line revealed a single point mutation within exon 21 that resulted in a nonconservative amino acid substitution (cysteine to phenylalanine) at codon 706. Stable expression of this mutant RB1 cDNA in a human cell line lacking endogenous RB1 demonstrated that this amino acid change was sufficient to inhibit phosphorylation. In addition, this cysteine-to-phenylalanine substitution also resulted in loss of RB1 binding to the simian virus 40 large tumor and adenovirus E1A transforming proteins. These results confirm the importance of exon 21 coding sequences and suggest that the cysteine residue at codon 706 may play a role in achieving a specific protein conformation essential for protein-protein interactions.

  9. 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.

  10. Switching kinetics of the ferroelectric transition in K2SeO4 studied by stroboscopic ?-ray diffraction

    NASA Astrophysics Data System (ADS)

    Leist, J.; Gibhardt, H.; Eckold, G.

    2013-11-01

    The kinetics of the ferroelectric lock-in transition in potassium selenate (K2SeO4) was studied on a millisecond timescale using high-resolution ?-ray diffraction. A large change of the line width and wavevector of the first order satellite is observed during the switching process. This is attributed to a loss of long-range order under the influence of the electric field. In addition, the incommensurate phase is stabilized by the pulsed field and the transition to the pure commensurate phase is shifted to lower temperatures. Strains that may build up during the rapid switching process are supposed to be the reason for this behaviour.

  11. Use of Composite Protein Database including Search Result Sequences for Mass Spectrometric Analysis of Cell Secretome

    PubMed Central

    Shin, Jihye; Kim, Gamin; Kabir, Mohammad Humayun; Park, Seong Jun; Lee, Seoung Taek; Lee, Cheolju

    2015-01-01

    Mass spectrometric (MS) data of human cell secretomes are usually run through the conventional human database for identification. However, the search may result in false identifications due to contamination of the secretome with fetal bovine serum (FBS) proteins. To overcome this challenge, here we provide a composite protein database including human as well as 199 FBS protein sequences for MS data search of human cell secretomes. Searching against the human-FBS database returned more reliable results with fewer false-positive and false-negative identifications compared to using either a human only database or a human-bovine database. Furthermore, the improved results validated our strategy without complex experiments like SILAC. We expect our strategy to improve the accuracy of human secreted protein identification and to also add value for general use. PMID:25822838

  12. Modeling how reproductive ecology can drive protein diversification and result in linkage disequilibrium between sperm and egg proteins.

    PubMed

    Tomaiuolo, Maurizio; Levitan, Don R

    2010-07-01

    Gamete-recognition proteins determine whether sperm and eggs are compatible at fertilization, and they often evolve rapidly. The source of selection driving the evolution of these proteins is still debated. It has been suggested that sexual conflict can result in proliferation of genetic variation and possibly linkage disequilibrium between sperm and egg proteins. Empirical evidence suggests that both male and female reproductive success can be predicted by their sperm ligand genotype, but why female success can be predicted by a protein expressed only in males is unknown. Here we use mathematical modeling to investigate the interaction between reproductive behavior and sperm availability on the evolution of sperm ligands and egg receptors. We consider haploid and diploid expression in gametes in two possible ecological scenarios, monogamous spawning and competitive spawning. Reproductive behavior plays an important role in determining possible outcomes resulting from sexual conflict. Sperm limitation selects for common genotypes regardless of mating behavior. Under conditions of sperm abundance, competitive spawning provides conditions for the persistence of allelic variation and gametic disequilibrium. With monogamous spawning, such conditions are more restrictive. PMID:20455709

  13. Bioactive protein-based nanofibers interact with intestinal biological components resulting in transepithelial permeation of a therapeutic protein.

    PubMed

    Stephansen, Karen; Garca-Daz, Mara; Jessen, Flemming; Chronakis, Ioannis S; Nielsen, Hanne Mrck

    2015-11-10

    Proteins originating from natural sources may constitute a novel type of material for use in drug delivery. However, thorough understanding of the behavior and effects of such a material when processed into a matrix together with a drug is crucial prior to further development into a drug product. In the present study the potential of using bioactive electrospun fish sarcoplasmic proteins (FSP) as a carrier matrix for small therapeutic proteins was demonstrated in relation to the interactions with biological components of the intestinal tract. The inherent structural and chemical properties of FSP as a biomaterial facilitated interactions with cells and enzymes found in the gastrointestinal tract and displayed excellent biocompatibility. More specifically, insulin was efficiently encapsulated into FSP fibers maintaining its conformation, and subsequent controlled release was obtained in simulated intestinal fluid. The encapsulation of insulin into FSP fibers provided protection against chymotrypsin degradation, and resulted in an increase in insulin transport to around 12% without compromising the cellular viability. This increased transport was driven by interactions upon contact between the nanofibers and the Caco-2 cell monolayer leading to the opening of the tight junction proteins. Overall, electrospun FSP may constitute a novel material for oral delivery of biopharmaceuticals. PMID:26320547

  14. 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

    ... 31 Money and Finance: Treasury 1 2010-07-01 2010-07-01 false Q-3: How are the SEOs and most highly compensated employees identified for purposes of compliance with this part? 30.3 Section 30.3 Money and Finance: Treasury Office of the Secretary of the Treasury TARP STANDARDS FOR COMPENSATION AND...

  15. 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. PMID:25528508

  16. Sr3Bi2(SeO3)6H2O: A novel anionic layer consisting of second-order Jahn-Teller (SOJT) distortive cations

    NASA Astrophysics Data System (ADS)

    Ahn, Hyun Sun; Lee, Eun Pyo; Chang, Hong-Young; Lee, Dong Woo; Ok, Kang Min

    2015-01-01

    A new layered bismuth selenite hydrate material, Sr3Bi2(SeO3)6H2O has been synthesized through a hydrothermal reaction using SrCO3, Bi2O3, SeO2, and water as reagents. The crystal structure of the reported material has been determined by single crystal X-ray diffraction analysis. The anionic layered framework of Sr3Bi2(SeO3)6H2O consists of polyhedra of second-order Jahn-Teller (SOJT) distortive cations, Bi3+ and Se4+. Attributable to the variable and asymmetric coordination geometry of the constituent cations, a rich structural chemistry including uni-dimensional bands and linkers is observed in the layer. The material is thermally stable up to about 390 C and starts decomposing due to the sublimation of SeO2 above the temperature. The UV-vis diffuse reflectance spectrum suggests a band gap of 3.3 eV. Elemental analysis, infrared spectrum, local dipole moment calculations, and electronic structure calculations are also reported.

  17. 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.

  18. Ordering of the O(2)D O(2) bonds near the phase transition in KD3(SeO3)2 single crystals by D nuclear magnetic resonance

    NASA Astrophysics Data System (ADS)

    Lim, Ae Ran; Jeong, Se-Young

    2013-01-01

    Deuterium resonance investigations of KD3(SeO3)2 single crystals have been performed near the phase transition temperature T C . There are two types of deuterium bonds in these crystals with different behaviors at this phase transition. Our experimental results show that there are significant changes in the D spinlattice relaxation time T 1 at T C ; the abrupt decrease in T 1 near T C can be explained by the critical slowing down of an overdamped soft pseudospin-type deuteron mode. Further, the ordering of the O(2)D O(2) bonds is affected by the phase transition, whereas the ordering of the O(1)-D O(3) bonds is unaffected. The D NMR measurements also show that the D(2) deuteron disordering above T C is dynamic and not static.

  19. Protein Radical Formation Resulting from Eosinophil Peroxidase-catalyzed Oxidation of Sulfite.

    PubMed

    Ranguelova, Kalina; Chatterjee, Saurabh; Ehrenshaft, Marilyn; Ramirez, Dario C; Summers, Fiona A; Kadiiska, Maria B; Mason, Ronald P

    2010-07-30

    Eosinophil peroxidase (EPO) is an abundant heme protein in eosinophils that catalyzes the formation of cytotoxic oxidants implicated in asthma, allergic inflammatory disorders, and cancer. It is known that some proteins with peroxidase activity (horseradish peroxidase and prostaglandin hydroperoxidase) can catalyze oxidation of bisulfite (hydrated sulfur dioxide), leading to the formation of sulfur trioxide anion radical ((.)SO(3)(-)). This free radical further reacts with oxygen to form peroxymonosulfate anion radical ((-)O(3)SOO(.)) and the very reactive sulfate anion radical (SO(4)()), which is nearly as strong an oxidant as the hydroxyl radical. However, the ability of EPO to generate reactive sulfur radicals has not yet been reported. Here we demonstrate that eosinophil peroxidase/H(2)O(2) is able to oxidize bisulfite, ultimately forming the sulfate anion radical (SO(4)()), and that these reactive intermediates can oxidize target proteins to protein radicals, thereby initiating protein oxidation. We used immuno-spin trapping and confocal microscopy to study protein oxidation by EPO/H(2)O(2) in the presence of bisulfite in a pure enzymatic system and in human promyelocytic leukemia HL-60 clone 15 cells, maturated to eosinophils. Polyclonal antiserum raised against the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) detected the presence of DMPO covalently attached to the proteins resulting from the DMPO trapping of protein free radicals. We found that sulfite oxidation mediated by EPO/H(2)O(2) induced the formation of radical-derived DMPO spin-trapped human serum albumin and, to a lesser extent, of DMPO-EPO. These studies suggest that EPO-dependent oxidative damage may play a role in tissue injury in bisulfite-exacerbated eosinophilic inflammatory disorders. PMID:20501663

  20. Oxidative stress status accompanying diabetic bladder cystopathy results in the activation of protein degradation pathways

    PubMed Central

    Kanika, Nirmala; Chang, Jinsook; Tong, Yuehong; Tiplitsky, Scott; Lin, Juan; Yohannes, Elizabeth; Tar, Moses; Chance, Mark; Christ, George J.; Melman, Arnold; Davies, Kelvin

    2010-01-01

    Objectives To investigate the role that oxidative stress plays in the development of diabetic cystopathy. Materials and methods Comparative gene expression in the bladder of non-diabetic and streptozotocin (STZ)-induced 2-month-old diabetic rats was carried out using microarray analysis. Evidence of oxidative stress was investigated in the bladder by analyzing glutathione S-transferase activity, lipid peroxidation, and carbonylation and nitrosylation of proteins. The activity of protein degradation pathways was assessed using western blot analysis. Results Analysis of global gene expression showed that detrusor smooth muscle tissue of STZ-induced diabetes undergoes significant enrichment in targets involved in the production or regulation of reactive oxygen species (P = 1.27 10?10). The microarray analysis was confirmed by showing that markers of oxidative stress were all significantly increased in the diabetic bladder. It was hypothesized that the sequelae to oxidative stress would be increased protein damage and apoptosis. This was confirmed by showing that two key proteins involved in protein degradation (Nedd4 and LC3B) were greatly up-regulated in diabetic bladders compared to controls by 12.2 0.76 and 4.4 1.0-fold, respectively, and the apoptosis inducing protein, BAX, was up-regulated by 6.76 0.76-fold. Conclusions Overall, the findings obtained in the present study add to the growing body of evidence showing that diabetic cystopathy is associated with oxidative damage of smooth muscle cells, and results in protein damage and activation of apoptotic pathways that may contribute to a deterioration in bladder function. PMID:21518418

  1. Ablation of retinal ciliopathy protein RPGR results in altered photoreceptor ciliary composition

    PubMed Central

    Rao, Kollu N.; Li, Linjing; Anand, Manisha; Khanna, Hemant

    2015-01-01

    Cilia regulate several developmental and homeostatic pathways that are critical to survival. Sensory cilia of photoreceptors regulate phototransduction cascade for visual processing. Mutations in the ciliary protein RPGR (retinitis pigmentosa GTPase regulator) are a prominent cause of severe blindness disorders due to degeneration of mature photoreceptors. However, precise function of RPGR is still unclear. Here we studied the involvement of RPGR in ciliary trafficking by analyzing the composition of photoreceptor sensory cilia (PSC) in Rpgrko retina. Using tandem mass spectrometry analysis followed by immunoblotting, we detected few alterations in levels of proteins involved in proteasomal function and vesicular trafficking in Rpgrko PSC, prior to onset of degeneration. We also found alterations in the levels of high molecular weight soluble proteins in Rpgrko PSC. Our data indicate RPGR regulates entry or retention of soluble proteins in photoreceptor cilia but spares the trafficking of key structural and phototransduction-associated proteins. Given a frequent occurrence of RPGR mutations in severe photoreceptor degeneration due to ciliary disorders, our results provide insights into pathways resulting in altered mature cilia function in ciliopathies. PMID:26068394

  2. 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

  3. Loss of Photoreceptors Results in Upregulation of Synaptic Proteins in Bipolar Cells and Amacrine Cells

    PubMed Central

    Dagar, Sushma; Nagar, Saumya; Goel, Manvi; Cherukuri, Pitchaiah; Dhingra, Narender K.

    2014-01-01

    Deafferentation is known to cause significant changes in the postsynaptic neurons in the central nervous system. Loss of photoreceptors, for instance, results in remarkable morphological and physiological changes in bipolar cells and horizontal cells. Retinal ganglion cells (RGCs), which send visual information to the brain, are relatively preserved, but show aberrant firing patterns, including spontaneous bursts of spikes in the absence of photoreceptors. To understand how loss of photoreceptors affects the circuitry presynaptic to the ganglion cells, we measured specific synaptic proteins in two mouse models of retinal degeneration. We found that despite the nearly total loss of photoreceptors, the synaptophysin protein and mRNA levels in retina were largely unaltered. Interestingly, the levels of synaptophysin in the inner plexiform layer (IPL) were higher, implying that photoreceptor loss results in increased synaptophysin in bipolar and/or amacrine cells. The levels of SV2B, a synaptic protein expressed by photoreceptors and bipolar cells, were reduced in whole retina, but increased in the IPL of rd1 mouse. Similarly, the levels of syntaxin-I and synapsin-I, synaptic proteins expressed selectively by amacrine cells, were higher after loss of photoreceptors. The upregulation of syntaxin-I was evident as early as one day after the onset of photoreceptor loss, suggesting that it did not require any massive or structural remodeling, and therefore is possibly reversible. Together, these data show that loss of photoreceptors results in increased synaptic protein levels in bipolar and amacrine cells. Combined with previous reports of increased excitatory and inhibitory synaptic currents in RGCs, these results provide clues to understand the mechanism underlying the aberrant spiking in RGCs. PMID:24595229

  4. Caspase-independent Mitochondrial Cell Death Results from Loss of Respiration, Not Cytotoxic Protein Release

    PubMed Central

    Lartigue, Lydia; Kushnareva, Yulia; Seong, Youngmo; Lin, Helen; Faustin, Benjamin

    2009-01-01

    In apoptosis, mitochondrial outer membrane permeabilization (MOMP) triggers caspase-dependent death. However, cells undergo clonogenic death even if caspases are blocked. One proposed mechanism involved the release of cytotoxic proteins (e.g., AIF and endoG) from mitochondria. To initiate MOMP directly without side effects, we created a tamoxifen-switchable BimS fusion protein. Surprisingly, even after MOMP, caspase-inhibited cells replicated DNA and divided for ?48 h before undergoing proliferation arrest. AIF and endoG remained in mitochondria. However, cells gradually lost mitochondrial membrane potential and ATP content, and DNA synthesis slowed to a halt by 72 h. These defects resulted from a partial loss of respiratory function, occurring 48 h after MOMP, that was not merely due to dispersion of cytochrome c. In particular, Complex I activity was completely lost, and Complex IV activity was reduced by ?70%, whereas Complex II was unaffected. Later, cells exhibited a more profound loss of mitochondrial protein constituents. Thus, under caspase inhibition, MOMP-induced clonogenic death results from a progressive loss of mitochondrial function, rather than the release of cytotoxic proteins from mitochondria. PMID:19793916

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

    PubMed Central

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

    2014-01-01

    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. PMID:24951553

  6. Enzymatic hydrolysis of brewers' spent grain proteins and technofunctional properties of the resulting hydrolysates.

    PubMed

    Celus, Inge; Brijs, Kristof; Delcour, Jan A

    2007-10-17

    Brewers' spent grain (BSG) is the insoluble residue of barley malt resulting from the manufacture of wort. Although it is the main byproduct of the brewing industry, it has received little attention as a marketable commodity and is mainly used as animal feed. Our work focuses on one of the main constituents of BSG, i.e., the proteins. The lack of solubility of BSG proteins is one of the limitations for their more extensive use in food processing. We therefore aimed to generate BSG protein hydrolysates with improved technofunctional properties. BSG protein concentrate (BPC) was prepared by alkaline extraction of BSG and subsequent acid precipitation. BPC was enzymatically hydrolyzed in a pH-stat setup by several commercially available proteases (Alcalase, Flavourzyme, and Pepsin) for different times and/or with different enzyme concentrations in order to obtain hydrolysates with different degrees of hydrolysis (DH). Physicochemical properties, such as molecular weight (MW) distribution and hydrophobicity, as well as technofunctional properties, such as solubility, color, and emulsifying and foaming properties, were determined. Enzymatic hydrolysis of BPC improved emulsion and/or foam-forming properties. However, for the hydrolysates prepared with Alcalase and Pepsin, an increasing DH generally decreased emulsifying and foam-forming capacities. Moreover, the type of enzyme impacted the resulting technofunctional properties. Hydrolysates prepared with Flavourzyme showed good technofunctional properties, independent of the DH. Physicochemical characterization of the hydrolysates indicated the importance of protein fragments with relatively high MW (exceeding 14.5 k) and high surface hydrophobicity for favorable technofunctional properties. PMID:17896813

  7. Syntheses, Crystal Structures, and Properties of New Layered Tungsten(VI)-Containing Materials Based on the Hexagonal-WO 3 Structure: M2(WO 3) 3SeO 3 ( M = NH 4, Rb, Cs)

    NASA Astrophysics Data System (ADS)

    Harrison, William T. A.; Dussack, Laurie L.; Vogt, Thomas; Jacobson, Allan J.

    1995-11-01

    The hydrothermal syntheses and crystal structures of (NH4)2(WO3)3SeO3 and Cs2(WO3)3SeO3, two new noncentrosymmetric, layered tungsten(VI)-containing phases are reported. Infrared, Raman, and thermogravimetric data are also presented. (NH4)2(WO3)3SeO3 and Cs2(WO3)3SeO3 are isostructural phases built up from hexagonal-tungsten-oxide-like, anionic layers of vertex-sharing WO6 octahedra, capped on one side by Se atoms (as selenite groups). Interlayer NH+4 or Cs+ cations provide charge balance. The full H-bonding scheme in (NH4)2(WO3)3SeO3 has been elucidated from Rietveld refinement against neutron powder diffraction data. The WO6 octahedra display a 3 short + 3 long W-O bond-distance distribution within the WO6 unit in both these phases. (NH4)2(WO3)3SeO3 and Cs2(WO3)3SeO3 are isostructural with their molybdenum(VI)-containing analogues (NH4)2(MoO3)3SeO3 and Cs2 (MoO3)3SeO3. Crystal data: (NH4)2(WO3)3SeO3, Mr = 858.58, hexagonal, space group P63 (No. 173), a = 7.2291(2) , c = 12.1486(3) , V = 549.82(3) 3, Z = 2, Rp = 1.81%, and Rwp = 2.29% (2938 neutron powder data). Cs2(WO3)3SeO3, Mr = 1088.31, hexagonal, space group P63 (no. 173), a = 7.2615(2) , c = 12.5426(3) , V = 572.75(3) 3, Z = 2, Rp = 4.84%, and Rwp = 5.98% (2588 neutron powder data).

  8. 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.

  9. 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.

  10. Local structure of Rb2Li4(SeO4)32H2O by the modeling of X-ray diffuse scattering from average-structure to microdomain model

    NASA Astrophysics Data System (ADS)

    Komornicka, Dorota; Wo?cyrz, Marek; Pietraszko, Adam

    2012-08-01

    Local structure of dirubidium tetralithium tris(selenate(VI)) dihydrate Rb2Li4(SeO4)3 2H2O has been determined basing on the modeling of X-ray diffuse scattering. The origin of observed structured diffuse streaks is SeO4 tetrahedra switching between two alternative positions in two quasi-planar layers existing in each unit cell and formation of domains with specific SeO4 tetrahedra configuration locally fulfilling condition for C-centering in the 2a2bc superstructure cell. The local structure solution is characterized by a uniform distribution of rather large domains (ca. thousand of unit cells) in two layers, but also monodomains can be taken into account. Inside a single domain SeO4 tetrahedra are ordered along ab-diagonal forming two-string ribbons. Inside the ribbons SeO4 and LiO4 tetrahedra share the oxygen corners, whereas ribbons are bound to each other by a net of hydrogen bonds and fastened by corner sharing SeO4 tetrahedra of the neighboring layers.

  11. Vibrational spectroscopic study of the uranyl selenite mineral derriksite Cu4UO2(SeO3)2(OH)6?H2O

    NASA Astrophysics Data System (ADS)

    Frost, Ray L.; ?ejka, Ji?; Scholz, Ricardo; Lpez, Andrs; Theiss, Frederick L.; Xi, Yunfei

    2014-01-01

    Raman spectrum of the mineral derriksite Cu4UO2(SeO3)2(OH)6?H2O was studied and complemented by the infrared spectrum of this mineral. Both spectra were interpreted and partly compared with the spectra of demesmaekerite, marthozite, larisaite, haynesite and piretite. Observed Raman and infrared bands were attributed to the (UO2)2+, (SeO3)2-, (OH)- and H2O vibrations. The presence of symmetrically distinct hydrogen bonded molecule of water of crystallization and hydrogen bonded symmetrically distinct hydroxyl ions was inferred from the spectra in the derriksite unit cell. Approximate U-O bond lengths in uranyl and O-H⋯O hydrogen bond lengths were calculated from the Raman and infrared spectra of derriksite.

  12. Structural and conductivity studies of CsK(SO4)0.32(SeO4)0.68Te(OH)6

    NASA Astrophysics Data System (ADS)

    Djemel, M.; Abdelhedi, M.; Zouari, N.; Dammak, M.; Kolsi, A. W.

    2012-12-01

    The compound CsK(SO4)0.32(SeO4)0.68Te(OH)6 crystallizes in the monoclinic P21/n space group. It was analyzed, at room temperature, using X-ray diffractometer data. The main feature of these atomic arrangements is the coexistence of three and different anions (SO42-, SeO42- and TeO66-groups) in the unit cell, connected by hydrogen bonds which make the building of the crystal. The thermal analysis of the title compound shows three distinct endothermal peaks at 435, 460 and 475 K. Complex impedance measurements are performed on this material as a function of both temperature and frequency. The electric conduction has been studied. The temperature dependence on the conductivity indicates that the sample became an ionic conductor at high temperature.

  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. Electrophoretic analysis of sheep plasma protein labeled with Na2 75SeO3 in vivo

    SciTech Connect

    Davidson, W.B.; McMurray, C.H.

    1987-05-01

    Following an intravenous injection of /sup 75/Se, sodium selenite plasma samples were analyzed by two-dimensional electrophoresis. /sup 75/Se was detected by indirect autoradiography. From 0.5 to 53 hr postinjection of /sup 75/Se, 21 /sup 75/Se peptides were detected. Both the isoelectric points and molecular weights of these peptides are reported. The molecular weights of the peptides ranged from 20,000 to 70,000 daltons.

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

    PubMed Central

    ?aikovski, 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

  17. 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.

  18. 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-?, IL-1?, 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

  19. Desulfurization of Cysteine-Containing Peptides Resulting from Sample Preparation for Protein Characterization by MS

    PubMed Central

    Wang, Zhouxi; Rejtar, Tomas; Zhou, Zhaohui Sunny; Karger, Barry L.

    2010-01-01

    In this paper, we have examined two cysteine modifications resulting from sample preparation for protein characterization by MS: (1) a previously observed conversion of cysteine to dehydroalanine, now found in the case of disulfide mapping and (2) a novel modification corresponding to conversion of cysteine to alanine. Using model peptides, the conversion of cysteine to dehydroalanine via ?-elimination of a disulfide bond was seen to result from the conditions of typical tryptic digestion (37 C, pH 7.0 9.0) without disulfide reduction and alkylation.. Furthermore, the surprising conversion of cysteine to alanine was shown to occur by heating cysteine containing peptides in the presence of a phosphine (TCEP). The formation of alanine from cysteine, investigated by performing experiments in H2O or D2O, suggested a radical-based desulfurization mechanism unrelated to ?-elimination. Importantly, an understanding of the mechanism and conditions favorable for cysteine desulfurization provides insight for the establishment of improved sample preparation procedures of protein analysis. PMID:20049891

  20. 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, sauting, 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 sauted 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

  1. Compromised Mitochondrial Fatty Acid Synthesis in Transgenic Mice Results in Defective Protein Lipoylation and Energy Disequilibrium

    PubMed Central

    Smith, Stuart; Witkowski, Andrzej; Moghul, Ayesha; Yoshinaga, Yuko; Nefedov, Michael; de Jong, Pieter; Feng, Dejiang; Fong, Loren; Tu, Yiping; Hu, Yan; Young, Stephen G.; Pham, Thomas; Cheung, Carling; Katzman, Shana M.; Brand, Martin D.; Quinlan, Casey L.; Fens, Marcel; Kuypers, Frans; Misquitta, Stephanie; Griffey, Stephen M.; Tran, Son; Gharib, Afshin; Knudsen, Jens; Hannibal-Bach, Hans Kristian; Wang, Grace; Larkin, Sandra; Thweatt, Jennifer; Pasta, Saloni

    2012-01-01

    A mouse model with compromised mitochondrial fatty acid synthesis has been engineered in order to assess the role of this pathway in mitochondrial function and overall health. Reduction in the expression of mitochondrial malonyl CoA-acyl carrier protein transacylase, a key enzyme in the pathway encoded by the nuclear Mcat gene, was achieved to varying extents in all examined tissues employing tamoxifen-inducible Cre-lox technology. Although affected mice consumed more food than control animals, they failed to gain weight, were less physically active, suffered from loss of white adipose tissue, reduced muscle strength, kyphosis, alopecia, hypothermia and shortened lifespan. The Mcat-deficient phenotype is attributed primarily to reduced synthesis, in several tissues, of the octanoyl precursors required for the posttranslational lipoylation of pyruvate and α-ketoglutarate dehydrogenase complexes, resulting in diminished capacity of the citric acid cycle and disruption of energy metabolism. The presence of an alternative lipoylation pathway that utilizes exogenous free lipoate appears restricted to liver and alone is insufficient for preservation of normal energy metabolism. Thus, de novo synthesis of precursors for the protein lipoylation pathway plays a vital role in maintenance of mitochondrial function and overall vigor. PMID:23077570

  2. Deficiency of a protein-repair enzyme results in the accumulation of altered proteins, retardation of growth, and fatal seizures in?mice

    PubMed Central

    Kim, Edward; Lowenson, Jonathan D.; MacLaren, Duncan C.; Clarke, Steven; Young, Stephen G.

    1997-01-01

    l-Asparaginyl and l-aspartyl residues in proteins are subject to spontaneous degradation reactions that generate isomerized and racemized aspartyl derivatives. Proteins containing l-isoaspartyl and d-aspartyl residues can have altered structures and diminished biological activity. These residues are recognized by a highly conserved cytosolic enzyme, the protein l-isoaspartate(d-aspartate) O-methyltransferase (EC 2.1.1.77). The enzymatic methyl esterification of these abnormal residues in vitro can lead to their conversion (i.e., repair) to normal l-aspartyl residues and should therefore prevent the accumulation of potentially dysfunctional proteins in vivo as cells and tissues age. Particularly high levels of the repair methyltransferase are present in the brain, although enyzme activity is present in all vertebrate tissues. To define the physiological relevance of this protein-repair pathway and to determine whether deficient protein repair would cause central nervous system dysfunction, we used gene targeting in mouse embryonic stem cells to generate protein l-isoaspartate(d-aspartate) O-methyltransferase-deficient mice. Analyses of tissues from methyltransferase knockout mice revealed a striking accumulation of protein substrates for this enzyme in the cytosolic fraction of brain, heart, liver, and erythrocytes. The knockout mice showed significant growth retardation and succumbed to fatal seizures at an average of 42 days after birth. These results suggest that the ability of mice to repair l-isoaspartyl- and d-aspartyl-containing proteins is essential for normal growth and for normal central nervous system function. PMID:9177182

  3. 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.

  4. Altered protein prenylation in Sertoli cells is associated with adult infertility resulting from childhood mumps infection

    PubMed Central

    Wang, Xiu-Xing; Ying, Pu; Diao, Fan; Wang, Qiang; Ye, Dan; Jiang, Chen; Shen, Ning; Xu, Na; Chen, Wei-Bo; Lai, Shan-Shan; Jiang, Shan; Miao, Xiao-Li; Feng, Jin; Tao, Wei-Wei; Zhao, Ning-Wei; Yao, Bing; Xu, Zhi-Peng; Sun, Hai-Xiang; Sha, Jia-Hao; Huang, Xing-Xu; Shi, Qing-Hua; Tang, Hong

    2013-01-01

    Mumps commonly affects children 59 yr of age, and can lead to permanent adult sterility in certain cases. However, the etiology of this long-term effect remains unclear. Mumps infection results in progressive degeneration of the seminiferous epithelium and, occasionally, Sertoli cellonly syndrome. Thus, the remaining Sertoli cells may be critical to spermatogenesis recovery after orchitis healing. Here, we report that the protein farnesylation/geranylgeranylation balance is critical for patients fertility. The expression of geranylgeranyl diphosphate synthase 1 (GGPPS) was decreased due to elevated promoter methylation in the testes of infertile patients with mumps infection history. When we deleted GGPPS in mouse Sertoli cells, these cells remained intact, whereas the adjacent spermatogonia significantly decreased after the fifth postnatal day. The proinflammatory MAPK and NF-?B signaling pathways were constitutively activated in GGPPS?/? Sertoli cells due to the enhanced farnesylation of H-Ras. GGPPS?/? Sertoli cells secreted an array of cytokines to stimulate spermatogonia apoptosis, and chemokines to induce macrophage invasion into the seminiferous tubules. Invaded macrophages further blocked spermatogonia development, resulting in a long-term effect through to adulthood. Notably, this defect could be rescued by GGPP administration in EMCV-challenged mice. Our results suggest a novel mechanism by which mumps infection during childhood results in adult sterility. PMID:23825187

  5. Hydrothermal synthesis, structures and optical properties of A2Zn3(SeO3)4·XH2O (A=Li, Na, K; X=2 or 0)

    NASA Astrophysics Data System (ADS)

    Liu, Yunsheng; Mei, Dajiang; Xu, Jingli; Wu, Yuandong

    2015-12-01

    New alkali metal zinc selenites, A2Zn3(SeO3)4·XH2O (A=Li, Na, K; X=2 or 0) were prepared through hydrothermal reactions. Li2Zn3(SeO3)4·2H2O (1) crystallizes in the monoclinic space group P21/c with lattice parameters a=8.123(4), b=9.139(4), c=7.938(3) Å, β=112.838(9)°. Na2Zn3(SeO3)4·2H2O (2) crystallizes in the monoclinic space group C2/c with lattice parameters a=15.7940(18), b=6.5744(8), c=14.6787(17) Å, β=107.396(3)°. K2Zn3(SeO3)4 (3) crystallizes in the monoclinic space group C2/c with lattice parameters a=11.3584(12), b=8.6091(9), c=13.6816(14) Å, β=93.456(2)°. The anionic structures are composed of [Zn3O12]18- sheets, chains, and "isolated" units in compound 1, 2, 3, respectively, and trigonal pyramids SeO32-. The compounds were characterized by the solid state UV-vis-NIR diffuse reflectance spectroscopy, infrared spectra and thermogravimetric analysis.

  6. Minced beef is more rapidly digested and absorbed than beef steak, resulting in greater postprandial protein retention in older men.

    TOXLINE Toxicology Bibliographic Information

    Pennings B; Groen BB; van Dijk JW; de Lange A; Kiskini A; Kuklinski M; Senden JM; van Loon LJ

    2013-07-01

    BACKGROUND: Older individuals generally experience a reduced food-chewing efficiency. As a consequence, food texture may represent an important factor that modulates dietary protein digestion and absorption kinetics and the subsequent postprandial protein balance.OBJECTIVE: We assessed the effect of meat texture on the dietary protein digestion rate, amino acid availability, and subsequent postprandial protein balance in vivo in older men.DESIGN: Ten older men (mean SEM age: 74 2 y) were randomly assigned to a crossover experiment that involved 2 treatments in which they consumed 135 g of specifically produced intrinsically L-[1-(13)C]phenylalanine-labeled beef, which was provided as beef steak or minced beef. Meat consumption was combined with continuous intravenous L-[ring-(2)H5]phenylalanine and L-[ring-(2)H2]tyrosine infusion to assess beef protein digestion and absorption kinetics as well as whole-body protein balance and skeletal muscle protein synthesis rates.RESULTS: Meat protein-derived phenylalanine appeared more rapidly in the circulation after minced beef than after beef steak consumption (P < 0.05). Also, its availability in the circulation during the 6-h postprandial period was greater after minced beef than after beef steak consumption (61 3% compared with 49 3%, respectively; P < 0.01). The whole-body protein balance was more positive after minced beef than after beef steak consumption (29 2 compared with 19 3 ?mol phenylalanine/kg, respectively; P < 0.01). Skeletal muscle protein synthesis rates did not differ between treatments when assessed over a 6-h postprandial period.CONCLUSIONS: Minced beef is more rapidly digested and absorbed than beef steak, which results in increased amino acid availability and greater postprandial protein retention. However, this does not result in greater postprandial muscle protein synthesis rates. This trial was registered at clinicaltrials.gov as http://clinicaltrials.gov/ct2/show/NCT01145131 target=new>NCT01145131.

  7. P185-M Protein Identification and Validation of Results in Workflows that Integrate over Various Instruments, Datasets, Search Engines

    PubMed Central

    Hufnagel, P.; Glandorf, J.; Krting, G.; Jabs, W.; Schweiger-Hufnagel, U.; Hahner, S.; Lubeck, M.; Suckau, D.

    2007-01-01

    Analysis of complex proteomes often results in long protein lists, but falls short in measuring the validity of identification and quantification results on a greater number of proteins. Biological and technical replicates are mandatory, as is the combination of the MS data from various workflows (gels, 1D-LC, 2D-LC), instruments (TOF/TOF, trap, qTOF or FTMS), and search engines. We describe a database-driven study that combines two workflows, two mass spectrometers, and four search engines with protein identification following a decoy database strategy. The sample was a tryptically digested lysate (10,000 cells) of a human colorectal cancer cell line. Data from two LC-MALDI-TOF/TOF runs and a 2D-LC-ESI-trap run using capillary and nano-LC columns were submitted to the proteomics software platform ProteinScape. The combined MALDI data and the ESI data were searched using Mascot (Matrix Science), Phenyx (GeneBio), ProteinSolver (Bruker and Protagen), and Sequest (Thermo) against a decoy database generated from IPI-human in order to obtain one protein list across all workflows and search engines at a defined maximum false-positive rate of 5%. ProteinScape combined the data to one LC-MALDI and one LC-ESI dataset. The initial separate searches from the two combined datasets generated eight independent peptide lists. These were compiled into an integrated protein list using the ProteinExtractor algorithm. An initial evaluation of the generated data led to the identification of approximately 1200 proteins. Result integration on a peptide level allowed discrimination of protein isoforms that would not have been possible with a mere combination of protein lists.

  8. Deficiency of circadian clock protein BMAL1 in mice results in a low bone mass phenotype.

    PubMed

    Samsa, William E; Vasanji, Amit; Midura, Ronald J; Kondratov, Roman V

    2016-03-01

    The circadian clock is an endogenous time keeping system that controls the physiology and behavior of many organisms. The transcription factor Brain and Muscle ARNT-like Protein 1 (BMAL1) is a component of the circadian clock and necessary for clock function. Bmal1(-/-) mice display accelerated aging and many accompanying age associated pathologies. Here, we report that mice deficient for BMAL1 have a low bone mass phenotype that is absent at birth and progressively worsens over their lifespan. Accelerated aging of these mice is associated with the formation of bony bridges occurring across the metaphysis to the epiphysis, resulting in shorter long bones. Using micro-computed tomography we show that Bmal1(-/-) mice have reductions in cortical and trabecular bone volume and other micro-structural parameters and a lower bone mineral density. Histology shows a deficiency of BMAL1 results in a reduced number of active osteoblasts and osteocytes in vivo. Isolation of bone marrow derived mesenchymal stem cells from Bmal1(-/-) mice demonstrate a reduced ability to differentiate into osteoblasts in vitro, which likely explains the observed reductions in osteoblasts and osteocytes, and may contribute to the observed osteopenia. Our data support the role of the circadian clock in the regulation of bone homeostasis and shows that BMAL1 deficiency results in a low bone mass phenotype. PMID:26789548

  9. Tau protein as a serum marker of brain damage in mild traumatic brain injury: preliminary results.

    PubMed

    Bulut, M; Koksal, O; Dogan, S; Bolca, N; Ozguc, H; Korfali, E; Ilcol, Y O; Parklak, M

    2006-01-01

    The objective of this study was to investigate the diagnostic value of serum tau protein in determining the severity of traumatic brain injury in patients with mild traumatic brain injury (mTBI) and high-risk patients. Adult patients who presented to our emergency department (ED) with mTBI over 1 year were prospectively enrolled. Patients underwent cranial computed tomography (CT) and were subdivided into high- and low-risk groups, according to the probability of resultant intracranial injury. Serum tau levels of 60 patients and 20 healthy volunteers, who served as a control group, were measured. The mean age of the 60 patients (45 males, 15 females) was 32.5 years (range, 15-66 y). Mean Glasgow Coma Scale (GCS) score was 14+/-0.6. CT scans demonstrated intracranial injury in 11 patients (18.3%) and depressed fracture in 4 patients (6.7%). Serum tau levels of patients (188+/-210 pg/mL), compared with those of controls (86+/-48 pg/mL), were relatively higher; however, differences were not statistically significant (P=.445). Also, serum tau levels of high-risk patients (307+/-246 pg/mL) were significantly higher than those of low-risk patients (77+/-61 pg/mL) (P=.001). A total of 48 patients (80%) were accessible for follow-up after 6 months. Postconcussive syndrome was observed in 8 patients, 5 of whom had serum tau protein levels that were higher than those of the other 3 patients. However, no statistically significant difference was observed (P>.05). Investigators of the present study noted that serum tau levels in patients with mTBI were increased. Therefore, it is believed that this biomarker may prove helpful in identifying high-risk patients with mTBI. However, additional studies are needed to establish the diagnostic value of serum tau in detecting traumatic brain injury in patients with mTBI. PMID:16644603

  10. Synthesis, crystal structure and characterization of Na3H(SO4)1.78(SeO4)0.22

    NASA Astrophysics Data System (ADS)

    Ben Hassen, C.; Boujelbene, M.; Mhiri, T.

    2013-05-01

    Synthesis, crystal structure, Raman, IR and TG/DTA characterization are given for Trisodium hydrogen bisulfate selenite Na3H(SO4)1.78(SeO4)0.22. This compound crystallizes in the monoclinic system with space group P21/c and cell parameters: a = 8.6787 (4) , b = 9.6631 (6) , c = 9.2070 (5) , = 108.825 (4), Z = 4 and V = 730.83 (7) 3. The refinement of 2492 observed reflections (I > 2?(I)) leads to R1 = 0.045 and wR2 = 0.125. The structure is characterized by S/SeO4 tetrahedra which are linked into isolated pairs by hydrogen bonds which form dimers of composition [H(SO)2]. The existence of O-H and (S/Se)-O bonds in the structure at room temperature has been confirmed by IR and Raman spectroscopy in the frequency ranges 50-1300 and 500-4000 cm-1, respectively. Thermogravimetric analysis (TGA) and differential thermal analysis (DTA) measurements have been carried out on Na3H(SO4)1.78(SeO4)0.22 crystal in the temperature range between 50 and 600 C. Water evolution and major thermal decomposition take place with onset temperatures of approximately 282 C and 395 C, respectively. A Raman study of the decomposition of Na3H(SO4)1.78(SeO4)0.22 as a function of temperature supports a reaction sequence and possible intermediates during the process.

  11. Antibiotic-induced ribosomal assembly defects result from changes in the synthesis of ribosomal proteins.

    PubMed

    Siibak, Triinu; Peil, Lauri; Dnhfer, Alexandra; Tats, Age; Remm, Maido; Wilson, Daniel N; Tenson, Tanel; Remme, Jaanus

    2011-04-01

    Inhibitors of protein synthesis cause defects in the assembly of ribosomal subunits. In response to treatment with the antibiotics erythromycin or chloramphenicol, precursors of both large and small ribosomal subunits accumulate. We have used a pulse-labelling approach to demonstrate that the accumulating subribosomal particles maturate into functional 70S ribosomes. The protein content of the precursor particles is heterogeneous and does not correspond with known assembly intermediates. Mass spectrometry indicates that production of ribosomal proteins in the presence of the antibiotics correlates with the amounts of the individual ribosomal proteins within the precursor particles. Thus, treatment of cells with chloramphenicol or erythromycin leads to an unbalanced synthesis of ribosomal proteins, providing the explanation for formation of assembly-defective particles. The operons for ribosomal proteins show a characteristic pattern of antibiotic inhibition where synthesis of the first proteins is inhibited weakly but gradually increases for the subsequent proteins in the operon. This phenomenon most likely reflects translational coupling and allows us to identify other putative coupled non-ribosomal operons in the Escherichia coli chromosome. PMID:21320180

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

    PubMed Central

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

    2010-01-01

    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. PMID:20855595

  13. 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. PMID:20855595

  14. Loss of Clcc1 Results in ER Stress, Misfolded Protein Accumulation, and Neurodegeneration

    PubMed Central

    Jia, Yichang; Jucius, Thomas J.; Cook, Susan A.

    2015-01-01

    Folding of transmembrane and secretory proteins occurs in the lumen of the endoplasmic reticulum (ER) before transportation to the cell surface and is monitored by the unfolded protein response (UPR) signaling pathway. The accumulation of unfolded proteins in the ER activates the UPR that restores ER homeostasis by regulating gene expression that leads to an increase in the protein-folding capacity of the ER and a decrease in the ER protein-folding load. However, prolonged UPR activity has been associated with cell death in multiple pathological conditions, including neurodegeneration. Here, we report a spontaneous recessive mouse mutation that causes progressive cerebellar granule cell death and peripheral motor axon degeneration. By positional cloning, we identify the mutation in this strain as a retrotransposon insertion in the Clcc1 gene, which encodes a putative chloride channel localized to the ER. Furthermore, we demonstrate that the C3H/HeSnJ inbred strain has late onset cerebellar degeneration due to this mutation. Interestingly, acute knockdown of Clcc1 expression in cultured cells increases sensitivity to ER stress. In agreement, GRP78, the major HSP70 family chaperone in the ER, is upregulated in Clcc1-deficient granule cells in vivo, and ubiquitinated proteins accumulate in these neurons before their degeneration. These data suggest that disruption of chloride homeostasis in the ER disrupts the protein-folding capacity of the ER, leading to eventual neuron death. PMID:25698737

  15. Vascular endothelial dysfunction resulting from l-arginine deficiency in a patient with lysinuric protein intolerance

    PubMed Central

    Kamada, Yoshihiro; Nagaretani, Hiroyuki; Tamura, Shinji; Ohama, Tohru; Maruyama, Takao; Hiraoka, Hisatoyo; Yamashita, Shizuya; Yamada, Akira; Kiso, Shinichi; Inui, Yoshiaki; Ito, Nobuyuki; Kayanoki, Yoshiro; Kawata, Sumio; Matsuzawa, Yuji

    2001-01-01

    Although L-arginine is the only substrate for nitric oxide (NO) production, no studies have yet been reported on the effect of an L-arginine deficiency on vascular function in humans. Lysinuric protein intolerance (LPI) is a rare autosomal recessive defect of dibasic amino acid transport caused by mutations in the SLC7A7 gene, resulting in an L-arginine deficiency. Vascular endothelial function was examined in an LPI patient who was shown to be a compound heterozygote for two mutations in the gene (5.3-kbp Alu-mediated deletion, IVS3+1G??). The lumen diameter of the brachial artery was measured in this patient and in healthy controls at rest, during reactive hyperemia (endothelium-dependent vasodilation [EDV]), and after sublingual nitroglycerin administration (endothelium-independent vasodilation [EIV]) using ultrasonography. Both EDV and NOx concentrations were markedly reduced in the patient compared with those for the controls. They became normal after an L-arginine infusion. EIV was not significantly different between the patient and controls. Positron emission tomography of the heart and a treadmill test revealed ischemic changes in the patient, which were improved by the L-arginine infusion. Thus, in the LPI patient, L-arginine deficiency caused vascular endothelial dysfunction via a decrease in NO production. PMID:11544277

  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 (3.50.6 wavelength) with fluxes up to 818 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 ?-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. High-resolution mass spectrometry analysis of protein oxidations and resultant loss of function

    PubMed Central

    Barnes, Stephen; Shonsey, Erin M; Eliuk, Shannon M; Stella, David; Barrett, Kerri; Srivastava, Om P.; Kim, Helen; Renfrow, Matthew B.

    2009-01-01

    Mass spectrometry with or without pre-analysis peptide fractionation can be used to decipher the residues on proteins where oxidative modifications caused by peroxynitrite, singlet oxygen and electrophilic lipids have occurred. Peroxynitrite nitrates tyrosine and tryptophan residues on the surface of actin. Singlet oxygen, formed by the interaction of UVA light with tryptophan, can oxidize neighboring cysteine, histidine, methionine, tyrosine and tryptophan residues. Dose-response inactivation by 4-hydroxynonenal (4HNE) of human bile acid CoA: amino acid N-acyltransferase (hBAT) and the cytosolic brain isoform of creatine kinase (CKBB) is associated with site-specific modifications. Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) using nanoLC-electrospray ionization-mass spectrometry (ESI-MS) or direct infusion-ESI-MS with gas phase fractionation identified 14 4HNE adducts on hBAT and 17 on CKBB, respectively. At 4HNE concentrations in the physiological range, one member of the catalytic triad of hBAT (His362) was modified; for CKBB, although all four residues in the active site that were modifiable by 4HNE were ultimately modified, only one, Cys283, occurred at physiological concentrations of 4HNE. These results suggest that future in vivo studies should carefully assess the critical sites that are modified rather than using antibodies that do not distinguish between different modified sites. PMID:18793185

  18. 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

  19. Moderate energy restriction with high protein diet results in healthier outcome in women

    PubMed Central

    2010-01-01

    Background The present study compares two different weight reduction regimens both with a moderately high protein intake on body composition, serum hormone concentration and strength performance in non-competitive female athletes. Methods Fifteen normal weighted women involved in recreational resistance training and aerobic training were recruited for the study (age 28.5 6.3 yr, height 167.0 7.0 cm, body mass 66.3 4.2 kg, body mass index 23.8 1.8, mean SD). They were randomized into two groups. The 1 KG group (n = 8; energy deficit 1100 kcal/day) was supervised to reduce body weight by 1 kg per week and the 0.5 KG group (n = 7; energy deficit 550 kcal/day) by 0.5 kg per week, respectively. In both groups protein intake was kept at least 1.4 g/kg body weight/day and the weight reduction lasted four weeks. At the beginning of the study the energy need was calculated using food and training diaries. The same measurements were done before and after the 4-week weight reduction period including total body composition (DXA), serum hormone concentrations, jumping ability and strength measurements Results During the 4-week weight reduction period there were no changes in lean body mass and bone mass, but total body mass, fat mass and fat percentage decreased significantly in both groups. The changes were greater in the 1 KG group than in the 0.5 KG group in total body mass (p < 0.001), fat mass (p < 0.001) and fat percentage (p < 0.01). Serum testosterone concentration decreased significantly from 1.8 1.0 to 1.4 0.9 nmol/l (p < 0.01) in 1 KG and the change was greater in 1 KG (30%, p < 0.001) than in 0.5 KG (3%). On the other hand, SHBG increased significantly in 1 KG from 63.4 17.7 to 82.4 33.0 nmol/l (p < 0.05) during the weight reducing regimen. After the 4-week period there were no changes in strength performance in 0.5 KG group, however in 1 KG maximal strength in bench press decreased (p < 0.05) while endurance strength in squat and counter movement jump improved (p < 0.05) Conclusion It is concluded that a weight reduction by 0.5 kg per week with ~1.4 g protein/kg body weight/day can be recommended to normal weighted, physically active women instead of a larger (e.g. 1 kg per week) weight reduction because the latter may lead to a catabolic state. Vertical jumping performance is improved when fat mass and body weight decrease. Thus a moderate weight reduction prior to a major event could be considered beneficial for normal built athletes in jumping events. PMID:20205751

  20. 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)

  1. Pooled Results From 5 Validation Studies of Dietary Self-Report Instruments Using Recovery Biomarkers for Energy and Protein Intake

    PubMed Central

    Freedman, Laurence S.; Commins, John M.; Moler, James E.; Arab, Lenore; Baer, David J.; Kipnis, Victor; Midthune, Douglas; Moshfegh, Alanna J.; Neuhouser, Marian L.; Prentice, Ross L.; Schatzkin, Arthur; Spiegelman, Donna; Subar, Amy F.; Tinker, Lesley F.; Willett, Walter

    2014-01-01

    We pooled data from 5 large validation studies of dietary self-report instruments that used recovery biomarkers as references to clarify the measurement properties of food frequency questionnaires (FFQs) and 24-hour recalls. The studies were conducted in widely differing US adult populations from 1999 to 2009. We report on total energy, protein, and protein density intakes. Results were similar across sexes, but there was heterogeneity across studies. Using a FFQ, the average correlation coefficients for reported versus true intakes for energy, protein, and protein density were 0.21, 0.29, and 0.41, respectively. Using a single 24-hour recall, the coefficients were 0.26, 0.40, and 0.36, respectively, for the same nutrients and rose to 0.31, 0.49, and 0.46 when three 24-hour recalls were averaged. The average rate of under-reporting of energy intake was 28% with a FFQ and 15% with a single 24-hour recall, but the percentages were lower for protein. Personal characteristics related to under-reporting were body mass index, educational level, and age. Calibration equations for true intake that included personal characteristics provided improved prediction. This project establishes that FFQs have stronger correlations with truth for protein density than for absolute protein intake, that the use of multiple 24-hour recalls substantially increases the correlations when compared with a single 24-hour recall, and that body mass index strongly predicts under-reporting of energy and protein intakes. PMID:24918187

  2. 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

  3. 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. PMID:26507269

  4. Mutations in the Myelin Protein Zero result in a spectrum of Charcot-Marie-Tooth phenotypes.

    PubMed

    Kocha?ski, A

    2004-05-01

    Initially the Myelin Protein Zero gene was shown to be mutated in the demyelinating form of Charcot-Marie-Tooth disease (CMT1). The vast majority of the mutations in the Myelin Protein Zero gene have been detected in the Charcot-Marie-Tooth (1B) disease, however, some of them were found in patients suffering from congenital hypomyelinating neuropathy and axonal type Charcot-Marie-Tooth disease. In this study, a Charcot-Marie-Tooth disease phenotype diversity associated with different mutations in the MPZ gene, is described. PMID:15298082

  5. Presynaptic Deletion of GIT Proteins Results in Increased Synaptic Strength at a Mammalian Central Synapse.

    PubMed

    Montesinos, Mónica S; Dong, Wei; Goff, Kevin; Das, Brati; Guerrero-Given, Debbie; Schmalzigaug, Robert; Premont, Richard T; Satterfield, Rachel; Kamasawa, Naomi; Young, Samuel M

    2015-12-01

    A cytomatrix of proteins at the presynaptic active zone (CAZ) controls the strength and speed of neurotransmitter release at synapses in response to action potentials. However, the functional role of many CAZ proteins and their respective isoforms remains unresolved. Here, we demonstrate that presynaptic deletion of the two G protein-coupled receptor kinase-interacting proteins (GITs), GIT1 and GIT2, at the mouse calyx of Held leads to a large increase in AP-evoked release with no change in the readily releasable pool size. Selective presynaptic GIT1 ablation identified a GIT1-specific role in regulating release probability that was largely responsible for increased synaptic strength. Increased synaptic strength was not due to changes in voltage-gated calcium channel currents or activation kinetics. Quantitative electron microscopy revealed unaltered ultrastructural parameters. Thus, our data uncover distinct roles for GIT1 and GIT2 in regulating neurotransmitter release strength, with GIT1 as a specific regulator of presynaptic release probability. PMID:26637799

  6. Diminished Self-Chaperoning Activity of the ?F508 Mutant of CFTR Results in Protein Misfolding

    PubMed Central

    Riordan, John R.; Dokholyan, Nikolay V.

    2008-01-01

    The absence of a functional ATP Binding Cassette (ABC) protein called the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) from apical membranes of epithelial cells is responsible for cystic fibrosis (CF). Over 90% of CF patients carry at least one mutant allele with deletion of phenylalanine at position 508 located in the N-terminal nucleotide binding domain (NBD1). Biochemical and cell biological studies show that the ?F508 mutant exhibits inefficient biosynthetic maturation and susceptibility to degradation probably due to misfolding of NBD1 and the resultant misassembly of other domains. However, little is known about the direct effect of the Phe508 deletion on the NBD1 folding, which is essential for rational design strategies of cystic fibrosis treatment. Here we show that the deletion of Phe508 alters the folding dynamics and kinetics of NBD1, thus possibly affecting the assembly of the complete CFTR. Using molecular dynamics simulations, we find that meta-stable intermediate states appearing on wild type and mutant folding pathways are populated differently and that their kinetic accessibilities are distinct. The structural basis of the increased misfolding propensity of the ?F508 NBD1 mutant is the perturbation of interactions in residue pairs Q493/P574 and F575/F578 found in loop S7-H6. As a proof-of-principle that the S7-H6 loop conformation can modulate the folding kinetics of NBD1, we virtually design rescue mutations in the identified critical interactions to force the S7-H6 loop into the wild type conformation. Two redesigned NBD1-?F508 variants exhibited significantly higher folding probabilities than the original NBD1-?F508, thereby partially rescuing folding ability of the NBD1-?F508 mutant. We propose that these observed defects in folding kinetics of mutant NBD1 may also be modulated by structures separate from the 508 site. The identified structural determinants of increased misfolding propensity of NBD1-?F508 are essential information in correcting this pathogenic mutant. PMID:18463704

  7. H295R expression of melanocortin 2 receptor accessory protein results in ACTH responsiveness.

    PubMed

    Nanba, Kazutaka; Chen, Andrew X; Turcu, Adina F; Rainey, William E

    2016-02-01

    The H295R adrenocortical cell line is widely used for molecular analysis of adrenal functions but is known to have only modest ACTH responsiveness. The lack of ACTH response was linked to a low expression of its receptor, melanocortin 2 receptor (MC2R). We hypothesized that increasing the MC2R accessory protein (MRAP), which is required to traffic MC2R from the endoplasmic reticulum to the cell surface, would increase ACTH responsiveness. Lentiviral particles containing human MRAP-open reading frame were generated and transduced in H295R cells. Using antibiotic resistance, 18 clones were isolated for characterization. The most ACTH-responsive steroidogenic clone, H295RA, was used for further experiments. Successful induction of MRAP and increased expression of MC2R in H295RA cells was confirmed by quantitative real-time RT-PCR and protein analysis. Treatment with ACTH significantly increased aldosterone, cortisol, and dehydroepiandrosterone production in H295RA cells. ACTH also significantly increased transcript levels for all of the steroidogenic enzymes required to produce aldosterone, cortisol, and dehydroepiandrosterone, as well as MC2R mRNA. Using liquid chromatography/tandem mass spectrometry, we further revealed that the main unconjugated steroids produced in H295RA cells were 11-deoxycortisol, cortisol, and androstenedione. Treatment of H295RA cells with ACTH also acutely increased cAMP production and cellular protein levels for total and phosphorylated steroidogenic acute regulatory protein. In summary, through genetic manipulation, we have developed an ACTH-responsive human adrenocortical cell line. The cell line will provide a powerful in vitro tool for molecular analysis of physiologic and pathologic conditions involving the hypothalamic-pituitary-adrenal axis. PMID:26576642

  8. Protein High-Force Pulling Simulations Yield Low-Force Results

    PubMed Central

    Lichter, Seth; Rafferty, Benjamin; Flohr, Zachary; Martini, Ashlie

    2012-01-01

    All-atom explicit-solvent molecular dynamics simulations are used to pull with extremely large constant force (7503000 pN) on three small proteins. The introduction of a nondimensional timescale permits direct comparison of unfolding across all forces. A crossover force of approximately 1100 pN divides unfolding dynamics into two regimes. At higher forces, residues sequentially unfold from the pulling end while maintaining the remainder of the protein force-free. Measurements of hydrodynamic viscous stresses are made easy by the high speeds of unfolding. Using an exact low-Reynolds-number scaling, these measurements can be extrapolated to provide, for the first time, an estimate of the hydrodynamic force on low-force unfolding. Below 1100 pN, but surprisingly still at extremely large applied force, intermediate states and cooperative unfoldings as seen at much lower forces are observed. The force-insensitive persistence of these structures indicates that decomposition into unfolded fragments requires a large fluctuation. This finding suggests how proteins are constructed to resist transient high force. The progression of helix and sheet unfolding is also found to be insensitive to force. The force-insensitivity of key aspects of unfolding opens the possibility that numerical simulations can be accelerated by high applied force while still maintaining critical features of unfolding. PMID:22529933

  9. X-ray diffraction, Raman study and electrical properties of the new mixed compound Rb1.7K0.3(SO4)0.88(SeO4)0.12Te(OH)6

    NASA Astrophysics Data System (ADS)

    Djemel, M.; Abdelhedi, M.; Ktari, L.; Dammak, M.

    2013-09-01

    At room temperature, the new compound Rb1.7K0.3(SO4)0.88(SeO4)0.12Te(OH)6 crystallizes in the monoclinic system with space group C2. The unit cell parameters are: a = 11.4168 (4), b = 6.6321 (4), c = 13.6078 (6), ? = 106.975 (3), V = 985.46 (8), Z = 4 and ?cal = 3.25 g cm-1. The title compound undergoes a superionic phase transition at T = 479 K. This transition was confirmed by an abrupt increase of conductivity. Differential scanning calorimetry of Rb1.7K0.3(SO4)0.88(SeO4)0.12Te(OH)6 material showed three anomalies at 411, 461, and 479 K, respectively. Raman and IR spectra of Rb1.7K0.3(SO4)0.88(SeO4)0.12Te(OH)6, recorded at room temperature in the frequency 50-4000 cm-1 show that the SO42-, SeO42- and TeO66- groups coexist in the crystal independently.

  10. Allelic variants of the amylose extender mutation of maize demonstrate phenotypic variation in starch structure resulting from modified protein-protein interactions.

    PubMed

    Liu, Fushan; Ahmed, Zaheer; Lee, Elizabeth A; Donner, Elizabeth; Liu, Qiang; Ahmed, Regina; Morell, Matthew K; Emes, Michael J; Tetlow, Ian J

    2012-02-01

    Amylose extender (ae(-)) starches characteristically have modified starch granule morphology resulting from amylopectin with reduced branch frequency and longer glucan chains in clusters, caused by the loss of activity of the major starch branching enzyme (SBE), which in maize endosperm is SBEIIb. A recent study with ae(-) maize lacking the SBEIIb protein (termed ae1.1 herein) showed that novel protein-protein interactions between enzymes of starch biosynthesis in the amyloplast could explain the starch phenotype of the ae1.1 mutant. The present study examined an allelic variant of the ae(-) mutation, ae1.2, which expresses a catalytically inactive form of SBEIIb. The catalytically inactive SBEIIb in ae1.2 lacks a 28 amino acid peptide (Val272-Pro299) and is unable to bind to amylopectin. Analysis of starch from ae1.2 revealed altered granule morphology and physicochemical characteristics distinct from those of the ae1.1 mutant as well as the wild-type, including altered apparent amylose content and gelatinization properties. Starch from ae1.2 had fewer intermediate length glucan chains (degree of polymerization 16-20) than ae1.1. Biochemical analysis of ae1.2 showed that there were differences in the organization and assembly of protein complexes of starch biosynthetic enzymes in comparison with ae1.1 (and wild-type) amyloplasts, which were also reflected in the composition of starch granule-bound proteins. The formation of stromal protein complexes in the wild-type and ae1.2 was strongly enhanced by ATP, and broken by phosphatase treatment, indicating a role for protein phosphorylation in their assembly. Labelling experiments with [γ-(32)P]ATP showed that the inactive form of SBEIIb in ae1.2 was phosphorylated, both in the monomeric form and in association with starch synthase isoforms. Although the inactive SBEIIb was unable to bind starch directly, it was strongly associated with the starch granule, reinforcing the conclusion that its presence in the granules is a result of physical association with other enzymes of starch synthesis. In addition, an Mn(2+)-based affinity ligand, specific for phosphoproteins, was used to show that the granule-bound forms of SBEIIb in the wild-type and ae1.2 were phosphorylated, as was the granule-bound form of SBEI found in ae1.2 starch. The data strongly support the hypothesis that the complement of heteromeric complexes of proteins involved in amylopectin synthesis contributes to the fine structure and architecture of the starch granule. PMID:22121198

  11. Total protein concentration and diagnostic test results for gray wolf (Canis lupus) serum using Nobuto filter paper strips.

    PubMed

    Jara, Roco F; Seplveda, Carolina; Ip, Hon S; Samuel, Michael D

    2015-04-01

    Nobuto filter paper strips are widely used for storing blood-serum samples, but the recovery of proteins from these strips following rehydration is unknown. Poor recovery of proteins could reduce the concentration of antibodies and antigens and reduce the sensitivity of diagnostic assays. We compared the protein concentration, and its association with test sensitivity, of eluted Nobuto strip samples with paired sera. We collected and froze serum from five gray wolves (Canis lupus) for 8 mo. When thawed, we used a spectrophotometer (absorbance 280 nm) to determine the serum protein concentration for paired sera and Nobuto eluates for each animal in 2-fold serial dilutions. Total protein concentration was similar for both sample storage methods (Nobuto eluates and control sera), except for the undiluted samples in which Nobuto eluates had higher total protein concentrations. Both sample storage methods appear to produce similar results using the SNAP 4Dx Test to detect antibodies against pathogens causing Lyme disease, anaplasmosis, and ehrlichiosis as well as antigen for canine heartworm disease. PMID:25574804

  12. Inactivation of the protein phosphatase 2A regulatory subunit A results in morphological and transcriptional defects in Saccharomyces cerevisiae.

    PubMed Central

    van Zyl, W; Huang, W; Sneddon, A A; Stark, M; Camier, S; Werner, M; Marck, C; Sentenac, A; Broach, J R

    1992-01-01

    We have determined that TPD3, a gene previously identified in a screen for mutants defective in tRNA biosynthesis, most likely encodes the A regulatory subunit of the major protein phosphatase 2A species in the yeast Saccharomyces cerevisiae. The predicted amino acid sequence of the product of TPD3 is highly homologous to the sequence of the mammalian A subunit of protein phosphatase 2A. In addition, antibodies raised against Tpd3p specifically precipitate a significant fraction of the protein phosphatase 2A activity in the cell, and extracts of tpd3 strains yield a different chromatographic profile of protein phosphatase 2A than do extracts of isogenic TPD3 strains. tpd3 deletion strains generally grow poorly and have at least two distinct phenotypes. At reduced temperatures, tpd3 strains appear to be defective in cytokinesis, since most cells become multibudded and multinucleate following a shift to 13 degrees C. This is similar to the phenotype obtained by overexpression of the protein phosphatase 2A catalytic subunit or by loss of CDC55, a gene that encodes a protein with homology to a second regulatory subunit of protein phosphatase 2A. At elevated temperatures, tpd3 strains are defective in transcription by RNA polymerase III. Consistent with this in vivo phenotype, extracts of tpd3 strains fail to support in vitro transcription of tRNA genes, a defect that can be reversed by addition of either purified RNA polymerase III or TFIIIB. These results reinforce the notion that protein phosphatase 2A affects a variety of biological processes in the cell and provide an initial identification of critical substrates for this phosphatase. Images PMID:1328868

  13. Mutations in the DI-DII Linker of Human Parainfluenza Virus Type 3 Fusion Protein Result in Diminished Fusion Activity

    PubMed Central

    Xie, Wenyan; Wen, Hongling; Chu, Fulu; Yan, Shaofeng; Lin, Bin; Xie, Wenli; Liu, Ying; Ren, Guijie; Zhao, Li; Song, Yanyan; Sun, Chengxi; Wang, Zhiyu

    2015-01-01

    Human parainfluenza virus type 3 (HPIV3) can cause severe respiratory tract diseases in infants and young children, but no licensed vaccines or antiviral agents are currently available for treatment. Fusing the viral and target cell membranes is a prerequisite for its entry into host cells and is directly mediated by the fusion (F) protein. Although several domains of F are known to have important effects on regulating the membrane fusion activity, the roles of the DI-DII linker (residues 369–374) of the HPIV3 F protein in the fusogenicity still remains ill-defined. To facilitate our understanding of the role of this domain might play in F-induced cell-cell fusion, nine single mutations were engineered into this domain by site-directed mutagenesis. A vaccinia virus-T7 RNA polymerase transient expression system was employed to express the wild-type or mutated F proteins. These mutants were analyzed for membrane fusion activity, cell surface expression, and interaction between F and HN protein. Each of the mutated F proteins in this domain has a cell surface expression level similar to that of wild-type F. All of them resulted in a significant reduction in fusogenic activity in all steps of membrane fusion. Furthermore, all these fusion-deficient mutants reduced the amount of the HN-F complexes at the cell surface. Together, the results of our work suggest that this region has an important effect on the fusogenic activity of F. PMID:26305905

  14. 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

  15. Synonymous modification results in high-fidelity gene expression of repetitive protein and nucleotide sequences

    PubMed Central

    Wu, Bin; Miskolci, Veronika; Sato, Hanae; Tutucci, Evelina; Kenworthy, Charles A.; Donnelly, Sara K.; Yoon, Young J.; Cox, Dianne

    2015-01-01

    Repetitive nucleotide or amino acid sequences are often engineered into probes and biosensors to achieve functional readouts and robust signal amplification. However, these repeated sequences are notoriously prone to aberrant deletion and degradation, impacting the ability to correctly detect and interpret biological functions. Here, we introduce a facile and generalizable approach to solve this often unappreciated problem by modifying the nucleotide sequences of the target mRNA to make them nonrepetitive but still functional (synonymous). We first demonstrated the procedure by designing a cassette of synonymous MS2 RNA motifs and tandem coat proteins for RNA imaging and showed a dramatic improvement in signal and reproducibility in single-RNA detection in live cells. The same approach was extended to enhancing the stability of engineered fluorescent biosensors containing a fluorescent resonance energy transfer (FRET) pair of fluorescent proteins on which a great majority of systems thus far in the field are based. Using the synonymous modification to FRET biosensors, we achieved correct expression of full-length sensors, eliminating the aberrant truncation products that often were assumed to be due to nonspecific proteolytic cleavages. Importantly, the biological interpretations of the sensor are significantly different when a correct, full-length biosensor is expressed. Thus, we show here a useful and generally applicable method to maintain the integrity of expressed genes, critical for the correct interpretation of probe readouts. PMID:25877922

  16. Morbid Obesity Resulting from Inactivation of the Ciliary Protein CEP19 in Humans and Mice

    PubMed Central

    Shalata, Adel; Ramirez, MariaC.; Desnick, RobertJ.; Priedigkeit, Nolan; Buettner, Christoph; Lindtner, Claudia; Mahroum, Mohammed; Abdul-Ghani, Muhammad; Dong, Feng; Arar, Nazik; Camacho-Vanegas, Olga; Zhang, Rui; Camacho, SandraC.; Chen, Ying; Ibdah, Mwafaq; DeFronzo, Ralph; Gillespie, Virginia; Kelley, Kevin; Dynlacht, BrianD.; Kim, Sehyun; Glucksman, MarcJ.; Borochowitz, ZviU.; Martignetti, JohnA.

    2013-01-01

    Obesity is a major public health concern, and complementary research strategies have been directed toward the identification of the underlying causative gene mutations that affect the normal pathways and networks that regulate energy balance. Here, we describe an autosomal-recessive morbid-obesity syndrome and identify the disease-causing gene defect. The average body mass index of affected family members was 48.7 (range = 36.761.0), and all had features of the metabolic syndrome. Homozygosity mapping localized the disease locus to a region in 3q29; we designated this region the morbid obesity 1 (MO1) locus. Sequence analysis identified a homozygous nonsense mutation in CEP19, the gene encoding the ciliary protein CEP19, in all affected family members. CEP19 is highly conserved in vertebrates and invertebrates, is expressed in multiple tissues, and localizes to the centrosome and primary cilia. Homozygous Cep19-knockout mice were morbidly obese, hyperphagic, glucose intolerant, and insulin resistant. Thus, loss of the ciliary protein CEP19 in humans and mice causes morbid obesity and defines a target for investigating the molecular pathogenesis of this disease and potential treatments for obesity and malnutrition. PMID:24268657

  17. Functional analysis of the novel TBX5 c.1333delC mutation resulting in an extended TBX5 protein

    PubMed Central

    Bhm, Johann; Heinritz, Wolfram; Craig, Alexander; Vujic, Mihailo; Ekman-Joelsson, Britt-Marie; Kohlhase, Jrgen; Froster, Ursula

    2008-01-01

    Background Autosomal dominant Holt-Oram syndrome (HOS) is caused by mutations in the TBX5 gene and is characterized by congenital heart and preaxial radial ray upper limb defects. Most of the TBX5 mutations found in patients with HOS cause premature truncation of the primary TBX5 transcript. TBX5 missense mutations alter the three-dimensional structure of the protein and result in failed nuclear localization or reduced binding to target DNA. In this study we present our functional analyses of the novel and unusual c.1333delC mutation found in a patient with classical HOS. Methods The functional impact of this novel mutation was assessed by investigating the intracellular localization of the resulting TBX5 protein and its ability to activate the expression of its downstream target ANF. Results The deletion of the cytosine is the first TBX5 frameshift mutation predicted to result in an elongated TBX5 protein with 74 miscoding amino acids and 62 supernumerary C-terminal amino acids. The c.1333delC mutation affects neither the nuclear localization, nor its colocalization with SALL4, but severely affects the activation of the ANF promoter. Conclusion The mutation c.1333delC does not locate within functional domains, but impairs the activation of the downstream target. This suggests that misfolding of the protein prevents its biological function. PMID:18828908

  18. Overexpression of Drosophila juvenile hormone esterase binding protein results in anti-JH effects and reduced pheromone abundance

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The titer of juvenile hormone (JH), which has wide ranging physiological effects in insects, is regulated in part by JH esterase (JHE). We show that overexpression in Drosophila melanogaster of the JHE binding protein, DmP29 results in a series of apparent anti-JH effects. We hypothesize that DmP29 ...

  19. In vitro translation of androgen receptor cRNA results in an activated androgen receptor protein.

    PubMed Central

    Kuiper, G G; de Ruiter, P E; Trapman, J; Jenster, G; Brinkmann, A O

    1993-01-01

    Translation of androgen receptor (AR) cRNA in a reticulocyte lysate and subsequent analysis of the translation products by SDS/PAGE showed a protein with an apparent molecular mass of 108 kDa. Scatchard-plot analysis revealed a single binding component with high affinity for R1881 (Kd = 0.3 nM). All AR molecules synthesized specifically bound steroid. No evidence for AR phosphorylation during in vitro synthesis was found. When AR was labelled with [3H]R1881 and analysed on sucrose-density gradients, a complex of approx. 6 S was observed. The complex was shifted to a higher sedimentation coefficient after incubation with a monoclonal AR antibody directed against an epitope in the DNA-binding domain. In the presence as well as the absence of hormone, AR molecules were able to bind to DNA-cellulose without an activation step. Gel retardation assays revealed that the AR forms complexes with a DNA element containing glucocorticoid-responsive element/androgen-responsive element sequences. Receptor-DNA interactions were stabilized by different polyclonal antibodies directed against either the N- or C-terminal part of the AR and were abolished by an antibody directed against the DNA-binding domain of the receptor. In conclusion, translation of AR cRNA in vitro yields an activated AR protein which binds steroid with high affinity. It is proposed that AR antibodies enhance AR-DNA binding by stabilizing AR dimers when bound to DNA. Images Figure 1 Figure 5 Figure 6 Figure 7 Figure 8 PMID:8250838

  20. 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. PMID:26232633

  1. Study of the Crystallization Fields of Vanadyl(IV) Selenites in the System VOSeO3 - SeO2 - H2O.

    PubMed

    Georgieva, Velyana; Tavlieva, Mariana; Vlaev, Lyubomir; Genieva, Svetlana

    2012-12-01

    The solubility of VOSeO3-SeO2-H2O system was studied within the temperature interval 50-300 C. The phase diagram of vanadyl(IV) selenites was plotted and the crystallization fields were determined for the different phases. Depending on the conditions for hydrothermal synthesis, several types of selenites were obtained - [VO(SeO3)(H2O)2] 0.5 H2O, VOSeO3 H2O, VOSeO3 and VOSe2O5. The different phases were established and characterized by chemical, thermal and powder X-ray diffraction analyses, as well as by IR spectroscopy. PMID:24061365

  2. Simultaneous removal of SO2 and trace SeO2 from flue gas: effect of SO2 on selenium capture and kinetics study.

    PubMed

    Li, Yuzhong; Tong, Huiling; Zhuo, Yuqun; Wang, Shujuan; Xu, Xuchang

    2006-12-15

    Sulfur dioxide (SO2) and trace elements are all pollutants derived from coal combustion. This study relates to the simultaneous removal of SO2 and trace selenium dioxide (SeO2) from flue gas by calcium oxide (CaO) adsorption in the moderate temperature range, especially the effect of SO2 presence on selenium capture. Experiments performed on a thermogravimetric analyzer (TGA) can reach the following conclusions. When the CaO conversion is relatively low and the reaction rate is controlled by chemical kinetics, the SO2 presence does not affect the selenium capture. When the CaO conversion is very high and the reaction rate is controlled by product layer diffusion, the SO2 presence and the product layer diffusion resistance jointly reduce the selenium capture. On the basis of the kinetics study, a method to estimate the trace selenium removal efficiency using kinetic parameters and the sulfur removal efficiency is developed. PMID:17256549

  3. 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.

  4. 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 trm9? 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 trm9? 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

  5. 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.

  6. Interaction of Zyxin, a Focal Adhesion Protein, with the E6 Protein from Human Papillomavirus Type 6 Results in Its Nuclear Translocation

    PubMed Central

    Degenhardt, Yan Yan; Silverstein, Saul

    2001-01-01

    Zyxin, a focal adhesion molecule, interacts specifically with the E6 protein from human papillomavirus (HPV) type 6 in a yeast two-hybrid screen of a cDNA library prepared from human keratinocytes. Zyxin does not interact significantly with E6 proteins from HPV types 11, 16, or 18. The interaction was confirmed by in vitro and in vivo analyses and it requires the LIM domains (Lin-11, Isl-1, and Mec-3 [G. Freyd, S. K. Kim, and H. R. Horvitz, Nature 344:876879, 1990]) found at the carboxyl terminus of zyxin. Cotransfection of E6 from HPV (6E6) and zyxin results in the accumulation of zyxin in the nucleus where it can function as a transcriptional activator. 6E6 can also mobilize endogenous zyxin to the nucleus. PMID:11689660

  7. 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

  8. 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)

  9. 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

  10. Inhibition of mitochondrial protein synthesis results in increased endothelial cell susceptibility to nitric oxide-induced apoptosis

    PubMed Central

    Ramachandran, Anup; Moellering, Douglas R.; Ceaser, Erin; Shiva, Sruti; Xu, Jun; Darley-Usmar, Victor

    2002-01-01

    Mutations in mitochondrial DNA, affecting the activity of respiratory complexes, have been implicated in many chronic degenerative diseases. Mitochondrial proteins coded for by both the mitochondrial and nuclear genes are known to have important signaling roles in apoptosis. However, the impact of the inhibition of mitochondrial protein synthesis on apoptosis is largely unknown. This inhibition is particularly important in NO-dependent cytotoxicity, which is believed to have a significant mitochondrial component and depend on other factors such as glycolysis. In this study we have examined whether the inhibition of mitochondrial protein synthesis by chloramphenicol increases the susceptibility of endothelial cells to undergo NO-dependent apoptosis in glucose-free media. Bovine aortic endothelial cells were treated with chloramphenicol, which resulted in a decreased ratio of mitochondrial complex IV to cytochrome c and increased oxidant production in the cell. Inhibition of mitochondrial protein synthesis was associated with a greater susceptibility of the cells to apoptosis induced by NO in glucose-free medium. PMID:12011428

  11. Protein

    MedlinePLUS

    ... Alike Protein is built from building blocks called amino acids. Our bodies make amino acids in two different ways: Either from scratch, or by modifying others. A few amino acids (known as the essential amino acids) must come ...

  12. 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

  13. EXPRESSION OF AN INSECT (DENDROIDES CANADENSIS) ANTIFREEZE PROTEIN IN ARABIDOPSIS THALIANA RESULTS IN A DECREASE IN PLANT FREEZING TEMPERATURE

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Transgenic Arabidopsis thaliana plants which express genes encoding insect, Dendroides canadensis, antifreeze proteins (AFP) were produced by Agrobacterium-mediated transformation. The antifreeze protein genes, both with and without the signal peptide sequence (for protein secretion), were expresse...

  14. Do endocrine disrupting chemicals threaten Mediterranean swordfish? Preliminary results of vitellogenin and Zona radiata proteins in Xiphias gladius.

    PubMed

    Fossi, M C; Casini, S; Ancora, S; Moscatelli, A; Ausili, A; Notarbartolo-di-Sciara, G

    2001-12-01

    Endocrine Disrupting Chemicals (EDCs) have the potential to alter hormone pathways that regulate reproductive processes in wildlife and fishes. In this research the hypothesis that Mediterranean top predator species (such as large pelagic fish) are potentially at risk due to EDCs is investigated. These marine organisms tend to accumulate high concentrations of EDCs such as polyhalogenated aromatic hydrocarbons (PHAHs). The potential effects of EDCs on a fish species of commercial interest, the top predator Xiphias gladius (swordfish), were investigated using vitellogenin (Vtg) and Zona radiata proteins (Zrp) as diagnostic and prognostic biomarkers. Dramatic induction of typically female proteins (Vtg and Zrp) was detected by ELISA and Western Blot in adult males of the species. These results are the first warning of the potential risk for reproductive function of Mediterranean top predators, and suggest the need for continuous monitoring of this fragile marine environment. PMID:11763150

  15. 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.

  16. Disrupted Proteolipid Protein Trafficking Results in Oligodendrocyte Apoptosis in an Animal Model of Pelizaeus-Merzbacher Disease

    PubMed Central

    Gow, Alexander; Southwood, Cherie M.; Lazzarini, Robert A.

    1998-01-01

    Abstract. Pelizaeus-Merzbacher disease (PMD) is a dysmyelinating disease resulting from mutations, deletions, or duplications of the proteolipid protein (PLP) gene. Distinguishing features of PMD include pleiotropy and a range of disease severities among patients. Previously, we demonstrated that, when expressed in transfected fibroblasts, many naturally occurring mutant PLP alleles encode proteins that accumulate in the endoplasmic reticulum and are not transported to the cell surface. In the present communication, we show that oligodendrocytes in an animal model of PMD, the msd mouse, accumulate Plp gene products in the perinuclear region and are unable to transport them to the cell surface. Another important aspect of disease in msd mice is oligodendrocyte cell death, which is increased by two- to threefold. We demonstrate in msd mice that this death occurs by apoptosis and show that at the time oligodendrocytes die, they have differentiated, extended processes that frequently contact axons and are expressing myelin structural proteins. Finally, we define a hypothesis that accounts for pathogenesis in most PMD patients and animal models of this disease and, moreover, can be used to develop potential therapeutic strategies for ameliorating the disease phenotype. PMID:9472043

  17. 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

  18. Chemical and structural evolution in the Th-SeO3(2-)/SeO4(2-) system: from simple selenites to cluster-based selenate compounds.

    PubMed

    Xiao, Bin; Langer, Eike; Dellen, Jakob; Schlenz, Hartmut; Bosbach, Dirk; Suleimanov, Evgeny V; Alekseev, Evgeny V

    2015-03-16

    While extensive success has been gained in the structural chemistry of the U-Se system, the synthesis and characterization of Th-based Se structures are widely unexplored. Here, four new Th-Se compounds, ?-Th(SeO3)2, ?-Th(SeO3)2, Th(Se2O5)2, and Th3O2(OH)2(SeO4)3, have been obtained from mild hydrothermal or low-temperature (180-220 C) flux conditions and were subsequently structurally and spectroscopically characterized. The crystal structures of ?-Th(SeO3)2 and ?-Th(SeO3)2 are based on ThO8 and SeO3 polyhedra, respectively, featuring a three-dimensional (3D) network with selenite anions filling in the Th channels along the a axis. Th(Se2O5)2 is a 3D framework composed of isolated ThO8 polyhedra interconnected by [Se2O5](2-) dimers. Th3O2(OH)2(SeO4)3 is also a 3D framework constructed by octahedral hexathorium clusters [Th6(?3-O)4(?3-OH)4](12+), which are interlinked by selenate groups SeO4(2-). The positions of the vibrational modes associated with both Se(IV)O3(2-) and Se(VI)O4(2-) units, respectively, were determined for four compounds, and the Raman spectra of ?- and ?-Th(SeO3)2 are compared and discussed in detail. PMID:25719971

  19. Enrichment of Functional Redox Reactive Proteins and Identification by Mass Spectrometry Results in Several Terminal Fe(III)-reducing Candidate Proteins in Shewanella oneidensis MR-1.

    SciTech Connect

    Elias, Dwayne A.; Yang, Feng; Mottaz, Heather M.; Beliaev, Alex S.; Lipton, Mary S.

    2007-02-01

    Identification of the proteins directly involved in microbial metal-reduction is important to understanding the biochemistry involved in heavy metal reduction/immobilization and the ultimate cleanup of DOE contaminated sites. Although previous strategies for the identification of these proteins have traditionally required laborious protein purification/characterization of metal-reducing capability, activity is often lost before the final purification step, thus creating a significant knowledge gap. In the current study, subcellular fractions of S. oneidensis MR-1 were enriched for Fe(III)-NTA reducing proteins in a single step using several orthogonal column matrices. The protein content of eluted fractions that demonstrated activity were determined by ultra high pressure liquid chromatography coupled with tandem mass spectrometry (LCMS/ MS). A comparison of the proteins identified from active fractions in all separations produced 30 proteins that may act as the terminal electron-accepting protein for Fe(III)-reduction. These include MtrA, MtrB, MtrC and OmcA as well as a number of other proteins not previously associated with Fe(III)-reduction. This is the first report of such an approach where the laborious procedures for protein purification are not required for identification of metal-reducing proteins. Such work provides the basis for a similar approach with other cultured organisms as well as analysis of sediment and groundwater samples from biostimulation efforts at contaminated sites.

  20. Crystal chemistry of selenates with mineral-like structures: VIII. Butlerite chains in the structure of K(UO2)(SeO4)(OH)(H2O)

    NASA Astrophysics Data System (ADS)

    Gurzhii, V. V.; Bessonov, A. A.; Krivovichev, S. V.; Tananaev, I. G.; Armbruster, T.; Myasoedov, B. F.

    2009-12-01

    A new potassium uranyl selenate compound K(UO2)(SeO4)(OH)(H2O) has been synthesized for the first time using the technique of evaporation from water solution. Its crystal structure has been solved by direct methods (monoclinic, P21/ c, a = 8.0413(9) , b = 8.0362(9) , c = 11.6032(14) , ? = 106.925(2), V = 717.34(14) 3) and refined to R 1 = 0.0319 ( wR 2 = 0.0824) for 1285 reflections with | F 0| > 4? F . The structure consists of [(UO2(SeO4)(OH)(H2O)]- chains extending along axis b. In the chains, the uranyl pentagonal bipyramids are linked via bridged hydroxyl anions and tetrahedral oxoanions [SeO4]2-. Potassium ions are situated between these chains. No chains of that type have been observed in uranyl compounds earlier, but they had been detected in the structures of butlerite, parabutlerite, uklonskovite, fibroferrite, and a number of synthetic compounds.

  1. Crystal chemistry of selenates with mineral-like structures. III. Heteropolyhedral chains in the crystal structure of [Mg(H2O)4(SeO4)]2(H2O)

    NASA Astrophysics Data System (ADS)

    Krivovichev, S. V.

    2007-12-01

    The crystal structure of a new compound [Mg(H2O)4(SeO4)]2(H2O) (monoclinic, P2 1/ a, a = 7.2549(12), b = 20.059(5), c = 10.3934(17) , ? = 101.989(13), V = 1479.5(5) 3) has been solved by direct methods and refined to R 1 = 0.059 for 2577 observed reflections with | F hkl | ? 4?| F hkl |. The structure consists of [Mg(H2O)4(SeO4)]0 chains formed by alternating corner-sharing Mg octahedrons and (SeO4)2- tetrahedrons. O atoms of Mg octahedrons that are shared with selenate tetrahedrons are in a trans orientation. The heteropoly-hedral octahedral-tetrahedral chains are parallel to the c axis and undulate within the (010) plane. The adjacent chains are linked by hydrogen bonds involving H2O molecules not bound with M2+ cations.

  2. 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. PMID:22696056

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

    TOXLINE Toxicology Bibliographic Information

    Toosi S; Orlow SJ; Manga P

    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.

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

    PubMed Central

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

    2012-01-01

    Vitiligo is characterized by depigmented skin patches due to loss of epidermal melanocytes. Oxidative stress may play 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 (4-TBP) and monobenzyl ether of hydroquinone (MBEH), known triggers of vitiligo. We show that expression of key UPR components, including the transcription factor X-box binding protein 1 (XBP1), are increased following exposure of melanocytes to phenols. XBP1 activation increases production of immune mediators interleukin-6 (IL6) and IL8. Co-treatment with XBP1 inhibitors reduced IL6 and IL8 production induced by phenols, while over-expression 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. PMID:22696056

  5. Proton Dynamics in the Anti-ferroelectric CsH3(SeO3)2 by using 1H NMR Measurements

    NASA Astrophysics Data System (ADS)

    Lee, Moohee; Ndiaye, B.; Kang, K.; Kim, H.; Sim, J.; Lim, Ae Ran

    2014-03-01

    1H NMR techniques have been employed on the anti-ferroelectric CsH3(SeO3)2 to measure spectrum, shift, T1 and T2 from 300 K down to 80 K at 4.85 T. The 1H NMR spectrum at 300 K shows a composite structure; one dominant broad peak and two small narrow peaks. From the temperature dependences of both intensity and T1 for each peak, we identify that the narrow peaks come from rapidly moving protons whereas the broad peaks originate from rigid protons. The spectra below 200 K show several peaks associated with six nonequivalent proton sites and also the T1 decays show a non-exponential curve coming from many proton sites. T1 is very long even at 300 K and becomes even longer at low temperature. By analyzing T1 decays with T1S and T1L, we confirm that 1/T1(T) show an activated behavior; the short component originates from proton dynamics with activation energy of ~ 140 K and the long component is associated with that of ~ 100 K. Further analysis suggests that some protons show an abrupt change in both shift and T1L across Tc and may be responsible for the phase transition.

  6. The failure to express a protein disulphide isomerase-like protein results in a floury endosperm and an endoplasmic reticulum stress response in rice

    PubMed Central

    Han, Xiaohua; Wang, Yihua; Liu, Xi; Jiang, Ling; Ren, Yulong; Liu, Feng; Peng, Cheng; Li, Jingjing; Jin, Ximing; Wu, Fuqing; Wang, Jiulin; Guo, Xiuping; Zhang, Xin; Cheng, Zhijun; Wan, Jianmin

    2012-01-01

    The rice somaclonal mutant T3612 produces small grains with a floury endosperm, caused by the loose packing of starch granules. The positional cloning of the mutation revealed a deletion in a gene encoding a protein disulphide isomerase-like enzyme (PDIL1-1). In the wild type, PDIL1-1 was expressed throughout the plant, but most intensely in the developing grain. In T3612, its expression was abolished, resulting in a decrease in the activity of plastidial phosphorylase and pullulanase, and an increase in that of soluble starch synthase I and ADP-glucose pyrophosphorylase. The amylopectin in the T3612 endosperm showed an increase in chains with a degree of polymerization 813 compared with the wild type. The expression in the mutant's endosperm of certain endoplasmic reticulum stress-responsive genes was noticeably elevated. PDIL1-1 appears to play an important role in starch synthesis. Its absence is associated with endoplasmic reticulum stress in the endosperm, which is likely to underlie the formation of the floury endosperm in the T3612 mutant. PMID:21984651

  7. Complex Expression of the UL136 Gene of Human Cytomegalovirus Results in Multiple Protein Isoforms with Unique Roles in Replication

    PubMed Central

    Caviness, Katie; Cicchini, Louis; Rak, Michael; Umashankar, Mahadevaiah

    2014-01-01

    ABSTRACT Human cytomegalovirus (HCMV) is a complex DNA virus with a 230-kb genome encoding 170 and up to 750 proteins. The upper limit of this coding capacity suggests the evolution of complex mechanisms to substantially increase the coding potential from the 230-kb genome. Our work examines the complexity of one gene, UL136, encoded within the ULb? region of the genome that is lost during serial passage of HCMV in cultured fibroblasts. UL136 is expressed as five protein isoforms. We mapped these isoforms and demonstrate that they originate from both a complex transcriptional profile and, possibly, the usage of multiple translation initiation sites. Intriguingly, the pUL136 isoforms exhibited distinct subcellular distributions with varying association with the Golgi apparatus. The subcellular localization of membrane-bound isoforms of UL136 differed between when they were expressed exogenously and when they were expressed in the context of viral infection, suggesting that the trafficking of these isoforms is mediated by infection-specific factors. While UL136, like most ULb? genes, was dispensable for replication in fibroblasts, the soluble 23- and 19-kDa isoforms suppressed virus replication. In CD34+ hematopoietic progenitor cells (HPCs) infected in vitro, disruption of the 23- and 19-kDa isoforms resulted in increased replication and a loss of the latency phenotype, similar to the effects of the UL138 latency determinant encoded within the same genetic locus. Our work suggests a complex interplay between the UL136 isoforms which balances viral replication in multiple cell types and likely contributes to the cell type-dependent phenotypes of the UL133/8 locus and the outcome of HCMV infection. IMPORTANCE HCMV is a significant cause of morbidity in immunocompromised individuals, including transplant patients. The lifelong persistence of the virus results in a high seroprevalence worldwide and may contribute to age-related pathologies, such as atherosclerosis. The mechanisms of viral persistence are poorly understood; however, understanding the molecular basis of persistence is imperative for the development of new treatments. In this work, we characterize a complex HCMV gene, UL136, which is expressed as five protein isoforms. These isoforms arise predominantly from complex transcriptional mechanisms, which contribute to an increased coding capacity of the virus. Further, the UL136 isoforms oppose the activity of one another to balance HCMV replication in multiple cell types. We identify soluble isoforms of UL136 that function to suppress virus replication in fibroblasts and in CD34+ HPCs for latency. PMID:25297993

  8. Perinatal Protein Malnutrition Affects Mitochondrial Function in Adult and Results in a Resistance to High Fat Diet-Induced Obesity

    PubMed Central

    Jousse, Cline; Muranishi, Yuki; Parry, Laurent; Montaurier, Christophe; Even, Patrick; Launay, Jean-Marie; Carraro, Valrie; Maurin, Anne-Catherine; Averous, Julien; Chaveroux, Cdric; Bruhat, Alain; Mallet, Jacques; Morio, Batrice; Fafournoux, Pierre

    2014-01-01

    Epidemiological findings indicate that transient environmental influences during perinatal life, especially nutrition, may have deleterious heritable health effects lasting for the entire life. Indeed, the fetal organism develops specific adaptations that permanently change its physiology/metabolism and that persist even in the absence of the stimulus that initiated them. This process is termed nutritional programming. We previously demonstrated that mothers fed a Low-Protein-Diet (LPD) during gestation and lactation give birth to F1-LPD animals presenting metabolic consequences that are different from those observed when the nutritional stress is applied during gestation only. Compared to control mice, adult F1-LPD animals have a lower body weight and exhibit a higher food intake suggesting that maternal protein under-nutrition during gestation and lactation affects the energy metabolism of F1-LPD offspring. In this study, we investigated the origin of this apparent energy wasting process in F1-LPD and demonstrated that minimal energy expenditure is increased, due to both an increased mitochondrial function in skeletal muscle and an increased mitochondrial density in White Adipose Tissue. Importantly, F1-LPD mice are protected against high-fat-diet-induced obesity. Clearly, different paradigms of exposure to malnutrition may be associated with differences in energy expenditure, food intake, weight and different susceptibilities to various symptoms associated with metabolic syndrome. Taken together these results demonstrate that intra-uterine environment is a major contributor to the future of individuals and disturbance at a critical period of development may compromise their health. Consequently, understanding the molecular mechanisms may give access to useful knowledge regarding the onset of metabolic diseases. PMID:25118945

  9. A Heuristic method for assigning a false-discovery rate for protein identifications from Mascot database search results.

    PubMed

    Weatherly, D Brent; Atwood, James A; Minning, Todd A; Cavola, Cameron; Tarleton, Rick L; Orlando, Ron

    2005-06-01

    MS/MS and database searching has emerged as a valuable technology for rapidly analyzing protein expression, localization, and post-translational modifications. The probability-based search engine Mascot has found widespread use as a tool to correlate tandem mass spectra with peptides in a sequence database. Although the Mascot scoring algorithm provides a probability-based model for peptide identification, the independent peptide scores do not correlate with the significance of the proteins to which they match. Herein, we describe a heuristic method for organizing proteins identified at a specified false-discovery rate using Mascot-matched peptides. We call this method PROVALT, and it uses peptide matches from a random database to calculate false-discovery rates for protein identifications and reduces a complex list of peptide matches to a nonredundant list of homologous protein groups. This method was evaluated using Mascot-identified peptides from a Trypanosoma cruzi epimastigote whole-cell lysate, which was separated by multidimensional LC and analyzed by MS/MS. PROVALT was then compared with the two traditional methods of protein identification when using Mascot, the single peptide score and cumulative protein score methods, and was shown to be superior to both in regards to the number of proteins identified and the inclusion of lower scoring nonrandom peptide matches. PMID:15703444

  10. RECENT RESULTS WITH A PLANT-BASED TROUT FEED AND REVIEW OF WORK ON NOVEL PROTEIN SOURCES FOR TROUT.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A study was conducted to evaluate the effect of protein source and nutrient density on fish growth, feed efficiency, digestibility and plasma amino acid concentrations. A four by two factorial treatment arrangement with four protein sources (fishmeal/barley, plant concentrates, plant meals, animal ...

  11. ACUTE EXPOSURE OF THE NEONATAL RAT TO TRIETHYLTIN RESULTS IN PERSISTENT CHANGES IN NEUROTYPIC AND GLIOTYPIC PROTEINS (JOURNAL VERSION)

    EPA Science Inventory

    Measurements of neuron-specific (neurotypic) and glia-specific (fliotypic) proteins were used to characterize the toxic effects of TET on the developing CNS. Six proteins, each of which is associated with specific aspects of neuronal and glial development, were evaluated as follo...

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

    PubMed

    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

    2016-04-01

    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 important role in protecting against carbonylation of mitochondrial proteins. PMID:26654979

  13. 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 important role in protecting against carbonylation of mitochondrial proteins. PMID:26654979

  14. Characterization of GPI14/YJR013w mutation that induces the cell wall integrity signalling pathway and results in increased protein production in Saccharomyces cerevisiae.

    PubMed

    Davydenko, Svetlana G; Feng, Dejiang; Jäntti, Jussi; Keränen, Sirkka

    2005-09-01

    We report here identification and characterization of a mutation in the GPI14 gene, the yeast homologue of the mammalian PIG-M that functions in the synthesis of the GPI moiety anchoring proteins to the plasma membrane. We show that the first putative transmembrane domain of Gpi14p is not essential for its function. Downregulation of GPI14 expression/reduced protein function due to an amino terminal deletion resulted in increased transcription and production of an endogenous and a heterologous secreted protein expressed from HSP150 and ADH1 promoter, respectively. In these cells, unfolded protein response was induced but was not responsible for the enhanced production of these proteins. A cell wall defect in the gpi14 mutant cells was suggested by cell aggregation phenotype, increased sensitivity to Calcofluor white, an increased release of Gas1p and total protein into the culture medium. In the gpi14 mutant cells, transcription of RLM1, a transcription factor participating in the cell wall integrity signalling pathway, was increased, and deletion of RLM1 resulted in a synthetic lethal phenotype with the gpi14 mutation. These results suggest that partial inactivation of Gpi14p causes defects in the cell wall structure and suggest that compromised GPI anchor synthesis results in enhanced protein production via the cell wall integrity signalling pathway. PMID:16134120

  15. Surfactant Protein D Binds to Coxiella burnetii and Results in a Decrease in Interactions with Murine Alveolar Macrophages

    PubMed Central

    Soltysiak, Kelly A.; van Schaik, Erin J.; Samuel, James E.

    2015-01-01

    Coxiella burnetii is a Gram-negative, obligate intracellular bacterium and the causative agent of Q fever. Infections are usually acquired after inhalation of contaminated particles, where C. burnetii infects its cellular target cells, alveolar macrophages. Respiratory pathogens encounter the C-type lectin surfactant protein D (SP-D) during the course of natural infection. SP-D is a component of the innate immune response in the lungs and other mucosal surfaces. Many Gram-negative pulmonary pathogens interact with SP-D, which can cause aggregation, bactericidal effects and aid in bacterial clearance. Here we show that SP-D binds to C. burnetii in a calcium-dependent manner with no detectable bacterial aggregation or bactericidal effects. Since SP-D interactions with bacteria often alter macrophage interactions, it was determined that SP-D treatment resulted in a significant decrease in C. burnetii interactions to a mouse alveolar macrophage model cell line MH-S indicating SP-D causes a significant decrease in phagocytosis. The ability of SP-D to modulate macrophage activation by C. burnetii was tested and it was determined that SP-D does not alter the correlates measured for macrophage activation. Taken together these studies support those demonstrating limited activation of alveolar macrophages with C. burnetii and demonstrate interactions with SP-D participate in reduction of phagocyte attachment and phagocytosis. PMID:26366725

  16. Surfactant Protein D Binds to Coxiella burnetii and Results in a Decrease in Interactions with Murine Alveolar Macrophages.

    PubMed

    Soltysiak, Kelly A; van Schaik, Erin J; Samuel, James E

    2015-01-01

    Coxiella burnetii is a Gram-negative, obligate intracellular bacterium and the causative agent of Q fever. Infections are usually acquired after inhalation of contaminated particles, where C. burnetii infects its cellular target cells, alveolar macrophages. Respiratory pathogens encounter the C-type lectin surfactant protein D (SP-D) during the course of natural infection. SP-D is a component of the innate immune response in the lungs and other mucosal surfaces. Many Gram-negative pulmonary pathogens interact with SP-D, which can cause aggregation, bactericidal effects and aid in bacterial clearance. Here we show that SP-D binds to C. burnetii in a calcium-dependent manner with no detectable bacterial aggregation or bactericidal effects. Since SP-D interactions with bacteria often alter macrophage interactions, it was determined that SP-D treatment resulted in a significant decrease in C. burnetii interactions to a mouse alveolar macrophage model cell line MH-S indicating SP-D causes a significant decrease in phagocytosis. The ability of SP-D to modulate macrophage activation by C. burnetii was tested and it was determined that SP-D does not alter the correlates measured for macrophage activation. Taken together these studies support those demonstrating limited activation of alveolar macrophages with C. burnetii and demonstrate interactions with SP-D participate in reduction of phagocyte attachment and phagocytosis. PMID:26366725

  17. Multiple genomic defects result in an alternative RNA splice creating a human gamma H chain disease protein.

    PubMed

    Guglielmi, P; Bakhshi, A; Cogne, M; Seligmann, M; Korsmeyer, S J

    1988-09-01

    Heavy chain diseases (HCD) are human lymphoproliferative disorders in which a clonal B cell population produces Ig molecules made of truncated H chains without associated L chain. We characterized the rearranged H chain gene and its mRNA from the leukemic cells of a patient (RIV) with gamma-HCD. The abnormal RIV serum Ig consisted of shortened, dimeric gamma 1-chains which had an amino terminus within the hinge region. RIV lymphoblasts possessed a foreshortened (1200 bp) gamma 1-mRNA which had sequences for only the leader, hinge, second, and third constant region domains (CH2 + CH3), but lacked variable (VH) and CH1 information. Sequence of the productive gamma 1 allele revealed it had undergone VH-JH and H chain class switch recombinations. However, normal RNA splice sites had been eliminated by a DNA insertion/deletion (VH acceptor site), mutations (JH donor site), or a large deletion (CH1 region). Inserted sequences were of non-Ig and apparently non-genomic origin. These DNA alterations resulted in aberrant mRNA processing in which the leader region was spliced directly to the hinge region, accounting for the HCD protein. PMID:3137265

  18. Pigment protein complexes and functional properties of tetratype resulting from crosses between CP1 and CP 2 less Chlamydomonas mutants.

    PubMed

    Picaud, A; Dubertret, G

    1986-01-01

    A chlorophyll b-less mutant of Chlamydomonas reinhardtii (Pg 27) was isolated after UV irradiation of the wild type cells. This photosynthetically competent mutant totally lacks chlorophyll b and the CP2 chlorophyll-protein complex. However, SDS-PAGE, proteolytic digestions and immunodetections demonstrated that the 24-25 Kd apoproteins of the lacking CP2 complex are still present in thylakoids of the Pg27 mutant. It is concluded that this CP2-less mutant is affected in the biosynthesis pathway of chlorophyll b.This CP2-less mutant was crossed with a CP1-less mutant (Fl5) Fluorescence emission spectra and fluorescence inductions in the presence of DCMU were analysed in the resulting (cp 2 (-) , cp 1 (+) ), (cp 2 (+) , cp 1 (-) ), (cp 2 (+) , cp 1 (+) ), cp 2 (-) , cp 1 (-) )tetratype. Differences in PS 2 optical cross section and in the relative amplitude or localisation of fluorescence emission peaks fit well with a quadripartite model where PS1 and PS2 would each correspond to a reaction centre core complex (CP1 and CP2 respectively) associated to a light harvesting antenna (LHC1 and LHC2 respectively). The occurrence of energy transfers from PS1 peripheral antenna to PS2 in the Fl 5 mutant shows that, in absence of CP1, at least a part of its associated PS1 light harvesting antenna migrates in the PS2 containing appressed thylakoids. PMID:24443119

  19. MAP kinase p38 inhibitors: clinical results and an intimate look at their interactions with p38alpha protein.

    PubMed

    Lee, Matthew R; Dominguez, Celia

    2005-01-01

    Mitogen-activated protein kinase p38 is a serine/threonine kinase originally isolated from lipopolysaccharide (LPS) stimulated monocytes. There are four isoforms p38alpha p38beta, p38gamma, and p38delta. The most thoroughly studied isoform is p38alpha, whose activation has been observed in many hematopoietic and non-hematopoietic cell types upon appropriate stimuli. Subsequently, p38alpha kinase has been shown to be involved in the biosynthesis of TNFalpha and IL-1beta at the translational and transcriptional level. MAP kinase p38alpha represents a point of convergence for multiple signaling processes that are activated in inflammation and thus a key potential target for the modulation of cytokine production. The discovery and publication of p38alpha and the pyridinyl-imidazole inhibitor initiated a huge effort by many companies to develop p38alpha inhibitors as potential treatment for inflammatory diseases. Herein we provide a brief overview of recent reported clinical results for AMG 548, BIRB 796, VX 702, SCIO 469, and SCIO 323. However, our focus will be on the binding modes of these inhibitors and other p38 inhibitors in the recent literature. PMID:16378500

  20. Nd 5O 4Cl[AsO 3] 2 and Gd 5O 4Br 3[SeO 3] 2: Two lanthanoid oxide halides with complex "lone-pair" oxoanions

    NASA Astrophysics Data System (ADS)

    Kang, Dong-Hee; Wontcheu, Joseph; Schleid, Thomas

    2009-02-01

    Both compounds, neodymium oxide chloride oxoarsenate(III) Nd 5O 4Cl[AsO 3] 2 and gadolinium oxide bromide oxoselenate(IV) Gd 5O 4Br 3[SeO 3] 2, were prepared by solid-state reactions from mixtures of the corresponding binary oxides and halides, and their crystal structures have been determined by X-ray diffraction of single crystals. They crystallize monoclinically ( a = 1241.62(9) pm, b = 565.78(4) pm, c = 902.03(7) pm, ? = 116.454(3) for Nd 5O 4Cl[AsO 3] 2 and a = 1243.70(9) pm, b = 549.91(4) pm, c = 1005.28(8) pm, ? = 91.869(3) for Gd 5O 4Br 3[SeO 3] 2) in space group C2/ m with two formula units per unit cell. The non-isotypic crystal structures contain three crystallographically different M 3+ cations (M = Nd and Gd). The coordination sphere of (M1) 3+ consists of eight oxygen atoms (CN = 8) exclusively, whereas (M2) 3+ carries six oxygen atoms and one X - anion (X = Cl and Br, CN = 7) in each case. For (M3) 3+, however, CN = 8 is realized by six oxygen atoms and two Cl - anions in Nd 5O 4Cl[AsO 3] 2, but five oxygen atoms and three Br - anions in Gd 5O 4Br 3[SeO 3] 2. The isolated pyramidal [AsO 3] 3-/[SeO 3] 2- anions ( d(As 3+-O 2-) = 175-179; d(Se 4+-O 2-) = 165-174 pm) originate from three oxygen atoms (O2 and two O3), which surround the As 3+/Se 4+ cations together with the stereochemically active non-bonding electron pair ( lone pair) ? 1-tetrahedrally (?(O-As-O) = 95-102; ?(O-Se-O) = 95-96). Both crystal structures are built up of corrugated two-dimensional lanthanoid-oxygen layers {[}?2 consisting of edge- and corner-shared [OM 4] 10+ tetrahedra ( d(O 2--Nd 3+) = 228-242; d(O 2--Gd 3+) = 226-235 pm). The single Cl - anion in the neodymium and the two crystallographically independent Br - anions in the gadolinium compound reside in between these sheets, where the lone-pair electrons at the As 3+/Se 4+ cations point into the center of channels, which are formed by lanthanoid-oxygen layers and halide chains.

  1. 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.

  2. N-Acetylcysteine treatment of dystrophic mdx mice results in protein thiol modifications and inhibition of exercise induced myofibre necrosis.

    PubMed

    Terrill, Jessica R; Radley-Crabb, Hannah G; Grounds, Miranda D; Arthur, Peter G

    2012-05-01

    Oxidative stress is implicated as a factor that increases necrosis of skeletal muscles in Duchenne Muscular Dystrophy (DMD) and the dystrophic mdx mouse. Consequently, drugs that minimize oxidative stress are potential treatments for muscular dystrophy. This study examined the in vivo benefits to mdx mice of an antioxidant treatment with the cysteine precursor N-acetylcysteine (NAC), administered in drinking water. NAC was completely effective in preventing treadmill exercise-induced myofibre necrosis (assessed histologically) and the increased blood creatine kinase levels (a measure of sarcolemma leakiness) following exercise were significantly lower in the NAC treated mice. While NAC had no effect on malondialdehyde level or protein carbonylation (two indicators of irreversible oxidative damage), treatment with NAC for one week significantly decreased the oxidation of glutathione and protein thiols, and enhanced muscle protein thiol content. These data provide in vivo evidence for protective benefits of NAC treatment on dystropathology, potentially via protein thiol modifications. PMID:22206641

  3. 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

  4. Alterations in c-Myc phenotypes resulting from dynamin-related protein 1 (Drp1)-mediated mitochondrial fission

    PubMed Central

    Sarin, M; Wang, Y; Zhang, F; Rothermund, K; Zhang, Y; Lu, J; Sims-Lucas, S; Beer-Stolz, D; Van Houten, B E; Vockley, J; Goetzman, E S; Anthony Graves, J; Prochownik, E V

    2013-01-01

    The c-Myc (Myc) oncoprotein regulates numerous phenotypes pertaining to cell mass, survival and metabolism. Glycolysis, oxidative phosphorylation (OXPHOS) and mitochondrial biogenesis are positively controlled by Myc, with myc−/− rat fibroblasts displaying atrophic mitochondria, structural and functional defects in electron transport chain (ETC) components, compromised OXPHOS and ATP depletion. However, while Myc influences mitochondrial structure and function, it is not clear to what extent the reverse is true. To test this, we induced a state of mitochondrial hyper-fission in rat fibroblasts by de-regulating Drp1, a dynamin-like GTPase that participates in the terminal fission process. The mitochondria from these cells showed reduced mass and interconnectivity, a paucity of cristae, a marked reduction in OXPHOS and structural and functional defects in ETC Complexes I and V. High rates of abortive mitochondrial fusion were observed, likely reflecting ongoing, but ultimately futile, attempts to normalize mitochondrial mass. Cellular consequences included reduction of cell volume, ATP depletion and activation of AMP-dependent protein kinase. In response to Myc deregulation, apoptosis was significantly impaired both in the absence and presence of serum, although this could be reversed by increasing ATP levels by pharmacologic means. The current work demonstrates that enforced mitochondrial fission closely recapitulates a state of Myc deficiency and that mitochondrial integrity and function can affect Myc-regulated cellular behaviors. The low intracellular ATP levels that are frequently seen in some tumors as a result of inadequate vascular perfusion could favor tumor survival by countering the pro-apoptotic tendencies of Myc overexpression. PMID:23764851

  5. 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 IRE1? (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

  6. The crystal structure of ilinskite, NaCu5O2(SeO3)2Cl3, and review of mixed-ligand CuOmCln coordination geometries in minerals and inorganic compounds

    NASA Astrophysics Data System (ADS)

    Krivovichev, Sergey V.; Filatov, Stanislav K.; Vergasova, Lidiya P.

    2013-04-01

    The crystal structure of ilinskite, NaCu5O2(SeO3)2Cl3, a rare copper selenite chloride from volcanic fumaroles of the Great fissure Tolbachik eruption (Kamchatka peninsula, Russia), has been solved by direct methods and refined to R 1 = 0.044 on the basis of 2720 unique observed reflections. The mineral is orthorhombic, Pnma, a = 17.769(7), b = 6.448(3), c = 10.522(4) , V = 1205.6(8) 3, Z = 4. The The CuOmCln coordination polyhedra share edges to form tetramers that have 'additional' O1 and O2 atoms as centers. The O1Cu4 and O2Cu4 tetrahedra share common Cu atoms to form [O2Cu5]6+ sheets. The SeO3 groups and Cl atoms are adjacent to the [O2Cu5]6+ sheets to form complex layers parallel to (100). The Na+ cations are located in between the layers. A review of mixed-ligand CuOmCln coordination polyhedra in minerals and inorganic compounds is given. There are in total 26 stereochemically different mixed-ligand Cu-O-Cl coordinations.

  7. Cationization of immunoglobulin G results in enhanced organ uptake of the protein after intravenous administration in rats and primate

    SciTech Connect

    Triguero, D.; Buciak, J.L.; Pardridge, W.M. )

    1991-07-01

    Cationization of proteins in general enhances the cellular uptake of these macromolecules, and cationized antibodies are known to retain antigen binding properties. Therefore, cationized antibodies may be therapeutic and allow for intracellular immunization. The present studies test the hypothesis that the tissue uptake of cationized immunoglobulin G (IgG) after intravenous administration may be greatly increased relative to the uptake of native proteins. The pharmacokinetics of cationized immunoglobulin G clearance from blood, and the volume of distribution of the cationized or native protein (albumin, IgG) for 10 organs was measured both in anesthetized rats and in an anesthetized adult Macaca irus cynomologous monkey. Initial studies on brain showed that serum factors inhibited uptake of 125I-cationized IgG, but not 3H-cationized IgG. The blood-brain barrier permeability surface area product for 3H-cationized IgG was 0.57 {plus minus} 0.04 microliters min-1 g-1. The ratio of the volume of distribution of the 3-H-cationized IgG compared to 3H-labeled native albumin ranged from 0.9 (testis) to 15.7 (spleen) in the rat at 3 hr after injection, and a similarly enhanced organ uptake was observed in the primate. In conclusion, these studies demonstrate that cationization of immunoglobulin greatly increases organ uptake of the plasma protein compared to native immunoglobulins, and suggest that cationization of monoclonal antibodies may represent a potential new strategy for enhancing the intracellular delivery of these proteins.

  8. Suppression of protein l-isoaspartyl (d-aspartyl) methyltransferase results in hyperactivation of EGF-stimulated MEK-ERK signaling in cultured mammalian cells.

    PubMed

    Kosugi, Sakurako; Furuchi, Takemitsu; Katane, Masumi; Sekine, Masae; Shirasawa, Takuji; Homma, Hiroshi

    2008-06-20

    l-Aspartyl (l-Asp) and l-asparaginyl residues in proteins isomerize or racemize to d,l-isoaspartyl (d,l-isoAsp) or d-aspartyl (d-Asp) residues during protein aging. These atypical aspartyl residues can interfere with the biological function of the protein and lead to cellular dysfunction. Protein l-isoaspartyl (d-aspartyl) methyltransferase (PIMT) is a repair enzyme that facilitates conversion of l-isoAsp and d-Asp to l-Asp. PIMT deficient mice exhibit accumulation of l-isoAsp in several tissues and die, on average, 12 days after birth from progressive epileptic seizures with grand mal and myoclonus features. However, little is known about the molecular mechanisms by which accumulation of the aberrant residues leads to cellular abnormalities. In this study, we established PIMT-knockdown cells using a short interfering RNA expression system and characterized the resultant molecular abnormalities in intracellular signaling pathways. PIMT-knockdown cells showed significant accumulation of proteins with isomerized residues, compared to control cells. In the PIMT-knockdown cells, Raf-1, MEK, and ERK, members of the MAPK cascade, were hyperphosphorylated after EGF stimulation compared to control cells. These results suggest that PIMT repair of abnormal proteins is necessary to maintain normal MAPK signaling. PMID:18381200

  9. B Lymphocyte-Specific Loss of Ric-8A Results in a Gα Protein Deficit and Severe Humoral Immunodeficiency.

    PubMed

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

    2015-09-01

    Resistance to inhibitors of cholinesterase 8A (Ric-8A) is a highly evolutionarily conserved cytosolic protein initially identified in Caenorhabditis 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 ric8 (fl/fl) vav1-cre and ric8 (fl/fl) mb1-cre mice. The major hematopoietic cell lineages developed in the ric8 (fl/fl) vav1-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 ric8 (fl/fl) mb1-cre B cells exhibited poor responses to chemokines, abnormal trafficking, improper in situ positioning, and loss of polarity components during B cell differentiation. The ric8 (fl/fl) mb1-cre mice had a severely disrupted lymphoid architecture and poor primary and secondary Ab responses. In B lymphocytes, Ric-8A is essential for normal Gα protein levels and is required for B cell differentiation, trafficking, and Ab responses. PMID:26232433

  10. RNAi-mediated silencing of the Arabidopsis thaliana ULCS1 gene, encoding a WDR protein, results in cell wall modification impairment and plant infertility.

    PubMed

    Beris, Despoina; Kapolas, Georgios; Livanos, Pantelis; Roussis, Andreas; Milioni, Dimitra; Haralampidis, Kosmas

    2016-04-01

    Ubiquitin mediated protein degradation constitutes one of the most complex post translational gene regulation mechanisms in eukaryotes. This fine-tuned proteolytic machinery is based on a vast number of E3 ubiquitin ligase complexes that mark target proteins with ubiquitin. The specificity is accomplished by a number of adaptor proteins that contain functional binding domains, including the WD40 repeat motif (WDRs). To date, only few of these proteins have been identified in plants. An RNAi mediated silencing approach was used here to functionally characterize the Arabidopsis thaliana ULCS1 gene, which encodes for a small molecular weight WDR protein. AtULCS1 interacts with the E3Cullin Ring Ligase subunit DDB1a, regulating most likely the degradation of specific proteins involved in the manifestation of diverse developmental events. Silencing of AtULCS1 results in sterile plants with pleiotropic phenotypes. Detailed analysis revealed that infertility is the outcome of anther indehiscence, which in turn is due to the impairment of the plants to accomplish secondary wall modifications. Furthermore, IREGULAR XYLEM gene expression and lignification is diminished in anther endothecium and the stem vascular tissue of the silenced plants. These data underline the importance of AtULCS1 in plant development and reproduction. PMID:26940493

  11. A novel mutation, cog, which results in production of a new porin protein (OmpG) of Escherichia coli K-12.

    PubMed Central

    Misra, R; Benson, S A

    1989-01-01

    A mutant of Escherichia coli K-12 which produces a new outer membrane protein, OmpG, was isolated and genetically and biochemically characterized. The presence of OmpG allows growth on maltodextrins in the absence of the LamB maltoporin. The data obtained from in vivo growth and uptake experiments suggested that the presence of the OmpG protein results in an increase in outer membrane permeability for small hydrophilic compounds. In light of these findings, we suggest that OmpG is a porinlike protein. The mutation which results in the expression of OmpG has been termed cog (for control of OmpG) and mapped to 29 min on the E. coli chromosome. Diploid analysis shows that the mutant cog-192 allele is recessive for both the Dex+ and OmpG+ phenotypes. We propose that the cog mutation destroys a negative regulatory function and therefore derepresses ompG expression. Images PMID:2473977

  12. Moderate alcohol induces stress proteins HSF1 and hsp70 and inhibits proinflammatory cytokines resulting in endotoxin tolerance.

    PubMed

    Muralidharan, Sujatha; Ambade, Aditya; Fulham, Melissa A; Deshpande, Janhavee; Catalano, Donna; Mandrekar, Pranoti

    2014-08-15

    Binge or moderate alcohol exposure impairs host defense and increases susceptibility to infection because of compromised innate immune responses. However, there is a lack of consensus on the molecular mechanism by which alcohol mediates this immunosuppression. In this study, we show that cellular stress proteins HSF1 and hsp70 play a mechanistic role in alcohol-mediated inhibition of the TLR4/MyD88 pathway. Alcohol exposure induced transcription factor HSF1 mRNA expression and DNA binding activity in primary human monocytes and murine macrophages. Furthermore, HSF1 target gene hsp70 mRNA and protein are upregulated by alcohol in monocytes. In vitro pre-exposure to moderate alcohol reduced subsequent LPS-induced NF-?B promoter activity and downstream TNF-?, IL-6 and IL-1? production in monocytes and macrophages, exhibiting endotoxin tolerance. Mechanistic analysis demonstrates that alcohol-induced HSF1 binds to the TNF-? promoter in macrophages at early time points, exerting transrepression and decreased TNF-? expression. Furthermore, association of hsp70 with NF-?B subunit p50 in alcohol-treated macrophages correlates with reduced NF-?B activation at later time points. Hsp70 overexpression in macrophages was sufficient to block LPS-induced NF-?B promoter activity, suggesting alcohol-mediated immunosuppression by hsp70. The direct crosstalk of hsp70 and HSF1 was further confirmed by the loss of alcohol-mediated endotoxin tolerance in hsp70- and HSF1-silenced macrophages. Our data suggest that alcohol-mediated activation of HSF1 and induction of hsp70 inhibit TLR4-MyD88 signaling and are required for alcohol-induced endotoxin tolerance. Using stress proteins as direct drug targets would be clinically relevant in alcohol abuse treatment and may serve to provide a better understanding of alcohol-mediated immunosuppression. PMID:25024384

  13. Cyclophilin A Binds to the Viral RNA and Replication Proteins, Resulting in Inhibition of Tombusviral Replicase Assembly

    PubMed Central

    Kovalev, Nikolay

    2013-01-01

    Replication of plus-stranded RNA viruses is greatly affected by numerous host-encoded proteins that act as restriction factors. Cyclophilins, which are a large family of cellular prolyl isomerases, have been found to inhibit Tomato bushy stunt tombusvirus (TBSV) replication in a Saccharomyces cerevisiae model based on genome-wide screens and global proteomics approaches. In this report, we further characterize single-domain cyclophilins, including the mammalian cyclophilin A and plant Roc1 and Roc2, which are orthologs of the yeast Cpr1p cyclophilin, a known inhibitor of TBSV replication in yeast. We found that recombinant CypA, Roc1, and Roc2 strongly inhibited TBSV replication in a cell-free replication assay. Additional in vitro studies revealed that CypA, Roc1, and Roc2 cyclophilins bound to the viral replication proteins, and CypA and Roc1 also bound to the viral RNA. These interactions led to inhibition of viral RNA recruitment, the assembly of the viral replicase complex, and viral RNA synthesis. A catalytically inactive mutant of CypA was also able to inhibit TBSV replication in vitro due to binding to the replication proteins and the viral RNA. Overexpression of CypA and its mutant in yeast or plant leaves led to inhibition of tombusvirus replication, confirming that CypA is a restriction factor for TBSV. Overall, the current work has revealed a regulatory role for the cytosolic single-domain Cpr1-like cyclophilins in RNA virus replication. PMID:24089553

  14. 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

  15. 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...

  16. Adenovirus-mediated factor VIII gene expression results in attenuated anti-factor VIII-specific immunity in hemophilia A mice compared with factor VIII protein infusion.

    PubMed

    Bristol, J A; Gallo-Penn, A; Andrews, J; Idamakanti, N; Kaleko, M; Connelly, S

    2001-09-01

    Hemophilia A patients are typically treated by factor VIII (FVIII) protein replacement, an expensive therapy that induces FVIII-specific inhibitors in approximately 30% of patients with severe hemophilia. FVIII gene therapy has the potential to improve the current treatment protocols. In this report, we used a hemophilia A mouse model to compare the humoral and cellular immune responses between an E1/E2a/E3-deficient adenovirus expressing human FVIII directed by a liver-specific albumin promoter and purified recombinant FVIII protein infusion. Adenovirus-mediated FVIII expression did not elicit detectable CD4+ or CD8+ T cell responses and induced a weak antibody immune response to FVIII. In contrast, FVIII protein administration resulted in a potent anti-FVIII antibody response and moderate CD4+ T cell response. Furthermore, hemophiliac mice preimmunized with FVIII protein infusion to induce anti-FVIII immunity, and subsequently treated by adenovirus-mediated FVIII gene therapy, expressed therapeutic levels of FVIII despite the presence of low levels of anti-FVIII antibodies. No FVIII was detected in the plasma of mice with intermediate or high antibody levels, although anti-FVIII antibody levels in some vector-treated animals declined. The data support the hypothesis that liver-specific gene therapy-mediated expression of FVIII may be less immunogenic than traditional protein replacement therapy. PMID:11535168

  17. 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

  18. Synthesis, structure, and characterization of novel two- and three-dimensional vanadates: Ba2.5(VO2)3(SeO3)4.H2O and La(VO2)3(TeO6).3H2O.

    PubMed

    Sivakumar, T; Ok, Kang Min; Halasyamani, P Shiv

    2006-05-01

    Two new vanadates, Ba(2.5)(VO2)3(SeO3)4.H2O and La(VO2)3(TeO6).3H2O, have been synthesized by hydrothermal methods using BaCO3, Ba(OH)2.H2O, La(NO3)3.6H2O, V2O5, TeO2, and H2SeO3 as reagents. The structures were determined by single-crystal X-ray diffraction. Ba(2.5)(VO2)3(SeO3)4.H2O exhibits a two-dimensional layered structure consisting of VO(5) square pyramids and SeO3 polyhedra, whereas La(VO2)3(TeO6).3H2O has a three-dimensional framework structure composed of VO(4) tetrahedra and TeO6 octahedra. Infrared and Raman spectroscopy, UV-vis diffuse reflectance spectroscopy, and thermogravimetric analysis are also presented. Crystal data: Ba(2.5)(VO2)3(SeO3)4.H2O, trigonal, space group P (No. 147) with a = b = 12.8279(15) A, c = 7.2631(9) A, V = 1035.1(2) A(3), and Z = 2; La(VO2)3(TeO6).3H2O, trigonal, space group R3c (No. 161) with a = b = 9.4577(16) A, c = 23.455(7) A, V = 1816.9(7) A3, and Z = 6. PMID:16634591

  19. Conjugation of an anti transferrin receptor IgG3-avidin fusion protein with biotinylated saporin results in significant enhancement of its cytotoxicity against malignant hematopoietic cells.

    PubMed

    Daniels, Tracy R; Ng, Patrick P; Delgado, Tracie; Lynch, Maureen R; Schiller, Gary; Helguera, Gustavo; Penichet, Manuel L

    2007-11-01

    We have previously developed an antibody fusion protein composed of a mouse/human chimeric IgG3 specific for the human transferrin receptor genetically fused to avidin (anti-hTfR IgG3-Av) as a universal delivery system for cancer therapy. This fusion protein efficiently delivers biotinylated FITC into cancer cells via TfR-mediated endocytosis. In addition, anti-hTfR IgG3-Av alone exhibits intrinsic cytotoxic activity and interferes with hTfR recycling, leading to the rapid degradation of the TfR and lethal iron deprivation in certain malignant B-cell lines. We now report on the cytotoxic effects of a conjugate composed of anti-hTfR IgG3-Av and biotinylated saporin 6 (b-SO6), a toxin derived from the plant Saponaria officinalis that inhibits protein synthesis. Conjugation of anti-hTfR IgG3-Av with b-SO6 enhances the cytotoxic effect of the fusion protein in sensitive cells and also overcomes the resistance of malignant cells that show low sensitivity to the fusion protein alone. Our results show for the first time that loading anti-hTfR IgG3-Av with a biotinylated toxin enhances the cytotoxicity of the fusion protein alone. These results suggest that anti-hTfR IgG3-Av has great potential as a therapeutic agent for a wide range of applications due to its intrinsic cytotoxic activity plus its ability to deliver biotinylated molecules into cancer cells. PMID:18025284

  20. Combined growth hormone/insulin-like growth factor I in addition to glutamine-supplemented TPN results in net protein anabolism in critical illness.

    PubMed

    Carroll, Paul V; Jackson, Nicola C; Russell-Jones, David L; Treacher, David F; Snksen, Peter H; Umpleby, A Margot

    2004-01-01

    Protein loss leading to reduced lean body mass is recognized to contribute to the high levels of morbidity and mortality seen in critical illness. This prospective, randomized, controlled study compared the effects of conventional parenteral nutrition (TPN), glutamine-supplemented (0.4 g.kg-1.day-1) TPN (TPNGLN), and TPNGLN with combined growth hormone (GH, 0.2 IU.kg-1.day-1) and IGF-I (160 microg.kg-1.day-1) on protein metabolism in critical illness. Nineteen mechanically ventilated subjects [64 +/- 3 yr, body mass index (BMI) 23.8 +/- 1.3, kg/m2] were initially studied in the fasting state (study 1) and subsequently after 3 days of nutritional with/without hormonal support (study 2). All had recently been admitted to the ICU and the majority were postemergency abdominal surgery (APACHE II 17.5 +/- 1.0). Protein metabolism was assessed using a primed constant infusion of [1-13C]leucine. Conventional TPN contained mixed amino acids, Intralipid, and 50% dextrose. TPNGLN, unlike TPN alone, resulted in an increase in plasma glutamine concentration ( approximately 50%, P < 0.05). Both TPN and TPNGLN decreased the rate of protein breakdown (TPN 15%, P < 0.002; TPNGLN 16%, P < 0.05), but during these treatments the patients remained in a net negative protein balance. Combined treatment with TPNGLN + GH/IGF-I increased plasma IGF-I levels (10.3 +/- 0.8 vs. 48.1 +/- 9.1 nmol/l, study 1 vs. study 2, P < 0.05), and in contrast to therapy with nutrition alone, resulted in net protein gain (-0.75 +/- 0.14 vs. 0.33 +/- 0.12 g protein.kg-1.day-1, study 1 vs. study 2, P < 0.05). Therapy with GH/IGF-I + TPNGLN, unlike nutrition alone, resulted in net positive protein balance in a group of critically ill patients. PMID:12759221

  1. Forkhead box protein C2 contributes to invasion and metastasis of extrahepatic cholangiocarcinoma, resulting in a poor prognosis.

    PubMed

    Watanabe, Akira; Suzuki, Hideki; Yokobori, Takehiko; Altan, Bolag; Kubo, Norio; Araki, Kenichiro; Wada, Satoshi; Mochida, Yasushi; Sasaki, Shigeru; Kashiwabara, Kenji; Hosouchi, Yasuo; Kuwano, Hiroyuki

    2013-11-01

    Extrahepatic cholangiocarcinoma (EHCC) is a cancer with a poor prognosis, and the postoperative survival of patients depends on the existence of invasion and metastasis. The epithelial-to-mesenchymal transition (EMT) is an important step in EHCC invasion and metastasis. Forkhead box protein C2 (FOXC2) is a transcription factor that has been reported to induce the EMT. Therefore we examined the correlation between FOXC2 expression and clinical pathological factors, and analysed the function of FOXC2. The expression of FOXC2 in 77 EHCC cases was investigated by immunohistochemical staining, and the relationship between FOXC2 expression and clinicopathological factor was assessed. Knockdown by small interfering RNA (siRNA) was performed to determine the roles of FOXC2 in EHCC cell line. FOXC2 expression correlated with lymph node metastasis (P = 0.0205). Patients in the high FOXC2 expression group had a poorer prognosis than the patients in the low FOXC2 expression group. Moreover, FOXC2 knockdown inhibited cell motility and invasion, and decreased the expression of EMT markers (N-cadherin, and matrix metalloproteinase (MMP) -2) and Angiopietin-2 (Ang-2). The EMT inducer FOXC2 contributes to a poor prognosis and cancer progression. FOXC2 may be a promising molecular target for regulating EHCC metastasis. PMID:23919841

  2. Study of recombinant antibody fragments and PAI-1 complexes combining protein-protein docking and results from site-directed mutagenesis.

    PubMed

    Novoa de Armas, Hector; Dewilde, Maarten; Verbeke, Koen; De Maeyer, Marc; Declerck, Paul J

    2007-09-01

    Elevated plasma levels of plasminogen activator inhibitor-1 (PAI-1) have been correlated with cardiovascular diseases such as myocardial infarction and venous thrombosis. PAI-1 has also been shown to play an important role in tumor development, diabetes, and obesitas. Monoclonal antibodies MA-8H9D4 and MA-56A7C10, and their single-chain variable fragments (scFv), exhibit PAI-1-neutralizing properties. In this study, a rigid-body docking approach is used to predict the binding geometry of two distinct conformations of PAI-1 (active and latent) in complex with these antibody fragments. Resulting models were initially refined by using the dead-end elimination algorithm. Different filtering criteria based on the mutagenesis studies and structural considerations were applied to select the final models. These were refined by using the slow-cooling torsion-angle dynamic annealing protocol. The docked structures reveal the respective epitopes and paratopes and their potential interactions. This study provides crucial information that is necessary for the rational development of low-molecular weight PAI-1 inhibitors. PMID:17850750

  3. The copper-transporting capacity of ATP7A mutants associated with Menkes disease is ameliorated by COMMD1 as a result of improved protein expression.

    PubMed

    Vonk, Willianne I M; de Bie, Prim; Wichers, Catharina G K; van den Berghe, Peter V E; van der Plaats, Rozemarijn; Berger, Ruud; Wijmenga, Cisca; Klomp, Leo W J; van de Sluis, Bart

    2012-01-01

    Menkes disease (MD) is an X-linked recessive disorder characterized by copper deficiency resulting in a diminished function of copper-dependent enzymes. Most MD patients die in early childhood, although mild forms of MD have also been described. A diversity of mutations in the gene encoding of the Golgi-resident copper-transporting P(1B)-type ATPase ATP7A underlies MD. To elucidate the molecular consequences of the ATP7A mutations, various mutations in ATP7A associated with distinct phenotypes of MD (L873R, C1000R, N1304S, and A1362D) were analyzed in detail. All mutants studied displayed changes in protein expression and intracellular localization parallel to a dramatic decline in their copper-transporting capacity compared to ATP7A the wild-type. We restored these observed defects in ATP7A mutant proteins by culturing the cells at 30C, which improves the quality of protein folding, similar to that which as has recently has been demonstrated for misfolded ATP7B, a copper transporter homologous to ATP7A. Further, the effect of the canine copper toxicosis protein COMMD1 on ATP7A function was examined as COMMD1 has been shown to regulate the proteolysis of ATP7B proteins. Interestingly, in addition to adjusted growth temperature, binding of COMMD1 partially restored the expression, subcellular localization, and copper-exporting activities of the ATP7A mutants. However, no effect of pharmacological chaperones was observed. Together, the presented data might provide a new direction for developing therapies to improve the residual exporting activity of unstable ATP7A mutant proteins, and suggests a potential role for COMMD1 in this process. PMID:21667063

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

    PubMed

    Gabaev, Ildar; Steinbrck, 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

  5. Absence of IE1 p72 protein function during low-multiplicity infection by human cytomegalovirus results in a broad block to viral delayed-early gene expression.

    PubMed

    Gawn, Jonathan M; Greaves, Richard F

    2002-05-01

    Human cytomegalovirus (HCMV) ie1 deletion mutant CR208 is profoundly growth deficient after low-multiplicity infection of primary fibroblasts. Previously, we showed that many fewer cells infected with CR208 at low multiplicity accumulated the delayed-early (DE) protein ppUL44 than accumulated the immediate-early 2 (IE2) p86 protein, indicating a high frequency of abortive infections. We now demonstrate that accumulation of all DE proteins tested was defective after low-multiplicity infection in the absence of IE1 p72. Accumulation of the DE proteins pUL57, pUL98, and pUL69 followed a pattern very similar to that of ppUL44 during low-multiplicity CR208 infection. Accumulation of the ppUL112-113 proteins occurred in a greater proportion of cells than other DE proteins during low-multiplicity CR208 infection, but was still deficient relative to wild-type virus. We also show for the first time that steady-state levels of many DE RNAs were reduced during low-multiplicity CR208 infection and that by in situ hybridization of the abundant cytoplasmic 2.7-kb TRL4 DE (beta2.7) RNA, a viral DE RNA followed a defective pattern of accumulation similar to that of ppUL44. Furthermore, transfected DE promoter-reporter constructs were found in transient assays to be considerably less responsive to CR208 infection than to infection by wild-type Towne virus. Our results indicate a general defect in DE gene expression following low-multiplicity HCMV infection in the absence of functional IE1 p72, most probably mediated by reduced transcription of DE genes and by the reduced accumulation of DE RNAs. PMID:11932411

  6. Anti-depressant medication use and C-reactive protein: Results from two population-based studies

    PubMed Central

    Hamer, Mark; Batty, G. David; Marmot, Michael G.; Singh-Manoux, Archana; Kivimki, Mika

    2010-01-01

    The use of anti-depressant medication has been linked to cardiovascular disease (CVD). We examined the association between anti-depressant medication use and a marker of low grade systemic inflammation as a potential pathway linking anti-depressant use and CVD in two population based studies. Data were collected in a representative sample of 8,131 community dwelling adults (aged 47.4 15.9 yrs, 46.7% male) from the Scottish Health Surveys (SHS). The use of anti-depressant medication was coded according to the British National Formulary and blood was drawn for the measurement of C-reactive protein (CRP). In a second study, we attempted to replicate our findings using longitudinal data from the Whitehall II study (n=4584, aged 55.5 5.9 yrs, mean follow-up 5.5 years). Antidepressants were used in 5.6% of the SHS sample, with selective serotonin reuptake inhibitors (SSRIs) being the most common. There was a higher risk of elevated CRP (>3 mg/L) in users of tricyclic antidepressant (TCA) medication (multivariate adjusted odds ratio (OR) = 1.52, 95% CI, 1.07 2.15), but not in SSRI users (multivariate adjusted OR = 1.07, 95% CI, 0.81 1.42). A longitudinal association between any antidepressant use and subsequent CRP was confirmed in the Whitehall cohort. In summary, the use of anti-depressants was associated with elevated levels of systemic inflammation independently from the symptoms of mental illness and cardiovascular co-morbidity. This might be a potential mechanism through which antidepressant medication increases CVD risk. Further data are required to explore the effects of dosage and duration of antidepressant treatment. PMID:20863880

  7. 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 1610 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 (AFP) treatment.

  8. Allelic variants of the amylose extender mutation of maize demonstrate phenotypic variation in starch structure resulting from modified protein–protein interactions

    PubMed Central

    Liu, Fushan; Ahmed, Zaheer; Lee, Elizabeth A.; Donner, Elizabeth; Liu, Qiang; Ahmed, Regina; Morell, Matthew K.; Emes, Michael J.; Tetlow, Ian J.

    2012-01-01

    amylose extender (ae−) starches characteristically have modified starch granule morphology resulting from amylopectin with reduced branch frequency and longer glucan chains in clusters, caused by the loss of activity of the major starch branching enzyme (SBE), which in maize endosperm is SBEIIb. A recent study with ae− maize lacking the SBEIIb protein (termed ae1.1 herein) showed that novel protein–protein interactions between enzymes of starch biosynthesis in the amyloplast could explain the starch phenotype of the ae1.1 mutant. The present study examined an allelic variant of the ae− mutation, ae1.2, which expresses a catalytically inactive form of SBEIIb. The catalytically inactive SBEIIb in ae1.2 lacks a 28 amino acid peptide (Val272–Pro299) and is unable to bind to amylopectin. Analysis of starch from ae1.2 revealed altered granule morphology and physicochemical characteristics distinct from those of the ae1.1 mutant as well as the wild-type, including altered apparent amylose content and gelatinization properties. Starch from ae1.2 had fewer intermediate length glucan chains (degree of polymerization 16–20) than ae1.1. Biochemical analysis of ae1.2 showed that there were differences in the organization and assembly of protein complexes of starch biosynthetic enzymes in comparison with ae1.1 (and wild-type) amyloplasts, which were also reflected in the composition of starch granule-bound proteins. The formation of stromal protein complexes in the wild-type and ae1.2 was strongly enhanced by ATP, and broken by phosphatase treatment, indicating a role for protein phosphorylation in their assembly. Labelling experiments with [γ-32P]ATP showed that the inactive form of SBEIIb in ae1.2 was phosphorylated, both in the monomeric form and in association with starch synthase isoforms. Although the inactive SBEIIb was unable to bind starch directly, it was strongly associated with the starch granule, reinforcing the conclusion that its presence in the granules is a result of physical association with other enzymes of starch synthesis. In addition, an Mn2+-based affinity ligand, specific for phosphoproteins, was used to show that the granule-bound forms of SBEIIb in the wild-type and ae1.2 were phosphorylated, as was the granule-bound form of SBEI found in ae1.2 starch. The data strongly support the hypothesis that the complement of heteromeric complexes of proteins involved in amylopectin synthesis contributes to the fine structure and architecture of the starch granule. PMID:22121198

  9. 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

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

    PubMed

    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

  11. 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

  12. 31 CFR 30.4 - Q-4: What actions are necessary for a TARP recipient to comply with the standards established...

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... risk officers the SEO compensation plans to ensure that the SEO compensation plans do not encourage... fiscal year, provide a narrative description of how the SEO compensation plans do not encourage the SEOs... these SEO compensation plans do not encourage behavior focused on short-term results rather than...

  13. 31 CFR 30.4 - Q-4: What actions are necessary for a TARP recipient to comply with the standards established...

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... risk officers the SEO compensation plans to ensure that the SEO compensation plans do not encourage... fiscal year, provide a narrative description of how the SEO compensation plans do not encourage the SEOs... these SEO compensation plans do not encourage behavior focused on short-term results rather than...

  14. 31 CFR 30.4 - Q-4: What actions are necessary for a TARP recipient to comply with the standards established...

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... risk officers the SEO compensation plans to ensure that the SEO compensation plans do not encourage... fiscal year, provide a narrative description of how the SEO compensation plans do not encourage the SEOs... these SEO compensation plans do not encourage behavior focused on short-term results rather than...

  15. 31 CFR 30.4 - Q-4: What actions are necessary for a TARP recipient to comply with the standards established...

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... risk officers the SEO compensation plans to ensure that the SEO compensation plans do not encourage... fiscal year, provide a narrative description of how the SEO compensation plans do not encourage the SEOs... these SEO compensation plans do not encourage behavior focused on short-term results rather than...

  16. 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...

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

    PubMed Central

    Lesburg, Charles A.; Duca, Jos S.

    2008-01-01

    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

  18. Ectodermal dysplasia-skin fragility syndrome resulting from a new homozygous mutation, 888delC, in the desmosomal protein plakophilin 1.

    PubMed

    Ersoy-Evans, Sibel; Erkin, Gl; Fassihi, Hiva; Chan, Ien; Paller, Amy S; Src, Seluk; McGrath, John A

    2006-07-01

    We report an unusual case of an inherited disorder of the desmosomal protein plakophilin 1, resulting in ectodermal dysplasia-skin fragility syndrome. The affected 6-year-old boy had red skin at birth and subsequently developed skin fragility, progressive plantar keratoderma, nail dystrophy, and alopecia. Skin biopsy revealed widening of intercellular spaces in the epidermis and a reduced number of small, poorly formed desmosomes. Mutation analysis of the plakophilin 1 gene PKP1 revealed a homozygous deletion of C at nucleotide 888 within exon 5. This mutation differs from the PKP1 gene pathology reported in 8 previously published individuals with this rare genodermatosis. However, all cases show similar clinical features, highlighting the importance of functional plakophilin 1 in maintaining desmosomal adhesion in skin, as well as the role of this protein in aspects of ectodermal development. PMID:16781314

  19. Depletion of the RNA-Binding Protein RBP33 Results in Increased Expression of Silenced RNA Polymerase II Transcripts in Trypanosoma brucei

    PubMed Central

    Fernndez-Moya, Sandra M.; Carrington, Mark; Estvez, Antonio M.

    2014-01-01

    We have characterized the RNA-binding protein RBP33 in Trypanosoma brucei, and found that it localizes to the nucleus and is essential for viability. The subset of RNAs bound to RBP33 was determined by immunoprecipitation of ribonucleoprotein complexes followed by deep sequencing. Most RBP33-bound transcripts are predicted to be non-coding. Among these, over one-third are located close to the end of transcriptional units (TUs) or have an antisense orientation within a TU. Depletion of RBP33 resulted in an increase in the level of RNAs derived from regions that are normally silenced, such as strand-switch regions, retroposon and repeat sequences. Our work provides the first example of an RNA-binding protein involved in the regulation of gene silencing in trypanosomes. PMID:25215501

  20. 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.

  1. Lack of dystrophin protein Dp71 results in progressive cataract formation due to loss of fiber cell organization

    PubMed Central

    Darche, Marie; Sahel, Jos-Alain; Rendon, Alvaro; Tadayoni, Ramin

    2014-01-01

    Purpose Dp71 is the main product of the Duchenne muscular dystrophy (DMD) gene in the central nervous system. While studying the impact of its absence on retinal functions, we discovered that mice lacking Dp71 also developed a progressive opacification of the crystalline lens. The purpose of this study was to perform a detailed characterization of the cataract formation in Dp71 knockout (KO-Dp71) mice. Methods Cataract formations in KO-Dp71 mice and wild-type (wt) littermates were assessed in vivo by slit-lamp examination and ex vivo by histological analysis as a function of aging. The expression and cellular localization of the DMD gene products were monitored by western blot and immunohistochemical analysis. Fiber cell integrity was assessed by analyzing the actin cytoskeleton as well as the expression of aquaporin-0 (AQP0). Results As expected, a slit-lamp examination revealed that only one of the 20 tested wt animals presented with a mild opacification of the lens and only at the most advanced age. However, a lack of Dp71 was associated with a 40% incidence of cataracts as early as 2 months of age, which progressively increased to full penetrance by 7 months. A subsequent histological analysis revealed an alteration in the structures of the lenses of KO-Dp71 mice that correlated with the severity of the lens opacity. An analysis of the expression of the different dystrophin gene products revealed that Dp71 was the major DMD gene product expressed in the lens, especially in fiber cells. The role of Dp71 in fiber cells was also suggested by the progressive disorganization of the lens fibers, which was observed in the absence of Dp71 and demonstrated by irregular staining of the actin network and the aqueous channel AQP0. Conclusions While its role in the retina has been well characterized, this study demonstrates for the first time the role played by Dp71 in a different ocular tissue: the crystalline lens. It primarily demonstrates the role that Dp71 plays in the maintenance of the integrity of the secondary lens fibers. PMID:25489223

  2. 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.

  3. Epigenetic and oncogenic regulation of SLC16A7 (MCT2) results in protein over-expression, impacting on signalling and cellular phenotypes in prostate cancer

    PubMed Central

    Pertega-Gomes, Nelma; Vizcaino, Jose R.; Felisbino, Sergio; Warren, Anne Y.; Shaw, Greg; Kay, Jonathan; Whitaker, Hayley; Lynch, Andy G.

    2015-01-01

    Monocarboxylate Transporter 2 (MCT2) is a major pyruvate transporter encoded by the SLC16A7 gene. Recent studies pointed to a consistent overexpression of MCT2 in prostate cancer (PCa) suggesting MCT2 as a putative biomarker and molecular target. Despite the importance of this observation the mechanisms involved in MCT2 regulation are unknown. Through an integrative analysis we have discovered that selective demethylation of an internal SLC16A7/MCT2 promoter is a recurrent event in independent PCa cohorts. This demethylation is associated with expression of isoforms differing only in 5?-UTR translational control motifs, providing one contributing mechanism for MCT2 protein overexpression in PCa. Genes co-expressed with SLC16A7/MCT2 also clustered in oncogenic-related pathways and effectors of these signalling pathways were found to bind at the SLC16A7/MCT2 gene locus. Finally, MCT2 knock-down attenuated the growth of PCa cells. The present study unveils an unexpected epigenetic regulation of SLC16A7/MCT2 isoforms and identifies a link between SLC16A7/MCT2, Androgen Receptor (AR), ETS-related gene (ERG) and other oncogenic pathways in PCa. These results underscore the importance of combining data from epigenetic, transcriptomic and protein level changes to allow more comprehensive insights into the mechanisms underlying protein expression, that in our case provide additional weight to MCT2 as a candidate biomarker and molecular target in PCa. PMID:26035357

  4. Epigenetic and oncogenic regulation of SLC16A7 (MCT2) results in protein over-expression, impacting on signalling and cellular phenotypes in prostate cancer.

    PubMed

    Pertega-Gomes, Nelma; Vizcaino, Jose R; Felisbino, Sergio; Warren, Anne Y; Shaw, Greg; Kay, Jonathan; Whitaker, Hayley; Lynch, Andy G; Fryer, Lee; Neal, David E; Massie, Charles E

    2015-08-28

    Monocarboxylate Transporter 2 (MCT2) is a major pyruvate transporter encoded by the SLC16A7 gene. Recent studies pointed to a consistent overexpression of MCT2 in prostate cancer (PCa) suggesting MCT2 as a putative biomarker and molecular target. Despite the importance of this observation the mechanisms involved in MCT2 regulation are unknown. Through an integrative analysis we have discovered that selective demethylation of an internal SLC16A7/MCT2 promoter is a recurrent event in independent PCa cohorts. This demethylation is associated with expression of isoforms differing only in 5'-UTR translational control motifs, providing one contributing mechanism for MCT2 protein overexpression in PCa. Genes co-expressed with SLC16A7/MCT2 also clustered in oncogenic-related pathways and effectors of these signalling pathways were found to bind at the SLC16A7/MCT2 gene locus. Finally, MCT2 knock-down attenuated the growth of PCa cells. The present study unveils an unexpected epigenetic regulation of SLC16A7/MCT2 isoforms and identifies a link between SLC16A7/MCT2, Androgen Receptor (AR), ETS-related gene (ERG) and other oncogenic pathways in PCa. These results underscore the importance of combining data from epigenetic, transcriptomic and protein level changes to allow more comprehensive insights into the mechanisms underlying protein expression, that in our case provide additional weight to MCT2 as a candidate biomarker and molecular target in PCa. PMID:26035357

  5. Switching an anti-IgG binding site between archaeal extremophilic proteins results in Affitins with enhanced pH stability.

    PubMed

    Bhar, Ghislaine; Pacheco, Sabino; Maillasson, Mike; Mouratou, Barbara; Pecorari, Frdric

    2014-12-20

    As a useful reagent for biotechnological applications, a scaffold protein needs to be as stable as possible to ensure longer lifetimes. We have developed archaeal extremophilic proteins from the 7 kDa DNA-binding family as scaffolds to derive affinity proteins (Affitins). In this study, we evaluated a rational structure/sequence-guided approach to stabilize an Affitin derived from Sac7d by transferring its human IgG binding site onto the framework of the more thermally stable Sso7d homolog. The chimera obtained was functional, well expressed in Escherichia coli, but less thermally stable than the original Affitin (T(m) = 74.2 C vs. T(m) = 80.4 C). Two single mutations described as thermally stabilizing wild type Sso7d were introduced into chimeras. Only the double mutation nearly restored thermal stability (T(m) = 76.9 C). Interestingly, the chimera and its double mutant were stable from pH 0 up to at least pH 13. Our results show that it is possible to increase further the stability of Affitins toward alkaline conditions (+2 pH units) while conserving their advantageous properties. As Affitins are based on a growing family of homologs from archaeal extremophiles, we conclude that this approach offers new potential for their improvement, which will be useful in demanding biotechnological applications. PMID:25450641

  6. Deletion mutagenesis within the dimerization initiation site of human immunodeficiency virus type 1 results in delayed processing of the p2 peptide from precursor proteins.

    PubMed

    Liang, C; Rong, L; Cherry, E; Kleiman, L; Laughrea, M; Wainberg, M A

    1999-07-01

    Previous work has shown that deletions of genomic segments at nucleotide (nt) positions +238 to +253, i.e., construct BH10-LD3, or nt positions +261 to +274, i.e., construct BH10-LD4, within the human immunodeficiency virus type 1 (HIV-1) dimerization initiation site (DIS) destroyed DIS secondary structure and dramatically reduced viral replication capacity. Surprisingly, two point mutations located within the viral peptide 2 (p2) and nucleocapsid (NC) protein termed MP2 and MNC, respectively, were able to compensate for this defect. Since the MP2 mutation involves an amino acid substitution near the cleavage site between p2 and NC, we investigated the effects of the above-mentioned deletions on the processing of Gag proteins. Immunoprecipitation assays performed with monoclonal antibodies against viral capsid (CA) (p24) protein showed that p2 was cleaved from CA with less efficiency in viruses that contained the LD3 and LD4 deletions than in wild-type viruses. The presence of the two compensatory mutations, MP2 and MNC, increased the efficiency of the cleavage of p2 from CA, but neither mutation alone had this effect or was sufficient to compensate for the observed impairment in infectiousness. A virus that contained both of the above-mentioned deletions within the DIS was also impaired in regard to processing and infectiousness, and it could likewise be compensated by the MP2 and MNC point mutations. These results suggest that the DIS region of HIV-1 RNA plays an important role in the processing of Gag proteins. PMID:10364374

  7. 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 within 2 of the crystallographic one can show dramatic differences in calculated relative protein-ligand energies. MM-GBSA rescoring of distinct poses overcomes some of the sensitivities of pose ranking experienced by the docking scoring functions due to protein preparation and binding site definition. PMID:25266271

  8. Genetic Deletion of Rnd3/RhoE Results in Mouse Heart Calcium Leakage Through Upregulation of Protein Kinase A Signaling

    PubMed Central

    Lin, Xi; Yue, Xiaojing; Wang, Qiongling; Wang, Guoliang; Fu, Qin; Ai, Xun; Chiang, David Y.; Miyake, Christina Y.; Wehrens, Xander H.T.; Chang, Jiang

    2014-01-01

    Rationale Rnd3, a small Rho GTPase, is involved in the regulation of cell actin cytoskeleton dynamics, cell migration, and proliferation. The biological function of Rnd3 in the heart remains unexplored. Objective To define the functional role of the Rnd3 gene in the animal heart and investigate the associated molecular mechanism. Methods and Results By loss-of-function approaches, we discovered that Rnd3 is involved in calcium regulation in cardiomyocytes. Rnd3-null mice died at the embryonic stage with fetal arrhythmias. The deletion of Rnd3 resulted in severe Ca2+ leakage through destabilized ryanodine receptor type 2 (RyR2) Ca2+ release channels. We further found that downregulation of Rnd3 attenuated ?2-adrenergic receptor (?2AR) lysosomal targeting and ubiquitination, which in turn resulted in the elevation of ?2AR protein levels leading to the hyperactivation of protein kinase A (PKA) signaling. The PKA activation destabilized RyR2 channels. This irregular spontaneous Ca2+ release can be curtailed by PKA inhibitor treatment. Increases in the PKA activity along with elevated cyclic adenosine monophosphate (cAMP) levels were detected in Rnd3-null embryos, in neonatal rat cardiomyocytes, and non-cardiac cell lines with Rnd3 knockdown, suggesting a general mechanism for Rnd3-mediated PKA signaling activation. ?2AR blocker treatment reduced arrhythmia and improved cardiac function. Conclusion Rnd3 is a novel factor involved in intracellular Ca2+ homeostasis regulation in the heart. Deficiency of the protein induces RyR2 dysfunction by a mechanism that attenuates Rnd3-mediated ?2AR ubiquitination, which leads to the activation of PKA signaling. Increased PKA signaling in turn promotes RyR2 hyperphosphorylation, which contributes to arrhythmogenesis and heart failure. PMID:25348166

  9. In vivo particle polymorphism results from deletion of a N-terminal peptide molecular switch in brome mosaic virus capsid protein.

    PubMed

    Calhoun, Shauni L; Speir, Jeffrey A; Rao, A L N

    2007-08-01

    The interaction between brome mosaic virus (BMV) coat protein (CP) and viral RNA is a carefully orchestrated process resulting in the formation of homogeneous population of infectious virions with T=3 symmetry. Expression in vivo of either wild type or mutant BMV CP through homologous replication never results in the assembly of aberrant particles. In this study, we report that deletion of amino acid residues 41-47 from the N-proximal region of BMV CP resulted in the assembly of polymorphic virions in vivo. Purified virions from symptomatic leaves remain non-infectious and Northern blot analysis of virion RNA displayed packaging defects. Biochemical characterization of variant CP by circular dichroism and MALDI-TOF, respectively, revealed that the engineered deletion affected the protein structure and capsid dynamics. Most significantly, CP subunits dissociated from polymorphic virions are incompetent for in vitro reassembly. Based on these observations, we propose a chaperon-mediated mechanism for the assembly of variant CP in vivo and also hypothesize that (41)KAIKAIA(47) N-proximal peptide functions as a molecular switch in regulating T=3 virion symmetry. PMID:17449079

  10. Results of a Phase II clinical trial with Id-protein-loaded dendritic cell vaccine in multiple myeloma: encouraging or discouraging?

    PubMed

    Garcia-Marquez, Maria A; Wennhold, Kerstin; Draube, Andreas; von Bergwelt-Baildon, Michael

    2012-10-01

    Recently gained insight into the role of dendritic cells (DCs) as APCs has attracted the attention of many researchers who hope to use them as a tool in immunotherapy for the induction of tumor-specific immunity in cancer settings. Despite high expectations, in multiple myeloma patients the results of DC-based vaccines in terms of clinical response have been disappointing. The findings of Zahradova et al. in a Phase II clinical trial with multiple myeloma patients corroborated these results. Although no clinical responses were observed, the investigators induced immunity after vaccination with Id-protein-loaded DC vaccine in some patients. These immunological results showed a trend towards a longer duration of stable disease in those patients that received the vaccination. Moreover, this study showed that Id-protein-loaded DC vaccines are safe and nontoxic and that they are able to induce immunity in some patients. Therefore, standardization of vaccination protocols appears to be the key to achieving a better clinical outcome. PMID:23148751

  11. Elimination of high-light-inducible polypeptides related to eukaryotic chlorophyll a/b-binding proteins results in aberrant photoacclimation in Synechocystis PCC6803.

    PubMed

    Havaux, Michel; Guedeney, Genevive; He, Qingfang; Grossman, Arthur R

    2003-03-01

    The hli genes, present in cyanobacteria, algae and vascular plants, encode small proteins [high-light-inducible polypeptides (HLIPs)] with a single membrane-spanning alpha-helix related to the first and third helices of eukaryotic chlorophyll a/b-binding proteins. The HLIPs are present in low amounts in low light and they accumulate transiently at high light intensities. We are investigating the function of those polypeptides in a Synechocystis PCC6803 mutant lacking four of the five hli genes. Growth of the quadruple hli mutant was adversely affected by high light intensities. The most striking effect of the quadruple hli mutation was an alteration of cell pigmentation. Pigment changes associated with cell acclimation to increasing light intensity [i.e. decrease in light-harvesting pigments, accumulation of the carotenoid myxoxanthophyll and decrease in photosystem I (PSI)-associated chlorophylls] were strongly exacerbated in the quadruple hli mutant, resulting in yellowish cultures that bleached in high light and died as light intensities exceeded (>500 micromol photon m(-2) s(-1)). However, these pigment changes were not associated with an inhibition of photosynthesis, as probed by in vivo chlorophyll fluorescence, photoacoustic and O(2)-evolution measurements. On the contrary, the HLIP deficiency was accompanied by a stimulation of the photochemical activity, especially in high-light-grown cells. Western blot analyses revealed that the PSI reaction center level (PsaA/B) was noticeably reduced in the quadruple hli mutant relative to the wild type, whereas the abundance of the PSII reaction center protein D1 was comparatively little affected. The hli mutations did not enhance photoinhibition and photooxidation when cells were exposed over a short term to a very high light intensity. Together, the results of this study indicate that HLIPs are critical in the adaptation of the cyanobacterium to variations in light intensity. The data are consistent with the idea that HLIPs are involved, through a direct or indirect means, in nonphotochemical dissipation of absorbed light energy. PMID:12615345

  12. The G protein-coupled estrogen receptor-1, GPER-1, promotes fibrillogenesis via a Shc-dependent pathway resulting in anchorage-independent growth.

    PubMed

    Magruder, Hilary T; Quinn, Jeffrey A; Schwartzbauer, Jean E; Reichner, Jonathan; Huang, Allan; Filardo, Edward J

    2014-12-01

    The G protein-coupled estrogen receptor-1, GPER-1, coordinates fibronectin (FN) matrix assembly and release of heparan-bound epidermal growth factor (HB-EGF). This mechanism of action results in the recruitment of FN-engaged integrin ?5?1 to fibrillar adhesions and the formation of integrin ?5?1-Shc adaptor protein complexes. Here, we show that GPER-1 stimulation of murine 4T1 or human SKBR3 breast cancer cells with 17?-estradiol (E2?) promotes the formation of focal adhesions and actin stress fibers and results in increased cellular adhesion and haptotaxis on FN, but not collagen. These actions are also induced by the xenoestrogen, bisphenol A, and the estrogen receptor (ER) antagonist, ICI 182, 780, but not the inactive stereoisomer, 17?-estradiol (E2?). In addition, we show that GPER-1 stimulation of breast cancer cells allows for FN-dependent, anchorage-independent growth and FN fibril formation in "hanging drop" assays, indicating that these GPER-1-mediated actions occur independently of adhesion to solid substrata. Stable expression of Shc mutant Y317F lacking its primary tyrosyl phosphorylation site disrupts E2?-induced focal adhesion and actin stress fiber formation and abolishes E2?-enhanced haptotaxis on FN and anchorage-dependent growth. Collectively, these data demonstrate that E2? action via GPER-1 enhances cellular adhesivity and FN matrix assembly and allows for anchorage-independent growth, cellular events that may allow for cellular survival, and tumor progression. PMID:25096985

  13. Loss of the acyl-CoA binding protein (Acbp) results in fatty acid metabolism abnormalities in mouse hair and skin

    PubMed Central

    Lee, Lance; DeBono, C. Anthony; Campagna, Dean R.; Young, David C.; Moody, D. Branch; Fleming, Mark D.

    2007-01-01

    Proper fatty acid metabolism is critical for hair and skin development and maintenance. The acyl-CoA binding protein (Acbp) is a widely expressed protein that binds long-chain fatty acyl-CoA esters and plays a role in fatty acyl-CoA transport and pool formation. However, loss of function of Acbp in the whole animal has not been investigated. Here, we show that deletion of Acbp in mouse results in sebocyte hyperplasia and sparse, matted hair with a greasy appearance. Consistent with these gross abnormalities, Acbp is highly expressed in the pilosebaceous units of mouse skin as determined by northern analysis and in situ hybridization. Loss of Acbp also results in fatty acid metabolism abnormalities, with hair lipid profiles showing altered levels of triacylglycerols and nearly co-migrating lipids. These data suggest that Acbp plays a role in triacylglycerol biosynthesis and that regulation of this process is important for proper hair and skin development and maintenance in the mouse. PMID:16902415

  14. Sugared water consumption by adult offspring of mothers fed a protein-restricted diet during pregnancy results in increased offspring adiposity: the second hit effect.

    PubMed

    Cervantes-Rodrguez, M; Martnez-Gmez, M; Cuevas, E; Nicols, L; Casteln, F; Nathanielsz, P W; Zambrano, E; Rodrguez-Antoln, J

    2014-02-01

    Poor maternal nutrition predisposes offspring to metabolic disease. This predisposition is modified by various postnatal factors. We hypothesised that coupled to the initial effects of developmental programming due to a maternal low-protein diet, a second hit resulting from increased offspring postnatal sugar consumption would lead to additional changes in metabolism and adipose tissue function. The objective of the present study was to determine the effects of sugared water consumption (5% sucrose in the drinking-water) on adult offspring adiposity as a 'second hit' following exposure to maternal protein restriction during pregnancy. We studied four offspring groups: (1) offspring of mothers fed the control diet (C); (2) offspring of mothers fed the restricted protein diet (R); (3) offspring of control mothers that drank sugared water (C-S); (4) offspring of restricted mothers that drank sugared water (R-S). Maternal diet in pregnancy was considered the first factor and sugared water consumption as the second factor - the second hit. Body weight and total energy consumption, before and after sugared water consumption, were similar in all the groups. Sugared water consumption increased TAG, insulin and cholesterol concentrations in both the sexes of the C-S and R-S offspring. Sugared water consumption increased leptin concentrations in the R-S females and males but not in the R offspring. There was also an interaction between sugared water and maternal diet in males. Sugared water consumption increased adipocyte size and adiposity index in both females and males, but the interaction with maternal diet was observed only in females. Adiposity index and plasma leptin concentrations were positively correlated in both the sexes. The present study shows that a second hit during adulthood can amplify the effects of higher adiposity arising due to poor maternal pregnancy diet in an offspring sex dependent fashion. PMID:24124655

  15. A Spontaneous Deletion within the Desmoglein 3 Extracellular Domain of Mice Results in Hypomorphic Protein Expression, Immunodeficiency, and a Wasting Disease Phenotype

    PubMed Central

    Kountikov, Evgueni I.; Poe, Jonathan C.; Maclver, Nancie J.; Rathmell, Jeffrey C.; Tedder, Thomas F.

    2016-01-01

    Desmoglein 3 is a transmembrane component of desmosome complexes that mediate epidermal cell-to-cell adhesion and tissue integrity. Antibody blockade of desmoglein 3 function in pemphigus vulgaris patients leads to skin blistering (acantholysis) and oral mucosa lesions. Desmoglein 3 deficiency in mice leads to a phenotype characterized by cyclic alopecia in addition to the dramatic skin and mucocutaneous acantholysis observed in pemphigus patients. In this study, mice that developed an overt squeaky (sqk) phenotype were identified with obstructed airways, cyclic hair loss, and severe immunodeficiency subsequent to the development of oral lesions and malnutrition. Single-nucleotide polymorphism–based quantitative trait loci mapping revealed a genetic deletion that resulted in expression of a hypomorphic desmoglein 3 protein with a truncation of an extracellular cadherin domain. Because hypomorphic expression of a truncated desmoglein 3 protein led to a spectrum of severe pathology not observed in mice deficient in desmoglein 3, similar human genetic alterations may also disrupt desmosome function and induce a disease course distinct from pathogenesis of pemphigus vulgaris. PMID:25542773

  16. Expression of human amyloid precursor protein in the skeletal muscles of Drosophila results in age- and activity-dependent muscle weakness

    PubMed Central

    2011-01-01

    Background One of the hallmarks of Alzheimer's disease, and several other degenerative disorders such as Inclusion Body Myositis, is the abnormal accumulation of amyloid precursor protein (APP) and its proteolytic amyloid peptides. To better understand the pathological consequences of inappropriate APP expression on developing tissues, we generated transgenic flies that express wild-type human APP in the skeletal muscles, and then performed anatomical, electrophysiological, and behavioral analysis of the adults. Results We observed that neither muscle development nor animal longevity was compromised in these transgenic animals. However, human APP expressing adults developed age-dependent defects in both climbing and flying. We could advance or retard the onset of symptoms by rearing animals in vials with different surface properties, suggesting that human APP expression-mediated behavioral defects are influenced by muscle activity. Muscles from transgenic animals did not display protein aggregates or structural abnormalities at the light or transmission electron microscopic levels. In agreement with genetic studies performed with developing mammalian myoblasts, we observed that co-expression of the ubiquitin E3 ligase Parkin could ameliorate human APP-induced defects. Conclusions These data suggest that: 1) ectopic expression of human APP in fruit flies leads to age- and activity-dependent behavioral defects without overt changes to muscle development or structure; 2) environmental influences can greatly alter the phenotypic consequences of human APP toxicity; and 3) genetic modifiers of APP-induced pathology can be identified and analyzed in this model. PMID:21518451

  17. 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...

  18. Solid-state synthesis, structure and properties of a novel open-framework cadmium selenite bromide: [Cd10(SeO3)8Br4]HBrH2O

    NASA Astrophysics Data System (ADS)

    Chen, Wen-Tong; Wang, Ming-Sheng; Wang, Guan-E.; Chen, Hui-Fen; Guo, Guo-Cong

    2013-08-01

    A novel open-framework cadmium selenite bromide, [Cd10(SeO3)8Br4]HBrH2O (1), has been obtained by a solid-state reaction at 450 C, and the structure has been determined by single-crystal X-ray diffraction analysis. Compound 1 crystallizes in Pbcm of the orthorhombic system: a=10.882(3), b=16.275(5), c=18.728(6) , V=3317(2) 3, R1/wR2=0.0411/0.0659. Compound 1 is characteristic of a novel 3-D open-framework structure, composing ?2[CdSeO3] layers and the pillars of edge-shared CdO3Br2 square pyramids. The lattice water molecules and the HBr molecules locate in the voids of the framework. Optical absorption spectrum of 1 reveals the presence of an optical gap of 1.65 eV. Solid-state photoluminescent study indicates that compound 1 exhibits strong violet emission. TG-DSC measurement shows that compound 1 is thermally stable up to 200 C.

  19. Secondary structure and shape of plasma sex steroid-binding protein--comparison with domain G of laminin results in a structural model of plasma sex steroid-binding protein.

    PubMed

    Beck, K; Gruber, T M; Ridgway, C C; Hughes, W; Sui, L; Ptra, P H

    1997-07-01

    We have analyzed the secondary structure, shape and dimensions of plasma sex steroid-binding protein (SBP) by CD, size-exclusion chromatography and electron microscopy. CD spectra show extrema at 186 nm and 216 nm characteristic for beta-sheet structures. Analysis with different algorithms indicates 15% alpha-helix, 43% beta-sheet and 10-16% beta-turn structures. An irreversible structural change is observed upon heating above 60 degrees C, which correlates with the loss of steroid-binding activity. As the SBP sequence shows similarity with domains of several multidomain proteins, including laminins, we evaluated the structure of domain G of laminin-1. The CD spectrum shows extrema at 200 nm and 216 nm. Deconvolution results in 13% alpha-helix, 32% beta-sheet and 15% beta-turn structures. Steroid-binding assays indicate that laminin and fragments thereof have no activity. Size-exclusion chromatography reveals that SBP has an extended shape and can be modeled as a cylinder with a length and diameter of 23 nm and 3 nm, respectively. This shape and the dimensions are in agreement with the appearance on electron micrographs. We propose a model for the structure of SBP in which two monomers assemble head to head with the steroid-binding site located in the center of the rod-like particle. PMID:9249045

  20. Heat shock protein 70 and nitric oxide concentrations in non-tumorous and neoplastic canine mammary tissues: preliminary results - Short communication.

    PubMed

    Szczubia?, Marek; Urban-Chmiel, Renata; ?opuszy?ski, Wojciech

    2015-06-01

    The concentrations of heat shock protein 70 (Hsp70) and nitric oxide ions (NO), measured as nitrite, were determined in canine mammary tumours and nontumorous mammary gland tissues. The concentrations of Hsp70 and NO were significantly higher in both benign and malignant tumours than in non-tumorous mammary tissues. Hsp70 concentration decreased with the increase in the grade of histological malignancy. A strong positive correlation was found between the concentrations of Hsp70 and NO in the benign tumours as well as in grade I and grade II malignant tumours. The results indicate that the process of neoplastic transformation in the canine mammary gland is related to a significant increase in Hsp70 and NO concentration in tumour tissues, and an interdependence between Hsp70 and nitrite ion production can be observed. PMID:26051259

  1. RNA Interference of Odorant-Binding Protein 2 (OBP2) of the Cotton Aphid, Aphis gossypii (Glover), Resulted in Altered Electrophysiological Responses.

    PubMed

    Rebijith, K B; Asokan, R; Hande, H Ranjitha; Kumar, N K Krishna; Krishna, V; Vinutha, J; Bakthavatsalam, N

    2016-01-01

    Aphis gossypii (Glover) (Hemiptera: Aphididae) is a highly invasive pest that feeds primarily on phloem resulting in severe economic loss to growers. A. gossypii has cosmopolitan distribution with broad host range, polyphenism, parthenogenetic mode of reproduction, vectoring abilities, and host alteration which has profound influence on its management. Odorant-binding proteins (OBPs) in insects are involved in olfaction, playing a key role in orienting the insect for feeding or oviposition. Recent studies revealed that OBP2 is found in both sensilla trichodea and sensilla basiconica and is preferentially binds to plant volatiles, thus playing crucial roles in host-seeking, detection of oviposition attractants, etc., However, information about the role of OBP2 in A. gossypii (AgOBP2) is still unavailable. In this study, we cloned and characterized OBP2, ortholog from A. gossypii, and the full-length AgOBP2 complementary DNA (cDNA) consisted of 859 bp with an open reading frame of 732 bp. Phylogenetic analysis resulted in grouping of AgOBP2 protein with members of the tribe Aphidini. Further, diet-mediated delivery of double-stranded RNA for AgOBP2 induced silencing, which was evaluated at 48 and 96 h. The reverse transcriptase real-time quantitative polymerase chain reaction (RTq-PCR) results revealed that the level of AgOBP2 messenger RNA (mRNA) was significantly reduced (55-77 %) in dsAgOBP2 treatment after 96 h as compared to the untreated control. The same was reiterated by the electrophysiological responses in the aphids which was reduced (>50 % at 0.25 μg/μl concentration) as compared to the untreated control. Thus, our results showed the potential of gene silencing, possibly to interfere with the odorant perception of A. gossypii for RNAi-mediated pest management. The results from our study provided the first evidence that AgOBP2 play crucial roles in host-seeking, detection of oviposition attractants, etc.; as a result, we suggests that OBP2 could potentially serve as a practicable target for RNAi-mediated gene silencing in hemipteran insect pest control. PMID:26432291

  2. Deletion of Hemojuvelin, an Iron-Regulatory Protein, in Mice Results in Abnormal Angiogenesis and Vasculogenesis in Retina Along With Reactive Gliosis

    PubMed Central

    Tawfik, Amany; Gnana-Prakasam, Jaya P.; Smith, Sylvia B.; Ganapathy, Vadivel

    2014-01-01

    Purpose. Loss-of-function mutations in hemojuvelin (HJV) cause juvenile hemochromatosis, an iron-overload disease. Deletion of Hjv in mice results in excessive iron accumulation and morphologic changes in the retina. Here, we studied the retinal vasculature in Hjv?/? mice. Methods. Age-matched wild-type and Hjv?/? mice were used for fluorescein angiography and preparation of retinal cryosections, flat-mounts, and trypsin-digested blood vessels. Retinal angiogenesis was monitored by immunofluorescent detection of isolectin-B4, endoglin, and VEGF. Retinal vasculogenesis was monitored by immunofluorescent detection of collagen IV. Reactive gliosis was assessed based on the expression of glial fibrillary acidic protein and vimentin and CD11b/c as markers for Mller cells and microglia. Results. Between 18 and 24 months of age, retinas of Hjv?/? mice displayed marked disruptions in angiogenesis and vasculogenesis. Blood vessels in Hjv?/? mice were tortuous and dilated, with a decrease in the tight-junction protein occludin. There was also evidence of neovascularization in Hjv?/? mice with blood vessels appearing in the vitreous, which were leaky. There was reactive gliosis in these mice involving both Mller cells and microglia. Such changes were not detected at 2 weeks of age. Even at the age of 4 months, retinas of Hjv?/? mice were almost normal with changes just beginning to appear. Thus, the vascular changes in Hjv?/? mouse retinas represent an age-dependent phenomenon. Conclusions. Deletion of Hjv in mice leads to abnormal retinal angiogenesis/vasculogenesis, with proliferation of new, leaky blood vessels in the vitreous. These changes are accompanied with reactive gliosis involving Mller cells and microglia. PMID:24812553

  3. Immune sensitization to methylene diphenyl diisocyanate (MDI) resulting from skin exposure: albumin as a carrier protein connecting skin exposure to subsequent respiratory responses

    PubMed Central

    2011-01-01

    Background Methylene diphenyl diisocyanate (MDI), a reactive chemical used for commercial polyurethane production, is a well-recognized cause of occupational asthma. The major focus of disease prevention efforts to date has been respiratory tract exposure; however, skin exposure may also be an important route for inducing immune sensitization, which may promote subsequent airway inflammatory responses. We developed a murine model to investigate pathogenic mechanisms by which MDI skin exposure might promote subsequent immune responses, including respiratory tract inflammation. Methods Mice exposed via the skin to varying doses (0.1-10% w/v) of MDI diluted in acetone/olive oil were subsequently evaluated for MDI immune sensitization. Serum levels of MDI-specific IgG and IgE were measured by enzyme-linked immunosorbant assay (ELISA), while respiratory tract inflammation, induced by intranasal delivery of MDI-mouse albumin conjugates, was evaluated based on bronchoalveolar lavage (BAL). Autologous serum IgG from "skin only" exposed mice was used to detect and guide the purification/identification of skin proteins antigenically modified by MDI exposure in vivo. Results Skin exposure to MDI resulted in specific antibody production and promoted subsequent respiratory tract inflammation in animals challenged intranasally with MDI-mouse albumin conjugates. The degree of (secondary) respiratory tract inflammation and eosinophilia depended upon the (primary) skin exposure dose, and was maximal in mice exposed to 1% MDI, but paradoxically limited in mice receiving 10-fold higher doses (e.g. 10% MDI). The major antigenically-modified protein at the local MDI skin exposure site was identified as albumin, and demonstrated biophysical changes consistent with MDI conjugation. Conclusions MDI skin exposure can induce MDI-specific immune sensitivity and promote subsequent respiratory tract inflammatory responses and thus, may play an important role in MDI asthma pathogenesis. MDI conjugation and antigenic modification of albumin at local (skin/respiratory tract) exposure sites may represent the common antigenic link connecting skin exposure to subsequent respiratory tract inflammation. PMID:21414210

  4. 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

    HUPO initiated the Plasma Proteome Project (PPP) in 2002. Its pilot phase has (1) evaluated advantages and limitations of many depletion, fractionation, and MS technology platforms; (2) compared PPP reference specimens of human serum and EDTA, heparin, and citrate-anticoagulated plasma; and (3) created a publicly-available knowledge base (www.bioinformatics. med.umich.edu/hupo/ppp; www.ebi.ac.uk/pride). Thirty-five participating laboratories in 13 countries submitted datasets. Working groups addressed (a) specimen stability and protein concentrations; (b) protein identifications from 18 MS/MS datasets; (c) independent analyses from raw MS-MS spectra; (d) search engine performance, subproteome analyses, and biological insights; (e) antibody arrays; and (f) direct MS/SELDI analyses. MS-MS datasets had 15 710 different International Protein Index (IPI) protein IDs; our integration algorithm applied to multiple matches of peptide sequences yielded 9504 IPI proteins identified with one or more peptides and 3020 proteins identified with two or more peptides (the Core Dataset). These proteins have been characterized with Gene Ontology, InterPro, Novartis Atlas, OMIM, and immunoassay based concentration determinations. The database permits examination of many other subsets, such as 1274 proteins identified with three or more peptides. Reverse protein to DNA matching identified proteins for 118 previously unidentified ORFs. We recommend use of plasma instead of serum, with EDTA (or citrate) for anticoagulation. To improve resolution, sensitivity and reproducibility of peptide identifications and protein matches, we recommend combinations of depletion, fractionation, and MS/MS technologies, with explicit criteria for evaluation of spectra, use of search algorithms, and integration of homologous protein matches. This Special Issue of PROTEOMICS presents papers integral to the collaborative analysis plus many reports of supplementary work on various aspects of the PPP workplan. 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.

  5. Immunization with a Lymphocytic Choriomeningitis Virus Peptide Mixed with Heat Shock Protein 70 Results in Protective Antiviral Immunity and Specific Cytotoxic T Lymphocytes

    PubMed Central

    Ciupitu, Anne-Marie T.; Petersson, Max; O'Donnell, Carey L.; Williams, Kevin; Jindal, Satish; Kiessling, Rolf; Welsh, Raymond M.

    1998-01-01

    Heat shock proteins (hsp's) isolated from murine cancer cells can elicit protective immunity and specific cytotoxic T lymphocytes (CTLs) by channeling tumor-derived peptides bound to hsp's to the major histocompatibility class I antigen presentation pathway. Here we have investigated if hsp70 can be used in a novel peptide vaccine for the induction of protective antiviral immunity and memory CTLs. A CTL epitope from the well-defined lymphocytic choriomeningitis virus (LCMV) system was mixed with recombinant hsp70 in vitro under conditions that optimize peptide binding to hsp70. Mice were immunized with the hsp70peptide mixture and challenged with LCMV. Virus titers were reduced 10100-fold in these mice compared to control mice. Immunization with the hsp70peptide mixture resulted in the development of CTL memory cells that could be reactivated during LCMV infection, and that in a 51Cr-release assay could lyse cells pulsed with the same peptide, but not cells pulsed with another LCMV peptide. These results show that hsp70 can be used with CTL epitopes to induce efficient protective antiviral immunity and the generation of peptide-specific CTLs. The results also demonstrate the usefulness of hsp70 as an alternative to adjuvants and DNA vectors for the delivery of CTL epitopes to antigen-presenting cells. PMID:9480978

  6. Reactive oxygen species derived from xanthine oxidase interrupt dimerization of breast cancer resistance protein, resulting in suppression of uric acid excretion to the intestinal lumen.

    PubMed

    Ogura, Jiro; Kuwayama, Kaori; Sasaki, Shunichi; Kaneko, Chihiro; Koizumi, Takahiro; Yabe, Keisuke; Tsujimoto, Takashi; Takeno, Reiko; Takaya, Atsushi; Kobayashi, Masaki; Yamaguchi, Hiroaki; Iseki, Ken

    2015-09-01

    The prevalence of hyperuricemia/gout increases with aging. However, the effect of aging on function for excretion of uric acid to out of the body has not been clarified. We found that ileal uric acid clearance in middle-aged rats (11-12 months) was decreased compared with that in young rats (2 months). In middle-aged rats, xanthine oxidase (XO) activity in the ileum was significantly higher than that in young rats. Inosine-induced reactive oxygen species (ROS), which are derived from XO, also decreased ileal uric acid clearance. ROS derived from XO decreased the active homodimer level of breast cancer resistance protein (BCRP), which is a uric acid efflux transporter, in the ileum. Pre-administration of allopurinol recovered the BCRP homodimer level, resulting in the recovering ileal uric acid clearance. Moreover, we investigated the effects of ROS derived from XO on BCRP homodimer level directly in Caco-2 cells using hypoxanthine. Treatment with hypoxanthine decreased BCRP homodimer level. Treatment with hypoxanthine induced mitochondrial dysfunction, suggesting that the decreasing BCRP homodimer level might be caused by mitochondrial dysfunction. In conclusion, ROS derived from XO decrease BCRP homodimer level, resulting in suppression of function for uric acid excretion to the ileal lumen. ROS derived from XO may cause the suppression of function of the ileum for the excretion of uric acid with aging. The results of our study provide a new insight into the causes of increasing hyperuricemia/gout prevalence with aging. PMID:26119820

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

    PubMed Central

    Sehnal, David; SvobodovVa?ekov, Radka; Pravda, Luk; Ionescu, Crina-Maria; Geidl, Stanislav; Horsk, Vladimr; Jaiswal, Deepti; Wimmerov, Michaela; Ko?a, 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

  8. 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...

  9. Downregulation of Cellular c-Jun N-Terminal Protein Kinase and NF-?B Activation by Berberine May Result in Inhibition of Herpes Simplex Virus Replication

    PubMed Central

    Song, Siwei; Qiu, Min; Chu, Ying; Chen, Deyan; Wang, Xiaohui; Su, Airong

    2014-01-01

    Berberine is a quaternary ammonium salt from the protoberberine group of isoquinoline alkaloids. Some reports show that berberine exhibits anti-inflammatory, antitumor, and antiviral properties by modulating multiple cellular signaling pathways, including p53, nuclear factor ?B (NF-?B), and mitogen-activated protein kinase. In the present study, we investigated the antiviral effect of berberine against herpes simplex virus (HSV) infection. Current antiherpes medicines such as acyclovir can lessen the recurring activation when used early at infection but are unable to prevent or cure infections where treatment has selected for resistant mutants. In searching for new antiviral agents against herpesvirus infection, we found that berberine reduced viral RNA transcription, protein synthesis, and virus titers in a dose-dependent manner. To elucidate the mechanism of its antiviral activity, the effect of berberine on the individual steps of viral replication cycle of HSV was investigated via time-of-drug addition assay. We found that berberine acted at the early stage of HSV replication cycle, between viral attachment/entry and genomic DNA replication, probably at the immediate-early gene expression stage. We further demonstrated that berberine significantly reduced HSV-induced NF-?B activation, as well as I?B-? degradation and p65 nuclear translocation. Moreover, we found that berberine also depressed HSV-induced c-Jun N-terminal kinase (JNK) phosphorylation but had little effect on p38 phosphorylation. Our results suggest that the berberine inhibition of HSV infection may be mediated through modulating cellular JNK and NF-?B pathways. PMID:24913175

  10. [Changing of filamentation dynamics of RecA protein, induced by D112R amino acid substitution or ATP to dATP replacement, results in filament steadiness TO THE RecX protein action].

    PubMed

    Dudkina, A V; Shvetsov, A V; Bakhlanova, I V; Ba?tin, D M

    2011-01-01

    It is known that RecX is a negative regulator of RecA protein. We found that the mutant RecA D112R protein exhibits increased resistance to RecX protein comparatively to wild-type RecA protein in vitro and in vivo. Using molecular modeling we showed, that amino acid located in position 112 can not approach RecX closer than 25-28 angstroms. Thus, direct contact between amino acid and RecX is impossible. RecA D112R protein more actively competes with SSB protein for the binding sites on ssDNA and, therefore, differs from the wild-type RecA protein by dynamics of filamentation on ssDNA. On the other hand, after the replacement of ATP by dATP, the wild-type RecA protein, changing the dynamics of filamentation on ssDNA, also becomes more resistant to RecX. Based on these data it is concluded that the dynamics of filamentation has a great, if not dominant role in the stability of RecA filament to RecX relative to the role of RecA-RecX protein-protein interactions discussed earlier. We also propose an improved model of regulation of RecA by RecX protein, where RecA filament elongation along ssDNA is blocked by RecX protein on the ssDNA region, located outside the filament. PMID:21790018

  11. Fusarochromanone-induced reactive oxygen species results in activation of JNK cascade and cell death by inhibiting protein phosphatases 2A and 5

    PubMed Central

    Gu, Ying; Barzegar, Mansoureh; Chen, Xin; Wu, Yang; Shang, Chaowei; Mahdavian, Elahe; Salvatore, Brian A.; Jiang, Shanxiang; Huang, Shile

    2015-01-01

    Recent studies have shown that fusarochromanone (FC101), a mycotoxin, is cytotoxic in a variety of cell lines. However, the molecular mechanism underlying its cytotoxicity remains elusive. Here we found that FC101 induced cell death in COS7 and HEK293 cells in part by activating JNK pathway. This is evidenced by the findings that inhibition of JNK with SP600125 or expression of dominant negative c-Jun partially prevented FC101-induced cell death. Furthermore, we observed that FC101-activated JNK pathway was attributed to induction of reactive oxygen species (ROS). Pretreatment with N-acetyl-L-cysteine (NAC), a ROS scavenger and antioxidant, suppressed FC101-induced activation of JNK and cell death. Moreover, we noticed that FC101 inhibited the serine/threonine protein phosphatases 2A (PP2A) and 5 (PP5) in the cells, which was abrogated by NAC. Overexpression of PP2A or PP5 partially prevented FC101-induced activation of JNK and cell death. The results indicate that FC101-induced ROS inhibits PP2A and PP5, leading to activation of JNK pathway and consequently resulting in cell death. PMID:26517353

  12. Dityrosine, 3,4-dihydroxyphenylalanine (DOPA), and radical formation from tyrosine residues on milk proteins with globular and flexible structures as a result of riboflavin-mediated photo-oxidation.

    PubMed

    Dalsgaard, Trine K; Nielsen, Jacob H; Brown, Bronwyn E; Stadler, Nadina; Davies, Michael J

    2011-07-27

    Riboflavin-mediated photo-oxidative damage to protein Tyr residues has been examined to determine whether protein structure influences competing protein oxidation pathways in single proteins and protein mixtures. EPR studies resulted in the detection of Tyr-derived o-semiquione radicals, with this species suggested to arise from oxidation of 3,4-dihydroxyphenylalanine (DOPA). The yield of this radical was lower in samples containing ?-casein than in samples containing only globular proteins. Consistent with this observation, the yield of DOPA detected on ?-casein was lower than that on two globular proteins, BSA and ?-lactoglobulin. In contrast, samples with ?-casein gave higher yields of dityrosine than samples containing BSA and ?-lactoglobulin. These results indicate that the flexible structure of ?-casein favors radical-radical termination of tyrosyl radicals to give dityrosine, whereas the less flexible structure of globular proteins decreases the propensity for tyrosyl radicals to dimerize, with this resulting in higher yields of DOPA and its secondary radical. PMID:21696221

  13. 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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Consumption of soy diet 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 ...

  14. Transfer of 15-lipoxygenase gene into rabbit iliac arteries results in the appearance of oxidation-specific lipid-protein adducts characteristic of oxidized low density lipoprotein.

    PubMed Central

    Yl-Herttuala, S; Luoma, J; Viita, H; Hiltunen, T; Sisto, T; Nikkari, T

    1995-01-01

    Oxidized low density lipoprotein (LDL) possesses several atherogenic properties. The mechanisms by which LDL becomes oxidized in vivo remain unknown, but previous studies have suggested that 15-lipoxygenase may be one of the factors involved in the initiation of LDL oxidation in the arterial wall. 3 wk after a retrovirus-mediated 15-lipoxygenase gene transfer into iliac arteries of normocholesterolemic rabbits there was a threefold increase in 15-lipoxygenase activity but no signs of LDL oxidation. However, when animals were made moderately hypercholesterolemic by feeding a 0.13% cholesterol diet for 2-3 wk starting from day 4 after the gene transfer, oxidation-specific lipid-protein adducts characteristic of oxidized LDL were detected in 15-lipoxygenase-transduced arteries. Control experiments in which contralateral iliac arteries were transduced with beta-galactosidase-containing retroviruses showed only occasional signs of the presence of oxidation-specific adducts. The results support the hypothesis that products derived from the 15-lipoxygenase activity are involved in the induction of LDL oxidation within the arterial wall, provided that sufficient concentrations of lipoproteins are present in the artery. Images PMID:7769108

  15. 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.

  16. A missense mutation in the transmembrane domain of CESA4 affects protein abundance in the plasma membrane and results in abnormal cell wall biosynthesis in rice.

    PubMed

    Zhang, Baocai; Deng, Lingwei; Qian, Qian; Xiong, Guangyan; Zeng, Dali; Li, Rui; Guo, Longbiao; Li, Jiayang; Zhou, Yihua

    2009-11-01

    Cellulose synthase (CESA) is a critical catalytic subunit of the cellulose synthase complex responsible for glucan chain elongation. Our knowledge about how CESA functions is still very limited. Here, we report the functional characterization of a rice mutant, brittle culm11, that shows growth retardation and dramatically reduced plant strength. Map-based cloning revealed that all the mutant phenotypes result from a missense mutation in OsCESA4 (G858R), a highly conserved residue at the end of the fifth transmembrane domain. The aberrant secondary cell wall of the mutant plants is attributed to significantly reduced cellulose content, abnormal secondary wall structure of sclerenchyma cells, and overall altered wall composition, as detected by chemical analyses and immunochemical staining. Importantly, we have found that this point mutation decreases the abundance of OsCESA4 in the plasma membrane, probably due to a defect in the process of CESA complex secretion. The data from our biochemical, genetic, and pharmacological analyses indicate that this residue is critical for maintaining the normal level of CESA proteins in the plasma membrane. PMID:19697141

  17. Immunohistochemical results of HER2/neu protein expression assessed by rabbit monoclonal antibodies SP3 and 4B5 in colorectal carcinomas

    PubMed Central

    Song, Zhangjuan; Deng, Yan; Zhuang, Kangmin; Li, Aimin; Liu, Side

    2014-01-01

    HER2/neu is an efficient target for cancer therapy. However, reports about its overexpression rate in colorectal carcinomas showed wide variability. This study aims to investigate HER2/neu expression in colorectal carcinomas using these two rabbit monoclonal HER2/neu antibodies, and to clarify the relationship between protein overexpression and gene amplification of HER2/neu and their clinicopathologic importance. Tissue microarray was performed from sections of 106 cases colorectal carcinomas. Their clinical data, including gender, age, stage, recurrence, lymph node metastasis, and follow-ups were collected. Immunohistochemistry for rabbit monoclonal antibody SP3 and 4B5 were performed, Fluorescent in situ hybridization was applied to detect the amplification of HER2/neu gene. The HER2/neu overexpression of (2+ and 3+) in our results were seen in 7.5% (8/106) for 4B5 and 3.8% (4/106) for SP3 respectively, the HER2/neu amplification was in 2.8% (3/106). All cases of overexpression for SP3 were included by those for 4B5. Both antibodies stained 3 cases of HER2/neu 3+, and FISH confirmed HER2/neu amplification did occurred in these cases. In our study, 4B5 was more sensitive to detect HER2/neu of colorectal carcinoma than SP3. 2.8% patients with colorectal patients might benefit from anti-HER2/neu therapy. PMID:25120833

  18. 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. Pitzers 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.

  19. Slow Proton Transfer Coupled to Unfolding Explains the Puzzling Results of Single-Molecule Experiments on BBL, a Paradigmatic Downhill Folding Protein

    PubMed Central

    Cerminara, Michele; Campos, Luis A.; Ramanathan, Ravishankar; Muñoz, Victor

    2013-01-01

    A battery of thermodynamic, kinetic, and structural approaches has indicated that the small α-helical protein BBL folds-unfolds via the one-state downhill scenario. Yet, single-molecule fluorescence spectroscopy offers a more conflicting view. Single-molecule experiments at pH 6 show a unique half-unfolded conformational ensemble at mid denaturation, whereas other experiments performed at higher pH show a bimodal distribution, as expected for two-state folding. Here we use thermodynamic and laser T-jump kinetic experiments combined with theoretical modeling to investigate the pH dependence of BBL stability, folding kinetics and mechanism within the pH 6–11 range. We find that BBL unfolding is tightly coupled to the protonation of one of its residues with an apparent pKa of ∼7. Therefore, in chemical denaturation experiments around neutral pH BBL unfolds gradually, and also converts in binary fashion to the protonated species. Moreover, under the single-molecule experimental conditions (denaturant midpoint and 279 K), we observe that proton transfer is much slower than the ∼15 microseconds folding-unfolding kinetics of BBL. The relaxation kinetics is distinctly biphasic, and the overall relaxation time (i.e. 0.2–0.5 ms) becomes controlled by the proton transfer step. We then show that a simple theoretical model of protein folding coupled to proton transfer explains quantitatively all these results as well as the two sets of single-molecule experiments, including their more puzzling features. Interestingly, this analysis suggests that BBL unfolds following a one-state downhill folding mechanism at all conditions. Accordingly, the source of the bimodal distributions observed during denaturation at pH 7–8 is the splitting of the unique conformational ensemble of BBL onto two slowly inter-converting protonation species. Both, the unprotonated and protonated species unfold gradually (one-state downhill), but they exhibit different degree of unfolding at any given condition because the native structure is less stable for the protonated form. PMID:24205082

  20. Copper deficiency in the young bovine results in dramatic decreases in brain copper concentration but does not alter brain prion protein biology.

    PubMed

    Legleiter, L R; Spears, J W; Liu, H C

    2008-11-01

    An Mn for Cu substitution on cellular prion proteins (PrP(c)) in the brain that results in biochemical changes to PrP(c) has been implicated in the pathogenesis of transmissible spongiform encephalopathies. Recent research in the mature bovine does not support this theory. The present study tested this hypothesis by using progeny from gestating cows receiving Cu-deficient diets or Cu-deficient diets coupled with high dietary Mn. Copper-adequate cows (n = 39) were assigned randomly to 1 of 3 treatments: 1) control (adequate in Cu and Mn), 2) Cu deficient (-Cu), or 3) Cu deficient plus high dietary Mn (-Cu+Mn). Cows assigned to treatments -Cu and -Cu+Mn received no supplemental Cu and were supplemented with Mo to further induce Cu deficiency. The -Cu+Mn treatment also received 500 mg of supplemental Mn/kg of dietary DM. Calves were weaned at 180 d and maintained on the same treatments as their respective dams for 260 d. Copper-deficient calves (-Cu and -Cu+Mn) had decreased (P = 0.001) brain (obex) Cu and tended to have increased (P = 0.09) obex Mn relative to control calves. Obex Mn:Cu ratios were substantially increased (P < 0.001) in calves receiving -Cu and -Cu+Mn treatments compared with control calves and were greater (P < 0.001) in -Cu+Mn calves than in -Cu calves. Obex prion protein characteristics, including proteinase K degradability, superoxide dismutase (SOD)-like activity, and glycoform distributions, were largely unaffected. Obex tissue antioxidant capacity was not compromised by perturbations in brain metals, but Cu-deficient calves tended to have decreased (P = 0.06) Cu:Zn SOD activity and increased (P = 0.06) Mn SOD activity. Although obex Cu was decreased because of Cu deficiency and Mn increased because of exposure to high dietary Mn, the obex metal imbalance had minimal effects on PrP(c) functional characteristics in the calves. PMID:18599661

  1. Increased Levels of Macrophage Inflammatory Proteins Result in Resistance to R5-Tropic HIV-1 in a Subset of Elite Controllers

    PubMed Central

    Walker, Wendy E.; Kurscheid, Sebastian; Joshi, Samit; Lopez, Charlie A.; Goh, Gerald; Choi, Murim; Barakat, Lydia; Francis, John; Fisher, Ann; Kozal, Michael; Zapata, Heidi; Shaw, Albert; Lifton, Richard; Fikrig, Erol

    2015-01-01

    ABSTRACT Elite controllers (ECs) are a rare group of HIV seropositive individuals who are able to control viral replication without antiretroviral therapy. The mechanisms responsible for this phenotype, however, have not been fully elucidated. In this study, we examined CD4+ T cell resistance to HIV in a cohort of elite controllers and explored transcriptional signatures associated with cellular resistance. We demonstrate that a subgroup of elite controllers possess CD4+ T cells that are specifically resistant to R5-tropic HIV while remaining fully susceptible to X4-tropic and vesicular stomatitis virus G (VSV-G)-pseudotyped viruses. Transcriptome analysis revealed 17 genes that were differentially regulated in resistant elite controllers relative to healthy controls. Notably, the genes encoding macrophage inflammatory protein 1? (MIP-1?), CCL3 and CCL3L1, were found to be upregulated. The MIP-1?, MIP-1?, and RANTES chemokines are natural ligands of CCR5 and are known to interfere with HIV replication. For three elite controllers, we observed increased production of MIP-1? and/or MIP-1? at the protein level. The supernatant from resistant EC cells contained MIP-1? and MIP-1? and was sufficient to confer R5-tropic resistance to susceptible CD4+ T cells. Additionally, this effect was reversed by using inhibitory anti-MIP antibodies. These results suggest that the T cells of these particular elite controllers may be naturally resistant to HIV infection by blocking R5-tropic viral entry. IMPORTANCE HIV is a pandemic health problem, and the majority of seropositive individuals will eventually progress to AIDS unless antiretroviral therapy (ART) is administered. However, rare patients, termed elite controllers, have a natural ability to control HIV infection in the absence of ART, but the mechanisms by which they achieve this phenotype have not been fully explored. This paper identifies one mechanism that may contribute to this natural resistance: some elite controllers have CD4+ T cells that produce high levels of MIP chemokines, which block R5-tropic HIV entry. This mechanism could potentially be exploited to achieve a therapeutic effect in other HIV-seropositive individuals. PMID:25740989

  2. 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; Krmer, 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

  3. Genetic variation in the vitamin D receptor (VDR) and the vitamin D binding protein (GC) and risk of colorectal cancer: Results from the Colon Cancer Family Registry

    PubMed Central

    Poynter, Jenny N.; Jacobs, Elizabeth T.; Figueiredo, Jane C.; Lee, Won H.; Conti, David V.; Campbell, Peter T.; Levine, A. Joan; Limburg, Paul; Le Marchand, Loic; Cotterchio, Michelle; Newcomb, Polly A.; Potter, John D.; Jenkins, Mark A.; Hopper, John L.; Duggan, David J.; Baron, John A.; Haile, Robert W.

    2009-01-01

    Epidemiologic evidence supports a role for vitamin D in colorectal cancer (CRC) risk. Variants in vitamin D-related genes might modify the association between vitamin D levels and CRC risk. In this analysis, we performed a comprehensive evaluation of common variants in the vitamin D receptor (VDR) and the vitamin D binding protein (GC, group-specific component) genes using a population-based case-unaffected sibling control design that included 1,750 sibships recruited into the Colon Cancer Family Registry (Colon CFR). We also evaluated whether any associations differed by calcium supplement use, family history of CRC, or tumor characteristics. Heterogeneity by calcium and vitamin D intake was evaluated for a subset of 585 cases and 837 sibling controls who completed a detailed food frequency questionnaire (FFQ). Age- and sex-adjusted associations were estimated using conditional logistic regression. Overall, we did not find evidence for an association between any SNP in VDR or GC and risk of CRC (range of unadjusted p-values 0.010.98 for VDR and 0.070.95 for GC). None of these associations was significant after adjustment for multiple comparisons. We also found no evidence that calcium or vitamin D intake (food and supplement) from the FFQ modified the association estimates between VDR and GC SNPs and CRC. We did observe associations between SNPs in GC and microsatellite unstable CRC, although these results should be confirmed in additional studies. Overall, our results do not provide evidence for a role of common genetic variants in VDR or GC in susceptibility to CRC. PMID:20086113

  4. 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. PMID:25561411

  5. High temperature redox reactions with uranium: Synthesis and characterization of Cs(UO2)Cl(SeO3), Rb2(UO2)3O2(SeO3)2, and RbNa5U2(SO4)7

    NASA Astrophysics Data System (ADS)

    Babo, Jean-Marie; Albrecht-Schmitt, Thomas E.

    2013-10-01

    Cs(UO2)Cl(SeO3) (1), Rb2(UO2)3O2(SeO3)3 (2), and RbNa5U2(SO4)7 (3) single crystals were synthesized using CsCl, RbCl, and a CuCl/NaCl eutectic mixture as fluxes, respectively. Their lattice parameters and space groups are as follows: P21/n (a=6.548(1) , b=11.052(2) , c=10.666(2) and ?=93.897(3)), P1bar (a=7.051(2) , b=7.198(2) , c=8.314(2) , ?=107.897(3), ?=102.687(3) and ?=100.564(3)) and C2/c (a=17.862(4) , b=6.931(1) , c=20.133(4) and ?=109.737(6). The small anionic building units found in these compounds are SeO32- and SO42- tetrahedra, oxide, and chloride. The crystal structure of the first compound is composed of [(UO2)2Cl2(SeO3)2]2- chains separated by Cs+ cations. The structure of (2) is constructed from [(UO2)3O11]16- chains further connected through selenite units into layers stacked perpendicularly to the [0 1 0] direction, with Rb+ cations intercalating between them. The structure of compound (3) is made of uranyl sulfate layers formed by edge and vertex connections between dimeric [U2O16] and [SO4] polyhedra. These layers contain unusual sulfate-metal connectivity as well as large voids.

  6. Results of a phase I/II open-label, safety and efficacy trial of coagulation factor IX (recombinant), albumin fusion protein in haemophilia B patients

    PubMed Central

    Martinowitz, U; Lissitchkov, T; Lubetsky, A; Jotov, G; Barazani-Brutman, T; Voigt, C; Jacobs, I; Wuerfel, T; Santagostino, E

    2015-01-01

    Introduction rIX-FP is a coagulation factor IX (recombinant), albumin fusion protein with more than fivefold half-life prolongation over other standard factor IX (FIX) products available on the market. Aim This prospective phase II, open-label study evaluated the safety and efficacy of rIX-FP for the prevention of bleeding episodes during weekly prophylaxis and assessed the haemostatic efficacy for on-demand treatment of bleeding episodes in previously treated patients with haemophilia B. Methods The study consisted of a 1014day evaluation of rIX-FP pharmacokinetics (PK), and an 11month safety and efficacy evaluation period with subjects receiving weekly prophylaxis treatment. Safety was evaluated by the occurrence of related adverse events, and immunogenic events, including development of inhibitors. Efficacy was evaluated by annualized spontaneous bleeding rate (AsBR), and the number of injections to achieve haemostasis. Results Seventeen subjects participated in the study, 13 received weekly prophylaxis and 4 received episodic treatment only. No inhibitors were detected in any subject. The mean and median AsBR were 1.25, and 1.13 respectively in the weekly prophylaxis arm. All bleeding episodes were treated with 1 or 2 injections of rIX-FP. Three prophylaxis subjects who were treated on demand prior to study entry had >85% reduction in AsBR compared to the bleeding rate prior to study entry. Conclusion This study demonstrated the efficacy for weekly routine prophylaxis of rIX-FP to prevent spontaneous bleeding episodes and for the treatment of bleeding episodes. In addition no safety issues were detected during the study and an improved PK profile was demonstrated. PMID:25990590

  7. Directed expression of the growth-associated protein B-50/GAP-43 to olfactory neurons in transgenic mice results in changes in axon morphology and extraglomerular fiber growth.

    PubMed

    Holtmaat, A J; Dijkhuizen, P A; Oestreicher, A B; Romijn, H J; Van der Lugt, N M; Berns, A; Margolis, F L; Gispen, W H; Verhaagen, J

    1995-12-01

    B-50/GAP-43, a neural growth-associated phosphoprotein, is thought to play a role in neuronal plasticity and nerve fiber formation since it is expressed at high levels in developing and regenerating neurons and in growth cones. Using a construct containing the coding sequence of B-50/GAP-43 under the control of regulatory elements of the olfactory marker protein (OMP) gene, transgenic mice were generated to study the effect of directed expression of B-50/GAP-43 in a class of neurons that does not normally express B-50/GAP-43, namely, mature OMP-positive olfactory neurons. Olfactory neurons have a limited lifespan and are replaced throughout adulthood by new neurons that migrate into the upper compartment of the epithelium following their formation from stem cells in the basal portion of this neuroepithelium. Thus, the primary olfactory pathway is exquisitely suited to examine a role of B-50/GAP-43 in neuronal migration, lifespan, and nerve fiber growth. We find that B-50/GAP-43 expression in adult olfactory neurons results in numerous primary olfactory axons with enlarged endings preferentially located at the rim of individual glomeruli. Furthermore, ectopic olfactory nerve fibers in between the juxtaglomerular neurons or in close approximation to blood vessels were frequently observed. This suggests that expression of B-50/GAP-43 in mature olfactory neurons alters their response to signals in the bulb. Other parameters examined, that is, migration and lifespan of olfactory neurons are normal in B-50/GAP-43 transgenic mice. These observations provide direct in vivo evidence for a role of B-50/GAP-43 in nerve fiber formation and in the determination of the morphology of axons. PMID:8613733

  8. The added value of C-reactive protein to clinical signs and symptoms in patients with obstructive airway disease: results of a diagnostic study in primary care

    PubMed Central

    Schneider, Antonius; Dinant, Geert-Jan; Maag, Inko; Gantner, Lutz; Meyer, Joachim Franz; Szecsenyi, Joachim

    2006-01-01

    Background To evaluate the diagnostic accuracy of clinical signs and symptoms, C-reactive protein (CRP) and spirometric parameters and determine their interrelation in patients suspected to have an obstructive airway disease (OAD) in primary care. Methods In a cross sectional diagnostic study, 60 adult patients coming to the general practitioner (GP) for the first-time with complaints suspicious for obstructive airway disease (OAD) underwent spirometry. Peak expiratory flow (PEF)-variability within two weeks was determined in patients with inconspicuous spirometry. Structured medical histories were documented and CRP was measured. The reference standard was the Tiffeneau ratio (FEV1/VC) in spirometry and the PEF-variability. OAD was diagnosed when FEV1/VC ? 70% or PEF-variability > 20%. Results 37 (62%) patients had OAD. The best cut-off value for CRP was found at 2 mg/l with a diagnostic odds ratio (OR) of 4.4 (95% CI 1.413.8). Self-reported wheezing was significantly related with OAD (OR 3.4; CI 1.110.3), whereas coughing was inversely related (OR 0.2; CI 0.10.7). The diagnostic OR of CRP increased when combined with dyspnea (OR 8.5; 95% CI 1.742.3) or smoking history (OR 8.4; 95% CI 1.548.9). CRP (p = 0.004), FEV1 (p = 0.001) and FIV1 (p = 0.023) were related with the severity of dyspnea. CRP increased with the number of cigarettes, expressed in pack years (p = 0.001). Conclusion The diagnostic accuracy of clinical signs and symptoms was low. The diagnostic accuracy of CRP improved in combination with dyspnea and smoking history. Due to their coherence with the severity of dyspnea and number of cigarettes respectively, CRP and spirometry might allow risk stratification of patients with OAD in primary care. Further studies need to be done to confirm these findings. PMID:16670014

  9. 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 with additional cytokines involved in inflammatory response to delineate the inflammatory profile. Subsequently, research on determinants of these associations and their role in development of chronic disease is needed. PMID:26900695

  10. F box protein FBXL2 exerts human lung tumor suppressor-like activity by ubiquitin-mediated degradation of cyclin D3 resulting in cell cycle arrest

    PubMed Central

    Chen, Bill B.; Glasser, Jennifer R.; Coon, Tiffany A.; Mallampalli, Rama K.

    2011-01-01

    Dyregulated behavior of cell cycle proteins and their control by ubiquitin E3 ligases is an emerging theme in human lung cancer. Here we identified and characterized the activity of a novel F box protein, termed FBXL2, belonging to the SCF (Skip-Cullin1-F-box protein) E3 ligase family. Ectopically expressed FBXL2 triggered G2/M phase arrest, induced chromosomal anomalies, and increased apoptosis of transformed lung epithelia by mediating polyubiquitination and degradation of the mitotic regulator, cyclin D3. Unlike other F box proteins that target phosphodegrons within substrates, FBXL2 uniquely recognizes a canonical calmodulin-binding motif within cyclin D3 to facilitate its polyubiquitination. Calmodulin bound and protected cyclin D3 from FBXL2 by direct intermolecular competition with the F box protein for access within this motif. The chemotherapeutic agent vinorelbine increased apoptosis of human lung carcinoma cells by inducing FBXL2 expression and cyclin D3 degradation, an effect accentuated by calmodulin knockdown. Depletion of endogenous FBXL2 stabilized cyclin D3 levels, accellerated cancer cell growth, and increased cell viability after vinorelbine treatment. Last, ectopic expression of FBXL2 significantly inhibited the growth and migration of tumorogenic cells and tumor formation in athymic nude mice. These observations implicate SCFFBXL2 as an indispensible regulator of mitosis that serves as a tumor suppressor. PMID:22020328

  11. Altered expression of an ankyrin-repeat protein results in leaf abnormalities, necrotic lesions, and the elaboration of a systemic signal.

    PubMed

    Wirdnam, Corina; Motoyama, Andrea; Arn-Bouldoires, Estelle; van Eeden, Sjoerd; Iglesias, Alejandro; Meins, Frederick

    2004-11-01

    The PR-like proteins, class I beta-1,3-glucanase (GLU I) and chitinase (CHN I), are induced as part of a stereotypic response that can provide protection against viral, bacterial, and fungal pathogens. We have identified two Nicotiana plumbaginifolia ankyrin-repeat proteins, designated Glucanohydrolase Binding Proteins (GBP) 1 and 2, that bind GLU I and CHN I both in vitro and when expressed in yeast cells. Sense as well as antisense transformants of tobacco carrying the GBP1 gene elaborated graft-transmissible acropetally moving signals that induced the downward curling of young leaves. This phenotype was associated with reduced starch, sucrose, and fructose accumulation; the formation of necrotic lesions; and, the induction of markers for the hypersensitive response. GBP1/2 are members of a conserved Plant- Specific Ankyrin- repeat (PANK) family that includes proteins implicated in carbohydrate allocation, reactive oxygen metabolism, hypersensitive cell death, rapid elicitor responses, virus pathogenesis, and auxin signaling. The similarity in phenotype of PANK transformants and transformants altered in carbohydrate metabolism leads us to propose that PANK family members are multifunctional proteins involved in linking plant defense responses and carbohydrate metabolism. PMID:15803410

  12. Microsomal triglyceride transfer protein -164 T?>?C gene polymorphism and risk of cardiovascular disease: results from the EPIC-Potsdam case-cohort study

    PubMed Central

    2013-01-01

    Background The microsomal triglyceride transfer protein (MTTP) is encoded by the MTTP gene that is regulated by cholesterol in humans. Previous studies investigating the effect of MTTP on ischemic heart disease have produced inconsistent results. Therefore, we have tested the hypothesis that the rare allele of the -164T?>?C polymorphism in MTTP alters the risk of cardiovascular disease (CVD), depending on the cholesterol levels. Methods The -164T > C polymorphism was genotyped in a case-cohort study (193 incident myocardial infarction (MI) and 131 incident ischemic stroke (IS) cases and 1 978 non-cases) nested within the European Prospective Investigation into Cancer and Nutrition (EPIC)Potsdam study, comprising 27 548 middle-aged subjects. The Heinz Nixdorf Recall study (30 CVD cases and 1 188 controls) was used to replicate our findings. Results Genotype frequencies were not different between CVD and CVD free subjects (P = 0.79). We observed an interaction between the -164T > C polymorphism and total cholesterol levels in relation to future CVD. Corresponding stratified analyses showed a significant increased risk of CVD (HRadditve = 1.38, 95% CI: 1.07 to 1.78) for individuals with cholesterol levels <200 mg/dL in the EPIC-Potsdam study. HRadditive was 1.06, 95% CI: 0.33 to 3.40 for individuals in the Heinz Nixdorf Recall study. A borderline significant decrease in CVD risk was observed in subjects with cholesterol levels ?200 mg/dL (HRadditve = 0.77, 95% CI: 0.58 to 1.03) in the EPIC-Potsdam study. A similar trend was observed in the independent cohort (HRadditve = 0.60, 95% CI: 0.29 to 1.25). Conclusions Our study suggests an interaction between MTTP -164T > C functional polymorphism with total cholesterol levels. Thereby risk allele carriers with low cholesterol levels may be predisposed to an increased risk of developing CVD, which seems to be abolished among risk allele carriers with high cholesterol levels. PMID:23356586

  13. Non-aqueous encapsulation of excipient-stabilized spray-freeze dried BSA into poly(lactide-co-glycolide) microspheres results in release of native protein.

    PubMed

    Carrasquillo, K G; Stanley, A M; Aponte-Carro, J C; De Jsus, P; Costantino, H R; Bosques, C J; Griebenow, K

    2001-10-19

    Encapsulation of the model protein bovine serum albumin (BSA) into poly(D,L lactide-co-glycolide) (PLG) microspheres was performed by a non-aqueous oil-in-oil (o/o) methodology. Powder formulations of BSA obtained by spray-freeze drying were first suspended in methylene chloride containing PLG followed by coacervation by adding silicon oil and microsphere hardening in heptane. The secondary structure of BSA was determined at relevant steps of the encapsulation procedure by employing Fourier-transform infrared (FTIR) spectroscopy. This fast and non-invasive method demonstrated the potential to rapidly screen pharmaceutically relevant protein delivery systems for their suitability. Structural perturbations in BSA were reduced during the spray-freeze drying step by employing the excipient trehalose. The protein was then encapsulated into PLG microspheres under various conditions without inducing significant structural perturbations. BSA released from these microspheres had a similar monomer content as unencapsulated BSA and also the same secondary structure. Upon blending of a poloxamer (Pluronic F-68) with the polymer phase, in vitro release was characterized by a small initial release and a prolonged and continuous sustained phase. In conclusion, the developed o/o methodology coupled with FTIR spectroscopic monitoring of protein structure is a powerful approach for the development of sustained release microspheres. PMID:11578736

  14. A single amino acid substitution in the hemagglutinin-neuraminidase protein of Newcastle disease virus results in increased fusion and decreased neuraminidase activities without changes in virus pathotype

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Newcastle disease virus (NDV) attachment to the host cell is mediated by the hemagglutinin-neuraminidase (HN), a multifunctional protein that has receptor recognition, neuraminidase and fusion promotion activities. The process that correlates receptor binding and fusion triggering is poorly understo...

  15. ORF3 protein of hepatitis E virus interacts with the Bbeta chain of fibrinogen resulting in decreased fibrinogen secretion from HuH-7 cells.

    PubMed

    Ratra, Ruchi; Kar-Roy, Anindita; Lal, Sunil K

    2009-06-01

    The ORF3 protein of hepatitis E virus (HEV), the precise cellular functions of which remain obscure, was used in a yeast two-hybrid screen to identify its cellular binding partners. One of the identified interacting partners was fibrinogen Bbeta protein. The ORF3-fibrinogen Bbeta interaction was verified by co-immunoprecipitation and fluorescence resonance energy transfer in mammalian cells. Fibrinogen is a hepatic acute-phase protein and serves as a central molecule that maintains host homeostasis and haemostasis during an acute-phase response. Metabolic labelling of ORF3-transfected HuH-7 cells showed that secreted as well as intracellular levels of fibrinogen were decreased in these cells compared with vector-transfected controls. Northern hybridization and RT-PCR analyses revealed that the mRNA levels of all three chains of fibrinogen, Aalpha, Bbeta and gamma, were transcriptionally downregulated in ORF3-transfected cells. The constitutive expression of fibrinogen genes can be significantly upregulated by interleukin (IL)-6, an important mediator of liver-specific gene expression during an acute-phase response. Transcription of fibrinogen genes after IL-6 stimulation was less in ORF3-expressing cells compared with controls. This report adds one more biological function to, and advances our understanding of, the cellular role of the ORF3 protein of HEV. The possible implications of these findings in the virus life cycle are discussed. PMID:19264644

  16. TAT-Mediated Transduction of MafA Protein In Utero Results in Enhanced Pancreatic Insulin Expression and Changes in Islet Morphology

    PubMed Central

    Vargas, Nancy; Fort, Nicholas M.; Cechin, Sirlene; Garca, Enrique; Espino-Grosso, Pedro; Fraker, Christopher A.; Ricordi, Camillo; Inverardi, Luca; Pastori, Ricardo L.; Domnguez-Bendala, Juan

    2011-01-01

    Alongside Pdx1 and Beta2/NeuroD, the transcription factor MafA has been shown to be instrumental in the maintenance of the beta cell phenotype. Indeed, a combination of MafA, Pdx1 and Ngn3 (an upstream regulator of Beta2/NeuroD) was recently reported to lead to the effective reprogramming of acinar cells into insulin-producing beta cells. These experiments set the stage for the development of new strategies to address the impairment of glycemic control in diabetic patients. However, the clinical applicability of reprogramming in this context is deemed to be poor due to the need to use viral vehicles for the delivery of the above factors. Here we describe a recombinant transducible version of the MafA protein (TAT-MafA) that penetrates across cell membranes with an efficiency of 100% and binds to the insulin promoter in vitro. When injected in utero into living mouse embryos, TAT-MafA significantly up-regulates target genes and induces enhanced insulin production as well as cytoarchitectural changes consistent with faster islet maturation. As the latest addition to our armamentarium of transducible proteins (which already includes Pdx1 and Ngn3), the purification and characterization of a functional TAT-MafA protein opens the door to prospective therapeutic uses that circumvent the use of viral delivery. To our knowledge, this is also the first report on the use of protein transduction in utero. PMID:21857924

  17. Feeding soy protein isolate (SPI) does not result in an estrogenic gene expression profile in the mammary of ovariectomized (OVX) female rats

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Concerns of increased breast cancer risk in women consuming soy exist because of the perceived estrogenicity of soy isoflavones. Female Sprague-Dawley rats (N equals 20/group) were fed AIN-93G diets with casein or SPI as the protein from PND30. On PND50 rats were OVX and 10/group infused s.c. with 5...

  18. Muscle Uncoupling Protein 3 Expression Is Unchanged by Chronic Ephedrine/Caffeine Treatment: Results of a Double Blind, Randomised Clinical Trial in Morbidly Obese Females

    PubMed Central

    Bracale, Renata; Petroni, Maria Letizia; Davinelli, Sergio; Bracale, Umberto; Scapagnini, Giovanni; Carruba, Michele O.; Nisoli, Enzo

    2014-01-01

    Ephedrine/caffeine combination (EC) has been shown to induce a small-to-moderate weight loss in obese patients. Several mechanisms have been proposed, among which an increased thermogenic capacity of skeletal muscle consequent to the EC-induced up-regulation of uncoupling protein 3 (UCP3) gene expression. We did a parallel group double-blind, placebo-controlled, 4-week trial to investigate this hypothesis. Thirteen morbidly obese women (2552 years of age, body-mass index 48.04.0 kg/m2, range 41.157.6) were randomly assigned to EC (200/20 mg, n?=?6) or to placebo (n?=?7) administered three times a day orally, before undergoing bariatric surgery. All individuals had an energy-deficit diet equal to about 70% of resting metabolic rate (RMR) diet (mean 57691105 kJ/day). The RMR analysed by intention to treat and the UCP3 (long and short isoform) mRNA levels in rectus abdominis were the primary outcomes. Body weight, plasma levels of adrenaline, noradrenaline, triglycerides, free fatty acids, glycerol, TSH, fT4, and fT3 were assessed, as well as fasting glucose, insulin and HOMA index, at baseline and at the end of treatments. Body weight loss was evident in both groups when compared to baseline values (overall ?5.23.2%, p<0.0001) without significant differences between the treated groups. EC treatment increased the RMR (+9.26.8%, p?=?0.020), differently from placebo which was linked to a reduction of RMR (?7.66.5%, p?=?0.029). No significant differences were seen in other metabolic parameters. Notably, no changes of either UCP3 short or UCP3 long isoform mRNA levels were evident between EC and placebo group. Our study provides evidence that 4-week EC administration resulted in a pronounced thermogenic effect not related to muscle UCP3 gene expression and weight loss in morbidly obese females under controlled conditions. Trial Registration ClinicalTrials.gov NCT02048215 PMID:24905629

  19. A Novel Form of DAP5 Protein Accumulates in Apoptotic Cells as a Result of Caspase Cleavage and Internal Ribosome Entry Site-Mediated Translation

    PubMed Central

    Henis-Korenblit, Sivan; Strumpf, Naomi Levy; Goldstaub, Dan; Kimchi, Adi

    2000-01-01

    Death-associated protein 5 (DAP5) (also named p97 and NAT1) is a member of the translation initiation factor 4G (eIF4G) family that lacks the eIF4E binding site. It was previously implicated in apoptosis, based on the finding that a dominant negative fragment of the protein protected against cell death. Here we address its function and two distinct levels of regulation during apoptosis that affect the protein both at translational and posttranslational levels. DAP5 protein was found to be cleaved at a single caspase cleavage site at position 790, in response to activated Fas or p53, yielding a C-terminal truncated protein of 86 kDa that is capable of generating complexes with eIF4A and eIF3. Interestingly, while the overall translation rate in apoptotic cells was reduced by 60 to 70%, in accordance with the simultaneous degradation of the two major mediators of cap-dependent translation, eIF4GI and eIF4GII, the translation rate of DAP5 protein was selectively maintained. An internal ribosome entry site (IRES) element capable of directing the translation of a reporter gene when subcloned into a bicistronic vector was identified in the 5? untranslated region of DAP5 mRNA. While cap-dependent translation from this transfected vector was reduced during Fas-induced apoptosis, the translation via the DAP5 IRES was selectively maintained. Addition of recombinant DAP5/p97 or DAP5/p86 to cell-free systems enhanced preferentially the translation through the DAP5 IRES, whereas neutralization of the endogenous DAP5 in reticulocyte lysates by adding a dominant negative DAP5 fragment interfered with this translation. The DAP5/p86 apoptotic form was more potent than DAP5/p97 in these functional assays. Altogether, the data suggest that DAP5 is a caspase-activated translation factor which mediates cap-independent translation at least from its own IRES, thus generating a positive feedback loop responsible for the continuous translation of DAP5 during apoptosis. PMID:10611228

  20. The stability of Taq DNA polymerase results from a reduced entropic folding penalty; identification of other thermophilic proteins with similar folding thermodynamics.

    PubMed

    Liu, Chin-Chi; LiCata, Vince J

    2014-05-01

    The thermal stability of Taq DNA polymerase is well known, and is the basis for its use in PCR. A comparative thermodynamic characterization of the large fragment domains of Taq (Klentaq) and E. coli (Klenow) DNA polymerases has been performed by obtaining full Gibbs-Helmholtz stability curves of the free energy of folding (?G) versus temperature. This analysis provides the temperature dependencies of the folding enthalpy and entropy (?H and ?S), and the heat capacity (?Cp ) of folding. If increased or enhanced non-covalent bonding in the native state is responsible for enhanced thermal stabilization of a protein, as is often proposed, then an enhanced favourable folding enthalpy should, in general, be observed for thermophilic proteins. However, for the Klenow-Klentaq homologous pair, the folding enthalpy (?Hfold ) of Klentaq is considerably less favorable than that of Klenow at all temperatures. In contrast, it is found that Klentaq's extreme free energy of folding (?Gfold ) originates from a significantly reduced entropic penalty of folding (?Sfold ). Furthermore, the heat capacity changes upon folding are similar for Klenow and Klentaq. Along with this new data, comparable extended analysis of available thermodynamic data for 17 other mesophilic-thermophilic protein pairs (where enough applicable thermodynamic data exists) shows a similar pattern in seven of the 18 total systems. When analyzed with this approach, the more familiar "reduced ?Cp mechanism" for protein thermal stabilization (observed in a different six of the 18 systems) frequently manifests as a temperature dependent shift from enthalpy driven stabilization to a reduced-entropic-penalty model. PMID:24174290

  1. Substitution of conserved methionines by leucines in chloroplast small heat shock protein results in loss of redox-response but retained chaperone-like activity

    PubMed Central

    Gustavsson, Niklas; Kokke, Bas P.A.; Anzelius, Björn; Boelens, Wilbert C.; Sundby, Cecilia

    2001-01-01

    During evolution of land plants, a specific motif occurred in the N-terminal domain of the chloroplast-localized small heat shock protein, Hsp21: a sequence with highly conserved methionines, which is predicted to form an amphipathic α-helix with the methionines situated along one side. The functional role of these conserved methionines is not understood. We have found previously that treatment, which causes methionine sulfoxidation in Hsp21, also leads to structural changes and loss of chaperone-like activity. Here, mutants of Arabidopsis thaliana Hsp21 protein were created by site-directed mutagenesis, whereby conserved methionines were substituted by oxidation-resistant leucines. Mutants lacking the only cysteine in Hsp21 were also created. Protein analyses by nondenaturing electrophoresis, size exclusion chromatography, and circular dichroism proved that sulfoxidation of the four highly conserved methionines (M49, M52, M55, and M59) is responsible for the oxidation-induced conformational changes in the Hsp21 oligomer. In contrast, the chaperone-like activity was not ultimately dependent on the methionines, because it was retained after methionine-to-leucine substitution. The functional role of the conserved methionines in Hsp21 may be to offer a possibility for redox control of chaperone-like activity and oligomeric structure dynamics. PMID:11514669

  2. Preliminary results on the effects of grape (Vitis vinifera) seed condensed tannins on in vitro intestinal digestibility of the lupin (Lupinus angustifolius) seed protein fraction in small ruminants.

    PubMed

    Bruno-Soares, A M; Soares-Pereira, A L; Matos, T J S; Ricardo-da-Silva, J M

    2011-08-01

    Condensed tannins (CT) from grape seeds (Vitis vinifera L.) were added to complex the protein fraction of Lupinus angustifolius seeds. Three CT/protein ratios were used: 96 mg/g (T(1)), 180 mg/g (T(2)) and 0 mg/g (T(0)). The CP losses in the rumen were assessed by the nylon-bag technique and CP intestinal digestibility (CPID) was estimated using an in vitro assay applying a three-step procedure: samples were subject to rumen degradation (in situ, 16 h) and the remaining residues were subject to the digestive enzymes of the abomasum and pancreas in vitro. A positive effect (p < 0.05) of the level of CT on the immediately soluble faction a and the insoluble degradable fraction b was observed between T(0) and T(2) . In the presence of CT the rumen degradation rate was reduced (p < 0.05) from 0.0763/h (T(0)) to 0.0443/h (T(2)). The application of CT showed a reduction (around 10% for T(1)) of effective rumen CP degradability. The CPID did not seem to be affected (p > 0.05) by the presence of CT. These findings suggest that the use of grape seed CT might have the potential to improve the efficiency of utilisation of the protein fraction from lupin seeds. PMID:21039934

  3. 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.

  4. 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

  5. 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.

  6. 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

  7. 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

    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

  8. 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

  9. 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

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

    PubMed

    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

  11. Recombination within the upstream gene of duplicated myelin basic protein genes of myelin deficient shimld mouse results in the production of antisense RNA.

    PubMed Central

    Okano, H; Ikenaka, K; Mikoshiba, K

    1988-01-01

    The myelin deficient shimld mouse is an autosomal recessive mutant, characterized by hypomyelination in the central nervous system. The expression of the myelin basic protein (MBP) gene is inhibited transcriptionally. The MBP gene is duplicated tandemly in mld, and exons 3 to 7 of the upstream copy is inverted. In the present studies, we determined the approximate position of the 5' boundary and the nucleotide sequence surrounding the 3' boundary of the inversion and found a number of sequences homologous to the switching regions of mouse immunoglobulin heavy chain gene and J regions of human T cell receptor genes. Antisense RNA complementary to exons 3 and 7, which correspond to the inverted segment, was detected by RNase protection studies. This abnormal transcript was also shown to elongate through the inverted segment to reach the transcription initiation site of the downstream gene. Images PMID:2463159

  12. Mutation of the elongin C binding domain of human respiratory syncytial virus non-structural protein 1 (NS1) results in degradation of NS1 and attenuation of the virus

    PubMed Central

    2011-01-01

    Background Human respiratory syncytial virus (RSV) is an important cause of lower respiratory tract disease in the paediatic population, immunocompromised individuals and the elderly worldwide. However, despite global efforts over the past several decades there are no commercially available vaccines. RSV encodes 2 non-structural proteins, NS1 and NS2, that are type I interferon antagonists. RSV restricts type I interferon signaling and the expression of antiviral genes by degrading STAT2. It has been proposed that NS1 binds to elongin C to form a ubiquitin ligase (E3) complex that targets STAT2 for ubiquitination and proteosomal degradation. Results Here, we have engineered a live recombinant RSV in which the 3 consensus amino acids of the NS1 elongin C binding domain have been replaced with alanine (NS1F-ELCmut). Mutation of this region of NS1 resulted in attenuation of RSV replication in A549 cells to levels similar to that observed when the NS1 gene is completely deleted (?NS1). This mutation also resulted in moderate attenuation in Vero cells. Attenuation was correlated to intracellular degradation of the mutated NS1 protein. Time course analysis showed that mutant NS1 protein accumulated in cytoplasmic bodies that contained the lysosomal marker LAMP1. However lack of cleavage of LC3 suggested that autophagy was not involved. Induction of IFN-? mRNA expression also was observed in association with the degradation of NS1 protein and attenuation of viral growth. Conclusions These results indicate that the elongin C binding region of NS1 is crucial for survival of the protein and that disruption of this region results in the degradation of NS1 and restriction of RSV replication. PMID:21600055

  13. Disruption of the Y-box binding protein-1 results in suppression of the epidermal growth factor receptor and HER-2.

    PubMed

    Wu, Joyce; Lee, Cathy; Yokom, Daniel; Jiang, Helen; Cheang, Maggie C U; Yorida, Erika; Turbin, Dmitry; Berquin, Isabelle M; Mertens, Peter R; Iftner, Thomas; Gilks, C Blake; Dunn, Sandra E

    2006-05-01

    The overexpression of the epidermal growth factor receptor (EGFR) and HER-2 underpin the growth of aggressive breast cancer; still, it is unclear what governs the regulation of these receptors. Our laboratories recently determined that the Y-box binding protein-1 (YB-1), an oncogenic transcription/translation factor, induced breast tumor cell growth in monolayer and in soft agar. Importantly, mutating YB-1 at Ser(102), which resides in the DNA-binding domain, prevented growth induction. We reasoned that the underlying cause for growth attenuation by YB-1(Ser(102)) is through the regulation of EGFR and/or HER-2. The initial link between YB-1 and these receptors was sought by screening primary tumor tissue microarrays. We determined that YB-1 (n = 389 cases) was positively associated with EGFR (P < 0.001, r = 0.213), HER-2 (P = 0.008, r = 0.157), and Ki67 (P < 0.0002, r = 0.219). It was inversely linked to the estrogen receptor (P < 0.001, r = -0.291). Overexpression of YB-1 in a breast cancer cell line increased HER-2 and EGFR. Alternatively, mutation of YB-1 at Ser(102) > Ala(102) prevented the induction of these receptors and rendered the cells less responsive to EGF. The mutant YB-1 protein was also unable to optimally bind to the EGFR and HER-2 promoters based on chromatin immunoprecipitation. Furthermore, knocking down YB-1 with small interfering RNA suppressed the expression of EGFR and HER-2. This was coupled with a decrease in tumor cell growth. In conclusion, YB-1(Ser(102)) is a point of molecular vulnerability for maintaining the expression of EGFR and HER-2. Targeting YB-1 or more specifically YB-1(Ser(102)) are novel approaches to inhibiting the expression of these receptors to ultimately suppress tumor cell growth. PMID:16651443

  14. 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 ...

  15. A Single Ala139-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

    PubMed Central

    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 msa1 and 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. PMID:12147498

  16. [The results of application of the rapid quantitative assay for fatty acid-binding protein at the onset of acute coronary syndrome].

    PubMed

    Kalinchenko, R M; Kopylov, F Iu; Syrkin, A L; Gitel', E P; Novikova, O V

    2013-01-01

    Fatty acid-binding protein (FABP) appearing in blood within a few hours of acute coronary syndrome (ACS) is a marker of myocardial necrosis. We estimated the diagnostic value of rapid immunochromatographic test for FABP in patients with ACS and compared it with other cardiomarkers: troponin 1 (Tn1), myoglobin and creatin phosphokinase-MB (CPK-MB). The study included 100 patients aged 61.3 +/- 12.9 yr hospitalized with ACS within 2 hr after beginning of anginous pain. FABP was detected by CardioFABP test, Tn1, myoglobin and CPK-MB by quantitative assays. Blood samples were taken 2, 6, and 24 hr after the onset of anginous pain. Acute myocardial infarction was diagnosed in 79 patients, unstable angina in 9, FC 3-4 angina of effort in 4, vasospastic angina in 1, non-coronary pathology in 7. Sensitivity of FABP, Tn1, myoglobin and CPK-MB 2 hr after onset of pain was 84.8; 34.2, 65.8; 22.8% respectively: it was 98.7: 92.4; 96.2; 82.3% in 6 hr and 56; 100; n/d; 86.7% in 24 hr. Specificity of FABP was 100% in all time intervals. It is concluded that FABP level determined by rapid qualitative assay within 2-6 hr after onset of ACS is a more sensitive cardiomarker than Tn1, myoglobin and CPK-MB for diagnostics of ACS. PMID:23659068

  17. 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. PMID:25940599

  18. 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; The Nebraska Center for Virology, University of Nebraska-Lincoln, Lincoln, Nebraska 68583-0900 ; 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.

  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. Mutation in the matrix protein of Newcastle disease virus can result in decreased fusion glycoprotein incorporation into particles and decreased infectivity.

    PubMed Central

    Peeples, M E; Bratt, M A

    1984-01-01

    Virus particles produced in eggs by the group D ts mutants of Newcastle disease virus at permissive temperature display low infectious and hemolytic activities (M.E. Peeples and M. A. Bratt , J. Virol. 42:440-446, 1982). These lower activities correlate with a decreased incorporation of F1+2 (fusion glycoprotein) into virus particles, compared with that for wild type. The incorporation of F1+2 into virus particles of the group D mutants is also lower than that for wild type when grown in chicken embryo cells in culture at either permissive or nonpermissive temperature. The infectivity of virions from these mutants correlates with the amounts of F1+2 in the virus particles, below a certain concentration, indicating that the quantity of F1+2 in virus particles is a determining factor in the infectivity of those particles. In addition, one of these mutants, D1, produces an M (matrix protein) which migrates at a faster rate in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Three of four revertants of D1 have coreverted to wild-type M electrophoretic mobility, associating M with the ts lesion and the other observed phenotypes. In each of these revertants, as well as in three revertants each from D2 and D3, there has been coreversion from the low specific infectious and hemolytic activities to greater, and often wild-type, activities. There is also a coreversion for F1+2 incorporation into virions. All of the revertants incorporate F1+2 into virions more efficiently than their mutant parents. The coreversions associate those phenotypes with the ts lesion and, in the case of D1, with the M lesion as well. Images PMID:6547186

  1. Lack of CD47 impairs bone cell differentiation and results in an osteopenic phenotype in vivo due to impaired signal regulatory protein ? (SIRP?) signaling.

    PubMed

    Koskinen, Cecilia; Persson, Emelie; Baldock, Paul; Stenberg, sa; Bostrm, Ingrid; Matozaki, Takashi; Oldenborg, Per-Arne; Lundberg, Pernilla

    2013-10-11

    Here, we investigated whether the cell surface glycoprotein CD47 was required for normal formation of osteoblasts and osteoclasts and to maintain normal bone formation activity in vitro and in vivo. In parathyroid hormone or 1?,25(OH)2-vitamin D3 (D3)-stimulated bone marrow cultures (BMC) from CD47(-/-) mice, we found a strongly reduced formation of multinuclear tartrate-resistant acid phosphatase (TRAP)(+) osteoclasts, associated with reduced expression of osteoclastogenic genes (nfatc1, Oscar, Trap/Acp, ctr, catK, and dc-stamp). The production of M-CSF and RANKL (receptor activator of nuclear factor ?? ligand) was reduced in CD47(-/-) BMC, as compared with CD47(+/+) BMC. The stromal cell phenotype in CD47(-/-) BMC involved a blunted expression of the osteoblast-associated genes osterix, Alp/Akp1, and ?-1-collagen, and reduced mineral deposition, as compared with that in CD47(+/+) BMC. CD47 is a ligand for SIRP? (signal regulatory protein ?), which showed strongly reduced tyrosine phosphorylation in CD47(-/-) bone marrow stromal cells. In addition, stromal cells lacking the signaling SIRP? cytoplasmic domain also had a defect in osteogenic differentiation, and both CD47(-/-) and non-signaling SIRP? mutant stromal cells showed a markedly reduced ability to support osteoclastogenesis in wild-type bone marrow macrophages, demonstrating that CD47-induced SIRP? signaling is critical for stromal cell support of osteoclast formation. In vivo, femoral bones of 18- or 28-week-old CD47(-/-) mice showed significantly reduced osteoclast and osteoblast numbers and exhibited an osteopenic bone phenotype. In conclusion, lack of CD47 strongly impairs SIRP?-dependent osteoblast differentiation, deteriorate bone formation, and cause reduced formation of osteoclasts. PMID:23990469

  2. 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, ShcGRB-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 and appears to depend on the activation of both the JAK-2 and PI-3 kinase pathways. Activation of the Src family of kinases or of the Ras/MAPK pathway can, however, be initiated by a kinase-impaired EGFR and is linked to survival. PMID:9819406

  3. Binding of selenium-75 to blood and liver cytosolic proteins in the preruminant calf

    SciTech Connect

    Jenkins, K.J.; Hidiroglou, M.

    1988-02-01

    Labeled selenite (75Se) administered to calves in milk replacer, containing .2 or 5 ppm Se, was rapidly absorbed with peak blood 75Se at 6 h. Gel filtration and dialysis treatment of plasma and erythrocyte hemolysates showed that initially 75Se was transported in blood as 75SeO3= or loosely bound to plasma and erythrocyte proteins. At high Se intake, albumin became a transport protein for some of the plasma 75Se, and proportionately more blood radioactivity was carried in the erythrocytes. At 72 h after dosing, most plasma 75Se was tightly bound to protein in glutathione peroxidase fraction with low peroxidase activity, possibly Se transport protein. At 72 h, distribution of 75Se in erythrocyte was 35 to 40% in glutathione peroxidase, 50% in hemoglobin, and 5% in a selenite plus selenopolypeptide fraction. Erythrocyte peroxidase activity was mostly in the glutathione peroxidase fraction (57%) and hemoglobin (38%). Molecular weight estimate for erythrocyte glutathione peroxidase was 84,200 daltons; about 90% of blood peroxidase activity was in erythrocytes. High Se intake had no marked effect on distribution of 75Se among liver cytosolic proteins. About 35% of 75Se was in glutathione peroxidase fraction, having most of the peroxidase activity, 25% in void volume, 11 to 18% in a selenite plus selenopolypeptide fraction, and small amounts in selenoproteins of about 12,000 and 50,000 daltons.

  4. 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

  5. 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. PMID:26304433

  6. 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

  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

    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 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

  9. 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

    We evaluated the effects of MP supply on growth performance before and after preconditioning and measurements of innate and humoral immune response of beef steers following vaccination. Angus steers ( = 36; BW = 231 21 kg; age = 184 18 d) were weaned on d -6, stratified by BW and age on d 0, and randomly assigned to 1 of 18 drylot pens (2 steers/pen). Treatments were assigned to pens (6 pens/treatment) and consisted of corn silage-based diets formulated to provide 85%, 100%, or 115% of the daily MP requirements of a beef steer gaining 1.1 kg/d from d 0 to 42. Steers were vaccinated against infectious bovine rhinotracheitis virus, bovine viral diarrhea (BVDV) types 1 and 2 viruses, and clostridium on d 14 and 28. Blood samples were collected on d 0, 14, 15, 17, 21, 28, 29, 30, 35, and 42. Body weight did not differ ( ? 0.17) among treatments from d 0 to 28. On d 42, 115% MP steers were heaviest, 100% MP steers were intermediate, and 85% MP steers were lightest ( = 0.05; 297, 290, and 278 7 kg, respectively). Overall, ADG and G:F did not differ ( ? 0.13) between 100% and 115% MP steers and were least ( < 0.01) for 85% MP steers (1.2, 1.4, and 0.8 0.07 kg/d and 0.23, 0.24, and 0.19 0.008, respectively). Plasma haptoglobin (Hp) concentrations did not differ among treatments ( ? 0.46), whereas plasma ceruloplasmin (Cp) concentrations were greatest ( ? 0.04) for 85% MP steers, intermediate for 100% MP steers, and least for 115% MP steers on d 30, 35, and 42. Plasma cortisol concentrations were greater ( ? 0.03) for 85% vs. 100% and 115% MP steers on d 14 and 28. Liver mRNA expression of Cp and Hp and muscle mRNA expression of m-calpain, mammalian target of rapamycin, and ubiquitin did not differ among treatments ( ? 0.17). Serum neutralization titers to BVDV-1b titers were greater ( ? 0.02) for 115% vs. 85% and 100% MP steers on d 42 (5.8, 3.0, and 3.7 0.60 log, respectively), whereas mean serum leukotoxin titers were greater for 85% vs. 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

  10. Calcium-energized motor protein forisome controls damage in phloem: potential applications as biomimetic "smart" material.

    PubMed

    Srivastava, Vineet Kumar; Tuteja, Renu; Tuteja, Narendra

    2015-06-01

    Forisomes are ATP independent, mechanically active proteins from the Fabaceae family (also called Leguminosae). These proteins are located in sieve tubes of phloem and function to prevent loss of nutrient-rich photoassimilates, upon mechanical injury/wounding. Forisomes are SEO (sieve element occlusion) gene family proteins that have recently been shown to be involved in wound sealing mechanism. Recent findings suggest that forisomes could act as an ideal model to study self assembly mechanism for the development of nanotechnological devices like microinstruments, the microfluidic system frequently used in space exploration missions. Technology enabling improvement in micro instruments has been identified as a key technology by NASA in future space exploration missions. Forisomes are designated as biomimetic smart materials which are calcium-energized motor proteins. Since forisomes are biomolecules from plant systems it can be doctored through genetic engineering. In contrast, "smart" materials which are not derived from plants are difficult to modify in their properties. Current levels of understanding about forisomes conformational shifts with respect to calcium ions and pH changes requires supplement of future advances with relation to its 3D structure to understand self assembly processes. In plant systems it forms blood clots in the form of occlusions to prevent nutrient fluid leakage and thus proves to be a unique damage control system of phloem tissue. PMID:24020505

  11. Detecting Protein Complexes from Signed Protein-Protein Interaction Networks.

    PubMed

    Ou-Yang, Le; Dai, Dao-Qing; Zhang, Xiao-Fei

    2015-01-01

    Identification of protein complexes is fundamental for understanding the cellular functional organization. With the accumulation of physical protein-protein interaction (PPI) data, computational detection of protein complexes from available PPI networks has drawn a lot of attentions. While most of the existing protein complex detection algorithms focus on analyzing the physical protein-protein interaction network, none of them take into account the "signs" (i.e., activation-inhibition relationships) of physical interactions. As the "signs" of interactions reflect the way proteins communicate, considering the "signs" of interactions can not only increase the accuracy of protein complex identification, but also deepen our understanding of the mechanisms of cell functions. In this study, we proposed a novel Signed Graph regularized Nonnegative Matrix Factorization (SGNMF) model to identify protein complexes from signed PPI networks. In our experiments, we compared the results collected by our model on signed PPI networks with those predicted by the state-of-the-art complex detection techniques on the original unsigned PPI networks. We observed that considering the "signs" of interactions significantly benefits the detection of protein complexes. Furthermore, based on the predicted complexes, we predicted a set of signed complex-complex interactions for each dataset, which provides a novel insight of the higher level organization of the cell. All the experimental results and codes can be downloaded from http://mail.sysu.edu.cn/home/stsddq@mail.sysu.edu.cn/dai/others/SGNMF.zip. PMID:26671805

  12. Inulin stimulates NO synthesis via activation of PKC-alpha and protein tyrosine kinase, resulting in the activation of NF-kappaB by IFN-gamma-primed RAW 264.7 cells.

    PubMed

    Koo, Hyun-Na; Hong, Seung-Heon; Seo, Han-Geuk; Yoo, Taek-Soo; Lee, Ki-Nam; Kim, Nam-Song; Kim, Cheorl-Ho; Kim, Hyung-Min

    2003-10-01

    Inulin, an active component of Chicorium intybus root, has been shown to stimulate the growth of bifidobacteria, and inhibit colon carcinogenesis. NO mediates a number of the host-defense functions of activated macrophages, including antimicrobial and tumoricidal activity. We examined the effect of inulin on the synthesis of NO in RAW 264.7 cells. Inulin alone had no effect, whereas inulin with IFN-gamma synergistically increased the NO production and inducible NO synthase (iNOS) expression in RAW 264.7 cells. Synergy between IFN-gamma and inulin was mainly dependent on inulin-induced TNF-alpha secretion. Also, protein kinase C (PKC)-alpha was involved in the inulin-induced NO production. Inulin-mediated NO production was inhibited by the protein tyrosine kinase (PTK) inhibitor, tyrphostin AG126. Since iNOS gene transcriptions have been shown to be under the control of the NF-kappaB/Rel family of transcription factors, we assessed the effect of inulin on NF-kappaB/Rel using an EMSA. Inulin produced strong induction of NF-kappaB/Rel binding, whereas AP-1 binding was slightly induced in RAW 264.7 cells. Inulin stimulated phosphorylation and degradation of IkappaB-alpha. These results suggest that in IFN-gamma-primed RAW 264.7 cells inulin might stimulate NO synthesis via activation of PKC-alpha and PTK, resulting in the activation of NF-kappaB. PMID:14559111

  13. 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.

  14. Protein folding.

    PubMed

    Rossmann, M G; Argos, P

    1981-01-01

    After some general remarks on protein structure, there follows a discussion on primary, secondary, and tertiary organization. The account of primary structure includes a discussion of the conformation of disulfide bonds. Types of helices, sheets, and turns are described in the section on secondary structure, followed by a discussion of super-secondary structure and the effects of metals and prosthetic groups of protein fold. The crux of the review lies in an examination of tertiary structure, or specifically of domains that are defined, in part, as functional units within a polypeptide chain. An assembly of domains can in turn result in a protein whose function is quite sophisticated. Some consideration of domain recognition is given in the section on taxonomy and in the appendix. The key part of the tertiary structure section concentrates on a taxonomic protein classification dependent not only on structure but also on function. A discussion of the requirements by quaternary structure on a fold are omitted in this review. Finally, no review of this kind can escape a discussion of evolutionary convergence and divergence. PMID:7023364

  15. Diets containing soy or rice protein isolate increase insulin sensitivity and improve lipid homeostasis in weanling rats fed high fat, high cholesterol Western diets as a result of activation of PPAR and LXR-mediated pathways

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The current study examined the effects of feeding soy protein isolate (SPI) and rice protein isolate (RPI) on insulin sensitivity and fat breakdown in weanling rats consuming high fat/high cholesterol diets. Male Sprague-Dawley rats were placed on semi-purified diets containing the milk protein case...

  16. Estimation of bar p/p Flux Ratio from Leaky Box and Closed Galaxy Models Using CERN Accelerator Results on bar p Production

    NASA Astrophysics Data System (ADS)

    Saha, R. K.; Majumdar, R.; Pal, P.; Bhattacharyya, D. P.

    Using steady state leaky box and closed galaxy models and the latest primary proton spectrum based on the directly measured data along with the accelerator data on bar p production, the bar p/p flux ratio in the interstellar medium has been estimated in the spectral range from 6 to 100 GeV/n. The derived result is in approximate agreement with the balloon borne calorimeter data of Golden et al. We have closed galaxy model for the estimation of the old component and young component of primary proton spectrum from the composite primary proton spectrum obtained from the recent experiments performed by Menn et al., Webber et al., Seo et al., JACEE and others. The derived results differ slightly from the results of Protheroe, Pal and Bhattacharyya.

  17. 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.

  18. 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.

  19. Surface Mediated Protein Disaggregation

    NASA Astrophysics Data System (ADS)

    Radhakrishna, Mithun; Kumar, Sanat K.

    2014-03-01

    Preventing protein aggregation is of both biological and industrial importance. Biologically these aggregates are known to cause amyloid type diseases like Alzheimer's and Parkinson's disease. Protein aggregation leads to reduced activity of the enzymes in industrial applications. Inter-protein interactions between the hydrophobic residues of the protein are known to be the major driving force for protein aggregation. In the current paper we show how surface chemistry and curvature can be tuned to mitigate these inter-protein interactions. Our results calculated in the framework of the Hydrophobic-Polar (HP) lattice model show that, inter-protein interactions can be drastically reduced by increasing the surface hydrophobicity to a critical value corresponding to the adsorption transition of the protein. At this value of surface hydrophobicity, proteins lose inter-protein contacts to gain surface contacts and thus the surface helps in reducing the inter-protein interactions. Further, we show that the adsorption of the proteins inside hydrophobic pores of optimal sizes are most efficient both in reducing inter-protein contacts and simultaneously retaining most of the native-contacts due to strong protein-surface interactions coupled with stabilization due to the confinement. Department of Energy (Grant No DE-FG02-11ER46811).

  20. Dietary Proteins

    MedlinePLUS

    ... animal products are complete proteins. This means they supply all of the amino acids the body can't make on its own. Plant proteins are incomplete. You must combine different types of plant proteins to get all of ...

  1. Total protein

    MedlinePLUS

    The total protein test measures the total amount of two classes of proteins found in the fluid portion of your blood. These are albumin and globulin. Proteins are important parts of all cells and tissues. ...

  2. 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 experiments 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.

  3. Results from tandem Phase 1 studies evaluating the safety, reactogenicity and immunogenicity of the vaccine candidate antigen Plasmodium falciparum FVO merozoite surface protein-1 (MSP142) administered intramuscularly with adjuvant system AS01

    PubMed Central

    2013-01-01

    Background The development of an asexual blood stage vaccine against Plasmodium falciparum malaria based on the major merozoite surface protein-1 (MSP1) antigen is founded on the protective efficacy observed in preclinical studies and induction of invasion and growth inhibitory antibody responses. The 42 kDa C-terminus of MSP1 has been developed as the recombinant protein vaccine antigen, and the 3D7 allotype, formulated with the Adjuvant System AS02A, has been evaluated extensively in human clinical trials. In preclinical rabbit studies, the FVO allele of MSP142 has been shown to have improved immunogenicity over the 3D7 allele, in terms of antibody titres as well as growth inhibitory activity of antibodies against both the heterologous 3D7 and homologous FVO parasites. Methods Two Phase 1 clinical studies were conducted to examine the safety, reactogenicity and immunogenicity of the FVO allele of MSP142 in the adjuvant system AS01 administered intramuscularly at 0-, 1-, and 2-months: one in the USA and, after evaluation of safety data results, one in Western Kenya. The US study was an open-label, dose escalation study of 10 and 50 μg doses of MSP142 in 26 adults, while the Kenya study, evaluating 30 volunteers, was a double-blind, randomized study of only the 50 μg dose with a rabies vaccine comparator. Results In these studies it was demonstrated that this vaccine formulation has an acceptable safety profile and is immunogenic in malaria-naïve and malaria-experienced populations. High titres of anti-MSP1 antibodies were induced in both study populations, although there was a limited number of volunteers whose serum demonstrated significant inhibition of blood-stage parasites as measured by growth inhibition assay. In the US volunteers, the antibodies generated exhibited better cross-reactivity to heterologous MSP1 alleles than a MSP1-based vaccine (3D7 allele) previously tested at both study sites. Conclusions Given that the primary effector mechanism for blood stage vaccine targets is humoral, the antibody responses demonstrated to this vaccine candidate, both quantitative (total antibody titres) and qualitative (functional antibodies inhibiting parasite growth) warrant further consideration of its application in endemic settings. Trial registrations Clinical Trials NCT00666380 PMID:23342996

  4. 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. PMID:15640517

  5. Lactose binding to human galectin-7 (p53-induced gene 1) induces long-range effects through the protein resulting in increased dimer stability and evidence for positive cooperativity

    PubMed Central

    Ermakova, Elena; Miller, Michelle C; Nesmelova, Irina V; Lpez-Merino, Lara; Berbs, Manuel Alvaro; Nesmelov, Yuri; Tkachev, Yaroslav V; Lagartera, Laura; Daragan, Vladimir A; Andr, Sabine; Caada, F Javier; Jimnez-Barbero, Jess; Sols, Dolores; Gabius, Hans-Joachim; Mayo, Kevin H

    2013-01-01

    The product of p53-induced gene 1 is a member of the galectin family, i.e., galectin-7 (Gal-7). To move beyond structural data by X-ray diffraction, we initiated the study of the lectin by nuclear magnetic resonance (NMR) and circular dichroism spectroscopies, and molecular dynamics (MD) simulations. In concert, our results indicate that lactose binding to human Gal-7 induces long-range effects (minor conformational shifts and changes in structural dynamics) throughout the protein that result in stabilization of the dimer state, with evidence for positive cooperativity. Monte Carlo fits of 15N-Gal-7 HSQC titrations with lactose using a two-site model yield K1 = 0.9 0.6 103 M?1 and K2 = 3.4 0.8 103 M?1. Ligand binding-induced stabilization of the Gal-7 dimer was supported by several lines of evidence: MD-based calculations of interaction energies between ligand-loaded and ligand-free states, gel filtration data and hetero-FRET spectroscopy that indicate a highly reduced tendency for dimer dissociation in the presence of lactose, CD-based thermal denaturation showing that the transition temperature of the lectin is significantly increased in the presence of lactose, and saturation transfer difference (STD) NMR using a molecular probe of the monomer state whose presence is diminished in the presence of lactose. MD simulations with the half-loaded ligand-bound state also provided insight into how allosteric signaling may occur. Overall, our results reveal long-range effects on Gal-7 structure and dynamics, which factor into entropic contributions to ligand binding and allow further comparisons with other members of the galectin family. PMID:23376190

  6. Protein solubility modeling.

    PubMed

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

    1999-07-20

    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. PMID:10397850

  7. Mechanism of protein decarbonylation.

    PubMed

    Wong, Chi-Ming; Marcocci, Lucia; Das, Dividutta; Wang, Xinhong; Luo, Haibei; Zungu-Edmondson, Makhosazane; Suzuki, Yuichiro J

    2013-12-01

    Ligand/receptor stimulation of cells promotes protein carbonylation that is followed by the decarbonylation process, which might involve thiol-dependent reduction (C.M. Wong et al., Circ. Res. 102:301-318; 2008). This study further investigated the properties of this protein decarbonylation mechanism. We found that the thiol-mediated reduction of protein carbonyls is dependent on heat-labile biologic components. Cysteine and glutathione were efficient substrates for decarbonylation. Thiols decreased the protein carbonyl content, as detected by 2,4-dinitrophenylhydrazine, but not the levels of malondialdehyde or 4-hydroxynonenal protein adducts. Mass spectrometry identified proteins that undergo thiol-dependent decarbonylation, which include peroxiredoxins. Peroxiredoxin-2 and -6 were carbonylated and subsequently decarbonylated in response to the ligand/receptor stimulation of cells. siRNA knockdown of glutaredoxin inhibited the decarbonylation of peroxiredoxin. These results strengthen the concept that thiol-dependent decarbonylation defines the kinetics of protein carbonylation signaling. PMID:24044890

  8. 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.

  9. Solitary Inhibition of the Breast Cancer Resistance Protein Efflux Transporter Results in a Clinically Significant Drug-Drug Interaction with Rosuvastatin by Causing up to a 2-Fold Increase in Statin Exposure.

    PubMed

    Elsby, Robert; Martin, Paul; Surry, Dominic; Sharma, Pradeep; Fenner, Katherine

    2016-03-01

    The intestinal efflux transporter breast cancer resistance protein (BCRP) restricts the absorption of rosuvastatin. Of the transporters important to rosuvastatin disposition, fostamatinib inhibited BCRP (IC50 = 50 nM) and organic anion-transporting polypeptide 1B1 (OATP1B1; IC50 > 10 μM), but not organic anion transporter 3, in vitro, predicting a drug-drug interaction (DDI) in vivo through inhibition of BCRP only. Consequently, a clinical interaction study between fostamatinib and rosuvastatin was performed (and reported elsewhere). This confirmed the critical role BCRP plays in statin absorption, as inhibition by fostamatinib resulted in a significant 1.96-fold and 1.88-fold increase in rosuvastatin area under the plasma concentration-time curve (AUC) and Cmax, respectively. An in vitro BCRP inhibition assay, using polarized Caco-2 cells and rosuvastatin as probe substrate, was subsequently validated with literature inhibitors and used to determine BCRP inhibitory potencies (IC50) of the perpetrator drugs eltrombopag, darunavir, lopinavir, clopidogrel, ezetimibe, fenofibrate, and fluconazole. OATP1B1 inhibition was also determined using human embryonic kidney 293-OATP1B1 cells versus estradiol 17β-glucuronide. Calculated parameters of maximum enterocyte concentration [Igut max], maximum unbound hepatic inlet concentration, transporter fraction excreted value, and determined IC50 value were incorporated into mechanistic static equations to compute theoretical increases in rosuvastatin AUC due to inhibition of BCRP and/or OATP1B1. Calculated theoretical increases in exposure correctly predicted the clinically observed changes in rosuvastatin exposure and suggested intestinal BCRP inhibition (not OATP1B1) to be the mechanism underlying the DDIs with these drugs. In conclusion, solitary inhibition of the intestinal BCRP transporter can result in clinically significant DDIs with rosuvastatin, causing up to a maximum 2-fold increase in exposure, which may warrant statin dose adjustment in clinical practice. PMID:26700956

  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. PMID:26539872

  11. Genetic and Molecular Analysis of the X Chromosomal Region 14b17-14c4 in Drosophila Melanogaster: Loss of Function in Nona, a Nuclear Protein Common to Many Cell Types, Results in Specific Physiological and Behavioral Defects

    PubMed Central

    Stanewsky, R.; Rendahl, K. G.; Dill, M.; Saumweber, H.

    1993-01-01

    We have performed a genetic analysis of the 14C region of the X chromosome of Drosophila melanogaster to isolate loss of function alleles of no-on-transient A (nonA; 14C1-2; 1-52.3). NONA is a nuclear protein common to many cell types, which is present in many puffs on polytene chromosomes. Sequence data suggest that the protein contains a pair of RNA binding motifs (RRM) found in many single-strand nucleic acid binding proteins. Hypomorphic alleles of this gene, which lead to aberrant visual and courtship song behavior, still contain normally distributed nonA RNA and NONA protein in embryos, and in all available alleles NONA protein is present in puffs of third instar larval polytene chromosomes. We find that complete loss of this general nuclear protein is semilethal in hemizygous males and homozygous cell lethal in the female germline. Surviving males show more extreme defects in nervous system function than have been described for the hypomorphic alleles. Five other essential genes that reside within this region have been partially characterized. PMID:8244005

  12. 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

  13. Protein-Protein Interfaces in Viral Capsids Are Structurally Unique.

    PubMed

    Cheng, Shanshan; Brooks, Charles L

    2015-11-01

    Viral capsids exhibit elaborate and symmetrical architectures of defined sizes and remarkable mechanical properties not seen with cellular macromolecular complexes. Given the uniqueness of the higher-order organization of viral capsid proteins in the virosphere, we explored the question of whether the patterns of protein-protein interactions within viral capsids are distinct from those in generic protein complexes. Our comparative analysis involving a non-redundant set of 551 inter-subunit interfaces in viral capsids from VIPERdb and 20,014 protein-protein interfaces in non-capsid protein complexes from the Protein Data Bank found 418 generic protein-protein interfaces that share similar physicochemical patterns with some protein-protein interfaces in the capsid set, using the program PCalign we developed for comparing protein-protein interfaces. This overlap in the structural space of protein-protein interfaces is significantly small, with a p-value <0.0001, based on a permutation test on the total set of protein-protein interfaces. Furthermore, the generic protein-protein interfaces that bear similarity in their spatial and chemical arrangement with capsid ones are mostly small in size with fewer than 20 interfacial residues, which results from the relatively limited choices of natural design for small interfaces rather than having significant biological implications in terms of functional relationships. We conclude based on this study that protein-protein interfaces in viral capsids are non-representative of patterns in the smaller, more compact cellular protein complexes. Our finding highlights the design principle of building large biological containers from repeated, self-assembling units and provides insights into specific targets for antiviral drug design for improved efficacy. PMID:26375252

  14. 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…

  15. 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

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

    SciTech Connect

    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.

  17. 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-Meja, 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 deviationSD=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

  18. Computational Prediction of Protein-Protein Interaction Networks: Algo-rithms and Resources.

    PubMed

    Zahiri, Javad; Bozorgmehr, Joseph Hannon; Masoudi-Nejad, Ali

    2013-09-01

    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

  19. Computational Prediction of ProteinProtein 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

  20. Protein folds and protein folding

    PubMed Central

    Schaeffer, R. Dustin; Daggett, Valerie

    2011-01-01

    The classification of protein folds is necessarily based on the structural elements that distinguish domains. Classification of protein domains consists of two problems: the partition of structures into domains and the classification of domains into sets of similar structures (or folds). Although similar topologies may arise by convergent evolution, the similarity of their respective folding pathways is unknown. The discovery and the characterization of the majority of protein folds will be followed by a similar enumeration of available protein folding pathways. Consequently, understanding the intricacies of structural domains is necessary to understanding their collective folding pathways. We review the current state of the art in the field of protein domain classification and discuss methods for the systematic and comprehensive study of protein folding across protein fold space via atomistic molecular dynamics simulation. Finally, we discuss our large-scale Dynameomics project, which includes simulations of representatives of all autonomous protein folds. PMID:21051320

  1. 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

  2. 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 Se0 through redox reactions by proteins released from bacterial cells. Sulfhydryl groups on peptides excreted outside the cells may also react directly with selenite. Furthermore, membrane reductases and the intracellular synthesis of low molecular weight thiols such as bacillithiols may also play a role in SeO32− reduction. Formation of SeNPs seems to be the result of an Ostwald ripening mechanism. PMID:24606965

  3. 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. PMID:25747476

  4. 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, Mirk Palla, Andrew Sun and Jung-Chi Liao The load dependence of the physical properties of a molecular motor Xianchao Meng, Min Yu and Yunxin Zhang Microtubule organization by kinesin motors and microtubule crosslinking protein MAP65 Joshua Pringle, Amutha Muthukumar, Amanda Tan, Laura Crankshaw, Leslie Conway and Jennifer L Ross Backtracking dynamics of RNA polymerase: pausing and error correction Mamata Sahoo and Stefan Klumpp First-passage problems in DNA replication: effects of template tension on stepping and exonuclease activities of a DNA polymerase motor Ajeet K Sharma and Debashish Chowdhury

  5. TWIST Results

    NASA Astrophysics Data System (ADS)

    Bueno, J.

    2009-12-01

    The TRIUMF Weak Interaction Symmetry Test (TWIST) has now completed its data collection period. These data represent the world's most precise measurement of the muon decay spectrum. The experiment expects to meet its ambitious goals of an order of magnitude improvement over pre-TWIST results for the muon decay parameters ?, ? and P???. Our most recent published results are presented. The measures that have been taken to reduce the systematic uncertainties for the upcoming final result are described.

  6. Exploring NMR ensembles of calcium binding proteins: Perspectives to design inhibitors of protein-protein interactions

    PubMed Central

    2011-01-01

    Background Disrupting protein-protein interactions by small organic molecules is nowadays a promising strategy employed to block protein targets involved in different pathologies. However, structural changes occurring at the binding interfaces make difficult drug discovery processes using structure-based drug design/virtual screening approaches. Here we focused on two homologous calcium binding proteins, calmodulin and human centrin 2, involved in different cellular functions via protein-protein interactions, and known to undergo important conformational changes upon ligand binding. Results In order to find suitable protein conformations of calmodulin and centrin for further structure-based drug design/virtual screening, we performed in silico structural/energetic analysis and molecular docking of terphenyl (a mimicking alpha-helical molecule known to inhibit protein-protein interactions of calmodulin) into X-ray and NMR ensembles of calmodulin and centrin. We employed several scoring methods in order to find the best protein conformations. Our results show that docking on NMR structures of calmodulin and centrin can be very helpful to take into account conformational changes occurring at protein-protein interfaces. Conclusions NMR structures of protein-protein complexes nowadays available could efficiently be exploited for further structure-based drug design/virtual screening processes employed to design small molecule inhibitors of protein-protein interactions. PMID:21569443

  7. How Proteins Bind Macrocycles

    PubMed Central

    Villar, Elizabeth A.; Beglov, Dmitri; Chennamadhavuni, Spandan; Porco, John A.; Kozakov, Dima; Vajda, Sandor; Whitty, Adrian

    2014-01-01

    The potential utility of synthetic macrocycles as drugs, particularly against low druggability targets such as protein-protein interactions, has been widely discussed. There is little information, however, to guide the design of macrocycles for good target protein-binding activity or bioavailability. To address this knowledge gap we analyze the binding modes of a representative set of macrocycle-protein complexes. The results, combined with consideration of the physicochemical properties of approved macrocyclic drugs, allow us to propose specific guidelines for the design of synthetic macrocycles libraries possessing structural and physicochemical features likely to favor strong binding to protein targets and also good bioavailability. We additionally provide evidence that large, natural product derived macrocycles can bind to targets that are not druggable by conventional, drug-like compounds, supporting the notion that natural product inspired synthetic macrocycles can expand the number of proteins that are druggable by synthetic small molecules. PMID:25038790

  8. Molecular modelling of protein-protein/protein-solvent interactions

    NASA Astrophysics Data System (ADS)

    Luchko, Tyler

    The inner workings of individual cells are based on intricate networks of protein-protein interactions. However, each of these individual protein interactions requires a complex physical interaction between proteins and their aqueous environment at the atomic scale. In this thesis, molecular dynamics simulations are used in three theoretical studies to gain insight at the atomic scale about protein hydration, protein structure and tubulin-tubulin (protein-protein) interactions, as found in microtubules. Also presented, in a fourth project, is a molecular model of solvation coupled with the Amber molecular modelling package, to facilitate further studies without the need of explicitly modelled water. Basic properties of a minimally solvated protein were calculated through an extended study of myoglobin hydration with explicit solvent, directly investigating water and protein polarization. Results indicate a close correlation between polarization of both water and protein and the onset of protein function. The methodology of explicit solvent molecular dynamics was further used to study tubulin and microtubules. Extensive conformational sampling of the carboxy-terminal tails of 8-tubulin was performed via replica exchange molecular dynamics, allowing the characterisation of the flexibility, secondary structure and binding domains of the C-terminal tails through statistical analysis methods. Mechanical properties of tubulin and microtubules were calculated with adaptive biasing force molecular dynamics. The function of the M-loop in microtubule stability was demonstrated in these simulations. The flexibility of this loop allowed constant contacts between the protofilaments to be maintained during simulations while the smooth deformation provided a spring-like restoring force. Additionally, calculating the free energy profile between the straight and bent tubulin configurations was used to test the proposed conformational change in tubulin, thought to cause microtubule destabilization. No conformational change was observed but a nucleotide dependent 'softening' of the interaction was found instead, suggesting that an entropic force in a microtubule configuration could be the mechanism of microtubule collapse. Finally, to overcome much of the computational costs associated with explicit soIvent calculations, a new combination of molecular dynamics with the 3D-reference interaction site model (3D-RISM) of solvation was integrated into the Amber molecular dynamics package. Our implementation of 3D-RISM shows excellent agreement with explicit solvent free energy calculations. Several optimisation techniques, including a new multiple time step method, provide a nearly 100 fold performance increase, giving similar computational performance to explicit solvent.

  9. Protein delivery with nanoscale precision

    NASA Astrophysics Data System (ADS)

    Tang, Qiling; Zhang, Yuexing; Chen, Liaohai; Yan, Funing; Wang, Rong

    2005-08-01

    A novel assay of protein delivery to a surface with nanoscale precision was established. This was achieved by combining recent advancements in atomic force microscopy (AFM) and bioconjugation. We utilized a heterobifunctional photocleavable cross linker to functionalize an AFM tip with proteins. Upon irradiation, the proteins were released from the tip due to a photolytic reaction of the cross linker. These proteins bound tightly to their binding partners on a substrate. When tip functionalization is carefully controlled, proteins can be locally delivered to a desired area. Importantly, the result of protein delivery can be examined immediately by high-resolution imaging in the same area using the protein-free tip. Successful protein delivery was also confirmed by fluorescence imaging and was proved to be reproducible. The approach allows protein delivery and subsequent imaging to be performed in the same local area with the same AFM tip, thus opening up the possibility of monitoring protein functions in living cells in real time.

  10. 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...

  11. 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 of developing ACPA-positive RA is associated with a strong gene-environment interaction between smoking and HLA-DRB1 SE alleles in a Malaysian multiethnic population of Asian descent. This interaction seems to apply also between smoking and the specific HLA-DRB1*04:05 SE allele, which is common in Asian populations but not in Caucasians. PMID:22537824

  12. Gradual Soil Water Depletion Results in Reversible Changes of Gene Expression, Protein Profiles, Ecophysiology, and Growth Performance in Populus euphratica, a Poplar Growing in Arid Regions1[W][OA

    PubMed Central

    Bogeat-Triboulot, Marie-Batrice; Brosch, Mikael; Renaut, Jenny; Jouve, Laurent; Le Thiec, Didier; Fayyaz, Payam; Vinocur, Basia; Witters, Erwin; Laukens, Kris; Teichmann, Thomas; Altman, Arie; Hausman, Jean-Franois; Polle, Andrea; Kangasjrvi, Jaakko; Dreyer, Erwin

    2007-01-01

    The responses of Populus euphratica Oliv. plants to soil water deficit were assessed by analyzing gene expression, protein profiles, and several plant performance criteria to understand the acclimation of plants to soil water deficit. Young, vegetatively propagated plants originating from an arid, saline field site were submitted to a gradually increasing water deficit for 4 weeks in a greenhouse and were allowed to recover for 10 d after full reirrigation. Time-dependent changes and intensity of the perturbations induced in shoot and root growth, xylem anatomy, gas exchange, and water status were recorded. The expression profiles of approximately 6,340 genes and of proteins and metabolites (pigments, soluble carbohydrates, and oxidative compounds) were also recorded in mature leaves and in roots (gene expression only) at four stress levels and after recovery. Drought successively induced shoot growth cessation, stomatal closure, moderate increases in oxidative stress-related compounds, loss of CO2 assimilation, and root growth reduction. These effects were almost fully reversible, indicating that acclimation was dominant over injury. The physiological responses were paralleled by fully reversible transcriptional changes, including only 1.5% of the genes on the array. Protein profiles displayed greater changes than transcript levels. Among the identified proteins for which expressed sequence tags were present on the array, no correlation was found between transcript and protein abundance. Acclimation to water deficit involves the regulation of different networks of genes in roots and shoots. Such diverse requirements for protecting and maintaining the function of different plant organs may render plant engineering or breeding toward improved drought tolerance more complex than previously anticipated. PMID:17158588

  13. INTEGRAL Results

    SciTech Connect

    Walter, Roland

    2005-11-22

    INTEGRAL is operational since more than two years and producing high quality data. The instruments are working almost perfectly. A selection of INTEGRAL results obtained on point sources are presented.

  14. Predicting Permanent and Transient Protein-Protein Interfaces

    PubMed Central

    La, David; Kong, Misun; Hoffman, William; Choi, Youn Im; Kihara, Daisuke

    2014-01-01

    Protein-protein interactions are involved in many diverse functions in a cell. To optimize functional roles of interactions, proteins interact with a spectrum of binding affinities. Interactions are conventionally classified into permanent and transient, where the former denotes tight binding between proteins that result in strong complexes, while the latter compose of relatively weak interactions that can dissociate after binding to regulate functional activity at specific time point. Knowing the type of interactions has significant implications for understanding the nature and function of protein-protein interactions. In this study, we constructed amino acid substitution models that capture mutation patterns at permanent and transient type of protein interfaces, which were found to be different with statistical significance. Using the substitution models, we developed a novel computational method that predicts permanent and transient protein binding interfaces in protein surfaces. Without knowledge of the interacting partner, the method employs a single query protein structure and a multiple sequence alignment of the sequence family. Using a large dataset of permanent and transient proteins, we show that our method performs very well in protein interface classification. A very high Area Under the Curve (AUC) value of 0.957 was observed when predicted protein binding sites were classified. Remarkably, near prefect accuracy was achieved with an AUC of 0.991 when actual binding sites were classified. The developed method will be also useful for protein design of permanent and transient protein binding interfaces. PMID:23239312

  15. How Many Protein-Protein Interactions Types Exist in Nature?

    PubMed Central

    Mitra, Pralay; Zhang, Yang

    2012-01-01

    Protein quaternary structure universe refers to the ensemble of all protein-protein complexes across all organisms in nature. The number of quaternary folds thus corresponds to the number of ways proteins physically interact with other proteins. This study focuses on answering two basic questions: Whether the number of protein-protein interactions is limited and, if yes, how many different quaternary folds exist in nature. By all-to-all sequence and structure comparisons, we grouped the protein complexes in the protein data bank (PDB) into 3,629 families and 1,761 folds. A statistical model was introduced to obtain the quantitative relation between the numbers of quaternary families and quaternary folds in nature. The total number of possible protein-protein interactions was estimated around 4,000, which indicates that the current protein repository contains only 42% of quaternary folds in nature and a full coverage needs approximately a quarter century of experimental effort. The results have important implications to the protein complex structural modeling and the structure genomics of protein-protein interactions. PMID:22719985

  16. The Halophile Protein Database

    PubMed Central

    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/ PMID:25468930

  17. Cutting Edge: Codeletion of the Ras GTPase-Activating Proteins (RasGAPs) Neurofibromin 1 and p120 RasGAP in T Cells Results in the Development of T Cell Acute Lymphoblastic Leukemia.

    PubMed

    Lubeck, Beth A; Lapinski, Philip E; Oliver, Jennifer A; Ksionda, Olga; Parada, Luis F; Zhu, Yuan; Maillard, Ivan; Chiang, Mark; Roose, Jeroen; King, Philip D

    2015-07-01

    Ras GTPase-activating proteins (RasGAPs) inhibit signal transduction initiated through the Ras small GTP-binding protein. However, which members of the RasGAP family act as negative regulators of T cell responses is not completely understood. In this study, we investigated potential roles for the RasGAPs RASA1 and neurofibromin 1 (NF1) in T cells through the generation and analysis of T cell-specific RASA1 and NF1 double-deficient mice. In contrast to mice lacking either RasGAP alone in T cells, double-deficient mice developed T cell acute lymphoblastic leukemia/lymphoma, which originated at an early point in T cell development and was dependent on activating mutations in the Notch1 gene. These findings highlight RASA1 and NF1 as cotumor suppressors in the T cell lineage. PMID:26002977

  18. Molecular simulations of lipid-mediated protein-protein interactions.

    PubMed

    de Meyer, Frdrick 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

  19. 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

  20. DONUT results

    SciTech Connect

    Furukawa, Tomoko

    2008-02-21

    The DONUT experiment succeeded in observing tau-neutrino CC interactions for the first time in 2000. The analysis using total sample is presented in this paper, based on 3.5x10{sup 17} protons on target. The number of identified {nu}{sub {tau}} CC interactions is 9 from 581 neutrino interactions located in the emulsion. The result of the first measurement of {nu}{sub {tau}} CC cross section is consistent with the expectation from the Standard Model.

  1. 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. PMID:11541767

  2. Wnt proteins.

    PubMed

    Willert, Karl; Nusse, Roel

    2012-09-01

    Wnt proteins comprise a major family of signaling molecules that orchestrate and influence a myriad of cell biological and developmental processes. Although our understanding of the role of Wnt signaling in regulating development and affecting disease, such as cancer, has been ever increasing, the study of the Wnt proteins themselves has been painstaking and slow moving. Despite advances in the biochemical characterization of Wnt proteins, many mysteries remain unsolved. In contrast to other developmental signaling molecules, such as fibroblast growth factors (FGF), transforming growth factors (TGF?), and Sonic hedgehog (Shh), Wnt proteins have not conformed to many standard methods of protein production, such as bacterial overexpression, and analysis, such as ligand-receptor binding assays. The reasons for their recalcitrant nature are likely a consequence of the complex set of posttranslational modifications involving several highly specialized and poorly characterized processing enzymes. With the recent description of the first Wnt protein structure, the time is ripe to uncover and possibly resolve many of the remaining issues surrounding Wnt proteins and their interactions. Here we describe the process of maturation of Wnt from its initial translation to its eventual release from a cell and interactions in the extracellular environment. PMID:22952392

  3. Quantification of the influence of protein-protein interactions on adsorbed protein structure and bioactivity.

    PubMed

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

    2013-10-01

    While protein-surface interactions have been widely studied, relatively little is understood at this time regarding how protein-surface interaction effects are influenced by protein-protein interactions and how these effects combine with the internal stability of a protein to influence its adsorbed-state structure and bioactivity. The objectives of this study were to develop a method to study these combined effects under widely varying protein-protein interaction conditions using hen egg-white lysozyme (HEWL) adsorbed on silica glass, poly(methyl methacrylate), and polyethylene as our model systems. In order to vary protein-protein interaction effects over a wide range, HEWL was first adsorbed to each surface type under widely varying protein solution concentrations for 2h to saturate the surface, followed by immersion in pure buffer solution for 15h to equilibrate the adsorbed protein layers in the absence of additionally adsorbing protein. Periodic measurements were made at selected time points of the areal density of the adsorbed protein layer as an indicator of the level of protein-protein interaction effects within the layer, and these values were then correlated with measurements of the adsorbed protein's secondary structure and bioactivity. The results from these studies indicate that protein-protein interaction effects help stabilize the structure of HEWL adsorbed on silica glass, have little influence on the structural behavior of HEWL on HDPE, and actually serve to destabilize HEWL's structure on PMMA. The bioactivity of HEWL on silica glass and HDPE was found to decrease in direct proportion to the degree of adsorption-induce protein unfolding. A direct correlation between bioactivity and the conformational state of adsorbed HEWL was less apparent on PMMA, thus suggesting that other factors influenced HEWL's bioactivity on this surface, such as the accessibility of HEWL's bioactive site being blocked by neighboring proteins or the surface itself. The developed methods provide an effective means to characterize the influence of protein-protein interaction effects and provide new molecular-level insights into how protein-protein interaction effects combine with protein-surface interaction and internal protein stability effects to influence the structure and bioactivity of adsorbed protein. PMID:23751416

  4. Protein aggregation and prionopathies.

    PubMed

    Renner, M; Melki, R

    2014-06-01

    Prion protein and prion-like proteins share a number of characteristics. From the molecular point of view, they are constitutive proteins that aggregate following conformational changes into insoluble particles. These particles escape the cellular clearance machinery and amplify by recruiting the soluble for of their constituting proteins. The resulting protein aggregates are responsible for a number of neurodegenerative diseases such as Creutzfeldt-Jacob, Alzheimer, Parkinson and Huntington diseases. In addition, there are increasing evidences supporting the inter-cellular trafficking of these aggregates, meaning that they are "transmissible" between cells. There are also evidences that brain homogenates from individuals developing Alzheimer and Parkinson diseases propagate the disease in recipient model animals in a manner similar to brain extracts of patients developing Creutzfeldt-Jacob's disease. Thus, the propagation of protein aggregates from cell to cell may be a generic phenomenon that contributes to the evolution of neurodegenerative diseases, which has important consequences on human health issues. Moreover, although the distribution of protein aggregates is characteristic for each disease, new evidences indicate the possibility of overlaps and crosstalk between the different disorders. Despite the increasing evidences that support prion or prion-like propagation of protein aggregates, there are many unanswered questions regarding the mechanisms of toxicity and this is a field of intensive research nowadays. PMID:24698014

  5. Protein Attachment on Nanodiamonds.

    PubMed

    Lin, Chung-Lun; Lin, Cheng-Huang; Chang, Huan-Cheng; Su, Meng-Chih

    2015-07-16

    A recent advance in nanotechnology is the scale-up production of small and nonaggregated diamond nanoparticles suitable for biological applications. Using detonation nanodiamonds (NDs) with an average diameter of ?4 nm as the adsorbents, we have studied the static attachment of three proteins (myoglobin, bovine serum albumin, and insulin) onto the nanoparticles by optical spectroscopy, mass spectrometry, and dynamic light scattering, and electrophoretic zeta potential measurements. Results show that the protein surface coverage is predominantly determined by the competition between protein-protein and protein-ND interactions, giving each protein a unique and characteristic structural configuration in its own complex. Specifically, both myoglobin and bovine serum albumin show a Langmuir-type adsorption behavior, forming 1:1 complexes at saturation, whereas insulin folds into a tightly bound multimer before adsorption. The markedly different adsorption patterns appear to be independent of the protein concentration and are closely related to the affinity of the individual proteins for the NDs. The present study provides a fundamental understanding for the use of NDs as a platform for nanomedical drug delivery. PMID:25815400

  6. Replication Proteins and Human Disease

    PubMed Central

    Jackson, Andrew P.; Laskey, Ronald A.; Coleman, Nicholas

    2014-01-01

    In this article, we discuss the significance of DNA replication proteins in human disease. There is a broad range of mutations in genes encoding replication proteins, which result in several distinct clinical disorders that share common themes. One group of replication proteins, the MCMs, has emerged as effective biomarkers for early detection of a range of common cancers. They offer practical and theoretical advantages over other replication proteins and have been developed for widespread clinical use. PMID:23881941

  7. 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 large molecular weight, net negative charge and hydrophilicity of synthetic small interfering RNAs makes it hard for the molecules to cross the plasma membrane and enter the cell cytoplasm. Immune responses can also diminish the effectiveness of this approach. In this issue, Shiri Weinstein and Dan Peer from Tel Aviv University provide an overview of the challenges and recent progress in the use of nanocarriers for delivering RNAi effector molecules into target tissues and cells more effectively [5]. Also in this issue, researchers in Korea report new results that demonstrate the potential of nanostructures in neural network engineering [6]. Min Jee Jang et al report directional growth of neurites along linear carbon nanotube patterns, demonstrating great progress in neural engineering and the scope for using nanotechnology to treat neural diseases. Modern medicine cannot claim to have abolished the pain and suffering that accompany disease. But a comparison between the ghastly and often ineffective iron implements of early medicine and the smart gadgets and treatments used in hospitals today speaks volumes for the extraordinary progress that has been made, and the motivation behind this research. References [1] Wallis F 2000 Signs and senses: diagnosis and prognosis in early medieval pulse and urine texts Soc. Hist. Med. 13 265-78 [2] Arntz Y, Seelig J D, Lang H P, Zhang J, Hunziker P, Ramseyer J P, Meyer E, Hegner M and Gerber Ch 2003 Label-free protein assay based on a nanomechanical cantiliever array Nanotechnology 14 86-90 [3] Gowtham S, Scheicher R H, Pandey R, Karna S P and Ahuja R 2008 First-principles study of physisorption of nucleic acid bases on small-diameter carbon nanotubes Nanotechnology 19 125701 [4] Wang H-N and Vo-Dinh T 2009 Multiplex detection of breast cancer biomarkers using plasmonic molecular sentinel nanoprobes Nanotechnology 20 065101 [5] Weinstein S and Peer D 2010 RNAi nanomedicines: challenges and opportunities within the immune system Nanotechnology 21 232001 [6] Jang M J, Namgung S, Hong S, and Nam Y 2010 Directional neurite growth using carbon nanotube patterned substrates as a biomimetic cue Nanotechnology 21 235102

  8. Electronic transport via proteins.

    PubMed

    Amdursky, Nadav; Marchak, Debora; Sepunaru, Lior; Pecht, Israel; Sheves, Mordechai; Cahen, David

    2014-11-12

    A central vision in molecular electronics is the creation of devices with functional molecular components that may provide unique properties. Proteins are attractive candidates for this purpose, as they have specific physical (optical, electrical) and chemical (selective binding, self-assembly) functions and offer a myriad of possibilities for (bio-)chemical modification. This Progress Report focuses on proteins as potential building components for future bioelectronic devices as they are quite efficient electronic conductors, compared with saturated organic molecules. The report addresses several questions: how general is this behavior; how does protein conduction compare with that of saturated and conjugated molecules; and what mechanisms enable efficient conduction across these large molecules? To answer these questions results of nanometer-scale and macroscopic electronic transport measurements across a range of organic molecules and proteins are compiled and analyzed, from single/few molecules to large molecular ensembles, and the influence of measurement methods on the results is considered. Generalizing, it is found that proteins conduct better than saturated molecules, and somewhat poorer than conjugated molecules. Significantly, the presence of cofactors (redox-active or conjugated) in the protein enhances their conduction, but without an obvious advantage for natural electron transfer proteins. Most likely, the conduction mechanisms are hopping (at higher temperatures) and tunneling (below ca. 150-200 K). PMID:25256438

  9. Whey Protein

    MedlinePLUS

    ... that taking whey protein daily while participating in resistance training does not reduce cholesterol levels or body ... not improve muscle function, walking speed, or other movement tests in people with polymyalgia rheumatica. Other conditions. ...

  10. Proteins : paradigms of complexity /

    SciTech Connect

    Frauenfelder, Hans,

    2001-01-01

    Proteins are the working machines of living systems. Directed by the DNA, of the order of a few hundred building blocks, selected from twenty different amino acids, are covalently linked into a linear polypeptide chain. In the proper environment, the chain folds into the working protein, often a globule of linear dimensions of a few nanometers. The biologist considers proteins units from which living systems are built. Many physical scientists look at them as systems in which the laws of complexity can be studied better than anywhere else. Some of the results of such studies will be sketched.

  11. 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

  12. Small Molecule Inhibitors to Disrupt Protein-protein Interactions of Heat Shock Protein 90 Chaperone Machinery

    PubMed Central

    Seo, Young Ho

    2015-01-01

    Heat shock protein 90 (Hsp90) is an adenosine triphosphate dependent molecular chaperone in eukaryotic cells that regulates the activation and maintenance of numerous regulatory and signaling proteins including epidermal growth factor receptor, human epidermal growth factor receptor 2, mesenchymal-epithelial transition factor, cyclin-dependent kinase-4, protein kinase B, hypoxia-inducible factor 1?, and matrix metalloproteinase-2. Since many of Hsp90 clients are oncogenic proteins, Hsp90 has become an attractive therapeutic target for treatment of cancer. To discover small molecule inhibitors targeting Hsp90 chaperone machinery, several strategies have been employed, which results in three classes of inhibitors such as N-terminal inhibitors, C-terminal inhibitors, and inhibitors disrupting protein-protein interactions of Hsp90 chaperone machinery. Developing small molecule inhibitors that modulate protein-protein interactions of Hsp90 is a challenging task, although it offers many alternative opportunities for therapeutic intervention. The lack of well-defined binding pocket and starting points for drug design challenges medicinal chemists to discover small molecule inhibitors disrupting protein-protein interactions of Hsp90. The present review will focus on the current studies on small molecule inhibitors disrupting protein-protein interactions of Hsp90 chaperone machinery, provide biological background on the structure, function and mechanism of Hsp90s protein-protein interactions, and discuss the challenges and promise of its small molecule modulations. PMID:25853099

  13. Balanced ProteinWater Interactions Improve Properties of Disordered Proteins and Non-Specific Protein Association

    PubMed Central

    2015-01-01

    Some frequently encountered deficiencies in all-atom molecular simulations, such as nonspecific proteinprotein interactions being too strong, and unfolded or disordered states being too collapsed, suggest that proteins are insufficiently well solvated in simulations using current state-of-the-art force fields. To address these issues, we make the simplest possible change, by modifying the short-range proteinwater pair interactions, and leaving all the waterwater and proteinprotein parameters unchanged. We find that a modest strengthening of proteinwater interactions is sufficient to recover the correct dimensions of intrinsically disordered or unfolded proteins, as determined by direct comparison with small-angle X-ray scattering (SAXS) and Frster resonance energy transfer (FRET) data. The modification also results in more realistic protein-protein affinities, and average solvation free energies of model compounds which are more consistent with experiment. Most importantly, we show that this scaling is small enough not to affect adversely the stability of the folded state, with only a modest effect on the stability of model peptides forming ?-helix and ?-sheet structures. The proposed adjustment opens the way to more accurate atomistic simulations of proteins, particularly for intrinsically disordered proteins, proteinprotein association, and crowded cellular environments. PMID:25400522

  14. Isolation of protein subpopulations undergoing protein-protein interactions.

    PubMed

    Nelson, Thomas J; Backlund, Peter S; Yergey, Alfred L; Alkon, Daniel L

    2002-03-01

    A new method is described for isolating and identifying proteins participating in protein-protein interactions in a complex mixture. The method uses a cyanogen bromide-activated Sepharose matrix to isolate proteins that are non-covalently bound to other proteins. Because the proteins are accessible to chemical manipulation, mass spectrometric identification of the proteins can yield information on specific classes of interacting proteins, such as calcium-dependent or substrate-dependent protein interactions. This permits selection of a subpopulation of proteins from a complex mixture on the basis of specified interaction criteria. The new method has the advantage of screening the entire proteome simultaneously, unlike the two-hybrid system or phage display, which can only detect proteins binding to a single bait protein at a time. The method was tested by selecting rat brain extract for proteins exhibiting calcium-dependent protein interactions. Of 12 proteins identified by mass spectrometry, eight were either known calcium-binding proteins or proteins with known calcium-dependent protein interactions, indicating that the method is capable of enriching a subpopulation of proteins from a complex mixture on the basis of a specific class of protein interactions. Because only naturally occurring interactions of proteins in their native state are observed, this method will have wide applicability to studies of protein interactions in tissue samples and autopsy specimens, for screening for perturbations of protein-protein interactions by signaling molecules, pharmacological agents or toxins, and screening for differences between cancerous and untransformed cells. PMID:12096125

  15. Predictions of Protein-Protein Interfaces within Membrane Protein Complexes

    PubMed Central

    Asadabadi, Ebrahim Barzegari; Abdolmaleki, Parviz

    2013-01-01

    Background Prediction of interaction sites within the membrane protein complexes using the sequence data is of a great importance, because it would find applications in modification of molecules transport through membrane, signaling pathways and drug targets of many diseases. Nevertheless, it has gained little attention from the protein structural bioinformatics community. Methods In this study, a wide variety of prediction and classification tools were applied to distinguish the residues at the interfaces of membrane proteins from those not in the interfaces. Results The tuned SVM model achieved the high accuracy of 86.95% and the AUC of 0.812 which outperforms the results of the only previous similar study. Nevertheless, prediction performances obtained using most employed models cannot be used in applied fields and needs more effort to improve. Conclusion Considering the variety of the applied tools in this study, the present investigation could be a good starting point to develop more efficient tools to predict the membrane protein interaction site residues. PMID:23919118

  16. Physical inactivity is correlated with levels of quantitative C-reactive protein in serum, independent of obesity: results of the national surveillance of risk factors of non-communicable diseases in Iran.

    PubMed

    Esteghamati, Alireza; Morteza, Afsaneh; Khalilzadeh, Omid; Anvari, Mehdi; Noshad, Sina; Zandieh, Ali; Nakhjavani, Manouchehr

    2012-03-01

    Increased C-reactive protein (CRP) levels are associated with coronary heart disease, stroke, and mortality. Physical activity prevents cardiovascular disorders, which can be partly mediated through reducing inflammation, including serum CRP levels. The association of different intensities of physical activity, sedentary behaviours, and C-reactive protein (CRP) levels in serum was examined after adjustment for markers of adiposity, including waist-circumference and body mass index (BMI), in a large population-based study. Using data of the SuRFNCD-2007 study, a large national representative population-based study in Iran, the relationship between quantitative CRP concentrations in serum and physical activity was examined in a sample of 3,001 Iranian adults. The global physical activity questionnaire (GPAQ) was used for evaluating the duration and intensity of physical activity. Total physical activity (TPA) was calculated using metabolic equivalents for the intensity of physical activity. Quantitative CRP concentrations in serum were measured with high-sensitivity enzyme immunoassay. The CRP levels in serum significantly correlated with TPA (r=-0.103, p=0.021 in men and r=-0.114, p=0.017 in women), duration of vigorous-intensity activity (r=-0.122, p=0.019 in men and r=-0.109, p=0.026 in women), duration of moderate-intensity activity (r=-0.107, p=0.031 in men and r=-0.118, p=0.020 in women), and duration of sedentary behaviours (r=0.092, p=0.029 in men and r=0.101, p=0.022 in women) after multiple adjustments for age, area of residence, BMI, waist-circumference, smoking, and diabetes mellitus. Physical activity (of both moderate and vigorous intensity) is inversely associated with the quantitative CRP levels in serum, independent of diabetes and body adiposity. PMID:22524121

  17. Physical Inactivity Is Correlated with Levels of Quantitative C-reactive Protein in Serum, Independent of Obesity: Results of the National Surveillance of Risk Factors of Non-communicable Diseases in Iran

    PubMed Central

    Morteza, Afsaneh; Khalilzadeh, Omid; Anvari, Mehdi; Noshad, Sina; Zandieh, Ali; Nakhjavani, Manouchehr

    2012-01-01

    Increased C-reactive protein (CRP) levels are associated with coronary heart disease, stroke, and mortality. Physical activity prevents cardiovascular disorders, which can be partly mediated through reducing inflammation, including serum CRP levels. The association of different intensities of physical activity, sedentary behaviours, and C-reactive protein (CRP) levels in serum was examined after adjustment for markers of adiposity, including waist-circumference and body mass index (BMI), in a large population-based study. Using data of the SuRFNCD-2007 study, a large national representative population-based study in Iran, the relationship between quantitative CRP concentrations in serum and physical activity was examined in a sample of 3,001 Iranian adults. The global physical activity questionnaire (GPAQ) was used for evaluating the duration and intensity of physical activity. Total physical activity (TPA) was calculated using metabolic equivalents for the intensity of physical activity. Quantitative CRP concentrations in serum were measured with high-sensitivity enzyme immunoassay. The CRP levels in serum significantly correlated with TPA (r=-0.103, p=0.021 in men and r=-0.114, p=0.017 in women), duration of vigorous-intensity activity (r=-0.122, p=0.019 in men and r=-0.109, p=0.026 in women), duration of moderate-intensity activity (r=-0.107, p=0.031 in men and r=-0.118, p=0.020 in women), and duration of sedentary behaviours (r=0.092, p=0.029 in men and r=0.101, p=0.022 in women) after multiple adjustments for age, area of residence, BMI, waist-circumference, smoking, and diabetes mellitus. Physical activity (of both moderate and vigorous intensity) is inversely associated with the quantitative CRP levels in serum, independent of diabetes and body adiposity. PMID:22524121

  18. 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.

  19. 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 with baseline CRP (P = 0.593), baseline DAS28-CRP (P = 0.540), or change in DAS28-CRP after treatment with an anti-TNF over a 6-month period (P = 0.302). Conclusions Although CRP genotype may influence baseline CRP levels, in patients with very active disease, no such association was found. This suggests that genetic variation at the CRP locus does not influence DAS28-CRP, which may continue to be used in determining eligibility for and response to anti-TNF treatment, without adjusting for CRP genotype. PMID:23039402

  20. Heat shock proteins: molecular chaperones of protein biogenesis.

    PubMed Central

    Craig, E A; Gambill, B D; Nelson, R J

    1993-01-01

    Heat shock proteins (Hsps) were first identified as proteins whose synthesis was enhanced by stresses such as an increase in temperature. Recently, several of the major Hsps have been shown to be intimately involved in protein biogenesis through a direct interaction with a wide variety of proteins. As a reflection of this role, these Hsps have been referred to as molecular chaperones. Hsp70s interact with incompletely folded proteins, such as nascent chains on ribosomes and proteins in the process of translocation from the cytosol into mitochondria and the endoplasmic reticulum. Hsp60 also binds to unfolded proteins, preventing aggregation and facilitating protein folding. Although less well defined, other Hsps such as Hsp90 also play important roles in modulating the activity of a number of proteins. The function of the proteolytic system is intertwined with that of molecular chaperones. Several components of this system, encoded by heat-inducible genes, are responsible for the degradation of abnormal or misfolded proteins. The budding yeast Saccharomyces cerevisiae has proven very useful in the analysis of the role of molecular chaperones in protein maturation, translocation, and degradation. In this review, results of experiments are discussed within the context of experiments with other organisms in an attempt to describe the current state of understanding of these ubiquitous and important proteins. PMID:8336673

  1. Predicting permanent and transient protein-protein interfaces.

    PubMed

    La, David; Kong, Misun; Hoffman, William; Choi, Youn Im; Kihara, Daisuke

    2013-05-01

    Protein-protein interactions (PPIs) are involved in diverse functions in a cell. To optimize functional roles of interactions, proteins interact with a spectrum of binding affinities. Interactions are conventionally classified into permanent and transient, where the former denotes tight binding between proteins that result in strong complexes, whereas the latter compose of relatively weak interactions that can dissociate after binding to regulate functional activity at specific time point. Knowing the type of interactions has significant implications for understanding the nature and function of PPIs. In this study, we constructed amino acid substitution models that capture mutation patterns at permanent and transient type of protein interfaces, which were found to be different with statistical significance. Using the substitution models, we developed a novel computational method that predicts permanent and transient protein binding interfaces (PBIs) in protein surfaces. Without knowledge of the interacting partner, the method uses a single query protein structure and a multiple sequence alignment of the sequence family. Using a large dataset of permanent and transient proteins, we show that our method, BindML+, performs very well in protein interface classification. A very high area under the curve (AUC) value of 0.957 was observed when predicted protein binding sites were classified. Remarkably, near prefect accuracy was achieved with an AUC of 0.991 when actual binding sites were classified. The developed method will be also useful for protein design of permanent and transient PBIs. PMID:23239312

  2. Chemically defined protein-free in vitro culture of mammalian embryo does not restrict its developmental potential for differentiation of skin appendages.

    PubMed

    Bulic-Jakus, F; Strahinic-Belovari, T; Maric, S; Jezek, D; Juric-Lekic, G; Vlahovic, M; Serman, D

    2001-01-01

    In a unique serum- and protein-free chemically defined in vitro culture model of postimplantation mammalian development the epidermis differentiates regularly, although the differentiation of other tissues is impaired due to the lack of the serum. The present study in that model was done to estimate more carefully the degree of epidermal differentiation in defined media supplemented with some growth- or differentiation-stimulating substances. The main objective was to discover by grafting in vivo to the richer environment whether simple protein-free culture conditions restrict an inherent embryonic potential for differentiation of skin appendages. Embryonic parts of E9.5 gastrulating Fischer rat embryos were cultivated for 2 weeks in the protein-free Eagle's minimum essential medium supplemented with holotransferrin, apotransferrin, insulin and/or Na(2)SeO(3) and in controls cultivated in protein-free medium or in serum-supplemented medium. In all experiments there was a high incidence of differentiation of the epidermis. A high level of epidermal differentiation was confirmed for the first time at the ultrastructural level. A well-differentiated cornified layer and cells connected with desmosomes containing keratohyaline masses and cytokeratin filaments were found. A strong immunohistochemical signal for the proliferating cell nuclear antigen was always detected in the basal layer of the epidermis showing that those cells were still able to proliferate. Finally, embryos precultivated for 1 or 2 weeks in the protein-free medium and media supplemented with apotransferrin or serum were grafted under the kidney capsule for an additional 2 weeks. It was discovered that even after spending 2 weeks in the simple protein-free medium in vitro, embryos retained their developmental potential for differentiation of skin appendages (hair and sebaceous glands). PMID:11399853

  3. Protein enriched pasta: structure and digestibility of its protein network.

    PubMed

    Laleg, Karima; Barron, Cécile; Santé-Lhoutellier, Véronique; Walrand, Stéphane; Micard, Valérie

    2016-02-17

    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

  4. 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

    2015-01-01

    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. PMID:25704442

  5. Occupational protein contact dermatitis.

    PubMed

    Barbaud, Annick; Poreaux, Claire; Penven, Emmanuelle; Waton, Julie

    2015-12-01

    Occupational contact dermatitis is generally caused by haptens but can also be induced by proteins causing mainly immunological contact urticaria (ICU); chronic hand eczema in the context of protein contact dermatitis (PCD). In a monocentric retrospective study, from our database, only 31 (0.41%) of patients with contact dermatitis had positive skin tests with proteins: 22 had occupational PCD, 3 had non-occupational PCD, 5 occupational ICU and 1 cook had a neutrophilic fixed food eruption (NFFE) due to fish. From these results and analysis of literature, the characteristics of PCD can be summarized as follows. It is a chronic eczematous dermatitis, possibly exacerbated by work, suggestive if associated with inflammatory perionyxix and immediate erythema with pruritis, to be investigated when the patient resumes work after a period of interruption. Prick tests with the suspected protein-containing material are essential, as patch tests have negative results. In case of multisensitisation revealed by prick tests, it is advisable to analyse IgE against recombinant allergens. A history of atopy, found in 56 to 68% of the patients, has to be checked for. Most of the cases are observed among food-handlers but PCD can also be due to non-edible plants, latex, hydrolysed proteins or animal proteins. Occupational exposure to proteins can thus lead to the development of ICU. Reflecting hypersensitivity to very low concentrations of allergens, investigating ICU therefore requires caution and prick tests should be performed with a diluted form of the causative protein-containing product. Causes are food, especially fruit peel, non-edible plants, cosmetic products, latex, animals. PMID:26242922

  6. Insulin-Like Growth Factor 1 and Insulin-Like Growth Factor-Binding Protein 3 in Relation to the Risk of Type 2 Diabetes Mellitus: Results From the EPIC-Potsdam Study.

    PubMed

    Drogan, Dagmar; Schulze, Matthias B; Boeing, Heiner; Pischon, Tobias

    2016-03-15

    Higher levels of insulin-like growth factor-binding protein 3 (IGFBP-3) might raise the risk of type 2 diabetes mellitus (T2DM) via binding of insulin-like growth factor 1 (IGF-1), an insulin-like hormone that is involved in glucose homeostasis. We investigated serum concentrations of IGF-1 and IGFBP-3 and their molar ratio in relation to T2DM incidence in a nested case-cohort study within the European Prospective Investigation Into Cancer and Nutrition-Potsdam Study. We included a randomly selected subcohort of persons without T2DM at the time of blood sampling (n = 2,269) and 776 individuals with incident T2DM identified between 1994 and 2005. For the highest quartile versus lowest, the multivariable-adjusted hazard rate ratios were 0.91 (95% confidence interval (CI): 0.68, 1.23; P for trend = 0.31) for IGF-1, 1.33 (95% CI: 1.00, 1.76; P for trend = 0.04) for IGFBP-3, and 0.77 (95% CI: 0.57, 1.03; P for trend = 0.03) for IGF-1:IGFBP-3 ratio. IGFBP-3 level remained positively associated with T2DM incidence-and the ratio of IGF-1 to IGFBP-3 was inversely related with T2DM incidence-in models that included adjustment for IGF-1 concentrations (P for trend < 0.05). Therefore, our findings do not confirm an association between total IGF-1 concentrations and risk of T2DM in the general study population, although higher IGFBP-3 levels might raise T2DM risk independent of IGF-1 levels. PMID:26880678

  7. Measurements of Protein-Protein Interactions in Solutions by Chromatography

    NASA Astrophysics Data System (ADS)

    Berejnov, V.; Bloustine, J.; Fraden, S.

    2003-03-01

    The protein-protein interaction in solutions have generated a great deal of interest among structural biologists since [1] showed a correlation between protein crystallisability and a virial coefficient B2 describing such interaction. The work [1] demonstrated that many proteins crystallize in conditions where the B2 becomes slightly negative, indicating net attractive interactions between protein molecules. We present a new efficient method for extracting second virial coefficients B2 of protein solutions from retention time measurements in size exclusion chromatography (SEC). We measure B2 by analyzing the concentration dependance of the chromatographic partition coefficient. We show the ability of this method to track the evolution of B2 from positive to negative values in lysozyme and bovine serum albumin solutions. Our SEC results agree quantitatively with data obtained by light scattering. 1. A.George and W.W.Wilson, Predicting protein crystallization from a dilute solution property. Acta. Cryst., D50:361--365, 1994.

  8. Expression of proteins with dimethylarginines in Escherichia coli for proteinprotein interaction studies

    PubMed Central

    Hsieh, Cheng-Hsilin; Huang, San-Yuan; Wu, Yu-Ching; Liu, Li-Fan; Han, Chau-Chung; Liu, Yi-Chen; Tam, Ming F.

    2007-01-01

    Protein arginine methylation often modulates proteinprotein interactions. To isolate a sufficient quantity of proteins enriched in methyl arginine(s) from natural sources for biochemical studies is laborious and difficult. We describe here an expression system that produces recombinant proteins that are enriched in ?-NG,NG-asymmetry dimethylarginines. A yeast type I arginine methyltransferase gene (HMT1) is put on a plasmid under the control of the Escherichia coli methionine aminopeptidase promoter for constitutive expression. The protein targeted for post-translational modification is put on the same plasmid behind a T7 promoter for inducible expression of His6-tagged proteins. Sbp1p and Stm1p were used as model proteins to examine this expression system. The 13 arginines within the arginine-glycine-rich motif of Sbp1p and the RGG sequence near the C terminus of Stm1p were methylated. Unexpectedly, the arginine residue on the thrombin cleavage site (LVPRGS) of the fusion proteins can also be methylated by Hmt1p. Sbp1p and Sbp1p/hmt1 were covalently attached to solid supports for the isolation of interacting proteins. The results indicate that arginine methylation on Sbp1p exerts both positive and negative effects on proteinprotein interaction. PMID:17456744

  9. Discover Protein Complexes in Protein-Protein Interaction Networks Using Parametric Local Modularity

    PubMed Central

    2010-01-01

    Background Recent advances in proteomic technologies have enabled us to create detailed protein-protein interaction maps in multiple species and in both normal and diseased cells. As the size of the interaction dataset increases, powerful computational methods are required in order to effectively distil network models from large-scale interactome data. Results We present an algorithm, miPALM (Module Inference by Parametric Local Modularity), to infer protein complexes in a protein-protein interaction network. The algorithm uses a novel graph theoretic measure, parametric local modularity, to identify highly connected sub-networks as candidate protein complexes. Using gold standard sets of protein complexes and protein function and localization annotations, we show our algorithm achieved an overall improvement over previous algorithms in terms of precision, recall, and biological relevance of the predicted complexes. We applied our algorithm to predict and characterize a set of 138 novel protein complexes in S. cerevisiae. Conclusions miPALM is a novel algorithm for detecting protein complexes from large protein-protein interaction networks with improved accuracy than previous methods. The software is implemented in Matlab and is freely available at http://www.medicine.uiowa.edu/Labs/tan/software.html. PMID:20958996

  10. Ultrafast hydration dynamics of proteins

    NASA Astrophysics Data System (ADS)

    Zhong, Dongping

    2004-03-01

    Hydration dynamics of the water layer at the protein surface is important to protein structure and function. Recent studies of water-protein interactions have merged into a cohesive picture: Biological water molecules are not static but dynamic in nature. Previously limited time resolution did not fully resolve hydration processes. Here, we report our direct mapping of the hydration dynamics at a protein surface. The intrinsic tryptophan residue will be engineered to scan the protein surface by site-directed mutagenesis. Spatial and temporal hydration heterogeneity will for the first time be characterized in both native and molten globule states. Dynamical hydration patterns will be evaluated in terms of the local topography and chemical identity. These results will provide a molecular basis for the understanding of protein-water interactions, a topic central to protein science.

  11. Refolding of inclusion body proteins.

    PubMed

    Mayer, Marcus; Buchner, Johannes

    2004-01-01

    Genome sequencing projects have led to the identification of an enormous number of open reading frames that code for unknown proteins. Elucidation of the structure and function of these proteins makes it necessary to produce proteins fast, in high yields and at low cost. The recombinant expression of proteins in bacterial hosts often results in the formation of inclusion bodies. Here, the protein accumulates in large quantities separated from the cellular protein. However, the protein is insoluble and inactive. Thus, it is necessary to establish efficient refolding protocols. Progress has been made recently in this field concerning refolding strategies, the use of low-molecular-weight additives as folding enhancers, and the determination of optimum refolding parameters. Here we present an overview of the refolding technology and give a standard protocol for inclusion body refolding. PMID:14959834

  12. Protein transduction assisted by polyethylenimine-cationized carrier proteins.

    PubMed

    Kitazoe, Midori; Murata, Hitoshi; Futami, Junichiro; Maeda, Takashi; Sakaguchi, Masakiyo; Miyazaki, Masahiro; Kosaka, Megumi; Tada, Hiroko; Seno, Masaharu; Huh, Nam-ho; Namba, Masayoshi; Nishikawa, Mitsuo; Maeda, Yoshitake; Yamada, Hidenori

    2005-06-01

    Previously, we have reported that cationized-proteins covalently modified with polyethylenimine (PEI) (direct PEI-cationization) efficiently enter cells and function in the cytosol [Futami et al. (2005) J. Biosci. Bioeng. 99, 95-103]. However, it may be more convenient if a protein could be delivered into cells just by mixing the protein with a PEI-cationized carrier protein having a specific affinity (indirect PEI-cationization). Thus, we prepared PEI-cationized avidin (PEI-avidin), streptavidin (PEI-streptavidin), and protein G (PEI-protein G), and examined whether they could deliver biotinylated proteins and antibodies into living cells. PEI-avidin (and/or PEI-streptavidin) carried biotinylated GFPs into various mammalian cells very efficiently. A GFP variant containing a nuclear localization signal was found to arrive even in the nucleus. The addition of a biotinylated RNase A derivative mixed with PEI-streptavidin to a culture medium of 3T3-SV-40 cells resulted in remarkable cell growth inhibition, suggesting that the biotinylated RNase A derivative entered cells and digested intracellular RNA molecules. Furthermore, the addition of a fluorescein-labeled anti-S100C (beta-actin binding protein) antibody mixed with PEI-protein G to human fibroblasts resulted in the appearance of a fluorescence image of actin-like filamentous structures in the cells. These results indicate that indirect PEI-cationization using non-covalent interaction is as effective as the direct PEI-cationization for the transduction of proteins into living cells and for expression of their functions in the cytosol. Thus, PEI-cationized proteins having a specific affinity for certain molecules such as PEI-streptavidin, PEI-avidin and PEI-protein G are concluded to be widely applicable protein transduction carrier molecules. PMID:16002991

  13. Protein intrinsic disorder toolbox for comparative analysis of viral proteins

    PubMed Central

    Goh, Gerard Kian-Meng; Dunker, A Keith; Uversky, Vladimir N

    2008-01-01

    To examine the usefulness of protein disorder predictions as a tool for the comparative analysis of viral proteins, a relational database has been constructed. The database includes proteins from influenza A and HIV-related viruses. Annotations include viral protein sequence, disorder prediction, structure, and function. Location of each protein within a virion, if known, is also denoted. Our analysis reveals a clear relationship between proximity to the RNA core and the percentage of predicted disordered residues for a set of influenza A virus proteins. Neuraminidases (NA) and hemagglutinin (HA) of major influenza A pandemics tend to pair in such a way that both proteins tend to be either ordered-ordered or disordered-disordered by prediction. This may be the result of these proteins evolving from being lipid-associated. High abundance of intrinsic disorder in envelope and matrix proteins from HIV-related viruses likely represents a mechanism where HIV virions can escape immune response despite the availability of antibodies for the HIV-related proteins. This exercise provides an example showing how the combined use of intrinsic disorder predictions and relational databases provides an improved understanding of the functional and structural behaviour of viral proteins. PMID:18831795

  14. 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.

  15. Protein Adsorption in Three Dimensions

    PubMed Central

    Vogler, Erwin A.

    2011-01-01

    Recent experimental and theoretical work clarifying the physical chemistry of blood-protein adsorption from aqueous-buffer solution to various kinds of surfaces is reviewed and interpreted within the context of biomaterial applications, especially toward development of cardiovascular biomaterials. The importance of this subject in biomaterials surface science is emphasized by reducing the “protein-adsorption problem” to three core questions that require quantitative answer. An overview of the protein-adsorption literature identifies some of the sources of inconsistency among many investigators participating in more than five decades of focused research. A tutorial on the fundamental biophysical chemistry of protein adsorption sets the stage for a detailed discussion of the kinetics and thermodynamics of protein adsorption, including adsorption competition between two proteins for the same adsorbent immersed in a binary-protein mixture. Both kinetics and steady-state adsorption can be rationalized using a single interpretive paradigm asserting that protein molecules partition from solution into a three-dimensional (3D) interphase separating bulk solution from the physical-adsorbent surface. Adsorbed protein collects in one-or-more adsorbed layers, depending on protein size, solution concentration, and adsorbent surface energy (water wettability). The adsorption process begins with the hydration of an adsorbent surface brought into contact with an aqueous-protein solution. Surface hydration reactions instantaneously form a thin, pseudo-2D interface between the adsorbent and protein solution. Protein molecules rapidly diffuse into this newly-formed interface, creating a truly 3D interphase that inflates with arriving proteins and fills to capacity within milliseconds at mg/mL bulk-solution concentrations CB. This inflated interphase subsequently undergoes time-dependent (minutes-to-hours) decrease in volume VI by expulsion of either-or-both interphase water and 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 adsorption to any surface by competing with adsorbing protein molecules. This Leading Opinion ends by proposing several changes to the protein-adsorption paradigm that might advance answers to the three core questions that frame the “protein-adsorption problem” that is so fundamental to biomaterials surface science. PMID:22088888

  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. PMID:24573470

  17. Protein structure modeling with MODELLER.

    PubMed

    Eswar, Narayanan; Eramian, David; Webb, Ben; Shen, Min-Yi; Sali, Andrej

    2008-01-01

    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 using experimental structure determination methods. Computational protein structure modeling techniques have the potential to bridge this sequence-structure gap. This chapter presents an example that illustrates the use of MODELLER to construct a comparative model for a protein with unknown structure. Automation of similar protocols (correction of protcols) has resulted in models of useful accuracy for domains in more than half of all known protein sequences. PMID:18542861

  18. Protein-protein interaction as a predictor of subcellular location

    PubMed Central

    Shin, Chang Jin; Wong, Simon; Davis, Melissa J; Ragan, Mark A

    2009-01-01

    Background Many biological processes are mediated by dynamic interactions between and among proteins. In order to interact, two proteins must co-occur spatially and temporally. As protein-protein interactions (PPIs) and subcellular location (SCL) are discovered via separate empirical approaches, PPI and SCL annotations are independent and might complement each other in helping us to understand the role of individual proteins in cellular networks. We expect reliable PPI annotations to show that proteins interacting in vivo are co-located in the same cellular compartment. Our goal here is to evaluate the potential of using PPI annotation in determining SCL of proteins in human, mouse, fly and yeast, and to identify and quantify the factors that contribute to this complementarity. Results Using publicly available data, we evaluate the hypothesis that interacting proteins must be co-located within the same subcellular compartment. Based on a large, manually curated PPI dataset, we demonstrate that a substantial proportion of interacting proteins are in fact co-located. We develop an approach to predict the SCL of a protein based on the SCL of its interaction partners, given sufficient confidence in the interaction itself. The frequency of false positive PPIs can be reduced by use of six lines of supporting evidence, three based on type of recorded evidence (empirical approach, multiplicity of databases, and multiplicity of literature citations) and three based on type of biological evidence (inferred biological process, domain-domain interactions, and orthology relationships), with biological evidence more-effective than recorded evidence. Our approach performs better than four existing prediction methods in identifying the SCL of membrane proteins, and as well as or better for soluble proteins. Conclusion Understanding cellular systems requires knowledge of the SCL of interacting proteins. We show how PPI data can be used more effectively to yield reliable SCL predictions for both soluble and membrane proteins. Scope exists for further improvement in our understanding of cellular function through consideration of the biological context of molecular interactions. PMID:19243629

  19. Arbitrary protein?protein docking targets biologically relevant interfaces

    PubMed Central

    2012-01-01

    Background Protein-protein recognition is of fundamental importance in the vast majority of biological processes. However, it has already been demonstrated that it is very hard to distinguish true complexes from false complexes in so-called cross-docking experiments, where binary protein complexes are separated and the isolated proteins are all docked against each other and scored. Does this result, at least in part, reflect a physical reality? False complexes could reflect possible nonspecific or weak associations. Results In this paper, we investigate the twilight zone of protein-protein interactions, building on an interesting outcome of cross-docking experiments: false complexes seem to favor residues from the true interaction site, suggesting that randomly chosen partners dock in a non-random fashion on protein surfaces. Here, we carry out arbitrary docking of a non-redundant data set of 198 proteins, with more than 300 randomly chosen "probe" proteins. We investigate the tendency of arbitrary partners to aggregate at localized regions of the protein surfaces, the shape and compositional bias of the generated interfaces, and the potential of this property to predict biologically relevant binding sites. We show that the non-random localization of arbitrary partners after protein-protein docking is a generic feature of protein structures. The interfaces generated in this way are not systematically planar or curved, but tend to be closer than average to the center of the proteins. These results can be used to predict biological interfaces with an AUC value up to 0.69 alone, and 0.72 when used in combination with evolutionary information. An appropriate choice of random partners and number of docking models make this method computationally practical. It is also noted that nonspecific interfaces can point to alternate interaction sites in the case of proteins with multiple interfaces. We illustrate the usefulness of arbitrary docking using PEBP (Phosphatidylethanolamine binding protein), a kinase inhibitor with multiple partners. Conclusions An approach using arbitrary docking, and based solely on physical properties, can successfully identify biologically pertinent protein interfaces. PMID:22559010

  20. Protein damage, repair and proteolysis.

    PubMed

    Chondrogianni, Niki; Petropoulos, Isabelle; Grimm, Stefanie; Georgila, Konstantina; Catalgol, Betul; Friguet, Bertrand; Grune, Tilman; Gonos, Efstathios S

    2014-02-01

    Proteins are continuously affected by various intrinsic and extrinsic factors. Damaged proteins influence several intracellular pathways and result in different disorders and diseases. Aggregation of damaged proteins depends on the balance between their generation and their reversal or elimination by protein repair systems and degradation, respectively. With regard to protein repair, only few repair mechanisms have been evidenced including the reduction of methionine sulfoxide residues by the methionine sulfoxide reductases, the conversion of isoaspartyl residues to L-aspartate by L-isoaspartate methyl transferase and deglycation by phosphorylation of protein-bound fructosamine by fructosamine-3-kinase. Protein degradation is orchestrated by two major proteolytic systems, namely the lysosome and the proteasome. Alteration of the function for both systems has been involved in all aspects of cellular metabolic networks linked to either normal or pathological processes. Given the importance of protein repair and degradation, great effort has recently been made regarding the modulation of these systems in various physiological conditions such as aging, as well as in diseases. Genetic modulation has produced promising results in the area of protein repair enzymes but there are not yet any identified potent inhibitors, and, to our knowledge, only one activating compound has been reported so far. In contrast, different drugs as well as natural compounds that interfere with proteolysis have been identified and/or developed resulting in homeostatic maintenance and/or the delay of disease progression. PMID:23107776

  1. Dynamic identifying protein functional modules based on adaptive density modularity in protein-protein interaction networks

    PubMed Central

    2015-01-01

    Background The identification of protein functional modules would be a great aid in furthering our knowledge of the principles of cellular organization. Most existing algorithms for identifying protein functional modules have a common defect -- once a protein node is assigned to a functional module, there is no chance to move the protein to the other functional modules during the follow-up processes, which lead the erroneous partitioning occurred at previous step to accumulate till to the end. Results In this paper, we design a new algorithm ADM (Adaptive Density Modularity) to detect protein functional modules based on adaptive density modularity. In ADM algorithm, according to the comparison between external closely associated degree and internal closely associated degree, the partitioning of a protein-protein interaction network into functional modules always evolves quickly to increase the density modularity of the network. The integration of density modularity into the new algorithm not only overcomes the drawback mentioned above, but also contributes to identifying protein functional modules more effectively. Conclusions The experimental result reveals that the performance of ADM algorithm is superior to many state-of-the-art protein functional modules detection techniques in aspect of the accuracy of prediction. Moreover, the identified protein functional modules are statistically significant in terms of "Biological Process" annotated in Gene Ontology, which provides substantial support for revealing the principles of cellular organization. PMID:26330105

  2. 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.

  3. Protein-Protein Interaction Detection: Methods and Analysis

    PubMed Central

    Rao, V. Srinivasa; Srinivas, K.; Sujini, G. N.; Kumar, G. N. Sunand

    2014-01-01

    Protein-protein interaction plays key role in predicting the protein function of target protein and drug ability of molecules. The majority of genes and proteins realize resulting phenotype functions as a set of interactions. The in vitro and in vivo methods like affinity purification, Y2H (yeast 2 hybrid), TAP (tandem affinity purification), and so forth have their own limitations like cost, time, and so forth, and the resultant data sets are noisy and have more false positives to annotate the function of drug molecules. Thus, in silico methods which include sequence-based approaches, structure-based approaches, chromosome proximity, gene fusion, in silico 2 hybrid, phylogenetic tree, phylogenetic profile, and gene expression-based approaches were developed. Elucidation of protein interaction networks also contributes greatly to the analysis of signal transduction pathways. Recent developments have also led to the construction of networks having all the protein-protein interactions using computational methods for signaling pathways and protein complex identification in specific diseases. PMID:24693427

  4. Dipolar response of hydrated proteins.

    PubMed

    Matyushov, Dmitry V

    2012-02-28

    The paper presents an analytical theory and numerical simulations of the dipolar response of hydrated proteins in solution. We calculate the effective dielectric constant representing the average dipole moment induced at the protein by a uniform external field. The dielectric constant shows a remarkable variation among the proteins, changing from 0.5 for ubiquitin to 640 for cytochrome c. The former value implies a negative dipolar susceptibility, that is a dia-electric dipolar response and negative dielectrophoresis. It means that ubiquitin, carrying an average dipole of ?240 D, is expected to repel from the region of a stronger electric field. This outcome is the result of a negative cross-correlation between the protein and water dipoles, compensating for the positive variance of the intrinsic protein dipole in the overall dipolar susceptibility. In contrast to the neutral ubiquitin, charged proteins studied here show para-electric dipolar response and positive dielectrophoresis. The study suggests that the dipolar response of proteins in solution is strongly affected by the coupling of the protein surface charge to the hydration water. The protein-water dipolar cross-correlations are long-ranged, extending ~2 nm from the protein surface into the bulk. A similar correlation length of about 1 nm is seen for the electrostatic potential produced by the hydration water inside the protein. The analysis of numerical simulations suggests that the polarization of the protein-water interface is highly heterogeneous and does not follow the standard dielectric results for cavities carved in dielectrics. The polarization of the water shell gains in importance, relative to the intrinsic protein dipole, at high frequencies, above the protein Debye peak. The induced interfacial dipole can be either parallel or antiparallel to the protein dipole, depending on the distribution of the protein surface charge. As a result, the high-frequency absorption of the protein solution can be either higher or lower than the absorption of water. Both scenarios have been experimentally observed in the THz window of radiation. PMID:22380065

  5. Essential protein identification based on essential protein-protein interaction prediction by Integrated Edge Weights.

    PubMed

    Jiang, Yuexu; Wang, Yan; Pang, Wei; Chen, Liang; Sun, Huiyan; Liang, Yanchun; Blanzieri, Enrico

    2015-07-15

    Essential proteins play a crucial role in cellular survival and development process. Experimentally, essential proteins are identified by gene knockouts or RNA interference, which are expensive and often fatal to the target organisms. Regarding this, an alternative yet important approach to essential protein identification is through computational prediction. Existing computational methods predict essential proteins based on their relative densities in a protein-protein interaction (PPI) network. Degree, betweenness, and other appropriate criteria are often used to measure the relative density. However, no matter what criterion is used, a protein is actually ordered by the attributes of this protein per se. In this research, we presented a novel computational method, Integrated Edge Weights (IEW), to first rank protein-protein interactions by integrating their edge weights, and then identified sub PPI networks consisting of those highly-ranked edges, and finally regarded the nodes in these sub networks as essential proteins. We evaluated IEW on three model organisms: Saccharomyces cerevisiae (S. cerevisiae), Escherichia coli (E. coli), and Caenorhabditis elegans (C. elegans). The experimental results showed that IEW achieved better performance than the state-of-the-art methods in terms of precision-recall and Jackknife measures. We had also demonstrated that IEW is a robust and effective method, which can retrieve biologically significant modules by its highly-ranked protein-protein interactions for S. cerevisiae, E. coli, and C. elegans. We believe that, with sufficient data provided, IEW can be used to any other organisms' essential protein identification. A website about IEW can be accessed from http://digbio.missouri.edu/IEW/index.html. PMID:25892709

  6. PocketQuery: protein-protein interaction inhibitor starting points from protein-protein interaction structure.

    PubMed

    Koes, David Ryan; Camacho, Carlos J

    2012-07-01

    PocketQuery (http://pocketquery.csb.pitt.edu) is a web interface for exploring the properties of protein-protein interaction (PPI) interfaces with a focus on the discovery of promising starting points for small-molecule design. PocketQuery rapidly focuses attention on the key interacting residues of an interaction using a 'druggability' score that provides an estimate of how likely the chemical mimicry of a cluster of interface residues would result in a small-molecule inhibitor of an interaction. These residue clusters are chemical starting points that can be seamlessly exported to a pharmacophore-based drug discovery workflow. PocketQuery is updated on a weekly basis to contain all applicable PPI structures deposited in the Protein Data Bank and allows users to upload their own custom structures for analysis. PMID:22523085

  7. Protein solubilization: a novel approach.

    PubMed

    Johnson, David H; Wilson, W William; DeLucas, Lawrence J

    2014-11-15

    Formulation development presents significant challenges with respect to protein therapeutics. One component of these challenges is to attain high protein solubility (>50mg/ml for immunoglobulins) with minimal aggregation. Protein-protein interactions contribute to aggregation and the integral sum of these interactions can be quantified by a thermodynamic parameter known as the osmotic second virial coefficient (B-value). The method presented here utilizes high-throughput measurement of B-values to identify the influence of additives on protein-protein interactions. The experiment design uses three tiers of screens to arrive at final solution conditions that improve protein solubility. The first screen identifies individual additives that reduce protein interactions. A second set of B-values are then measured for different combinations of these additives via an incomplete factorial screen. Results from the incomplete factorial screen are used to train an artificial neural network (ANN). The "trained" ANN enables predictions of B-values for more than 4000 formulations that include additive combinations not previously experimentally measured. Validation steps are incorporated throughout the screening process to ensure that (1) the protein's thermal and aggregation stability characteristics are not reduced and (2) the artificial neural network predictive model is accurate. The ability of this approach to reduce aggregation and increase solubility is demonstrated using an IgG protein supplied by Minerva Biotechnologies, Inc. PMID:25270058

  8. Phylogenomics of Prokaryotic Ribosomal Proteins

    PubMed Central

    Yutin, Natalya; Puigb, 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

  9. 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.

  10. 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

    2016-02-29

    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. PMID:26633836

  11. PSAIA – Protein Structure and Interaction Analyzer

    PubMed Central

    Mihel, Josip; Šikić, Mile; Tomić, Sanja; Jeren, Branko; Vlahoviček, Kristian

    2008-01-01

    Background PSAIA (Protein Structure and Interaction Analyzer) was developed to compute geometric parameters for large sets of protein structures in order to predict and investigate protein-protein interaction sites. Results In addition to most relevant established algorithms, PSAIA offers a new method PIADA (Protein Interaction Atom Distance Algorithm) for the determination of residue interaction pairs. We found that PIADA produced more satisfactory results than comparable algorithms implemented in PSAIA. Particular advantages of PSAIA include its capacity to combine different methods to detect the locations and types of interactions between residues and its ability, without any further automation steps, to handle large numbers of protein structures and complexes. Generally, the integration of a variety of methods enables PSAIA to offer easier automation of analysis and greater reliability of results. PSAIA can be used either via a graphical user interface or from the command-line. Results are generated in either tabular or XML format. Conclusion In a straightforward fashion and for large sets of protein structures, PSAIA enables the calculation of protein geometric parameters and the determination of location and type for protein-protein interaction sites. XML formatted output enables easy conversion of results to various formats suitable for statistic analysis. Results from smaller data sets demonstrated the influence of geometry on protein interaction sites. Comprehensive analysis of properties of large data sets lead to new information useful in the prediction of protein-protein interaction sites. PMID:18400099

  12. 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 1265years with severe hemophilia A were randomized to twice-weekly prophylaxis (2030IUkg?1), 3-times-weekly prophylaxis (3040IUkg?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) andimmunogenicity 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. MeanSD ABR was significantly lower with prophylaxis (twice-weekly, 5.77.2; 3-times-weekly, 4.36.5; combined, 4.96.8) vs. on-demand treatment (57.724.6; 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

  13. 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 complexes while allowing any remaining unbound dye/antibody pairs to flow away. The retained dye/antibody/protein complexes are transferred to a cuvette, wherein they are irradiated with light from a miniature near-infrared laser delivered via a fiber-optic cable. The resulting fluorescence from the dye(s) is measured by use of a miniature spectrometer, the output of which is digitized, then analyzed by laptop computer. The software running in the computer identifies the protein species by the wavelengths of their spectral peaks and determines the amounts of the proteins, and thus, one day, microbes of the various species from the intensities of the peaks. The abovementioned removal of the unbound dye/antibody pairs during centrifugation prevents false positive readings. The process proves successful in detecting proteins in solution and thus can now be employed for use in microbe detection.

  14. Nucleation precursors in protein crystallization

    PubMed Central

    Vekilov, Peter G.; Vorontsova, Maria A.

    2014-01-01

    Protein crystal nucleation is a central problem in biological crystallography and other areas of science, technology and medicine. Recent studies have demonstrated that protein crystal nuclei form within crucial precursors. Here, methods of detection and characterization of the precursors are reviewed: dynamic light scattering, atomic force microscopy and Brownian microscopy. Data for several proteins provided by these methods have demonstrated that the nucleation precursors are clusters consisting of protein-dense liquid, which are metastable with respect to the host protein solution. The clusters are several hundred nanometres in size, the cluster population occupies from 10−7 to 10−3 of the solution volume, and their properties in solutions supersaturated with respect to crystals are similar to those in homogeneous, i.e. undersaturated, solutions. The clusters exist owing to the conformation flexibility of the protein molecules, leading to exposure of hydrophobic surfaces and enhanced intermolecular binding. These results indicate that protein conformational flexibility might be the mechanism behind the metastable mesoscopic clusters and crystal nucleation. Investigations of the cluster properties are still in their infancy. Results on direct imaging of cluster behaviors and characterization of cluster mechanisms with a variety of proteins will soon lead to major breakthroughs in protein biophysics. PMID:24598910

  15. The Biological Value of Protein.

    PubMed

    Moore, Daniel R; Soeters, Peter B

    2015-01-01

    The biological value of a protein extends beyond its amino-acid composition and digestibility, and can be influenced by additional factors in a tissue-specific manner. In healthy individuals, the slow appearance of dietary amino acids in the portal vein and subsequently in the systemic circulation in response to bolus protein ingestion improves nitrogen retention and decreases urea production. This is promoted by slow absorption when only protein is ingested (e.g. casein). When a full meal is ingested, whey achieves slightly better nitrogen retention than soy or casein, which is very likely achieved by its high content of essential amino acids (especially leucine). Elderly people exhibit 'anabolic resistance' implying that more protein is required to reach maximal rates of muscle protein synthesis compared to young individuals. Protein utilization in inflammatory or traumatic conditions increases substantially in the splanchnic tissues containing most of the immune system, and in wounds and growing tissues. This happens especially in the elderly, which often suffer from chronic inflammatory activity due to disease, physical inactivity and/or the aging process itself. Consequently, the proportion of protein absorbed in the gut and utilized for muscle protein synthesis decreases in these situations. This compromises dietary-protein-induced stimulation of muscle protein synthesis and ultimately results in increased requirements of protein (∼1.2 g/kg body weight/day) to limit gradual muscle loss with age. To optimally preserve muscle mass, physical exercise is required. Exercise has both direct effects on muscle mass and health, and indirect effects by increasing the utilization of dietary protein (especially whey) to enhance rates of muscle protein synthesis. PMID:26545252

  16. GECluster: a novel protein complex prediction method

    PubMed Central

    Su, Lingtao; Liu, Guixia; Wang, Han; Tian, Yuan; Zhou, Zhihui; Han, Liang; Yan, Lun

    2014-01-01

    Identification of protein complexes is of great importance in the understanding of cellular organization and functions. Traditional computational protein complex prediction methods mainly rely on the topology of proteinprotein interaction (PPI) networks but seldom take biological information of proteins (such as Gene Ontology (GO)) into consideration. Meanwhile, the environment relevant analysis of protein complex evolution has been poorly studied, partly due to the lack of high-precision protein complex datasets. In this paper, a combined PPI network is introduced to predict protein complexes which integrate both GO and expression value of relevant protein-coding genes. A novel protein complex prediction method GECluster (Gene Expression Cluster) was proposed based on a seed node expansion strategy, in which a combined PPI network was utilized. GECluster was applied to a training combined PPI network and it predicted more credible complexes than peer methods. The results indicate that using a combined PPI network can efficiently improve protein complex prediction accuracy. In order to study protein complex evolution within cells due to changes in the living environment surrounding cells, GECluster was applied to seven combined PPI networks constructed using the data of a test set including yeast response to stress throughout a wine fermentation process. Our results showed that with the rise of alcohol concentration, protein complexes within yeast cells gradually evolve from one state to another. Besides this, the number of core and attachment proteins within a protein complex both changed significantly. PMID:26019559

  17. Gel protein capillary extraction apparatus. electronic protein transfer.

    PubMed

    Cooper, Jonathan W; Gao, Jun; Lee, Cheng S

    2002-03-01

    A gel protein capillary extraction apparatus is developed and demonstrated for its rapid and effective transfer of SDS-protein complexes from polyacrylamide gel to a fused-silica capillary. The small dimensions of capillary columns permit the application of high voltages for achieving rapid and effective transfer of gel proteins. Furthermore, the fused-silica capillaries are internally coated with polyacrylamide for the elimination of electroosmotic pumping and protein adsorption onto the capillary wall. The extracted proteins are present in a highly concentrated solution plug as the result of field amplification and sample stacking during the extraction process. Three model proteins, including cytochrome c (14 kDa), ovalbumin (45 kDa), and beta-galactosidase (116 kDa), are visualized using coomassie blue staining and electrophoretically extracted from the gels with protein loading as low as 50 ng. The SDS-cytochrome c complexes extracted from a 50-ng protein loading are concentrated in a 30-nL solution plug inside the capillary with an estimated concentration of 0. 1 mg/mL or 10(-5) M. The capillary format allows the straightforward integration of a miniaturized trypsin-membrane reactor for on-line proteolytic digestion and ESI-MS analysis for protein/peptide identification. PMID:11924982

  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. 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. PMID:26637825

  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. PMID:26675729

  1. Manipulating protein adsorption using a patchy protein-resistant brush.

    PubMed

    Gon, Saugata; Bendersky, Marina; Ross, Jennifer L; Santore, Maria M

    2010-07-20

    Toward the development of surfaces for the precise manipulation of proteins, this study explores the fabrication and protein-interactive behavior of a new type of surface containing extremely small (on the order of 10 nm or less) flat adhesive "patches" or islands embedded in and partially concealed by a protein-repellant PEG (poly(ethylene glycol)) brush. The adsorption of fibrinogen, the model protein chosen to probe the biomaterial interactions of these surfaces, is very sensitive to the surface density of the adhesive patches, occurring only above a threshold. This suggests that two or more adhesive patches are needed to capture each protein. When the average spacing of the adhesive patches exceeds the fibrinogen length, no adsorption occurs because individual patches are too weakly binding for protein capture, as a result of being at least partially obstructed by the brush. The small size of the adhesive patches relative to the 47 nm fibrinogen length thus defines a limiting regime of surface design, distinct from surfaces where larger features can adhere single isolated proteins or multiple proteins together. The restricted protein-surface contact may comprise a means of preserving protein structure and function in the adsorbed state. This article demonstrates several additional interesting features of PEG brushes relevant to biomaterial design. First a moderate amount of adhesive material can be buried at the base of a brush without a measurable impact on the corona density. Second, a different amount of material at the base of a brush can be rendered ineffective to capturing adhesive proteins, despite a modest compromise of the brush corona. From this will follow insight into the design of patterned biomaterial surfaces, the bioactivity of the edges of patterned features, and an understanding of how flaws in brushes compromise protein resistance or allow access to small adhesive sites. PMID:20557060

  2. Protein-protein and protein-salt interactions in aqueous protein solutions containing concentrated electrolytes

    SciTech Connect

    Curtis, R.A.; Blanch, H.W.; Prausnitz, J.M.

    1998-01-05

    Protein-protein and protein-salt interactions have been obtained for ovalbumin in solutions of ammonium sulfate and for lysozyme in solutions of ammonium sulfate, sodium chloride, potassium isothiocyanate, and potassium chloride. The two-body interactions between ovalbumin molecules in concentrated ammonium-sulfate solutions can be described by the DLVO potentials plus a potential that accounts for the decrease in free volume available to the protein due to the presence of the salt ions. The interaction between ovalbumin and ammonium sulfate is unfavorable, reflecting the kosmotropic nature of sulfate anions. Lysozyme-lysozyme interactions cannot be described by the above potentials because anion binding to lysozyme alters these interactions. Lysozyme-isothiocyanate complexes are strongly attractive due to electrostatic interactions resulting from bridging by the isothiocyanate ion. Lysozyme-lysozyme interactions in sulfate solutions are more repulsive than expected, possibly resulting from a larger excluded volume of a lysozyme-sulfate bound complex or perhaps, hydration forces between the lysozyme-sulfate complexes.

  3. Protein Folding Dynamics

    NASA Astrophysics Data System (ADS)

    Gass, Stephen Francis

    1990-01-01

    The dynamics of the process of protein folding from a native to a denatured state is studied in this thesis. Chapter One consists of an introduction to the area and rational for the study of the dynamics. Chapter Two is an exploration of the static and dynamical behavior of a Spin-Glass like model of protein folding. The range of physically reasonable parameters is studied and the model is seen to reproduce the general character of experimental results. Chapter Three analyzes the properties of diffusion in a complex one dimensional potential. The mean first passage time for crossing the potential is found to be fit well by existing theoretical models. The effects of the roughness of the potential on the temperature dependence are seen to have the same form as the presence of an "Arrhenius" type barrier. Chapter four examines the implications of the work of the previous chapters on the folding transition in proteins, and details the experimental results available for comparison with the model. It is found that the limiting factor in protein folding is "diffusive" searching for the native state rather than a single rate limiting step. Because of this increasing the stability of the folded state has little beneficial effect on the folding rate and can in fact slow the process by increasing the stability of local minima on the folding pathway thus inhibiting diffusive searching. An optimal window of stability of 5-30 kcal/mole is therefore found to exist where folding will occur in a biologically relevant time scale. In this chapter the model is applied to fit the experimental data on folding stability and rates for various proteins and is found to replicate the experimental results for physically reasonable parameter choices, adding credibility to the assertion that the model accurately represents physical behavior.

  4. Are protein-protein interfaces special regions on a protein's surface?

    PubMed

    Tonddast-Navaei, Sam; Skolnick, Jeffrey

    2015-12-28

    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. PMID:26723634

  5. 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.

  6. Length, protein protein interactions, and complexity

    NASA Astrophysics Data System (ADS)

    Tan, Taison; Frenkel, Daan; Gupta, Vishal; Deem, Michael W.

    2005-05-01

    The evolutionary reason for the increase in gene length from archaea to prokaryotes to eukaryotes observed in large-scale genome sequencing efforts has been unclear. We propose here that the increasing complexity of protein-protein interactions has driven the selection of longer proteins, as they are more able to distinguish among a larger number of distinct interactions due to their greater average surface area. Annotated protein sequences available from the SWISS-PROT database were analyzed for 13 eukaryotes, eight bacteria, and two archaea species. The number of subcellular locations to which each protein is associated is used as a measure of the number of interactions to which a protein participates. Two databases of yeast protein-protein interactions were used as another measure of the number of interactions to which each S. cerevisiae protein participates. Protein length is shown to correlate with both number of subcellular locations to which a protein is associated and number of interactions as measured by yeast two-hybrid experiments. Protein length is also shown to correlate with the probability that the protein is encoded by an essential gene. Interestingly, average protein length and number of subcellular locations are not significantly different between all human proteins and protein targets of known, marketed drugs. Increased protein length appears to be a significant mechanism by which the increasing complexity of protein-protein interaction networks is accommodated within the natural evolution of species. Consideration of protein length may be a valuable tool in drug design, one that predicts different strategies for inhibiting interactions in aberrant and normal pathways.

  7. 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.

  8. Protein requirements in the elderly.

    PubMed

    Volkert, Dorothee; Sieber, Cornel Christian

    2011-03-01

    Adequate protein intake and the maintenance of nitrogen equilibrium are of particular importance in the elderly because this age group is at increased risk of illness and malnutrition. The current recommendation for protein intake of healthy elderly subjects is 0.8 g/kg body weight/day, the same as for younger adults. Nitrogen balance studies in the elderly, however, revealed conflicting results; some studies suggest that not all elderly can achieve a nitrogen balance with a protein intake of 0.8 g/kg body weight/day, particularly if energy supply is not adequate. Beyond the amount of protein needed for nitrogen balance, the optimal protein intake for preservation of lean body mass, body functions, and health is of paramount interest. At present, there is insufficient longer-term research with defined health outcomes to derive recommendations in this regard. Very little is also known about the protein needs of frail and unhealthy elderly. Until more evidence is available, it seems reasonable to ensure a protein intake of at least 0.8 g/kg body weight/day in all elderly persons, particularly in those at risk of malnutrition (e.g., frail and multimorbid elderly). In addition to ascertaining adequate protein and energy intake, physical activity should be encouraged in order to increase energy expenditure and food intake and to facilitate muscle protein anabolism. PMID:22139561

  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 102? 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 205? 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 for protein engineering and the creation of site-specific protein conjugates. PMID:24946229

  10. A Proteomic Approach to Characterize Protein Shedding

    SciTech Connect

    Ahram, Mamoun; Adkins, Joshua N.; Auberry, Deanna L.; Wunschel, David S.; Springer, David L.

    2005-01-01

    Shedding (i.e., proteolysis of ectodomains of membrane proteins) plays an important pathophysiological role. In order to study the feasibility of identifying shed proteins, we analyzed serum-free media of human mammary epithelial cells by mass spectrometry following induction of shedding by the phorbol ester, 4?-phorbol 12-myristate 13-acetate (PMA). Different means of sample preparation, including biotinylation of cell surface proteins, isolation of glycosylated proteins, and preparation of crude protein fraction, were carried out to develop the optimal method of sample processing. The collected proteins were digested with trypsin and analyzed by reversed-phase capillary liquid chromatography interfaced to an ion-trap mass spectrometer. The resulting peptide spectra were interpreted using the program SEQUEST. Analyzing the sample containing the crude protein mixture without chemical modification or separation resulted in the greatest number of identifications, including putatively shed proteins. Overall, 93 membrane-associated proteins were identified including 57 that contain at least one transmembrane domain and 36 that indirectly associate with the extracellular surface of the plasma membrane. Of the 57 transmembrane proteins, 43 were identified by extracellular peptides providing strong evidence for them originating from regulated proteolysis or shedding processes. We combined results from the different experiments and used a peptide count method to estimate changes in protein abundance. Using this approach, we identified 2 proteins, syndecan-4 and hepatoma-derived growth factor, whose abundances increased in media of cells treated with PMA. We also detected proteins whose abundances decreased after PMA treatment such as 78-kDa glucose-regulated protein and calreticulin. Further analysis using immunoblotting validated the abundance changes for syndecan-4 and 78-kDa glucose-regulated protein as a result of PMA treatment. These results demonstrate that mass spectrometry can be used to identify low-abundance shed proteins and to estimate changes in protein abundances.

  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 modifications. Conclusion PRO provides a framework for the formal representation of protein classes and protein forms in the OBO Foundry. It is designed to enable data retrieval and integration and machine reasoning at the molecular level of proteins, thereby facilitating cross-species comparisons, pathway analysis, disease modeling and the generation of new hypotheses. PMID:19426460

  12. Prediction of Protein-Protein Interaction Sites Based on Naive Bayes Classifier

    PubMed Central

    Geng, Haijiang; Lu, Tao; Lin, Xiao; Liu, Yu; Yan, Fangrong

    2015-01-01

    Protein functions through interactions with other proteins and biomolecules and these interactions occur on the so-called interface residues of the protein sequences. Identifying interface residues makes us better understand the biological mechanism of protein interaction. Meanwhile, information about the interface residues contributes to the understanding of metabolic, signal transduction networks and indicates directions in drug designing. In recent years, researchers have focused on developing new computational methods for predicting protein interface residues. Here we creatively used a 181-dimension protein sequence feature vector as input to the Naive Bayes Classifier- (NBC-) based method to predict interaction sites in protein-protein complexes interaction. The prediction of interaction sites in protein interactions is regarded as an amino acid residue binary classification problem by applying NBC with protein sequence features. Independent test results suggested that Naive Bayes Classifier-based method with the protein sequence features as input vectors performed well. PMID:26697220

  13. Protein release from hippocampus in vitro.

    PubMed

    Hesse, G W; Hofstein, R; Shashoua, V E

    1984-07-01

    Physiologically viable slices of rat hippocampus in vitro continuously release protein into the superfusion medium at a rate of about 2 micrograms/mg tissue/h. Assays of a cytoplasmic marker enzyme (lactate dehydrogenase) indicate that this material is not the result of cell lysis. Pulse-chase experiments using [3H]valine indicate that a substantial fraction of the newly synthesized proteins eventually appear in the incubation medium (18.7% +/- 3% of the total TCA precipitable radioactivity during a 6-h superfusion) and that the releasable protein pool has an apparent half-life of about 4 h. Simultaneous labeling of newly synthetized proteins with [3H]fucose and [14C]valine showed a 3-fold higher ratio of [3H]fucose to [14C]valine in the released protein fraction compared to the soluble cytoplasmic protein and to the crude membrane protein fraction, suggesting that the soluble released proteins are more highly glycosylated than the proteins retained in the tissue. Electrophoretic migration patterns on SDS-polyacrylamide gels with both labeled and unlabeled proteins show differences between the released proteins and the soluble cytoplasmic proteins of the tissue. Several molecular weights between 14 kdalton and 86 kdalton appear to be characteristic of the released protein fraction. These results suggest that a distinct group of proteins and glycoproteins exists in hippocampal tissue which is destined to be selectively released into the extracellular space. PMID:6744061

  14. 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

  15. Potential Interference of Protein-Protein Interactions by Graphyne.

    PubMed

    Luan, Binquan; Huynh, Tien; Zhou, Ruhong

    2016-03-10

    Graphyne has attracted tremendous attention recently due to its many potentially superior properties relative to those of graphene. Although extensive efforts have been devoted to explore the applicability of graphyne as an alternative nanomaterial for state-of-the-art nanotechnology (including biomedical applications), knowledge regarding its possible adverse effects to biological cells is still lacking. Here, using large-scale all-atom molecular dynamics simulations, we investigate the potential toxicity of graphyne by interfering a protein-protein interaction (ppI). We found that graphyne could indeed disrupt the ppIs by cutting through the protein-protein interface and separating the protein complex into noncontacting ones, due to graphyne's dispersive and hydrophobic interaction with the hydrophobic residues residing at the dimer interface. Our results help to elucidate the mechanism of interaction between graphyne and ppI networks within a biological cell and provide insights for its hazard reduction. PMID:26885561

  16. Protein mediated membrane adhesion

    NASA Astrophysics Data System (ADS)

    Carlson, Andreas; Mahadevan, L.

    2015-05-01

    Adhesion in the context of mechanical attachment, signaling, and movement in cellular dynamics is mediated by the kinetic interactions between membrane-embedded proteins in an aqueous environment. Here, we present a minimal theoretical framework for the dynamics of membrane adhesion that accounts for the kinetics of protein binding, the elastic deformation of the membrane, and the hydrodynamics of squeeze flow in the membrane gap. We analyze the resulting equations using scaling estimates to characterize the spatiotemporal features of the adhesive patterning and corroborate them using numerical simulations. In addition to characterizing aspects of cellular dynamics, our results might also be applicable to a range of phenomena in physical chemistry and materials science where flow, deformation, and kinetics are coupled to each other in slender geometries.

  17. Photolytic Crosslinking to Probe Protein-Protein and Protein-Matrix Interactions In Lyophilized Powders

    PubMed Central

    Iyer, Lavanya K.; Moorthy, Balakrishnan S.; Topp, Elizabeth M.

    2015-01-01

    Protein structure and local environment in lyophilized formulations were probed using high-resolution solid-state photolytic crosslinking with mass spectrometric analysis (ssPC-MS). In order to characterize structure and microenvironment, protein-protein, protein-excipient and protein-water interactions in lyophilized powders were identified. Myoglobin (Mb) was derivatized in solution with the heterobifunctional probe succinimidyl 4,4’-azipentanoate (SDA) and the structural integrity of the labeled protein (Mb-SDA) confirmed using CD spectroscopy and liquid chromatography / mass spectrometry (LC-MS). Mb-SDA was then formulated with and without excipients (raffinose, guanidine hydrochloride (Gdn HCl)) and lyophilized. The freeze-dried powder was irradiated with ultraviolet light at 365 nm for 30 min to produce crosslinked adducts that were analyzed at the intact protein level and after trypsin digestion. SDA-labeling produced Mb carrying up to 5 labels, as detected by LC-MS. Following lyophilization and irradiation, crosslinked peptide-peptide, peptide-water and peptide-raffinose adducts were detected. The exposure of Mb side chains to the matrix was quantified based on the number of different peptide-peptide, peptide-water and peptide-excipient adducts detected. In the absence of excipients, peptide-peptide adducts involving the CD, DE and EF loops and helix H were common. In the raffinose formulation, peptide-peptide adducts were more distributed throughout the molecule. The Gdn HCl formulation showed more protein-protein and protein-water adducts than the other formulations, consistent with protein unfolding and increased matrix interactions. The results demonstrate that ssPC-MS can be used to distinguish excipient effects and characterize the local protein environment in lyophilized formulations with high resolution. PMID:26204425

  18. 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.

  19. Protein tyrosine phosphorylation in Mycobacterium tuberculosis.

    PubMed

    Chow, K; Ng, D; Stokes, R; Johnson, P

    1994-12-01

    Crude cell extracts from three strains of Mycobacterium tuberculosis were analyzed for the presence of proteins possessing phosphorylated tyrosine residues. A protein migrating at approximately 55 kDa was detected using an antiphosphotyrosine monoclonal antibody. In addition, less predominant bands were observed between 50 kDa and 60 kDa. That M. tuberculosis contains specific tyrosine phosphorylated proteins implies that M. tuberculosis has tyrosine kinase activity. Examination of other, non-pathogenic mycobacterium species yielded no major antiphosphotyrosine reactive proteins. This suggests that the antiphosphotyrosine reactive protein is specific to M. tuberculosis strains. These results provide evidence that M. tuberculosis contains an antiphosphotyrosine reactive protein. PMID:7529204

  20. Protein thin film machines.

    PubMed

    Federici, Stefania; Oliviero, Giulio; Hamad-Schifferli, Kimberly; Bergese, Paolo

    2010-12-01

    We report the first example of microcantilever beams that are reversibly driven by protein thin film machines fueled by cycling the salt concentration of the surrounding solution. We also show that upon the same salinity stimulus the drive can be completely reversed in its direction by introducing a surface coating ligand. Experimental results are throughout discussed within a general yet simple thermodynamic model. PMID:20936224

  1. Nonenzymatic Protein Acetylation Detected by NAPPA Protein Arrays.

    PubMed

    Olia, Adam S; Barker, Kristi; McCullough, Cheryl E; Tang, Hsin-Yao; Speicher, David W; Qiu, Ji; LaBaer, Joshua; Marmorstein, Ronen

    2015-09-18

    Acetylation is a post-translational modification that occurs on thousands of proteins located in many cellular organelles. This process mediates many protein functions and modulates diverse biological processes. In mammalian cells, where acetyl-CoA is the primary acetyl donor, acetylation in the mitochondria is thought to occur by chemical means due to the relatively high concentration of acetyl-CoA located in this organelle. In contrast, acetylation outside of the mitochondria is thought to be mediated predominantly by acetyltransferase enzymes. Here, we address the possibility that nonenzymatic chemical acetylation outside of the mitochondria may be more common than previously appreciated. We employed the Nucleic Acid Programmable Protein Array platform to perform an unbiased screen for human proteins that undergo chemical acetylation, which resulted in the identification of a multitude of proteins with diverse functions and cellular localization. Mass spectrometry analysis revealed that basic residues typically precede the acetylated lysine in the -7 to -3 position, and we show by mutagenesis that these basic residues contribute to chemical acetylation capacity. We propose that these basic residues lower the pKa of the substrate lysine for efficient chemical acetylation. Many of the identified proteins reside outside of the mitochondria and have been previously demonstrated to be acetylated in vivo. As such, our studies demonstrate that chemical acetylation occurs more broadly throughout the eukaryotic cell than previously appreciated and suggests that this post-translational protein modification may have more diverse roles in protein function and pathway regulation. PMID:26083674

  2. Protein patterns and proteins that identify subtypes of glioblastoma multiforme.

    PubMed

    Furuta, Makoto; Weil, Robert J; Vortmeyer, Alexander O; Huang, Steve; Lei, Jingqi; Huang, Tai-Nan; Lee, Youn-Soo; Bhowmick, Deb A; Lubensky, Irina A; Oldfield, Edward H; Zhuang, Zhengping

    2004-09-01

    Glioblastoma multiforme (GBM) has been subdivided into two types based on clinical and genetic findings: primary tumors, which arise de novo, and secondary tumors, which progress from lower grade gliomas to GBMs. To analyse this dichotomy at the protein level, we employed selective tissue microdissection to obtain pure populations of tumor cells, which we studied using two-dimensional protein gel electrophoresis (2-DGE) and protein sequencing of select target proteins. Protein patterns were analysed in a blinded manner from the clinical and genetic data. 2-DGE clearly identified two distinct populations of tumors. 2-DGE was reproducible and reliable, as multiple samples analysed from the same patient gave identical results. In addition, we isolated and sequenced 11 proteins that were uniquely expressed in either the primary or the secondary GBMs, but not both. We demonstrate that specific proteomic patterns can be reproducibly identified by two-dimensional gel electrophoresis from limited numbers of selectively procured, microdissected tumor cells and that two patterns of GBMs, primary versus secondary, previously distinguished by clinical and genetic differences, can be recognized at the protein level. Proteins that are expressed distinctively may have important implications for the diagnosis, prognosis, and treatment of patients with GBM. PMID:15286718

  3. Non-enzymatic protein acetylation detected by NAPPA protein arrays*

    PubMed Central

    Olia, Adam S.; Barker, Kristi; McCullough, Cheryl E.; Tang, Hsin-Yao; Speicher, David W.; Qiu, Ji; LaBaer, Joshua; Marmorstein, Ronen

    2015-01-01

    Acetylation is a post-translational modification that occurs on thousands of proteins located in many cellular organelles. This process mediates many protein functions and modulates diverse biological processes. In mammalian cells, where acetyl-CoA is the primary acetyl donor, acetylation in the mitochondria is thought to occur by chemical means due to the relatively high concentration of acetyl-CoA located in this organelle. In contrast, acetylation outside of the mitochondria is thought to be mediated predominantly by acetyltransferase enzymes. Here we address the possibility that non-enzymatic chemical acetylation outside of the mitochondria may be more common than previously appreciated. We employed the Nucleic Acid Programmable Protein Array platform to perform an unbiased screen for human proteins that undergo chemical acetylation, which resulted in the identification of a multitude of proteins with diverse functions and cellular localization. Mass spectrometry analysis revealed that basic residues typically precede the acetylated lysine in the ?7 to ?3 position, and we show by mutagenesis that these basic residues contribute to chemical acetylation capacity. We propose that these basic residues lower the pKa of the substrate lysine for efficient chemical acetylation. Many of the identified proteins reside outside of the mitochondria, and have been previously demonstrated to be acetylated in vivo. As such, our studies demonstrate that chemical acetylation occurs more broadly throughout the eukaryotic cell than previously appreciated, and suggests that this post-translational protein modification may have more diverse roles in protein function and pathway regulation. PMID:26083674

  4. Mining physical protein-protein interactions from the literature

    PubMed Central

    Huang, Minlie; Ding, Shilin; Wang, Hongning; Zhu, Xiaoyan

    2008-01-01

    Background: Deciphering physical protein-protein interactions is fundamental to elucidating both the functions of proteins and biological processes. The development of high-throughput experimental technologies such as the yeast two-hybrid screening has produced an explosion in data relating to interactions. Since manual curation is intensive in terms of time and cost, there is an urgent need for text-mining tools to facilitate the extraction of such information. The BioCreative (Critical Assessment of Information Extraction systems in Biology) challenge evaluation provided common standards and shared evaluation criteria to enable comparisons among different approaches. Results: During the benchmark evaluation of BioCreative 2006, all of our results ranked in the top three places. In the task of filtering articles irrelevant to physical protein interactions, our method contributes a precision of 75.07%, a recall of 81.07%, and an AUC (area under the receiver operating characteristic curve) of 0.847. In the task of identifying protein mentions and normalizing mentions to molecule identifiers, our method is competitive among runs submitted, with a precision of 34.83%, a recall of 24.10%, and an F1 score of28.5%. In extracting protein interaction pairs, our profile-based method was competitive on the SwissProt-only subset (precision = 36.95%, recall = 32.68%, and F1 score = 30.40%) and on the entire dataset (30.96%, 29.35%, and26.20%, respectively). From the biologist's point of view, however, these findings are far from satisfactory. The error analysis presented in this report provides insight into how performance could be improved: three-quarters of false negatives were due to protein normalization problems (532/698), and about one-quarter were due to problems with correctly extracting interactions for this system. Conclusion: We present a text-mining framework to extract physical protein-protein interactions from the literature. Three key issues are addressed, namely filtering irrelevant articles, identifying protein names and normalizing them to molecule identifiers, and extracting protein-protein interactions. Our system is among the top three performers in the benchmark evaluation of BioCreative 2006. The tool will be helpful for manual interaction curation and can greatly facilitate the process of extracting protein-protein interactions. PMID:18834490

  5. Genome-wide Protein-protein Interaction Screening by Protein-fragment Complementation Assay (PCA) in Living Cells

    PubMed Central

    Filteau, Marie; Leducq, Jean-Baptiste; Dub, Alexandre K.; Landry, Christian R.

    2015-01-01

    Proteins are the building blocks, effectors and signal mediators of cellular processes. A proteins 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. PMID:25867901

  6. 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, significantly increasing the docking success rate. PMID:26650466

  7. Health Benefits of Texturized Whey Proteins

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Whey proteins are an important class of food ingredients used in many functional foods to boost protein content. Using the extrusion texturization process to partially open the native globular structures of whey proteins changed their conformation to the molten globular state, resulting in a new cla...

  8. Assessment of the reliability of protein-protein interactions and protein function prediction.

    PubMed

    Deng, Minghua; Sun, Fengzhu; Chen, Ting

    2003-01-01

    As more and more high-throughput protein-protein interaction data are collected, the task of estimating the reliability of different data sets becomes increasingly important. In this paper, we present our study of two groups of protein-protein interaction data, the physical interaction data and the protein complex data, and estimate the reliability of these data sets using three different measurements: (1) the distribution of gene expression correlation coefficients, (2) the reliability based on gene expression correlation coefficients, and (3) the accuracy of protein function predictions. We develop a maximum likelihood method to estimate the reliability of protein interaction data sets according to the distribution of correlation coefficients of gene expression profiles of putative interacting protein pairs. The results of the three measurements are consistent with each other. The MIPS protein complex data have the highest mean gene expression correlation coefficients (0.256) and the highest accuracy in predicting protein functions (70% sensitivity and specificity), while Ito's Yeast two-hybrid data have the lowest mean (0.041) and the lowest accuracy (15% sensitivity and specificity). Uetz's data are more reliable than Ito's data in all three measurements, and the TAP protein complex data are more reliable than the HMS-PCI data in all three measurements as well. The complex data sets generally perform better in function predictions than do the physical interaction data sets. Proteins in complexes are shown to be more highly correlated in gene expression. The results confirm that the components of a protein complex can be assigned to functions that the complex carries out within a cell. There are three interaction data sets different from the above two groups: the genetic interaction data, the in-silico data and the syn-express data. Their capability of predicting protein functions generally falls between that of the Y2H data and that of the MIPS protein complex data. The supplementary information is available at the following Web site: http://www-hto.usc.edu/-msms/AssessInteraction/. PMID:12603024

  9. 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.

  10. 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.

  11. 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.

  12. Protein compressibility, dynamics, and pressure.

    PubMed

    Kharakoz, D P

    2000-07-01

    The relationship between the elastic and dynamic properties of native globular proteins is considered on the basis of a wide set of reported experimental data. The formation of a small cavity, capable of accommodating water, in the protein interior is associated with the elastic deformation, whose contribution to the free energy considerably exceeds the heat motion energy. Mechanically, the protein molecule is a highly nonlinear system. This means that its compressibility sharply decreases upon compression. The mechanical nonlinearity results in the following consequences related to the intramolecular dynamics of proteins: 1) The sign of the electrostriction effect in the protein matrix is opposite that observed in liquids-this is an additional indication that protein behaves like a solid particle. 2) The diffusion of an ion from the solvent to the interior of a protein should depend on pressure nonmonotonically: at low pressure diffusion is suppressed, while at high pressure it is enhanced. Such behavior is expected to display itself in any dynamic process depending on ion diffusion. Qualitative and quantitative expectations ensuing from the mechanical properties are concordant with the available experimental data on hydrogen exchange in native proteins at ambient and high pressure. PMID:10866977

  13. 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. PMID:18339603

  14. Protein compressibility, dynamics, and pressure.

    PubMed Central

    Kharakoz, D P

    2000-01-01

    The relationship between the elastic and dynamic properties of native globular proteins is considered on the basis of a wide set of reported experimental data. The formation of a small cavity, capable of accommodating water, in the protein interior is associated with the elastic deformation, whose contribution to the free energy considerably exceeds the heat motion energy. Mechanically, the protein molecule is a highly nonlinear system. This means that its compressibility sharply decreases upon compression. The mechanical nonlinearity results in the following consequences related to the intramolecular dynamics of proteins: 1) The sign of the electrostriction effect in the protein matrix is opposite that observed in liquids-this is an additional indication that protein behaves like a solid particle. 2) The diffusion of an ion from the solvent to the interior of a protein should depend on pressure nonmonotonically: at low pressure diffusion is suppressed, while at high pressure it is enhanced. Such behavior is expected to display itself in any dynamic process depending on ion diffusion. Qualitative and quantitative expectations ensuing from the mechanical properties are concordant with the available experimental data on hydrogen exchange in native proteins at ambient and high pressure. PMID:10866977

  15. Dual targeting of peroxisomal proteins

    PubMed Central

    Ast, Julia; Stiebler, Alina C.; Freitag, Johannes; Blker, 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

  16. Calcium-binding proteins in Aplysia neurons.

    PubMed

    Hermann, A; Pauls, T L; Heizmann, C W

    1991-08-01

    1. Calcium (Ca)-binding proteins of neuronal ganglia and of single, identified neurons of the marine mollusk, Aplysia californica, were investigated. Using transblot/45Ca overlays two proteins, at Mr 45,000 and Mr 23,000, with a high Ca-binding ability were found. 2. Western blot analysis revealed that the protein at Mr 45,000 could be separated by 2D-PAGE into proteins with Mr 40,000 and Mr 43,000. The protein at Mr 40,000 immunocross-reacted with antisera directed against parvalbumin and rat calbindin D-28K, indicating a novel Ca-binding protein sharing common antigenic determinants for both proteins. 3. The protein at Mr 23,000 could be separated into a group of proteins with Mr 13,000-20,000 which showed a high degree of similarity to sarcoplasmatic calcium-binding proteins (SCP). 4. We further investigated the protein pattern of single, identified neurons of different electrical activity (bursting, beating, and silent) by 2D-PAGE. Major differences were found in the range of low Mr and low pI, where Ca-binding proteins are generally located. A protein at high concentrations characteristic for silent cells migrated at a position similar to crayfish SCP. 5. The results show that various Ca-binding proteins are characteristic for neurons in the Aplysia nervous system and support the idea that they may effect the electrical behavior of nerve cells. PMID:1751962

  17. 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.

  18. Identification of candidate residues for interaction of protein S with C4b binding protein and activated protein C.

    PubMed Central

    Greengard, J S; Fernandez, J A; Radtke, K P; Griffin, J H

    1995-01-01

    Protein S is a plasma factor essential for prevention of thrombosis, partly due to its activity as a cofactor for the plasma anticoagulant protease-activated protein C. To expand knowledge about structure-function relationships in homologous protein S molecules, studies of protein S from different species have been performed. Protein S anti-coagulant activity in human, monkey, bovine, and porcine plasma has been inactivated by purified human C4b binding protein (C4BP) with dose-dependence, suggesting that each protein S can bind human C4BP and that only the free form of each is anti-coagulantly active. Purified porcine protein S has a 10-fold higher Kd for human C4BP than has human protein S. Protein S residues 420-434 provide an essential binding site for the negative regulator C4BP. cDNA sequences show that protein S residues 420-434 are highly conserved in all four species with the notable exception of Lys-429-Ile in porcine protein S. Differences between porcine and human protein S, e.g. Lys-429-Ile, Lys-43-Ala, Ser-197-Leu, Ser 199-Phe, Glu-463-Gly, Lys-571-Glu, Asn-602-Ile, Gln-607-Pro, may contribute to the decreased affinity of porcine protein S for human C4BP. Moreover, the species specificity of cofactor activities of various species of protein S is determined for human versus bovine-activated protein C, and these results, combined with sequence comparisons, agree with previous evidence that the thrombin-sensitive region and the first epidermal growth factor domain of protein S, i.e. residues 47-116, are responsible for recognition of activated protein C. Images Figure 1 Figure 3 PMID:7832752

  19. PPIevo: protein-protein interaction prediction from PSSM based evolutionary information.

    PubMed

    Zahiri, Javad; Yaghoubi, Omid; Mohammad-Noori, Morteza; Ebrahimpour, Reza; Masoudi-Nejad, Ali

    2013-10-01

    Protein-protein interactions regulate a variety of cellular processes. There is a great need for computational methods as a complement to experimental methods with which to predict protein interactions due to the existence of many limitations involved in experimental techniques. Here, we introduce a novel evolutionary based feature extraction algorithm for protein-protein interaction (PPI) prediction. The algorithm is called PPIevo and extracts the evolutionary feature from Position-Specific Scoring Matrix (PSSM) of protein with known sequence. The algorithm does not depend on the protein annotations, and the features are based on the evolutionary history of the proteins. This enables the algorithm to have more power for predicting protein-protein interaction than many sequence based algorithms. Results on the HPRD database show better performance and robustness of the proposed method. They also reveal that the negative dataset selection could lead to an acute performance overestimation which is the principal drawback of the available methods. PMID:23747746

  20. Solubility of commercial milk protein concentrates and milk protein isolates.

    PubMed

    Sikand, V; Tong, P S; Roy, S; Rodriguez-Saona, L E; Murray, B A

    2011-12-01

    High-protein milk protein concentrate (MPC) and milk protein isolate (MPI) powders may have lower solubility than low-protein MPC powders, but information is limited on MPC solubility. Our objectives in this study were to (1) characterize the solubility of commercially available powder types with differing protein contents such as MPC40, MPC80, and MPI obtained from various manufacturers (sources), and (2) determine if such differences could be associated with differences in mineral, protein composition, and conformational changes of the powders. To examine possible predictors of solubility as measured by percent suspension stability (%SS), mineral analysis, Fourier transform infrared (FTIR) spectroscopy, and quantitative protein analysis by HPLC was performed. After accounting for overall differences between powder types, %SS was found to be strongly associated with the calcium, magnesium, phosphorus, and sodium content of the powders. The FTIR score plots were in agreement with %SS results. A principal component analysis of FTIR spectra clustered the highly soluble MPC40 separately from the rest of samples. Furthermore, 2 highly soluble MPI samples were clustered separately from the rest of the MPC80 and MPI samples. We found that the 900 to 1,200 cm? region exhibited the highest discriminating power, with dominant bands at 1,173 and 968 cm?, associated with phosphate vibrations. The 2 highly soluble MPI powders were observed to have lower ?-casein and ?-(S1)-casein contents and slightly higher whey protein contents than the other powders. The differences in the solubility of MPC and MPI were associated with a difference in mineral composition, which may be attributed to differences in processing conditions. Additional studies on the role of minerals composition on MPC80 solubility are warranted. Such a study would provide a greater understanding of factors associated with differences in solubility and can provide insight on methods to improve solubility of high-protein milk protein concentrates. PMID:22118108

  1. 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

  2. Evolutionary Optimization of Protein Folding

    PubMed Central

    Debès, Cédric; Wang, Minglei; Caetano-Anollés, Gustavo; Gräter, Frauke

    2013-01-01

    Nature has shaped the make up of proteins since their appearance, 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 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

  3. Manganese elevates manganese superoxide dismutase protein level through protein kinase C and protein tyrosine kinase.

    PubMed

    Li, Sufen; Lu, Lin; Liao, Xiudong; Gao, Tianquan; Wang, Funing; Zhang, Liyang; Xi, Lin; Liu, Songbai; Luo, Xugang

    2016-04-01

    Three experiments were conducted to investigate the effects of inorganic and organic Mn sources on MnSOD mRNA, protein and enzymatic activity and the possible signal pathways. The primary broiler myocardial cells were treated with MnCl2 (I) or one of organic chelates of Mn and amino acids with weak, moderate (M) or strong (S) chelation strength for 12 and 48 h. Cells were preincubated with superoxide radical anions scavenger N-acetylcysteine (NAC) or specific inhibitors for MAPKs and protein tyrosine kinase (PTK) or protein kinase C (PKC) for 30 min before treatments of I and M. The MnSOD mRNA, protein and enzymatic activity, phosphorylated MAPKs or protein kinases activations were examined. The results showed that additions of Mn increased (P < 0.05) MnSOD mRNA levels and M was more effective than I. Additions of Mn elevated (P < 0.05) MnSOD protein levels and enzymatic activities, and no differences were found among I and M. Addition of NAC did not decrease (P > 0.05) Mn-induced MnSOD mRNA and protein levels. None of the three MAPKs was phosphorylated (P > 0.05) by Mn. Additions of Mn decreased (P < 0.05) the PTK activities and increased (P < 0.05) the membrane PKC contents. Inhibitors for PTK or PKC decreased (P < 0.05) Mn-induced MnSOD protein levels. The results suggested that Mn-induced MnSOD mRNA and protein expressions be not related with NAC, and MAPK pathways might not involve in Mn-induced MnSOD mRNA expression. PKC and PTK mediated the Mn-induced MnSOD protein expression. PMID:26857738

  4. Computational approaches for detecting protein complexes from protein interaction networks: a survey

    PubMed Central

    2010-01-01

    Background Most proteins form macromolecular complexes to perform their biological functions. However, experimentally determined protein complex data, especially of those involving more than two protein partners, are relatively limited in the current state-of-the-art high-throughput experimental techniques. Nevertheless, many techniques (such as yeast-two-hybrid) have enabled systematic screening of pairwise protein-protein interactions en masse. Thus computational approaches for detecting protein complexes from protein interaction data are useful complements to the limited experimental methods. They can be used together with the experimental methods for mapping the interactions of proteins to understand how different proteins are organized into higher-level substructures to perform various cellular functions. Results Given the abundance of pairwise protein interaction data from high-throughput genome-wide experimental screenings, a protein interaction network can be constructed from protein interaction data by considering individual proteins as the nodes, and the existence of a physical interaction between a pair of proteins as a link. This binary protein interaction graph can then be used for detecting protein complexes using graph clustering techniques. In this paper, we review and evaluate the state-of-the-art techniques for computational detection of protein complexes, and discuss some promising research directions in this field. Conclusions Experimental results with yeast protein interaction data show that the interaction subgraphs discovered by various computational methods matched well with actual protein complexes. In addition, the computational approaches have also improved in performance over the years. Further improvements could be achieved if the quality of the underlying protein interaction data can be considered adequately to minimize the undesirable effects from the irrelevant and noisy sources, and the various biological evidences can be better incorporated into the detection process to maximize the exploitation of the increasing wealth of biological knowledge available. PMID:20158874

  5. HMPAS: Human Membrane Protein Analysis System

    PubMed Central

    2013-01-01

    Background Membrane proteins perform essential roles in diverse cellular functions and are regarded as major pharmaceutical targets. The significance of membrane proteins has led to the developing dozens of resources related with membrane proteins. However, most of these resources are built for specific well-known membrane protein groups, making it difficult to find common and specific features of various membrane protein groups. Methods We collected human membrane proteins from the dispersed resources and predicted novel membrane protein candidates by using ortholog information and our membrane protein classifiers. The membrane proteins were classified according to the type of interaction with the membrane, subcellular localization, and molecular function. We also made new feature dataset to characterize the membrane proteins in various aspects including membrane protein topology, domain, biological process, disease, and drug. Moreover, protein structure and ICD-10-CM based integrated disease and drug information was newly included. To analyze the comprehensive information of membrane proteins, we implemented analysis tools to identify novel sequence and functional features of the classified membrane protein groups and to extract features from protein sequences. Results We constructed HMPAS with 28,509 collected known membrane proteins and 8,076 newly predicted candidates. This system provides integrated information of human membrane proteins individually and in groups organized by 45 subcellular locations and 1,401 molecular functions. As a case study, we identified associations between the membrane proteins and diseases and present that membrane proteins are promising targets for diseases related with nervous system and circulatory system. A web-based interface of this system was constructed to facilitate researchers not only to retrieve organized information of individual proteins but also to use the tools to analyze the membrane proteins. Conclusions HMPAS provides comprehensive information about human membrane proteins including specific features of certain membrane protein groups. In this system, user can acquire the information of individual proteins and specified groups focused on their conserved sequence features, involved cellular processes, and diseases. HMPAS may contribute as a valuable resource for the inference of novel cellular mechanisms and pharmaceutical targets associated with the human membrane proteins. HMPAS is freely available at http://fcode.kaist.ac.kr/hmpas. PMID:24564858

  6. Enzymatic protein depalmitoylation by acyl protein thioesterases.

    PubMed

    Lin, David T S; Conibear, Elizabeth

    2015-04-01

    Protein palmitoylation is a dynamic post-translational modification, where the 16-carbon fatty acid, palmitate, is added to cysteines of proteins to modulate protein sorting, targeting and signalling. Palmitate removal from proteins is mediated by acyl protein thioesterases (APTs). Although initially identified as lysophospholipases, increasing evidence suggests APT1 and APT2 are the major APTs that mediate the depalmitoylation of diverse cellular substrates. Here, we describe the conserved functions of APT1 and APT2 across organisms and discuss the possibility that these enzymes are members of a larger family of depalmitoylation enzymes. PMID:25849916

  7. 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 be performing a structural role [2]. These findings suggest that adaptation of microbes to their geochemical environment is accompanied by minimization of the energetic costs of protein biosynthesis, which can be tested further by including methanogens in other environments like hot springs, submarine hydrothermal vents and peatlands. Comparative analyses will reveal which proteins and protein regions follow this energy-minimization strategy and which are excluded. It will then be possible to characterize proteins in terms of the extent to which their sequences are influenced by external geochemical forces. 1. Dick, J. M. (2008). Calculation of the relative metastabilities of proteins using the CHNOSZ software package. Geochem. Trans, 9(10). DOI: 10.1186/1467-4866-9-10. 2. Zwickl P., Grziwa A., Puehler G., Dahlmann B., Lottspeich F. and Baumeister W. (1992) Primary structure of the Thermoplasma proteasome and its implications for the structure, function, and evolution of the multicatalytic proteinase. Biochemistry 31, 964-972. DOI: 10.1021/bi00119a004.

  8. 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

  9. Multifunctional proteins revealed by overlapping clustering in protein interaction network

    PubMed Central

    Chapple, Charles E.; Gunoche, Alain; Brun, Christine

    2012-01-01

    Motivation: Multifunctional proteins perform several functions. They are expected to interact specifically with distinct sets of partners, simultaneously or not, depending on the function performed. Current graph clustering methods usually allow a protein to belong to only one cluster, therefore impeding a realistic assignment of multifunctional proteins to clusters. Results: Here, we present Overlapping Cluster Generator (OCG), a novel clustering method which decomposes a network into overlapping clusters and which is, therefore, capable of correct assignment of multifunctional proteins. The principle of OCG is to cover the graph with initial overlapping classes that are iteratively fused into a hierarchy according to an extension of Newman's modularity function. By applying OCG to a human proteinprotein interaction network, we show that multifunctional proteins are revealed at the intersection of clusters and demonstrate that the method outperforms other existing methods on simulated graphs and PPI networks. Availability: This software can be downloaded from http://tagc.univ-mrs.fr/welcome/spip.php?rubrique197 Contact: brun@tagc.univ-mrs.fr Supplementary information: Supplementary data are available at Bioinformatics online. PMID:22080466

  10. Protein-protein interaction site predictions with three-dimensional probability distributions of interacting atoms on protein surfaces.

    PubMed

    Chen, Ching-Tai; Peng, Hung-Pin; Jian, Jhih-Wei; Tsai, Keng-Chang; Chang, Jeng-Yih; Yang, Ei-Wen; Chen, Jun-Bo; Ho, Shinn-Ying; Hsu, Wen-Lian; Yang, An-Suei

    2012-01-01

    Protein-protein interactions are key to many biological processes. Computational methodologies devised to predict protein-protein interaction (PPI) sites on protein surfaces are important tools in providing insights into the biological functions of proteins and in developing therapeutics targeting the protein-protein interaction sites. One of the general features of PPI sites is that the core regions from the two interacting protein surfaces are complementary to each other, similar to the interior of proteins in packing density and in the physicochemical nature of the amino acid composition. In this work, we simulated the physicochemical complementarities by constructing three-dimensional probability density maps of non-covalent interacting atoms on the protein surfaces. The interacting probabilities were derived from the interior of known structures. Machine learning algorithms were applied to learn the characteristic patterns of the probability density maps specific to the PPI sites. The trained predictors for PPI sites were cross-validated with the training cases (consisting of 432 proteins) and were tested on an independent dataset (consisting of 142 proteins). The residue-based Matthews correlation coefficient for the independent test set was 0.423; the accuracy, precision, sensitivity, specificity were 0.753, 0.519, 0.677, and 0.779 respectively. The benchmark results indicate that the optimized machine learning models are among the best predictors in identifying PPI sites on protein surfaces. In particular, the PPI site prediction accuracy increases with increasing size of the PPI site and with increasing hydrophobicity in amino acid composition of the PPI interface; the core interface regions are more likely to be recognized with high prediction confidence. The results indicate that the physicochemical complementarity patterns on protein surfaces are important determinants in PPIs, and a substantial portion of the PPI sites can be predicted correctly with the physicochemical complementarity features based on the non-covalent interaction data derived from protein interiors. PMID:22701576

  11. Protein-Protein Interaction Site Predictions with Three-Dimensional Probability Distributions of Interacting Atoms on Protein Surfaces

    PubMed Central

    Chen, Ching-Tai; Peng, Hung-Pin; Jian, Jhih-Wei; Tsai, Keng-Chang; Chang, Jeng-Yih; Yang, Ei-Wen; Chen, Jun-Bo; Ho, Shinn-Ying; Hsu, Wen-Lian; Yang, An-Suei

    2012-01-01

    Protein-protein interactions are key to many biological processes. Computational methodologies devised to predict protein-protein interaction (PPI) sites on protein surfaces are important tools in providing insights into the biological functions of proteins and in developing therapeutics targeting the protein-protein interaction sites. One of the general features of PPI sites is that the core regions from the two interacting protein surfaces are complementary to each other, similar to the interior of proteins in packing density and in the physicochemical nature of the amino acid composition. In this work, we simulated the physicochemical complementarities by constructing three-dimensional probability density maps of non-covalent interacting atoms on the protein surfaces. The interacting probabilities were derived from the interior of known structures. Machine learning algorithms were applied to learn the characteristic patterns of the probability density maps specific to the PPI sites. The trained predictors for PPI sites were cross-validated with the training cases (consisting of 432 proteins) and were tested on an independent dataset (consisting of 142 proteins). The residue-based Matthews correlation coefficient for the independent test set was 0.423; the accuracy, precision, sensitivity, specificity were 0.753, 0.519, 0.677, and 0.779 respectively. The benchmark results indicate that the optimized machine learning models are among the best predictors in identifying PPI sites on protein surfaces. In particular, the PPI site prediction accuracy increases with increasing size of the PPI site and with increasing hydrophobicity in amino acid composition of the PPI interface; the core interface regions are more likely to be recognized with high prediction confidence. The results indicate that the physicochemical complementarity patterns on protein surfaces are important determinants in PPIs, and a substantial portion of the PPI sites can be predicted correctly with the physicochemical complementarity features based on the non-covalent interaction data derived from protein interiors. PMID:22701576

  12. The natural immune response to inhaled soluble protein antigens involves major histocompatibility complex (MHC) class I-restricted CD8+ T cell-mediated but MHC class II-restricted CD4+ T cell-dependent immune deviation resulting in selective suppression of immunoglobulin E production.

    PubMed

    McMenamin, C; Holt, P G

    1993-09-01

    The immunological basis for atopy is currently ascribed to an inherent bias in the CD4+ T cell response to nonreplicating antigens presented at mucosal surfaces, resulting in dominance of the T helper 2 (Th2) interleukin 4 (IL-4)-producing phenotype, which favors IgE production. In contrast, the "normal" response to such antigens involves a predominance of interferon gamma (IFN-gamma)-producing Th1 clones. This difference has been suggested to be the result of active selection in atopics for Th2 (and hence against Th1) clones at the time of initial antigen presentation. In the study below, we demonstrate that the natural immune response to inhaled protein antigens, particularly in animals expressing the low immunoglobulin E (IgE) responder phenotype, includes a major histocompatibility complex (MHC) class I-restricted CD8+ T cell component, the appearance of which is associated with active suppression of IgE antibody production. Thus, continued exposure of rats to aerosolized ovalbumin (OVA) antigen elicits a transient IgE response, that is terminated by the onset of a state of apparent "tolerance" to further challenge, and this tolerant state is transferable to naive animals with CD8+ T cells. Kinetic studies on in vitro T cell reactivity in these aerosol-exposed rats demonstrated biphasic CD4+ Th2 responses which terminated, together with IgE antibody production, and coincident with the appearance of MHC class I-restricted OVA-specific IFN-gamma-producing CD8+ T cells. However, the latter were not autonomous in vitro and required a source of exogenous IL-2 for initial activation, which in CD(8+)-enriched splenocyte cultures could be provided by small numbers of contaminating OVA-specific CD4+ T cells. This represents the first formal evidence for the induction of an MHC class I-restricted T cell response to natural mucosal exposure to an inert protein antigen, and is consistent with a growing literature demonstrating sensitization of MHC class I-restricted CD8+ T cells by deliberate immunization with soluble proteins. We suggest that crossregulation of MHC class II-restricted CD4+ T cells via cytokine signals generated in parallel CD8+ T cell responses represents a covert and potentially important selection pressure that can shape the nature of host responses to nonreplicating antigens presented at mucosal surfaces. PMID:8102390

  13. 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

  14. Distinguishing Proteins From Arbitrary Amino Acid Sequences

    PubMed Central

    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

  15. 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

  16. Small Water Islands in Proteins

    NASA Astrophysics Data System (ADS)

    Helms, V.; Wade, R. C.; McCammon, J. A.

    1998-03-01

    Proteins often contain water-filled cavities. Their presence may have functional reasons or may be related to protein folding. In any case, they are integral parts of the protein. Here, results from molecular dynamics simulations for two systems are presented. First, we analyzed the hydration of an empty buried enzyme active site, cytochrome P450cam. A hydration free energy landscape was obtained by calculating free energy differences for hydrating the active site with 5 to 8 water molecules. In agreement with the crystal structure and with experiments performed under high hydrostatic pressure, 6 water molecules were found to be most favourable thermodynamically [Helms & Wade, Proteins, submited]. Long-lived hydrogen bond networks exist between the water molecules in the active site and result in a significant ordering. Secondly, results are presented from 5 simulations of green fluorescent protein (GFP) of 1 ns length each. The crystal structures of different forms of GFP contain a cluster of five water molecules next to the chromophore. The water cluster seems to play a crucial part in allowing the protein to switch between a fluorescent and a dark state [Dickson et al., Nature, 388, 385-8]. In the simulations, the water molecules are again strongly ordered by a long-lived hydrogen bond network. Both scenarios are discussed in the context of bulk liquid water.

  17. Graph pyramids for protein function prediction

    PubMed Central

    2015-01-01

    Background Uncovering the hidden organizational characteristics and regularities among biological sequences is the key issue for detailed understanding of an underlying biological phenomenon. Thus pattern recognition from nucleic acid sequences is an important affair for protein function prediction. As proteins from the same family exhibit similar characteristics, homology based approaches predict protein functions via protein classification. But conventional classification approaches mostly rely on the global features by considering only strong protein similarity matches. This leads to significant loss of prediction accuracy. Methods Here we construct the Protein-Protein Similarity (PPS) network, which captures the subtle properties of protein families. The proposed method considers the local as well as the global features, by examining the interactions among 'weakly interacting proteins' in the PPS network and by using hierarchical graph analysis via the graph pyramid. Different underlying properties of the protein families are uncovered by operating the proposed graph based features at various pyramid levels. Results Experimental results on benchmark data sets show that the proposed hierarchical voting algorithm using graph pyramid helps to improve computational efficiency as well the protein classification accuracy. Quantitatively, among 14,086 test sequences, on an average the proposed method misclassified only 21.1 sequences whereas baseline BLAST score based global feature matching method misclassified 362.9 sequences. With each correctly classified test sequence, the fast incremental learning ability of the proposed method further enhances the training model. Thus it has achieved more than 96% protein classification accuracy using only 20% per class training data. PMID:26044522

  18. 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.

  19. Datamining protein structure databanks for crystallization patterns of proteins.

    PubMed

    Valafar, Homayoun; Prestegard, James H; Valafar, Faramarz

    2002-12-01

    A study of 345 protein structures selected among 1,500 structures determined by nuclear magnetic resonance (NMR) methods, revealed useful correlations between crystallization properties and several parameters for the studied proteins. NMR methods of structure determination do not require the growth of protein crystals, and hence allow comparison of properties of proteins that have or have not been the subject of crystallographic approaches. One- and two-dimensional statistical analyses of the data confirmed a hypothesized relation between the size of the molecule and its crystallization potential. Furthermore, two-dimensional Bayesian analysis revealed a significant relationship between relative ratio of different secondary structures and the likelihood of success for crystallization trials. The most immediate result is an apparent correlation of crystallization potential with protein size. Further analysis of the data revealed a relationship between the unstructured fraction of proteins and the success of its crystallization. Utilization of Bayesian analysis on the latter correlation resulted in a prediction performance of about 64%, whereas a two-dimensional Bayesian analysis succeeded with a performance of about 75%. PMID:12594078

  20. Non-classical protein secretion in bacteria

    PubMed Central

    Bendtsen, Jannick D; Kiemer, Lars; Fausbøll, Anders; Brunak, Søren

    2005-01-01

    Background We present an overview of bacterial non-classical secretion and a prediction method for identification of proteins following signal peptide independent secretion pathways. We have compiled a list of proteins found extracellularly despite the absence of a signal peptide. Some of these proteins also have known roles in the cytoplasm, which means they could be so-called "moon-lightning" proteins having more than one function. Results A thorough literature search was conducted to compile a list of currently known bacterial non-classically secreted proteins. Pattern finding methods were applied to the sequences in order to identify putative signal sequences or motifs responsible for their secretion. We have found no signal or motif characteristic to any majority of the proteins in the compiled list of non-classically secreted proteins, and conclude that these proteins, indeed, seem to be secreted in a novel fashion. However, we also show that the apparently non-classically secreted proteins are still distinguished from cellular proteins by properties such as amino acid composition, secondary structure and disordered regions. Specifically, prediction of disorder reveals that bacterial secretory proteins are more structurally disordered than their cytoplasmic counterparts. Finally, artificial neural networks were used to construct protein feature based methods for identification of non-classically secreted proteins in both Gram-positive and Gram-negative bacteria. Conclusion We present a publicly available prediction method capable of discriminating between this group of proteins and other proteins, thus allowing for the identification of novel non-classically secreted proteins. We suggest candidates for non-classically secreted proteins in Escherichia coli and Bacillus subtilis. The prediction method is available online. PMID:16212653

  1. Bone morphogenetic proteins: periodontal regeneration.

    PubMed

    Rao, Subramaniam M; Ugale, Gauri M; Warad, Shivaraj B

    2013-03-01

    Periodontitis is an infectious inflammatory disease that results in attachment loss and bone loss. Regeneration of the periodontal tissues entails de novo formation of cementum, periodontal ligament, and alveolar bone. Several different approaches are currently being explored to achieve complete, reliable, and reproducible regeneration of periodontal tissues. The therapeutic management of new bone formation is one of the key issues in successful periodontal regeneration. Bone morphogenetic proteins form a unique group of proteins within the transforming growth factor superfamily of genes and have a vital role in the regulation in the bone induction and maintenance. The activity of bone morphogenetic proteins was first identified in the 1960s, but the proteins responsible for bone induction were unknown until the purification and cloning of human bone morphogenetic proteins in the 1980s, because of their osteoinductive potential. Bone morphogenetic proteins have gained a lot of interest as therapeutic agents for treating periodontal defects. A systematic search for data related to the use of bone morphogenetic proteins for the regeneration of periodontal defects was performed to recognize studies on animals and human (PUBMED, MEDLINE, COCHRANE, and Google search). All the studies included showed noticeable regeneration of periodontal tissues with the use of BMP. PMID:23626951

  2. 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,