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Sieve element occlusion (SEO) genes encode structural phloem proteins involved in wound sealing of the phloem.  


The sieve element occlusion (SEO) gene family originally was delimited to genes encoding structural components of forisomes, which are specialized crystalloid phloem proteins found solely in the Fabaceae. More recently, SEO genes discovered in various non-Fabaceae plants were proposed to encode the common phloem proteins (P-proteins) that plug sieve plates after wounding. We carried out a comprehensive characterization of two tobacco (Nicotiana tabacum) SEO genes (NtSEO). Reporter genes controlled by the NtSEO promoters were expressed specifically in immature sieve elements, and GFP-SEO fusion proteins formed parietal agglomerates in intact sieve elements as well as sieve plate plugs after wounding. NtSEO proteins with and without fluorescent protein tags formed agglomerates similar in structure to native P-protein bodies when transiently coexpressed in Nicotiana benthamiana, and the analysis of these protein complexes by electron microscopy revealed ultrastructural features resembling those of native P-proteins. NtSEO-RNA interference lines were essentially devoid of P-protein structures and lost photoassimilates more rapidly after injury than control plants, thus confirming the role of P-proteins in sieve tube sealing. We therefore provide direct evidence that SEO genes in tobacco encode P-protein subunits that affect translocation. We also found that peptides recently identified in fascicular phloem P-protein plugs from squash (Cucurbita maxima) represent cucurbit members of the SEO family. Our results therefore suggest a common evolutionary origin for P-proteins found in the sieve elements of all dicotyledonous plants and demonstrate the exceptional status of extrafascicular P-proteins in cucurbits. PMID:22733783

Ernst, Antonia M; Jekat, Stephan B; Zielonka, Sascia; Müller, Boje; Neumann, Ulla; Rüping, Boris; Twyman, Richard M; Krzyzanek, Vladislav; Prüfer, Dirk; Noll, Gundula A



Identification of the Cadmium-Inducible Hansenula polymorpha SEO1 Gene Promoter by Transcriptome Analysis and Its Application to Whole-Cell Heavy-Metal Detection Systems? †  

PubMed Central

The genomewide gene expression profiling of the methylotrophic yeast Hansenula polymorpha exposed to cadmium (Cd) allowed us to identify novel genes responsive to Cd treatment. To select genes whose promoters can be useful for construction of a cellular Cd biosensor, we further analyzed a set of H. polymorpha genes that exhibited >6-fold induction upon treatment with 300 ?M Cd for 2 h. The putative promoters, about 1,000-bp upstream fragments, of these genes were fused with the yeast-enhanced green fluorescence protein (GFP) gene. The resultant reporter cassettes were introduced into H. polymorpha to evaluate promoter strength and specificity. The promoter derived from the H. polymorpha SEO1 gene (HpSEO1) was shown to drive most strongly the expression of GFP upon Cd treatment among the tested promoters. The Cd-inducible activity was retained in the 500-bp deletion fragment of the HpSEO1 promoter but was abolished in the further truncated 250-bp fragment. The 500-bp HpSEO1 promoter directed specific expression of GFP upon exposure to Cd in a dose-dependent manner, with Cd detection ranging from 1 to 900 ?M. Comparative analysis of the Saccharomyces cerevisiae SEO1 (ScSEO1) promoter revealed that the ScSEO1 promoter has a broader specificity for heavy metals and is responsive to arsenic and mercury in addition to Cd. Our data demonstrate the potential use of the HpSEO1 promoter as a bioelement in whole-cell biosensors to monitor heavy metal contamination, particularly Cd. PMID:17660305

Park, Jeong-Nam; Sohn, Min Jeong; Oh, Doo-Byoung; Kwon, Ohsuk; Rhee, Sang Ki; Hur, Cheol-Goo; Lee, Sang Yup; Gellissen, Gerd; Kang, Hyun Ah



CEOS SEO and GISS Meeting  

NASA Technical Reports Server (NTRS)

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.

Killough, Brian; Stover, Shelley



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

NASA Technical Reports Server (NTRS)

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.

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



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


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

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



EPS dilution after SEOs and earnings management  

Microsoft Academic Search

Purpose – Firms are concerned about earnings per share (EPS) dilution after equity issues. The purpose of this paper is to investigate whether firms manage upward their discretionary accruals around seasoned equity offerings (SEOs) to mitigate the impact of dilution on reported earnings. Design\\/methodology\\/approach – The authors employ adjusted discretionary accruals from cash flow statements, normalized by the average common

Hui Di; Dalia Marciukaityte; Eugenie A. Goodwin



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


Two new quaternary sodium tungsten selenites, Na2(WO3)3(SeO3)·2H2O (P31c) and Na6(W6O19)(SeO3)2 (C2), have been synthesized and characterized. The former exhibits a hexagonal tungsten oxide layered structure, whereas the latter has a one-dimensional "ribbon" structure. The layers and "ribbons" consist of distorted WO6 and asymmetric SeO3 polyhedra. The layers in Na2(WO3)3(SeO3)·2H2O and the "ribbons" in Na6(W6O19)(SeO3)2 are separated by Na(+) cations. Powder second-harmonic-generation (SHG) measurements on Na2(WO3)3(SeO3)·2H2O and Na6(W6O19)(SeO3)2 using 1064 nm radiation reveal SHG efficiencies of approximately 450× and 20× ?-SiO2, respectively. Particle size versus SHG efficiency measurements indicate that the materials are type 1 non-phase-matchable. Converse piezoelectric measurements result in d33 values of approximately 23 and 12 pm/V, whereas pyroelectric measurements reveal coefficients of -0.41 and -1.10 ?C/m(2)·K at 60 °C for Na2(WO3)3(SeO3)·2H2O and Na6(W6O19)(SeO3)2, respectively. Frequency-dependent polarization measurements confirm that the materials are nonferroelectric; i.e., the macroscopic polarization is not reversible, or "switchable". IR and UV-vis spectroscopy, thermogravimetric and differential thermal analysis measurements, and electron localization function calculations were also done for the materials. Crystal data: Na2(WO3)3(SeO3)·2H2O, trigonal, space group P31c (No. 159), a = 7.2595(6) Å, b = 7.2595(6) Å, c = 12.4867(13) Å, V = 569.89(9) Å(3), Z = 2; Na6(W6O19)(SeO3)2, monoclinic, space group C2 (No. 5), a = 42.169(8) Å, b = 7.2690(15) Å, c = 6.7494(13) Å, ? = 98.48(3)°, V = 2046.2(7) Å(3), Z = 4. PMID:23425251

Nguyen, Sau Doan; Halasyamani, P Shiv



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

NASA Astrophysics Data System (ADS)

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.

Nafisi, Shohreh; Manouchehri, Firouzeh; Montazeri, Maryam



Seos - EARSEL'S Project on Science Education Through Earth Observation for High Schools  

NASA Astrophysics Data System (ADS)

SEOS is an initiative for using remote sensing in science education curricula in high schools funded under the 6th Framework Programme of the European Commission (EC). Eleven partners from several European countries, in cooperation with the European Space Agency (ESA) and teachers from European high schools, created e-learning tutorials for science students in high schools. The tutorials cover many disciplines such as physics, biology, geography, mathematics and engineering, emphasising the interdisciplinary character of remote sensing. They are the core element of the SEOS Learning Management System, allowing teachers to create their own courses, to distribute already available or new worksheets to the students for homework and to collect the results. Forums are available for teachers, students and other users to exchange information and discuss topics relevant for their study.

Reuter, R.



Twitter SEO Tip Sheet Feinberg Office of Communications  

E-print Network

Twitter SEO Tip Sheet Feinberg Office of Communications Twitter is a great tool for concisely are interested in having a Twitter presence, you want those users who are interested in your tweet topics. These terms, preceded by a pound sign, are how Twitter searchers find content they want; connect your tweets

Engman, David M.


Resource Seminar 2007: Introduction to Linux Eun-seo Choi  

E-print Network

Resource Seminar 2007: Introduction to Linux Eun-seo Choi Oct 11, 2007 1 Resources · Senior grads and magazines ­ Google, Wikipedia, etc. ­ Linux Journal (, Linux Magazine (http://www., Linux Format (, and many others (

Choi, Eunseo


J protein mutations and resulting proteostasis collapse  

PubMed Central

Despite a century of intensive investigation the effective treatment of protein aggregation diseases remains elusive. Ordinarily, molecular chaperones ensure that proteins maintain their functional conformation. The appearance of misfolded proteins that aggregate implies the collapse of the cellular chaperone quality control network. That said, the cellular chaperone network is extensive and functional information regarding the detailed action of specific chaperones is not yet available. J proteins (DnaJ/Hsp40) are a family of chaperone cofactors that harness Hsc70 (heat shock cognate protein of 70 kDa) for diverse conformational cellular tasks and, as such, represent novel clinically relevant targets for diseases resulting from the disruption of proteostasis. Here we review incisive reports identifying mutations in individual J protein chaperones and the proteostasis collapse that ensues. PMID:25071450

Koutras, Carolina; Braun, Janice E. A.



Automatic Modeling of Virtual Humans and Body Clothing Nadia Magnenat-Thalmann, Hyewon Seo, Frederic Cordier  

E-print Network

Automatic Modeling of Virtual Humans and Body Clothing Nadia Magnenat-Thalmann, Hyewon Seo-mail: {thalmann, seo, cordier} Abstract Highly realistic virtual human models are rapidly and solutions to automatic modeling of animatable virtual humans. Methods for capturing the shape of real people

Cordier, Frederic


An empirical analysis of health care IPOs and SEOs.  


This article reviews the extant literature regarding the three new issues phenomena: hot issue markets, first-day underpricing, and poor long-run performance as they apply to the heath care industry. Given the "creeping corporatization" of the heath care industry and the unique influence of nonmarket forces on it, we examine whether the three IPO phenomena exist within the industry. We find that hot issue markets, initial underpricing, and negative long-run abnormal returns and sales growth occur among both heath care IPOs and SEOs. Of particular interest, we find that firms are able to issue during times of excess heath care spending and subsequently underperform the market, apparently exploiting windows of opportunity. PMID:20515009

Brau, James C; Holloway, Jonathan M



Crystal structures of Na2SeO4·1.5H2O and Na2SeO4·10H2O.  


The crystal structures of Na2SeO4·1.5H2O (sodium selenate sesquihydrate) and Na2SeO4·10H2O (sodium selenate deca-hydrate) are isotypic with those of Na2CrO4·1.5H2O and Na2 XSeO4·10H2O (X = S, Cr), respectively. The asymmetric unit of the sesquihydrate contains two Na(+) cations, one SeO4 tetra-hedron and one and a half water mol-ecules, the other half being generated by twofold rotation symmetry. The coordination polyhedra of the cations are a distorted monocapped octa-hedron and a square pyramid; these [NaO x ] polyhedra are linked through common edges and corners into a three-dimensional framework structure, the voids of which are filled with the Se atoms of the SeO4 tetra-hedra. The structure is consolidated by O-H?O hydrogen bonds between coordinating water mol-ecules and framework O atoms. The asymmetric unit of the deca-hydrate consists of two Na(+) cations, one SeO4 tetra-hedron and ten water mol-ecules. Both Na(+) cations are octa-hedrally surrounded by water mol-ecules and by edge-sharing condensed into zigzag chains extending parallel to [001]. The SeO4 tetra-hedra and two uncoordinating water mol-ecules are situated between the chains and are connected to the chains through an intricate network of medium-strength O-H?O hydrogen bonds. PMID:25249853

Weil, Matthias; Bonneau, Barbara



Ambipolar rubrene thin film transistors Soonjoo Seo, Byoung-Nam Park, and Paul G. Evansa  

E-print Network

Ambipolar rubrene thin film transistors Soonjoo Seo, Byoung-Nam Park, and Paul G. Evansa Materials ambipolar field-effect transistors fabricated from rubrene thin films on SiO2/Si substrates. The mobilities channel of the transistor follows the geometric percolation of rubrene islands. © 2006 American Institute

Evans, Paul G.


Sodium selenite penta­hydrate, Na2SeO3·5H2O  

PubMed Central

In the crystal structure of Na2SeO3·5H2O [disodium selen­ate(IV) penta­hydrate], two Se, two selenite O atoms and one water O atom are located on a mirror plane, and one water O atom is located on a twofold rotation axis. The coordination of one Na+ cation is distorted trigonal bipyramidal, formed by three equatorial H2O ligands and two axial selenite O atoms. The other Na+ cation has an octa­hedral coordination by six water mol­ecules. The two independent SeO3 groups form almost undistorted trigonal pyramids, with Se—O bond lengths in the range 1.6856?(7)–1.7202?(10)?Å and O—Se—O angles in the range 101.98?(3)–103.11?(5)°, and both are ?2-O:O-bonded to a pair of Na+ cations. Hydrogen bonds involving all water molecules and selenite O atoms consolidate the crystal packing. Although anhydrous Na2SeO3 and Na2TeO3 are isotypic, the title compound is surprisingly not isotypic with Na2TeO3·5H2O. In the tellurite hydrate, all Na+ cations have an octa­hedral coordination and the TeO3 groups are bonded to Na+ only via one of their three O atoms. PMID:24454013

Mereiter, Kurt



Sodium selenite penta-hydrate, Na2SeO3·5H2O.  


In the crystal structure of Na2SeO3·5H2O [disodium selen-ate(IV) penta-hydrate], two Se, two selenite O atoms and one water O atom are located on a mirror plane, and one water O atom is located on a twofold rotation axis. The coordination of one Na(+) cation is distorted trigonal bipyramidal, formed by three equatorial H2O ligands and two axial selenite O atoms. The other Na(+) cation has an octa-hedral coordination by six water mol-ecules. The two independent SeO3 groups form almost undistorted trigonal pyramids, with Se-O bond lengths in the range 1.6856?(7)-1.7202?(10)?Å and O-Se-O angles in the range 101.98?(3)-103.11?(5)°, and both are ?2-O:O-bonded to a pair of Na(+) cations. Hydrogen bonds involving all water molecules and selenite O atoms consolidate the crystal packing. Although anhydrous Na2SeO3 and Na2TeO3 are isotypic, the title compound is surprisingly not isotypic with Na2TeO3·5H2O. In the tellurite hydrate, all Na(+) cations have an octa-hedral coordination and the TeO3 groups are bonded to Na(+) only via one of their three O atoms. PMID:24454013

Mereiter, Kurt



Factors controlling the variations in the mid-ocean ridge segmentation Eun-seo Choi1  

E-print Network

T31B-0449 Factors controlling the variations in the mid-ocean ridge segmentation Eun-seo Choi1 of the orthogonal pattern characterizing mid-ocean ridges and transform faults are studied using numerical models. Thermal stresses are isotropic, but mid-ocean ridges themselves and numerous ridge parallel faults can

Choi, Eunseo


Proteins in electric fields and pressure fields: experimental results.  


Experimental results obtained by Stark effect and pressure tuning optical spectroscopy are discussed with the emphasis on studies aimed at unraveling the coupling of prosthetic groups to proteins. A comparative, detailed analysis is given concerning the coupling of the heme group to the apoprotein in various heme proteins based on spectral hole burning data. Electrochromism and electric dichroism experiments related to the coupling problem are also discussed in the context of other protein systems. PMID:9733987

Fidy, J; Balog, E; Köhler, M



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

NASA Astrophysics Data System (ADS)

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.

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



P-proteins in Arabidopsis are heteromeric structures involved in rapid sieve tube sealing  

PubMed Central

Structural phloem proteins (P-proteins) are characteristic components of the sieve elements in all dicotyledonous and many monocotyledonous angiosperms. Tobacco P-proteins were recently confirmed to be encoded by the widespread sieve element occlusion (SEO) gene family, and tobacco SEO proteins were shown to be directly involved in sieve tube sealing thus preventing the loss of photosynthate. Analysis of the two Arabidopsis SEO proteins (AtSEOa and AtSEOb) indicated that the corresponding P-protein subunits do not act in a redundant manner. However, there are still pending questions regarding the interaction properties and specific functions of AtSEOa and AtSEOb as well as the general function of structural P-proteins in Arabidopsis. In this study, we characterized the Arabidopsis P-proteins in more detail. We used in planta bimolecular fluorescence complementation assays to confirm the predicted heteromeric interactions between AtSEOa and AtSEOb. Arabidopsis mutants depleted for one or both AtSEO proteins lacked the typical P-protein structures normally found in sieve elements, underlining the identity of AtSEO proteins as P-proteins and furthermore providing the means to determine the role of Arabidopsis P-proteins in sieve tube sealing. We therefore developed an assay based on phloem exudation. Mutants with reduced AtSEO expression levels lost twice as much photosynthate following injury as comparable wild-type plants, confirming that Arabidopsis P-proteins are indeed involved in sieve tube sealing. PMID:23840197

Jekat, Stephan B.; Ernst, Antonia M.; von Bohl, Andreas; Zielonka, Sascia; Twyman, Richard M.; Noll, Gundula A.; Prufer, Dirk



Hydroxocobalamin association during cell culture results in pink therapeutic proteins  

PubMed Central

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

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



Extended networks, porous sheets, and chiral frameworks. Thorium materials containing mixed geometry anions: Structures and properties of Th(SeO 3)(SeO 4), Th(IO 3) 2(SeO 4)(H 2O) 3·H 2O, and Th(CrO 4)(IO 3) 2  

Microsoft Academic Search

Three novel Th(IV) compounds containing heavy oxoanions, Th(SeO3)(SeO4) (1), Th(IO3)2(SeO4)(H2O)3·H2O (2), and Th(CrO4)(IO3)2 (3), have been synthesized under mild hydrothermal conditions. Each of these three distinct structures contain trigonal pyramidal and tetrahedral oxoanions. Compound 1 adopts a three-dimensional structure formed from ThO9 tricapped trigonal prisms, trigonal pyramidal selenite, SeO32–, anions containing Se(IV), and tetrahedral selenate, SeO42–, anions containing Se(VI). The

Tyler A. Sullens; Philip M. Almond; Jessica A. Byrd; James V. Beitz; Travis H. Bray; Thomas E. Albrecht-Schmitt



Extended networks, porous sheets, and chiral frameworks. Thorium materials containing mixed geometry anions: Structures and properties of Th(SeO3)(SeO4), Th(IO3)2(SeO4)(H2O)3·H2O, and Th(CrO4)(IO3)2  

Microsoft Academic Search

Three novel Th(IV) compounds containing heavy oxoanions, Th(SeO3)(SeO4) (1), Th(IO3)2(SeO4)(H2O)3·H2O (2), and Th(CrO4)(IO3)2 (3), have been synthesized under mild hydrothermal conditions. Each of these three distinct structures contain trigonal pyramidal and tetrahedral oxoanions. Compound 1 adopts a three-dimensional structure formed from ThO9 tricapped trigonal prisms, trigonal pyramidal selenite, SeO32 , anions containing Se(IV), and tetrahedral selenate, SeO42 , anions containing

Tyler A. Sullens; Philip M. Almond; Jessica A. Byrd; James V. Beitz; Travis H. Bray; Thomas E.. Albrecht-Schmitt



Protein-protein interactions: analysis of a false positive GST pulldown result.  


One of the most common ways to demonstrate a direct protein-protein interaction in vitro is the glutathione-S-transferse (GST)-pulldown. Here we report the detailed characterization of a putative interaction between two transcription factor proteins, GATA-1 and Krüppel-like factor 3 (KLF3/BKLF) that show robust interactions in GST-pulldown experiments. Attempts to map the interaction interface of GATA-1 on KLF3 using a mutagenic screening approach did not yield a contiguous binding face on KLF3, suggesting that the interaction might be non-specific. NMR experiments showed that the proteins do not interact at protein concentrations of 50-100 ?M. Rather, the GST tag can cause part of KLF3 to misfold. In addition to misfolding, the fact that both proteins are DNA-binding domains appears to introduce binding artifacts (possibly nucleic acid bridging) that cannot be resolved by the addition of nucleases or ethidium bromide (EtBr). This study emphasizes the need for caution in relying on GST-pulldown results and related methods, without convincing confirmation from different approaches. PMID:21638332

Wissmueller, Sandra; Font, Josep; Liew, Chu Wai; Cram, Edward; Schroeder, Thilo; Turner, Jeremy; Crossley, Merlin; Mackay, Joel P; Matthews, Jacqueline M



Organically templated zinc selenites: MIL-86 or [H 2N(CH 2) 2NH 2] 2·Zn 4(SeO 3) 4 and MIL-87 or [H 3N(CH 2) 3NH 3] 4·Zn 4(SeO 3) 8  

NASA Astrophysics Data System (ADS)

Two new organically templated zinc selenites MIL-86 or [H 2N(CH 2) 2NH 2] 2·Zn 4(SeO 3) 4 and MIL-87 or [H 3N(CH 2) 3NH 3] 4·Zn 4(SeO 3) 8 have been prepared by the reaction of ZnO with SeO 2 under hydrothermal conditions in the presence of organic amines. Crystal data: MIL-86, monoclinic, space group P2 1/ n, a=6.7712(4) Å, b=9.4520(5) Å, c=8.0295(4) Å, ?=113.601(1)°, V=470.91(4) Å 3; MIL-87, orthorhombic, space group Pbcm, a=8.8311(3) Å, b=7.8391(2) Å, c=16.2992(4) Å, V=1128.36(6) Å 3. MIL-86 combines the structural features of templated networks and coordination polymers. Its structure is built up from ZnO 3N tetrahedra and SeO 3 pseudo-pyramids as the polyhedral building units. The Zn?N bond corresponds to a direct link between zinc and the ethylenediamine template. In the other hand, MIL-87 is built up from chains of vertex-linked ZnO 4 and SeO 3 building units crosslinked by 1,3-diammonium propane cations. These structures are understood by comparison with the already known organically templated zinc phosphites.

Millange, Franck; Serre, Christian; Cabourdin, Tony; Marrot, Jérôme; Férey, Gérard



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

PubMed Central

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

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



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

NASA Technical Reports Server (NTRS)

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.

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.



Casein protein results in higher prandial and exercise induced whole body protein anabolism than whey protein in Chronic Obstructive Pulmonary Disease  

PubMed Central

Objective Exercise is known to improve physical functioning and health status in Chronic Obstructive Pulmonary Disease (COPD). Recently, disturbances in protein turnover and amino acid kinetics have been observed after exercise in COPD. The objective was to investigate which dairy protein is able to positively influence the protein metabolic response to exercise in COPD. Materials and Methods 8 COPD patients and 8 healthy subjects performed a cycle test on two days while ingesting casein or whey protein. Whole body protein breakdown (WbPB), synthesis (WbPS), splanchnic amino acid extraction (SPE), and NetWbPS (=WbPS–WbPB) were measured using stable isotope methodology during 20 minutes of exercise (at 50% peak work load of COPD group). The controls performed a second exercise test at the same relative workload. Exercise was followed by 1 hour of recovery. Results In the healthy group, WbPS, SPE, and NetPS were higher during casein than during whey feeding (p<0.01). WbPS and NetPS were higher during exercise, independent of exercise intensity (p<0.01). NetPS was higher during casein feeding in COPD due to lower WbPB (p<0.05). Higher SPE was found during exercise during casein and whey feeding in COPD (p<0.05). Lactate levels during exercise were higher in COPD (p<0.05) independent of the protein. Post-exercise, lower NetPS values were found independent of protein type in both groups. Conclusion Casein resulted in more protein anabolism than whey protein which was maintained during and following exercise in COPD. Optimizing protein intake might be of importance for muscle maintenance during daily physical activities in COPD. PMID:22512824

Engelen, Mariëlle PKJ; Rutten, Erica PA; De Castro, Carmen LN; Wouters, Emiel FM; Schols, Annemie MWJ; Deutz, Nicolaas EP



Overexpression of a Gluten Protein in Transgenic Wheat Results in Greatly Increased Dough Strength  

Microsoft Academic Search

The transgenic wheat line B73-6-1 contains additional genes encoding a gluten protein called HMW subunit 1Dx5, resulting in a four-fold increase in the proportion of this component in the seed proteins and corresponding increases in the proportions of total HMW subunits and total glutenins. This is associated with a dramatic increase in dough strength, as measured using a small scale

L Rooke; F Békés; R Fido; F Barro; P Gras; A. S Tatham; P Barcelo; P Lazzeri; P. R Shewry



Pseudomorphic InGaAs base ballistic hotelectron device K. Seo, M. Heiblum, C. M. Knoedler, WP. Hong, and P. Bhattacharya  

E-print Network

Pseudomorphic InGaAs base ballistic hotelectron device K. Seo, M. Heiblum, C. M. Knoedler, WP. Hong. Phys. 69, 4454 (1991); 10.1063/1.348378 Enhanced ballistic transport in InGaAs/InAlAs hotelectron:// Downloaded to IP: On: Thu, 10 Apr 2014 16:10:12 #12;Pseudomorphic InGaAs base ballistic hot

Heiblum, Mordehai "Moty"


Synthesis, Crystal Structure and Thermal Decomposition of the New Cadmium Selenite Chloride, Cd4(SeO3)2OCl2  

PubMed Central

A synthetic study in the Cd-Se-O-Cl system led to formation of the new oxochloride compound Cd4(SeO3)2OCl2 via solid state reactions. The compound crystallizes in the orthorhombic space group Fmmm with cell parameters a?=?7.3610(3) Å, b?=?15.4936(2) Å, c?=?17.5603(3) Å, Z?=?8, S?=?0.969, F(000)?=?2800, R?=?0.0185, Rw?=?0.0384. Single crystal X-ray data were collected at 293 K. The crystal structure can be considered as layered and the building units are distorted [Cd(1)O6] octahedra, distorted [Cd(2)O8] cubes, irregular [Cd(3)O4Cl2] polyhedra and SeO3E trigonal pyramids. There are two crystallographically unique Cl atoms that both are half occupied. Thermogravimetric studies show that the compound starts to decompose at 500°C. The crystal structure of the new compound is closely related to the previously described compound Cd4(SeO3)2Cl4(H2O). PMID:24844633

Rabbani, Faiz; Ajaz, Humayun; Zimmermann, Iwan; Johnsson, Mats



Size- and shape-controlled synthesis of ZnSe nanocrystals using SeO2 as selenium precursor.  


Here we report a low-cost and "green" phosphine-free route for the size- and shape-controlled synthesis of high-quality zinc blende (cubic) ZnSe nanocrystals. To avoid the use of expensive and toxic solvents such as trioctylphosphine (TOP) or tributylphosphine (TBP), SeO(2) was dispersed in 1-octadecene (ODE) as a chalcogen precursor. It has been found that the temperature and the surface ligand influenced the nucleation, the reaction speed and the formation of different shapes. Absorption spectroscopy, fluorescence spectroscopy, powder X-ray diffraction (XRD) and transmission electron microscopy (TEM) were used for the characterization of the as-synthesized ZnSe nanocrystals. The size-dependent photoluminescence (PL) range of the as-prepared ZnSe nanocrystals was between 390 and 450 nm, with the PL full width at half-maximum (FWHM) well controlled between 14 and 18 nm and PL quantum yields reached up to 40% at room temperature. Moreover, this new selenium precursor can be used to form tetrapod-shaped ZnSe nanocrystals when zinc acetylacetonate was introduced as the zinc precursor with a one-pot method. PMID:20976341

Shen, Huaibin; Niu, Jin Zhong; Wang, Hongzhe; Li, Xiaomin; Li, Lin Song; Chen, Xia



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

PubMed Central

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 (·SO3?). This free radical further reacts with oxygen to form peroxymonosulfate anion radical (?O3SOO·) and the very reactive sulfate anion radical (SO4??), 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/H2O2 is able to oxidize bisulfite, ultimately forming the sulfate anion radical (SO4??), 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/H2O2 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/H2O2 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

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



Recombinant human protein C derivatives: altered response to calcium resulting in enhanced activation by thrombin.  

PubMed Central

Calcium plays a dual role in the activation of protein C: it inhibits protein C activation by alpha-thrombin, whereas it is required for protein C activation by the thrombomodulin-thrombin complex. Available information suggests that these calcium effects are mediated through calcium induced structural changes in protein C. In this paper, we demonstrate that substitution of Asp167 (located in the activation peptide of human protein C, occupying position P3 relative to the peptide bond Arg169-Leu170 which is susceptible to hydrolysis by thrombin) by either Gly or Phe results in protein C derivatives which are characterized by an altered response to calcium. At 3 mM calcium, alpha-thrombin activated the derivatives 5- to 8-fold faster compared with the wild-type, an effect which was shown to be caused by a decreased inhibitory effect of calcium on the reaction. These same single amino acid substitutions enhanced the affinity of the thrombomodulin-thrombin complex for the substrate at 3 mM calcium 3-(Gly-substitution) to 6-(Phe-substitution) fold, either without influencing kcat (Gly-substitution) or with a 2.5-fold decrease of kcat. For both derivatives, the calcium concentrations resulting in half maximal inhibition of activation by alpha-thrombin and in half maximal stimulation of activation by the thrombomodulin-thrombin complex increased from 0.3 mM to 0.6 mM. It is concluded that Asp167 is involved in the calcium induced inhibition of protein C activation by thrombin. Moreover, our studies demonstrate that it is feasible to enhance the efficiency of enzymatic reactions by introducing point mutations in the substrate. Images Fig. 2. PMID:2369894

Ehrlich, H J; Grinnell, B W; Jaskunas, S R; Esmon, C T; Yan, S B; Bang, N U




NSDL National Science Digital Library

Laboratory manual and supplemental resources that were developed for a college laboratory course in protein purification. The enzyme, Beta-galactosidase, is purified in two steps, with analysis and verification of results. Course materials are divided into four units: Why Proteins, Assays, The Purification Process, and Analysis and Verification. Powerpoint lectures and study guides are provided.

Mowery, Jeanette; Seidman, Lisa A.



Increasing habitual protein intake results in reduced postprandial efficiency of peripheral, anabolic wheat protein nitrogen use in  

Microsoft Academic Search

Background: The postprandial retention of dietary protein de- creases when the prevailing protein intake increases. Objective: We investigated the influence of the prevailing protein intake on the regional utilization and anabolic use of wheat protein during the postprandial non-steady state in humans. Design: Healthy adults (n 8) were adapted for 7 d, first to a normal-proteindiet(NP:1g kg1 d1)andthentoahigh-protein diet(HP:2g kg1

Barbara Juillet; Hélène Fouillet; Cécile Bos; Francois Mariotti; Nicolas Gausserès; Robert Benamouzig; Daniel Tomé; Claire Gaudichon


Analyzing Proteomes and Protein Function Using Graphical Comparative Analysis of Tandem Mass Spectrometry Results  

Microsoft Academic Search

Although generating large amounts of proteomic data us- ing tandem mass spectrometry has become routine, there is currently no single set of comprehensive tools for the rigorous analysis of tandem mass spectrometry results given the large variety of possible experimental aims. Cur- rently available applications are typically designed for dis- playing proteins and posttranslational modifications from the point of view

K. Jill McAfee; Dexter T. Duncan; Michael Assink; Andrew J. Link



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

PubMed Central

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

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



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

PubMed Central

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

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



Loss of photoreceptors results in upregulation of synaptic proteins in bipolar cells and amacrine cells.  


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

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



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


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

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



Aging results in an unusual expression of Drosophila heat shock proteins  

SciTech Connect

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

Fleming, J.E.; Walton, J.K.; Dubitsky, R.; Bensch, K.G. (Linus Pauling Institute of Science and Medicine, Palo Alto, CA (USA))



Crystal chemistry of selenates with mineral-like structures: V. Crystal structures of (H 3 O) 2 [(UO 2 )(SeO 4 ) 2 (H 2 O)](H 2 O) 2 and (H 3 O) 2 [(UO 2 )(SeO 4 ) 2 (H 2 O)](H 2 O), new compounds with rhomboclase and goldichite topology  

Microsoft Academic Search

The crystal structures of two new compounds (H3O)2[(UO2)(SeO4)2(H2O)](H2O)2 (1, orthorhombic, Pnma, a = 14.0328(18), b = 11.6412(13), c = 8.2146(13) Å, V = 134.9(3) Å3) and (H3O)2[(UO2)(SeO4)2(H2O)](H2O) (2, monoclinic, P21\\/c, a = 7.8670(12), b = 7.5357(7), c = 21.386(3) Å, ? = 101.484(12)°, V = 1242.5(3) Å3) have been solved by direct methods and refined to R\\u000a 1 = 0.076

S. V. Krivovichev



Mutual Fund Trading Pressure: Firm-Level Stock Price Impact and Timing of SEOs  

E-print Network

We use price pressure resulting from purchases by mutual funds with large capital inflows to identify overvalued equity. This is a relatively exogenous overvaluation indicator as it is associated with who is buying—buyers ...

Khan, Mozaffar Nayim


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

NASA Astrophysics Data System (ADS)

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) Å, b = 9.6631 (6) Å, c = 9.2070 (5) Å, ß = 108.825 (4)°, Z = 4 and V = 730.83 (7) Å3. 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.

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



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

PubMed Central

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

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



Heterogeneous N-terminal Acylation of Retinal Proteins Results from the Retina's Unusual Lipid Metabolism†,§  

PubMed Central

Protein N-myristoylation occurs by a covalent attachment of a C14:0 fatty acid to the N-terminal Gly residue. This reaction is catalyzed by a N-myristoyltransferase that uses myristoyl-coenzyme A as substrate. But proteins in the retina also undergo heterogeneous N-acylation with C14:2, C14:1 and C12:0 fatty acids. The basis and the role of this retina-specific phenomenon are poorly understood. We studied guanylate cyclase-activating protein 1 (GCAP1) as an example of retina-specific heterogeneously N-acylated protein. The types and the abundance of fatty acids bound to bovine retinal GCAP1 were: C14:2, 37.0%; C14:0, 32.4%; C14:1, 22.3%; and C12:0, 8.3% as quantified by liquid chromatography coupled mass spectrometry. We also devised a method for N-acylating proteins in vitro and used it to modify GCAP1 with acyl moieties of different lengths. Analysis of these GCAPs both confirmed that N-terminal acylation of GCAP1 is critical for its high activity and proper Ca2+-dependent response and revealed comparable functionality for GCAP1 with acyl moieties of various lengths. We also tested the hypothesis that retinal heterogeneous N-acylation results from retinal enrichment of unusual N-myristoyltransferase substrates. Thus, acyl-coenzyme A esters were purified from both bovine retina and brain and analyzed by liquid chromatography coupled mass spectrometry. Substantial differences in acyl-coenzyme A profiles between the retina and brain were detected. Importantly, the ratios of uncommon N-acylation substrates; C14:2- and C14:1-coenyzme A to C14:0-coenzyme A were higher in the retina than in the brain. Thus, our results suggest that heterogeneous N-acylation, responsible for expansion of retinal proteome, reflects the unique character of retinal lipid metabolism. Additionally, we propose a new hypothesis explaining the physiological relevance of elevated retinal ratios of C14:2- and C14:1-coenzyme A to C14:0-coenzyme A. PMID:21449552

Bereta, Grzegorz; Palczewski, Krzysztof



The sieve element occlusion gene family in dicotyledonous plants  

PubMed Central

Sieve element occlusion (SEO) genes encoding forisome subunits have been identified in Medicago truncatula and other legumes. Forisomes are structural phloem proteins uniquely found in Fabaceae sieve elements. They undergo a reversible conformational change after wounding, from a condensed to a dispersed state, thereby blocking sieve tube translocation and preventing the loss of photoassimilates. Recently, we identified SEO genes in several non-Fabaceae plants (lacking forisomes) and concluded that they most probably encode conventional non-forisome P-proteins. Molecular and phylogenetic analysis of the SEO gene family has identified domains that are characteristic for SEO proteins. Here, we extended our phylogenetic analysis by including additional SEO genes from several diverse species based on recently published genomic data. Our results strengthen the original assumption that SEO genes seem to be widespread in dicotyledonous angiosperms, and further underline the divergent evolution of SEO genes within the Fabaceae. PMID:21422825

Jekat, Stephan B; Nordzieke, Steffen; Reineke, Anna R; Muller, Boje; Bornberg-Bauer, Erich; Noll, Gundula A



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

PubMed Central

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

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



Cleavage of human inhibitor of apoptosis protein XIAP results in fragments with distinct specificities for caspases.  


Several human inhibitor of apoptosis (IAP) family proteins function by directly inhibiting specific caspases in a mechanism that does not require IAP cleavage. In this study, however, we demonstrate that endogenous XIAP is cleaved into two fragments during apoptosis induced by the tumor necrosis factor family member Fas (CD95). The two fragments produced comprise the baculoviral inhibitory repeat (BIR) 1 and 2 domains (BIR1-2) and the BIR3 and RING (BIR3-Ring) domains of XIAP. Overexpression of the BIR1-2 fragment inhibits Fas-induced apoptosis, albeit at significantly reduced efficiency compared with full-length XIAP. In contrast, overexpression of the BIR3-Ring fragment results in a slight enhancement of Fas-directed apoptosis. Thus, cleavage of XIAP may be one mechanism by which cell death programs circumvent the anti-apoptotic barrier posed by XIAP. Interestingly, ectopic expression of the BIR3-Ring fragment resulted in nearly complete protection from Bax-induced apoptosis. Use of purified recombinant proteins revealed that BIR3-Ring is a specific inhibitor of caspase-9 whereas BIR1-2 is specific for caspases 3 and 7. Therefore XIAP possesses two different caspase inhibitory activities which can be attributed to distinct domains within XIAP. These data may provide an explanation for why IAPs have evolved with multiple BIR domains. PMID:10508158

Deveraux, Q L; Leo, E; Stennicke, H R; Welsh, K; Salvesen, G S; Reed, J C



Crystallisation of alpha-crustacyanin, the lobster carapace astaxanthin-protein: results from EURECA  

NASA Astrophysics Data System (ADS)

Crystallisation of alpha-crustacyanin, the lobster carapace astaxanthin-protein was attempted under microgravity conditions in EURECA satellite using liquid-liquid diffusion with polyethyleneglycol (PEG) as precipitant; in a second reaction chamber phenol and dioxan were used as additives to prevent composite crystal growth. Crystals of alpha-crustacyanin grown under microgravity from PEG were larger than those grown terrestrially in the same apparatus under otherwise identical conditions. On retrieval, the crystals from PEG were shown to be composite and gave a powder diffraction pattern. The second reaction chamber showed leakage on retrieval and had also been subjected to rapid temperature variation during flight. Crystal fragments were nevertheless recovered but showed a powder diffraction pattern. It is concluded, certainly for liquid-liquid diffusion using PEG alone, that, for crustacyanin, although microgravity conditions resulted in an increase in dimensions of crystals, a measurable improvement in molecular ordering was not achieved.

Zagalsky, P. F.; Wright, C. E.; Parsons, M.



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

NASA Astrophysics Data System (ADS)

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.

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



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


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

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



Experimental validation of FINDSITEcomb virtual ligand screening results for eight proteins yields novel nanomolar and micromolar binders  

PubMed Central

Background Identification of ligand-protein binding interactions is a critical step in drug discovery. Experimental screening of large chemical libraries, in spite of their specific role and importance in drug discovery, suffer from the disadvantages of being random, time-consuming and expensive. To accelerate the process, traditional structure- or ligand-based VLS approaches are combined with experimental high-throughput screening, HTS. Often a single protein or, at most, a protein family is considered. Large scale VLS benchmarking across diverse protein families is rarely done, and the reported success rate is very low. Here, we demonstrate the experimental HTS validation of a novel VLS approach, FINDSITEcomb, across a diverse set of medically-relevant proteins. Results For eight different proteins belonging to different fold-classes and from diverse organisms, the top 1% of FINDSITEcomb’s VLS predictions were tested, and depending on the protein target, 4%-47% of the predicted ligands were shown to bind with ?M or better affinities. In total, 47 small molecule binders were identified. Low nanomolar (nM) binders for dihydrofolate reductase and protein tyrosine phosphatases (PTPs) and micromolar binders for the other proteins were identified. Six novel molecules had cytotoxic activity (<10 ?g/ml) against the HCT-116 colon carcinoma cell line and one novel molecule had potent antibacterial activity. Conclusions We show that FINDSITEcomb is a promising new VLS approach that can assist drug discovery. PMID:24936211



Fatty Acid Accumulation and Resulting PPAR? Activation in Fibroblasts due to Trifunctional Protein Deficiency  

PubMed Central

To examine fatty acid accumulation and its toxic effects in cells, we analyzed skin fibroblasts from six patients with mitochondrial trifunctional protein deficiency, who had abnormalities in the second through fourth reactions in fatty acid ?-oxidation system. We found free fatty acid accumulation, enhanced three acyl-CoA dehydrogenases, catalyzing the first reaction in the ?-oxidation system and being assumed to have normal activities in these patients, and PPAR? activation that was confirmed in the experiments using MK886, a PPAR? specific antagonist and fenofibrate, a PPAR? specific agonist. These novel findings suggest that the fatty acid accumulation and the resulting PPAR? activation are major causes of the increase in the ?-oxidation ability as probable compensation for fatty acid metabolism in the patients' fibroblasts, and that enhanced cell proliferation and increased oxidative stress due to the PPAR? activation relate to the development of specific clinical features such as hypertrophic cardiomyopathy, slight hepatomegaly, and skeletal myopathy. Additionally, significant suppression of the PPAR? activation by means of MK886 treatment is assumed to provide a new method of treating this deficiency. PMID:22654897

Wakabayashi, Masato; Kamijo, Yuji; Nakajima, Takero; Tanaka, Naoki; Sugiyama, Eiko; Yangyang, Tian; Kimura, Takefumi; Aoyama, Toshifumi



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


The 19ID undulator beamline of the Structure Biology Center has been designed and built to take full advantage of the high flux, brilliance and quality of X-ray beams delivered by the Advanced Photon Source. The beamline optics are capable of delivering monochromatic X-rays with photon energies from 3.5 to 20 keV (3.5-0.6 A wavelength) with fluxes up to 8-18 x 10(12) 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 x 1.0 mm (horizontal x vertical, unfocused) to 0.083 mm x 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 kappa-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 x 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

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



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

PubMed Central

The 19ID undulator beamline of the Structure Biology Center has been designed and built to take full advantage of the high flux, brilliance and quality of X-ray beams delivered by the Advanced Photon Source. The beamline optics are capable of delivering monochromatic X-rays with photon energies from 3.5 to 20 keV (3.5–0.6 Å wavelength) with fluxes up to 8–18 × 1012 photons s?1 (depending on photon energy) onto cryogenically cooled crystal samples. The size of the beam (full width at half-maximum) at the sample position can be varied from 2.2 mm × 1.0 mm (horizontal × vertical, unfocused) to 0.083 mm × 0.020 mm in its fully focused configuration. Specimen-to-detector distances of between 100 mm and 1500 mm can be used. The high flexibility, inherent in the design of the optics, coupled with a ?-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

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



Effect of decreasing ideal protein levels on performance results and nitrogen efficiency of growing-finishing gilts.  


This study examined the effect of decreasing ideal protein concentrations on performance and nutrient efficiency. The experimental diets contained 100%, 90%, 80% or 70% of the ideal dietary protein level (Diet 100%, 90%, 80% and 70%, respectively) estimated in previous experiments with pigs of the same genetic background. The four different treatments were divided among 16 pens of six pigs each. The average initial and final body weight were 20.8 +/- 1.1 and 107 +/- 3 kg, respectively. Three-phase feeding was applied (BW 20-40 kg, 40-70 kg and 70-110 kg). The dietary ileal digestible (ID) methionine + cystine, threonine, tryptophan, isoleucine, valine, and leucine contents expressed as percent of ID lysine were 63, 72, 22, 60, 68, and > 100%, respectively. The lysine to protein ratio was kept constant at 6.8%. Between 21 and 106 kg BW the best performance was achieved on Diet 90%. Diet 70% led to significantly worse results. Although lean meat percentage did not differ, protein content of the carcass was lower on Diet 70% than on Diets 90% and 100%. Decreased protein concentrations increased crude protein efficiency and consequently decreased nitrogen excretion most at the 80% level. If protein varies together with digestible amino acid content, it can be concluded that Diet 80% may be the best choice for the environment and for profitability. If protein content stays at a fixed level, Diet 90% may be the safest choice. PMID:20496857

Millet, Sam; Aluwé, Marijke; De Paepe, Marc; De Brabander, Daniël L; Van Oeckel, Monique J



The results help explain differences in the efficiency of microbial protein synthesis.  

E-print Network

tissue' curve represents degrada- tion where access is restricted by other tis- sue. PROTEIN METABOLISM mass. Strained rumen fluid was diluted with 2 volumes of buffer and incubated with glucose, starch

Paris-Sud XI, Université de


Multiple protein structures and multiple ligands: effects on the apparent goodness of virtual screening results  

Microsoft Academic Search

As an extension to a previous published study (McGaughey et al., J Chem Inf Model 47:1504–1519, 2007) comparing 2D and 3D\\u000a similarity methods to docking, we apply a subset of those virtual screening methods (TOPOSIM, SQW, ROCS-color, and Glide)\\u000a to a set of protein\\/ligand pairs where the protein is the target for docking and the cocrystallized ligand is the target for

Robert P. Sheridan; Georgia B. McGaughey; Wendy D. Cornell




ERIC Educational Resources Information Center

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)

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



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

PubMed Central

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

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



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


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

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



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


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

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



Effect of a High-Protein Diet on Kidney Function in Healthy Adults: Results From the OmniHeart Trial  

PubMed Central

Background Consumption of a diet high in protein can cause glomerular hyperfiltration, a potentially maladaptive response, which may accelerate the progression of kidney disease. Study Design An ancillary study of the OmniHeart trial, a randomized 3-period crossover feeding trial testing the effects of partial replacement of carbohydrate with protein on kidney function. Setting & Participants Healthy adults (N=164) with prehypertension or stage 1 hypertension at a community-based research clinic with a metabolic kitchen. Intervention Participants were fed each of 3 diets for 6 weeks. Feeding periods were separated by a 2- to 4-week washout period. Weight was held constant on each diet. The 3 diets emphasized carbohydrate, protein, or unsaturated fat; dietary protein was either 15% (carbohydrate and unsaturated fat diets) or 25% (protein diet) of energy intake. Outcomes Fasting serum creatinine, cystatin C, and ?2-microglobulin levels, estimated glomerular filtration rate (eGFR). Measurements Serum creatinine, cystatin C, and ?2-microglobulin collected at the end of each feeding period. Results Baseline cystatin C-based eGFR was 92.0±16.3 (SD) mL/min/1.73 m2. Compared with the carbohydrate and unsaturated fat diets, the protein diet increased cystatin C-based eGFR by ~4 mL/min/1.73 m2 (P < 0.001). The effects of the protein diet on kidney function were independent of changes in blood pressure. There was no significant difference between the carbohydrate and unsaturated fat diets. Limitations Participants did not have kidney disease at baseline. Conclusions A healthy diet rich in protein increased eGFR. Whether long-term consumption of a high-protein diet leads to kidney disease is uncertain. PMID:23219108

Juraschek, Stephen P.; Appel, Lawrence J.; Anderson, Cheryl A.M.; Miller, Edgar R.



Vaccinia Virus Penetration Requires Cholesterol and Results in Specific Viral Envelope Proteins Associated with Lipid Rafts  

Microsoft Academic Search

Vaccinia virus infects a wide variety of mammalian cells from different hosts, but the mechanism of virus entry is not clearly defined. The mature intracellular vaccinia virus contains several envelope proteins medi- ating virion adsorption to cell surface glycosaminoglycans; however, it is not known how the bound virions initiate virion penetration into cells. For this study, we investigated the importance

Che-Sheng Chung; Cheng-Yen Huang; Wen Chang



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


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 [?-(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

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



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

Microsoft Academic Search

Both compounds, neodymium oxide chloride oxoarsenate(III) Nd5O4Cl[AsO3]2 and gadolinium oxide bromide oxoselenate(IV) Gd5O4Br3[SeO3]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 Nd5O4Cl[AsO3]2 and a=1243.70(9)pm, b=549.91(4)pm, c=1005.28(8)pm, ?=91.869(3)° for Gd5O4Br3[SeO3]2) in space group C2\\/m

Dong-Hee Kang; Joseph Wontcheu; Thomas Schleid



A Deep Intronic Mutation in the Ankyrin-1 Gene Causes Diminished Protein Expression Resulting in Hemolytic Anemia in Mice  

PubMed Central

Linkage between transmembrane proteins and the spectrin-based cytoskeleton is necessary for membrane elasticity of red blood cells. Mutations of the proteins that mediate this linkage result in various types of hemolytic anemia. Here we report a novel N-ethyl-N-nitrosourea?induced mutation of ankyrin-1, named hema6, which causes hereditary spherocytosis in mice through a mild reduction of protein expression. The causal mutation was traced to a single nucleotide transition located deep into intron 13 of gene Ank1. In vitro minigene splicing assay revealed two abnormally spliced transcripts containing cryptic exons from fragments of Ank1 intron 13. The inclusion of cryptic exons introduced a premature termination codon, which leads to nonsense-mediated decay of the mutant transcripts in vivo. Hence, in homozygous mice, only wild-type ankyrin-1 is expressed, albeit at 70% of the level in wild-type mice. Heterozygotes display a similar hereditary spherocytosis phenotype stemming from intermediate protein expression level, indicating the haploinsufficiency of the mutation. Weakened linkage between integral transmembrane protein, band 3, and underlying cytoskeleton was observed in mutant mice as the result of reduced high-affinity binding sites provided by ankyrin-1. Hema6 is the only known mouse mutant of Ank1 allelic series that expresses full-length canonical ankyrin-1 at a reduced level, a fact that makes it particularly useful to study the functional impact of ankyrin-1 quantitative deficiency. PMID:23934996

Huang, Hua; Zhao, PengXiang; Arimatsu, Kei; Tabeta, Koichi; Yamazaki, Kazuhisa; Krieg, Lara; Fu, Emily; Zhang, Tian; Du, Xin



Recombinant PNPLA3 protein shows triglyceride hydrolase activity and its I148M mutation results in loss of function.  


The patatin-like phospholipase domain containing 3 (PNPLA3, also called adiponutrin, ADPN) is a membrane-bound protein highly expressed in the liver. The genetic variant I148M (rs738409) was found to be associated with progression of chronic liver disease. We aimed to establish a protein purification protocol in a yeast system (Pichia pastoris) and to examine the human PNPLA3 enzymatic activity, substrate specificity and the I148M mutation effect. hPNPLA3 148I wild type and 148M mutant cDNA were cloned into P. pastoris expression vectors. Yeast cells were grown in 3L fermentors. PNPLA3 protein was purified from membrane fractions by Ni-affinity chromatography. Enzymatic activity was assessed using radiolabeled substrates. Both 148I wild type and 148M mutant proteins are localized to the membrane. The wild type protein shows a predominant lipase activity with mild lysophosphatidic acid acyl transferase activity (LPAAT) and the I148M mutation results in a loss of function of both these activities. Our data show that PNPLA3 has a predominant lipase activity and I148M mutation results in a loss of function. PMID:24369119

Pingitore, Piero; Pirazzi, Carlo; Mancina, Rosellina M; Motta, Benedetta M; Indiveri, Cesare; Pujia, Arturo; Montalcini, Tiziana; Hedfalk, Kristina; Romeo, Stefano



Multiple Interactions between Polyphenols and a Salivary Proline-Rich Protein Repeat Result in Complexation and Precipitation  

E-print Network

in the gut, resulting in a variety of antinutritive and toxic effects. Salivary proline-rich proteins (PRPs as tannins, are a distinct family of secondary metabolites present in the leaves, bark, and fruit of many, usually with D-glucose, of gallic acid and its derivatives (Haslam, 1989). Polyphenols are described

Williamson, Mike P.


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

SciTech Connect

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.

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



Depletion of the C. elegans NAC Engages the Unfolded Protein Response, Resulting in Increased Chaperone Expression and Apoptosis  

PubMed Central

The nascent polypeptide-associated complex (NAC) is a highly conserved heterodimer important for metazoan development, but its molecular function is not well understood. Recent evidence suggests the NAC is a component of the cytosolic chaperone network that interacts with ribosomal complexes and their emerging nascent peptides, such that the loss of the NAC in chaperone-depleted cells results in an increase in misfolded protein stress. We tested whether the NAC functions similarly in Caeonorhabditis (C.) elegans and found that its homologous NAC subunits, i.e. ICD-1 and -2, have chaperone-like characteristics. Loss of the NAC appears to induce misfolded protein stress in the ER triggering the unfolded protein response (UPR). Depletion of the NAC altered the response to heat stress, and led to an up-regulation of hsp-4, a homologue of the human chaperone and ER stress sensor GRP78/BiP. Worms lacking both ICD-1 and the UPR transcription factor XBP-1 generated a higher proportion of defective embryos, showed increased embryonic apoptosis and had a diminished survival rate relative to ICD-1-depleted animals with an intact UPR. Up-regulation of hsp-4 in NAC-depleted animals was specific to certain regions of the embryo; in embryos lacking ICD-1, the posterior region of the embryo showed strong up-regulation of hsp-4, while the anterior region did not. Furthermore, loss of ICD-1 produced prominent lysosomes in the gut region of adults and embryos putatively containing lipofuscins, lipid/protein aggregates associated with cellular aging. These results are the first set of evidence consistent with a role for C. elegans NAC in protein folding and localization during translation. Further, these findings confirm C. elegans as a valuable model for studying organismal and cell-type specific responses to misfolded protein stress. PMID:22957041

Arsenovic, Paul T.; Maldonado, Anthony T.; Colleluori, Vaughn D.; Bloss, Tim A.




ERIC Educational Resources Information Center

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)

Doolittle, Russell F.



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

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 NCT01998074. PMID:24914098

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



Upregulation of elastase proteins results in aortic dilatation in mucopolysaccharidosis I mice  

Microsoft Academic Search

Mucopolysaccharidosis I (MPS I), known as Hurler syndrome in the severe form, is a lysosomal storage disease due to ?-l-iduronidase (IDUA) deficiency. It results in fragmentation of elastin fibers in the aorta and heart valves via mechanisms that are unclear, but may result from the accumulation of the glycosaminoglycans heparan and dermatan sulfate. Elastin fragmentation causes aortic dilatation and valvular

Xiucui Ma; Mindy Tittiger; Russell H. Knutsen; Attila Kovacs; Laura Schaller; Robert P. Mecham; Katherine P. Ponder



Upregulation of elastase proteins results in aortic dilatation in mucopolysaccharidosis I mice  

E-print Network

-L-iduronidase (IDUA) deficiency. It results in fragmentation of elastin fibers in the aorta and heart valves via for patients with MPS I, and might reduce aortic aneurism formation in other disorders. Ã? 2008 Elsevier Inc have elastin fragmentation of the ascending aorta and heart valves, which can result in aortic

Ponder, Katherine P.


Does the dopamine transporter protein allele predict growth hormone testing results or response to growth hormone therapy?  

Microsoft Academic Search

Animal studies have shown dopamine transporter protein (DAT1) knock out mice are growth retarded and hyperactive. DAT1 has\\u000a been researched in several human psychiatric studies with varying results regarding phenotype and DAT1 alleles. However, the\\u000a relationship between DAT1 and short stature in humans has not been explored. Buccal swabs were collected from patients receiving\\u000a growth hormone (GH) therapy and were

Maala Daniel; Lucy D. Mastrandrea; Robbert J. Salis; Richard Erbe; Teresa Quattrin



RNA editing in wheat mitochondria results in the conservation of protein sequences  

Microsoft Academic Search

RNA editing is a process that results in the production of a messenger RNA with nucleotide sequences that differ from those of the template DNA1, and provides another mechanism for modulating gene expression. The phenomenon was initially described in the mitochondria of protozoa2, 3. Here we report that RNA editing is also required for the correct expression of plant mitochondria!

José M. Gualberto; Lorenzo Lamattina; Géraldine Bonnard; Jacques-Henry Weil; Jean-Michel Grienenberger



Targeting of C-terminal binding protein (CtBP) by ARF results in p53-independent apoptosis.  


ARF encodes a potent tumor suppressor that antagonizes MDM2, a negative regulator of p53. ARF also suppresses the proliferation of cells lacking p53, and loss of ARF in p53-null mice, compared with ARF or p53 singly null mice, results in a broadened tumor spectrum and decreased tumor latency. To investigate the mechanism of p53-independent tumor suppression by ARF, potential interacting proteins were identified by yeast two-hybrid screen. The antiapoptotic transcriptional corepressor C-terminal binding protein 2 (CtBP2) was identified, and ARF interactions with both CtBP1 and CtBP2 were confirmed in vitro and in vivo. Interaction with ARF resulted in proteasome-dependent CtBP degradation. Both ARF-induced CtBP degradation and CtBP small interfering RNA led to p53-independent apoptosis in colon cancer cells. ARF induction of apoptosis was dependent on its ability to interact with CtBP, and reversal of ARF-induced CtBP depletion by CtBP overexpression abrogated ARF-induced apoptosis. CtBP proteins represent putative targets for p53-independent tumor suppression by ARF. PMID:16508011

Paliwal, Seema; Pande, Sandhya; Kovi, Ramesh C; Sharpless, Norman E; Bardeesy, Nabeel; Grossman, Steven R



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


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

Carlini, David B; Stephan, Wolfgang



GAS41 amplification results in overexpression of a new spindle pole protein.  


Amplification is a hallmark of many human tumors but the role of most amplified genes in human tumor development is not yet understood. Previously, we identified a frequently amplified gene in glioma termed glioma-amplified sequence 41 (GAS41). Using the TCGA data portal and performing experiments on HeLa and TX3868, we analyzed the role of GAS41 amplification on GAS41 overexpression and the effect on the cell cycle. Here we show that GAS41 amplification is associated with overexpression in the majority of cases. Both induced and endogenous overexpression of GAS41 leads to an increase in multipolar spindles. We showed that GAS41 is specifically associated with pericentrosome material. As result of an increased GAS41 expression we found bipolar spindles with misaligned chromosomes. This number was even increased by a combined overexpression of GAS41 and a reduced expression of NuMA. We propose that GAS41 amplification may have an effect on the highly altered karyotype of glioblastoma via its role during spindle pole formation. PMID:22619067

Schmitt, Jana; Fischer, Ulrike; Heisel, Sabrina; Strickfaden, Hilmar; Backes, Christina; Ruggieri, Alessia; Keller, Andreas; Chang, Paul; Meese, Eckart



MEK1 protein kinase inhibition protects against damage resulting from focal cerebral ischemia  

PubMed Central

The MEK1 (MAP kinase/ERK kinase)/ERK (extracellular-signal-responsive kinase) pathway has been implicated in cell growth and differentiation [Seger, R. & Krebs, E. G. (1995) FASEB J. 9, 726–735]. Here we show that the MEK/ERK pathway is activated during focal cerebral ischemia and may play a role in inducing damage. Treatment of mice 30 min before ischemia with the MEK1-specific inhibitor PD98059 [Alessi, D. R., Cuenda, A., Cohen, P., Dudley, D. T. & Saltiel, A. R. (1995) J. Biol. Chem. 270, 27489–27494] reduces focal infarct volume at 22 hr after ischemia by 55% after transient occlusion of the middle cerebral artery. This is accompanied by a reduction in phospho-ERK1/2 immunohistochemical staining. MEK1 inhibition also results in reduced brain damage 72 hr after ischemia, with focal infarct volume reduced by 36%. This study indicates that the MEK1/ERK pathway contributes to brain injury during focal cerebral ischemia and that PD98059, a MEK1-specific antagonist, is a potent neuroprotective agent. PMID:10536014

Alessandrini, Alessandro; Namura, Shobu; Moskowitz, Michael A.; Bonventre, Joseph V.



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


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

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



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)

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.

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



Pb2TiOF(SeO3)2Cl and Pb2NbO2(SeO3)2Cl: small changes in structure induced a very large SHG enhancement.  


The replacement of NbO6 octahedra in Pb2NbO2(SeO3)2Cl by the TiO5F octahedra in Pb2TiOF(SeO3)2Cl induced a very large SHG enhancement from 2.3 × to 9.6 × KDP (KH2PO4), and both materials are type-I phase matchable. Theoretical calculations based on DFT methods indicate that the inclusion of F(-) anions in the d(0)-TM octahedral coordination unit has a great impact on the band structure and the SHG enhancement of the material. PMID:24042185

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



Disruption of the basal body protein POC1B results in autosomal-recessive cone-rod dystrophy.  


Exome sequencing revealed a homozygous missense mutation (c.317C>G [p.Arg106Pro]) in POC1B, encoding POC1 centriolar protein B, in three siblings with autosomal-recessive cone dystrophy or cone-rod dystrophy and compound-heterozygous POC1B mutations (c.199_201del [p.Gln67del] and c.810+1G>T) in an unrelated person with cone-rod dystrophy. Upon overexpression of POC1B in human TERT-immortalized retinal pigment epithelium 1 cells, the encoded wild-type protein localized to the basal body of the primary cilium, whereas this localization was lost for p.Arg106Pro and p.Gln67del variant forms of POC1B. Morpholino-oligonucleotide-induced knockdown of poc1b translation in zebrafish resulted in a dose-dependent small-eye phenotype, impaired optokinetic responses, and decreased length of photoreceptor outer segments. These ocular phenotypes could partially be rescued by wild-type human POC1B mRNA, but not by c.199_201del and c.317C>G mutant human POC1B mRNAs. Yeast two-hybrid screening of a human retinal cDNA library revealed FAM161A as a binary interaction partner of POC1B. This was confirmed in coimmunoprecipitation and colocalization assays, which both showed loss of FAM161A interaction with p.Arg106Pro and p.Gln67del variant forms of POC1B. FAM161A was previously implicated in autosomal-recessive retinitis pigmentosa and shown to be located at the base of the photoreceptor connecting cilium, where it interacts with several other ciliopathy-associated proteins. Altogether, this study demonstrates that POC1B mutations result in a defect of the photoreceptor sensory cilium and thus affect cone and rod photoreceptors. PMID:25018096

Roosing, Susanne; Lamers, Ideke J C; de Vrieze, Erik; van den Born, L Ingeborgh; Lambertus, Stanley; Arts, Heleen H; Peters, Theo A; Hoyng, Carel B; Kremer, Hannie; Hetterschijt, Lisette; Letteboer, Stef J F; van Wijk, Erwin; Roepman, Ronald; den Hollander, Anneke I; Cremers, Frans P M



Genetic properties of medium (M) and small (S) genomic RNA segments of Seoul hantavirus isolated from Rattus norvegicus and antigenicity analysis of recombinant nucleocapsid protein  

Microsoft Academic Search

A novel isolate of Seoul (SEO) hantaviruses was detected and identified in Rattus norvegicus in Shandong Province, China and designated as JUN5-14. The partial M segment and the coding region of nucleocapsid protein\\u000a (NP) in the S segment of JUN5-14 were PCR-amplified and sequenced. Nucleotide sequence analysis of the partial M segment (300 bp)\\u000a revealed that JUN5-14 isolate was closely related

Zexin Tao; Zhiyu Wang; Shaoxia Song; Hongling Wen; Guijie Ren; Guiting Wang



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


Equine infectious anemia (EIA) is an important viral infection affecting horses worldwide. The course of infection is accompanied generally by three characteristic stages: acute, chronic and inapparent. There is no effective EIA vaccine or treatment, and the control of the disease is based currently on identification of EIAV inapparent carriers by laboratory tests. Recombinant envelope protein (rgp90) was expressed in Escherichia coli and evaluated via enzyme-linked immunosorbent assay (ELISA). There was an excellent agreement (95.42%) between the ELISA results using rgp90 and agar gel immunodiffusion test results. AGID is considered the "gold-standard" serologic test for equine infectious anemia (EIA). After 1160 serum samples were tested, the relative sensitivity and specificity of the ELISA were 96.1% and 96.4%, respectively. Moreover, analysis diagnostic accuracy of the ELISA was performed. The ELISA proved robust. Furthermore, good reproducibility was observed for the negative controls and, positive controls for all plates tested. PMID:22227617

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



Disruption of srtA gene in Streptococcus suis results in decreased interactions with endothelial cells and extracellular matrix proteins.  


Streptococcus suis, a major pathogen of swine, is an emerging zoonotic agent which causes meningitis and septic shock. In this study, we investigated the ability of S. suis mutant strain (SRTDeltaA) lacking the sortase A gene (srtA) to interact with host cells and extracellular matrix (ECM) proteins, as well as its virulence in a mouse infection model. We demonstrated that mutant SRTDeltaA had reduced capacity to adhere to and invade porcine brain microvascular endothelial cells compared to the wild-type strain. In addition, mutant SRTDeltaA also showed significantly less adherence to plasma fibronectin, cellular fibronectin and collagen type I. However, disruption of srtA had little effect on the virulence of S. suis in a mouse intraperitoneal model of infection. These results indicate that surface proteins anchored by sortase A are required for a normal level of bacterial binding. However, other factors may also be important for S. suis virulence and interaction with host tissues. PMID:17954016

Vanier, G; Sekizaki, T; Domínguez-Punaro, M C; Esgleas, M; Osaki, M; Takamatsu, D; Segura, M; Gottschalk, M



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


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 8-13 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

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



Search Engine Optimization: SEO Book  

NSDL National Science Digital Library

The strange and wondrous ways in which search engines gather their indexes is made a little clearer in this tutorial. Written by a web designer disappointed with how difficult his pages were to find with the standard search engines, this page gives insight into how Infoseek, Lycos, Alta Vista, Excite, Web Crawler, and Open Text catalog web pages. The search strategy of each engine is described, along with tips for how web designers can increase their site's chances of being among the hits returned when users enter relevant search criteria. Although indexing algorithms are constantly being updated, this site presents common-sense guidelines that web designers interested in reaching a wider audience will find useful.

Wall, Aaron



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

PubMed Central

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

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



Tamm-Horsfall protein in patients with kidney dama...[Urol Res. 2004] -PubMed Result Urol Res. 2004 May;32(2):79-83. Links  

E-print Network

Tamm-Horsfall protein in patients with kidney dama...[Urol Res. 2004] - PubMed Result Urol Res. 2004 May;32(2):79-83. Links Tamm-Horsfall protein in patients with kidney damage and diabetes in diabetic and control kidney tissue specimens with or without kidney damage. Immunogold labeling

Abraham, Nader G.


Collision coupling, crosstalk, and compartmentalization in G-protein coupled receptor systems: Can a single model explain disparate results?  

PubMed Central

The collision coupling model describes interactions between receptors and G-proteins as first requiring the molecules to find each other by diffusion. A variety of experimental data on G-protein activation have been interpreted as suggesting (or not) the compartmentalization of receptors and/or G-proteins in addition to a collision coupling mechanism. In this work, we use a mathematical model of G-protein activation via collision coupling but without compartmentalization to demonstrate that these disparate observations do not imply the existence of such compartments. In experiments with GTP analogs (commonly GTP?S), the extent of G-protein activation is predicted to be a function of both receptor number and the rate of GTP analog hydrolysis. The sensitivity of G-protein activation to receptor number is shown to be dependent upon the assay used, with the sensitivity of phosphate production assays (GTPase) > GTP?S binding assays > cAMP inhibition assays. Finally, the amount of competition or crosstalk between receptor species activating the same type of G-proteins is predicted to depend on receptor and G-protein number, but in some (common) experimental regimes this dependence is expected to be minimal. Taken together, these observations suggest that the collision coupling model, without compartments of receptors and/or G-proteins, is sufficient to explain a variety of observations in literature data. PMID:18761019

Brinkerhoff, Christopher J.; Traynor, John R.; Linderman, Jennifer J.



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


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

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



Association of overactive bladder and C-reactive protein levels. Results from the Boston Area Community Health (BACH) Survey  

PubMed Central

OBJECTIVE To investigate the association between overactive bladder (OAB) and C-reactive protein (CRP) in a population-based sample of men and women. SUBJECTS AND METHODS Epidemiological survey of urological symptoms among men and women aged 30–79 years. A multi-stage stratified cluster design was used to randomly sample 5503 adults from the city of Boston. Analyses were conducted on 1898 men and 1854 women with available CRP levels. The International Continence Society defines OAB as ‘Urgency with or without urge incontinence, usually with frequency and nocturia.’ OAB was defined as: (1) urgency, (2) urgency with frequency, and (3) urgency with frequency and nocturia. Odds ratios (OR) and 95% confidence intervals (95% CI) of the CRP and OAB association were estimated using logistic regression. RESULTS Prevalence of OAB increased with CRP levels in both men and women. In men, adjusted ORs (95% CI) per log10(CRP) levels were 1.90 (1.26–2.86) with OAB defined as urgency, 1.65 (1.06–2.58) with OAB defined as urgency and frequency, and 1.92 (1.13–3.28) with OAB defined as urgency, frequency and nocturia. The association was more modest in women with ORs (95% CI) of 1.53 (1.07–2.18) for OAB as defined urgency, 1.51 (1.02–2.23) for OAB defined as urgency and frequency, and 1.34 (0.85–2.12) for OAB defined as urgency, frequency and nocturia. CONCLUSIONS Results show a consistent association of increasing CRP levels and OAB among both men and women. These results support our hypothesis for the role of inflammation in the development of OAB and a possible role for anti-inflammatory agents in its treatment. PMID:22176817

Kupelian, Varant; Rosen, Raymond C.; Roehrborn, Claus G.; Tyagi, Pradeep; Chancellor, Michael B.; McKinlay, John B.



Crowding Induces Differences in the Diffusion of Thermophilic and Mesophilic Proteins: A New Look at Neutron Scattering Results  

PubMed Central

The dynamical basis underlying the increased thermal stability of thermophilic proteins remains uncertain. Here, we challenge the new paradigm established by neutron scattering experiments in solution, in which the adaptation of thermophilic proteins to high temperatures lies in the lower sensitivity of their flexibility to temperature changes. By means of a combination of molecular dynamics and Brownian dynamics simulations, we report a reinterpretation of those experiments and show evidence that under crowding conditions, such as in vivo, thermophilic and homolog mesophilic proteins have diffusional properties with different thermal behavior. PMID:22261067

Marcos, Enrique; Mestres, Pau; Crehuet, Ramon



Screening for abnormalities of the protein C anticoagulant pathway using the ProC Global assay. Results of a European multicenter evaluation.  


ProC Global is a new global clotting assay designed to evaluate the functionality of the protein C anticoagulant pathway. It is based on the ability of endogenous activated protein C, generated by activation of protein C by Protac, to prolong an activated partial thromboplastin time, and the results are expressed in protein C activation time normalized ratio (PCAT-NR), after normalization. This multicenter trial involving five European laboratories was designed in order to determine the ability of the ProC Global assay to distinguish patients with and without abnormalities of the protein C pathway. The PCAT-NR was significantly lower in the patients with a thrombotic history not on oral anticoagulant treatment (n = 627) than in the healthy controls (n = 148), even after exclusion from both groups of the patients with abnormality of the protein C pathway. Using receiver operator characteristics analysis, the cut-off level of PCAT-NR = 0.80 was found to provide the best sensitivity-specificity ratio. All the carriers of the factor V Leiden mutation (n = 73), as well as all the patients with activated protein C resistance (n = 42), had a PCAT-NR below 0.80. The ProC Global assay performed well in patients with combined defects (97.0%, n = 33) or protein C deficiency (91.3%, n = 46), but it failed to detect all of them, and one patient with combined defects as well as four patients with a low protein C level had a PCAT-NR above the cut-off level. The sensitivity of the assay for protein S deficiency (n = 58) was weak (only 69.0%) and, surprisingly, more than 40% of the 375 patients without any of these abnormalities of the protein C pathway had a PCAT-NR below the cut-off level. PMID:10937806

Toulon, P; Halbmeyer, W M; Hafner, G; Schmitt, Y; Randgard, B; Odpadlik, M; Van Den Eynden, C; Wagner, C



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

PubMed Central

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

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



Anaerobic growth of Salmonella typhimurium results in increased uptake by Henle 407 epithelial and mouse peritoneal cells in vitro and repression of a major outer membrane protein.  

PubMed Central

The growth of Salmonella typhimurium under anaerobic conditions resulted in its greater ability to invade Henle 407 epithelial cells and in greater uptake by mouse peritoneal cells in vitro. Anaerobic growth also resulted in the repression of at least one major outer membrane protein. Images PMID:1987056

Schiemann, D A; Shope, S R



C-reactive protein as an adverse prognostic marker for men with castration-resistant prostate cancer (CRPC): Confirmatory results  

Microsoft Academic Search

We previously reported that higher serum concentrations of C-reactive protein (CRP) are associated with shorter survival in men with castration-resistant prostate cancer (CRPC). To confirm this finding in an independent data set, we used 119 CRPC patients enrolled in 6 phase II clinical trials and examined the relationship of CRP, alkaline phosphatase, hemoglobin, age, ECOG PS, and prostate specific antigen

Renee C. Prins; Brooks L. Rademacher; Solange Mongoue-Tchokote; Joshi J. Alumkal; Julie N. Graff; Kristine M. Eilers; Tomasz M. Beer


A unique vertebrate histone H1-related protamine-like protein results in an unusual sperm chromatin  

E-print Network

`giques II, Facultat de Medicina, Universitat de Barcelona, L'Hospitalet de Llobregat, Spain 3 Departamento. In this process, the somatic histones from the stem cells are replaced by highly specialized sperm nuclear basic of sperm nuclear basic proteins that have been shown to be related to somatic linker histones (histone H1

Eirin Lopez, Jose Maria


NCYM, a Cis-Antisense Gene of MYCN, Encodes a De Novo Evolved Protein That Inhibits GSK3? Resulting in the Stabilization of MYCN in Human Neuroblastomas  

PubMed Central

The rearrangement of pre-existing genes has long been thought of as the major mode of new gene generation. Recently, de novo gene birth from non-genic DNA was found to be an alternative mechanism to generate novel protein-coding genes. However, its functional role in human disease remains largely unknown. Here we show that NCYM, a cis-antisense gene of the MYCN oncogene, initially thought to be a large non-coding RNA, encodes a de novo evolved protein regulating the pathogenesis of human cancers, particularly neuroblastoma. The NCYM gene is evolutionally conserved only in the taxonomic group containing humans and chimpanzees. In primary human neuroblastomas, NCYM is 100% co-amplified and co-expressed with MYCN, and NCYM mRNA expression is associated with poor clinical outcome. MYCN directly transactivates both NCYM and MYCN mRNA, whereas NCYM stabilizes MYCN protein by inhibiting the activity of GSK3?, a kinase that promotes MYCN degradation. In contrast to MYCN transgenic mice, neuroblastomas in MYCN/NCYM double transgenic mice were frequently accompanied by distant metastases, behavior reminiscent of human neuroblastomas with MYCN amplification. The NCYM protein also interacts with GSK3?, thereby stabilizing the MYCN protein in the tumors of the MYCN/NCYM double transgenic mice. Thus, these results suggest that GSK3? inhibition by NCYM stabilizes the MYCN protein both in vitro and in vivo. Furthermore, the survival of MYCN transgenic mice bearing neuroblastoma was improved by treatment with NVP-BEZ235, a dual PI3K/mTOR inhibitor shown to destabilize MYCN via GSK3? activation. In contrast, tumors caused in MYCN/NCYM double transgenic mice showed chemo-resistance to the drug. Collectively, our results show that NCYM is the first de novo evolved protein known to act as an oncopromoting factor in human cancer, and suggest that de novo evolved proteins may functionally characterize human disease. PMID:24391509

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



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

PubMed Central

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

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



Dietary Protein Intake and Coronary Heart Disease in a Large Community Based Cohor: Results from the Atherosclerosis Risk in Communities (ARIC) Study  

PubMed Central

Background Prospective data examining the relationship between dietary protein intake and incident coronary heart disease (CHD) are inconclusive. Most evidence is derived from homogenous populations such as health professionals. Large community-based analyses in more diverse samples are lacking. Methods We studied the association of protein type and major dietary protein sources and risk for incident CHD in 12,066 middle-aged adults (aged 45–64 at baseline, 1987–1989) from four U.S. communities enrolled in the Atherosclerosis Risk in Communities (ARIC) Study who were free of diabetes mellitus and cardiovascular disease at baseline. Dietary protein intake was assessed at baseline and after 6 years of follow-up by food frequency questionnaire. Our primary outcome was adjudicated coronary heart disease events or deaths with following up through December 31, 2010. Cox proportional hazard models with multivariable adjustment were used for statistical analyses. Results During a median follow-up of 22 years, there were 1,147 CHD events. In multivariable analyses total, animal and vegetable protein were not associated with an increased risk for CHD before or after adjustment. In food group analyses of major dietary protein sources, protein intake from red and processed meat, dairy products, fish, nuts, eggs, and legumes were not significantly associated with CHD risk. The hazard ratios [with 95% confidence intervals] for risk of CHD across quintiles of protein from poultry were 1.00 [ref], 0.83 [0.70–0.99], 0.93 [0.75–1.15], 0.88 [0.73–1.06], 0.79 [0.64–0.98], P for trend ?=?0.16). Replacement analyses evaluating the association of substituting one source of dietary protein for another or of decreasing protein intake at the expense of carbohydrates or total fats did not show any statistically significant association with CHD risk. Conclusion Based on a large community cohort we found no overall relationship between protein type and major dietary protein sources and risk for CHD. PMID:25303709

Haring, Bernhard; Gronroos, Noelle; Nettleton, Jennifer A.; Wyler von Ballmoos, Moritz C.; Selvin, Elizabeth; Alonso, Alvaro



SPINS: a laboratory information management system for organizing and archiving intermediate and final results from NMR protein structure determinations.  


Recent technological advances and experimental techniques have contributed to an increasing number and size of NMR datasets. In order to scale up productivity, laboratory information management systems for handling these extensive data need to be designed and implemented. The SPINS (Standardized ProteIn Nmr Storage) Laboratory Information Management System (LIMS) addresses these needs by providing an interface for archival of complete protein NMR structure determinations, together with functionality for depositing these data to the public BioMagResBank (BMRB). The software tracks intermediate files during each step of an NMR structure-determination process, including: data collection, data processing, resonance assignments, resonance assignment validation, structure calculation, and structure validation. The underlying SPINS data dictionary allows for the integration of various third party NMR data processing and analysis software, enabling users to launch programs they are accustomed to using for each step of the structure determination process directly out of the SPINS user interface. PMID:16395675

Baran, Michael C; Moseley, Hunter N B; Aramini, James M; Bayro, Marvin J; Monleon, Daniel; Locke, Jessica Y; Montelione, Gaetano T



Multicopy Suppressors of Phenotypes Resulting from the Absence of Yeast VDAC Encode a VDAC-Like Protein  

Microsoft Academic Search

The permeability of the outer mitochondrial membrane to most metabolites is believed to be based in an outer membrane, channel-forming protein known as VDAC (voltage-dependent anion channel). Although multiple isoforms of VDAC have been identified in multicellular organisms, the yeast Saccharomyces cerevisiae has been thought to contain a single VDAC gene, designated POR1. However, cells missing the POR1 gene (Dpor1)




Reduced amount of the glucose-regulated protein GRP94 in skeletal myoblasts results in loss of fusion competence  

Microsoft Academic Search

We previously showed that skeletal myocytes of the adult rabbit do not accumulate the endoplasmic reticulum glucose-regulated protein GRP94, neither constitutively nor inducibly, at vari- ance with skeletal myocytes during perinatal devel- opment (5). Here we show that C2C12 cells up- regulate GRP94 during differentiation and, similarly to primary cultures of murine skeletal myocytes, specifically display GRP94 immunoreactivity on the



Prenatal protein malnutrition results in increased frequency of miniature inhibitory synaptic currents in rat CA1 pyramidal cells.  


There is growing evidence for an effect of prenatal protein malnutrition on the GABAergic neurotransmitter system in the rat hippocampus and associated structures. In the present study, we examined the functional electrophysiological consequences of observed alterations in GABA(A) and benzodiazepine receptor systems. Whole-cell patch clamp recordings of spontaneous and of miniature inhibitory postsynaptic currents (mIPSCs) generated by CA1 pyramidal cells were performed in in vitro hippocampal slices prepared from control and prenatally protein malnourished adult male rats. The characteristics of spontaneous synaptic currents were unaltered by the prenatal insult, as were the amplitudes and kinetics of GABA(A) receptor-mediated mIPSCs. The frequency of mIPSCs, however, was significantly increased in CA1 pyramidal cells in slices prepared from prenatally malnourished vs. control rats. The effect of the benzodiazepine receptor agonist chlordiazepoxide on the characteristics of mIPSCs was also examined and found to be the same in cells from both nutritional groups. The increased frequency of mIPSCs together with the lack of a change in amplitude, kinetics, or modulation by benzodiazepines of mIPSCs in response to prenatal protein malnutrition indicate a presynaptic locus of effect of this insult. PMID:10842348

Luebke, J; St John, J; Galler, J R



Targeting heat shock proteins by phenethyl isothiocyanate results in cell-cycle arrest and apoptosis of human breast cancer cells.  


Heat shock proteins (HSPs) are chaperones for several client proteins involved in transcriptional regulation, signal transduction, and cell cycle control. HSPs (27, 70, and 90) are abundantly expressed in a wide range of cancers and are transcriptionally regulated by heat shock factor (HSF1). Most of the synthetic HSP inhibitors exhibit toxicity, therefore, searching for inhibitors with limited or no toxicity will be of help. The objective of the present study was to determine the effect of natural isothiocyanate (phenethyl isothiocyanate; PEITC) on different HSPs (27, 70, and 90) and HSF1 in 2 breast cancer cell lines, namely breast adenocarcinoma MCF-7 (with wild type p53) and highly metastatic breast cancer cell MDA-MB-231 (with mutated p53). PEITC significantly inhibited the expression of HSPs (particularly HSP 90) and HSF1. Molecular consequences due to HSP inhibition were downregulation of cell-cycle regulatory proteins like Cyclin B1, CDK1, Cdc25C, PLK-1, and upregulation of p21 irrespective of p53 status. These modulations were accompanied by cell-cycle arrest at G2/M phase and apoptosis by activation of caspases 3 and 9. PEITC therefore may be regarded as a potent HSP inhibitor and an antitumor agent in the treatment of breast cancer. PMID:23530648

Sarkars, Ruma; Mukherjee, Sutapa; Roy, Madhumita



Life-long protein malnutrition in the rat (Rattus norvegicus) results in altered patterns of craniofacial growth and smaller individuals  

PubMed Central

Dietary protein is a limiting factor in mammalian growth, significantly affecting the non-linear trajectories of skeletal growth. Young females may be particularly vulnerable to protein malnutrition if the restriction is not lifted before they become reproductive. With such early malnutrition, limited amino acids would be partitioned between two physiological objectives, successful reproduction vs. continued growth. Thus, the consequences of protein malnutrition could affect more than one generation. However, few studies have quantified these cross-generational effects. Our objective was to test for differences in skeletal growth in a second generation of malnourished rats compared with rats malnourished only post-weaning, the first generation and with controls. In this longitudinal study we modelled the growth of 22 craniofacial measurements with the logistic Gompertz equation, and tested for differences in the equation's parameters among the diet groups. The female offspring of post-weaning malnourished dams did not catch up in size to the first generation or to controls, although certain aspects of their craniofacial skeleton were less affected than others. The second generation's growth trajectories resembled the longer and slower growth of the first malnourished generation. There was a complex interaction between developmental processes and early nutritional environment, which affected variation of adult size. PMID:16761979

Lobe, Shannon L; Bernstein, Marica C; German, Rebecca Z



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

PubMed Central

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

Kovalev, Nikolay



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


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

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



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

PubMed Central

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

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



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


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

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



Absence of iron-regulatory protein Hfe results in hyperproliferation of retinal pigment epithelium: role of cystine/glutamate exchanger.  


Haemochromatosis is an iron-overload disorder with age-dependent oxidative stress and dysfunction in a variety of tissues. Mutations in HFE (histocompatability leucocyte antigen class I-like protein involved in iron homoeostasis) are responsible for most cases of haemochromatosis. We demonstrated recently that HFE is expressed exclusively in the basal membrane of RPE (retinal pigment epithelium). In the present study, we used Hfe-/- mice to examine ferritin levels (an indirect readout for iron levels) and morphological changes in retina. We found increased ferritin accumulation in retina in 18-month-old, but not in 2-month-old, mice with considerable morphological damage compared with age-matched controls. The retinal phenotype included hypertrophy and hyperplasia of RPE. RPE cells isolated from Hfe-/- mice exhibited a hyperproliferative phenotype. We also compared the gene expression profile between wild-type and Hfe-/- RPE cells by microarray analysis. These studies showed that many cell cycle-related genes were differentially regulated in Hfe-/- RPE cells. One of the genes up-regulated in Hfe-/- RPE cells was Slc7a11 (where Slc is solute carrier) which codes for the 'transporter proper' xCT in the heterodimeric cystine/glutamate exchanger (xCT/4F2hc). This transporter plays a critical role in cellular glutathione status and cell-cycle progression. We confirmed the microarrray data by monitoring xCT mRNA levels by RT (reverse transcription)-PCR and also by measuring transport function. We also found increased levels of glutathione and the transcription factor/cell-cycle promoter AP1 (activator protein 1) in Hfe-/- RPE cells. Wild-type mouse RPE cells and human RPE cell lines, when loaded with iron by exposure to ferric ammonium citrate, showed increased expression and activity of xCT, reproducing the biochemical phenotype observed with Hfe-/- RPE cells. PMID:19715555

Gnana-Prakasam, Jaya P; Thangaraju, Muthusamy; Liu, Kebin; Ha, Yonju; Martin, Pamela M; Smith, Sylvia B; Ganapathy, Vadivel



Positive Selection Results in Frequent Reversible Amino Acid Replacements in the G Protein Gene of Human Respiratory Syncytial Virus  

PubMed Central

Human respiratory syncytial virus (HRSV) is the major cause of lower respiratory tract infections in children under 5 years of age and the elderly, causing annual disease outbreaks during the fall and winter. Multiple lineages of the HRSVA and HRSVB serotypes co-circulate within a single outbreak and display a strongly temporal pattern of genetic variation, with a replacement of dominant genotypes occurring during consecutive years. In the present study we utilized phylogenetic methods to detect and map sites subject to adaptive evolution in the G protein of HRSVA and HRSVB. A total of 29 and 23 amino acid sites were found to be putatively positively selected in HRSVA and HRSVB, respectively. Several of these sites defined genotypes and lineages within genotypes in both groups, and correlated well with epitopes previously described in group A. Remarkably, 18 of these positively selected tended to revert in time to a previous codon state, producing a “flip-flop” phylogenetic pattern. Such frequent evolutionary reversals in HRSV are indicative of a combination of frequent positive selection, reflecting the changing immune status of the human population, and a limited repertoire of functionally viable amino acids at specific amino acid sites. PMID:19119418

Botosso, Viviane F.; de A. Zanotto, Paolo M.; Ueda, Mirthes; Arruda, Eurico; Gilio, Alfredo E.; Vieira, Sandra E.; Stewien, Klaus E.; Peret, Teresa C. T.; Jamal, Leda F.; Pardini, Maria I. de M. C.; Pinho, Joao R. R.; Massad, Eduardo; Sant'Anna, Osvaldo A.; Holmes, Eddie C.; Durigon, Edison L.



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

PubMed Central

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



Placing the RPL32 Promoter Upstream of a Second Promoter Results in a Strongly Increased Number of Stably Transfected Mammalian Cell Lines That Display High Protein Expression Levels.  


The use of high stringency selection systems commonly results in a strongly diminished number of stably transfected mammalian cell lines. Here we placed twelve different promoters upstream of an adjacent primary promoter and tested whether this might result in an increased number of colonies; this is in the context of a stringent selection system. We found that only the promoter of the human ribosomal protein, RPL32, induced a high number of colonies in CHO-DG44 cells. This phenomenon was observed when the RPL32 promoter was combined with the CMV, SV40, EF1-?, and the ?-actin promoters. In addition, these colonies displayed high protein expression levels. The RPL32 promoter had to be functionally intact, since the deletion of a small region upstream of the transcription start site demolished its positive action. We conclude that adding the RPL32 promoter to an expression cassette in cis may be a powerful tool to augment gene expression levels. PMID:21350661

Hoeksema, F; Hamer, K; Siep, M; Verhees, J A; Otte, A P



Placing the RPL32 Promoter Upstream of a Second Promoter Results in a Strongly Increased Number of Stably Transfected Mammalian Cell Lines That Display High Protein Expression Levels  

PubMed Central

The use of high stringency selection systems commonly results in a strongly diminished number of stably transfected mammalian cell lines. Here we placed twelve different promoters upstream of an adjacent primary promoter and tested whether this might result in an increased number of colonies; this is in the context of a stringent selection system. We found that only the promoter of the human ribosomal protein, RPL32, induced a high number of colonies in CHO-DG44 cells. This phenomenon was observed when the RPL32 promoter was combined with the CMV, SV40, EF1-?, and the ?-actin promoters. In addition, these colonies displayed high protein expression levels. The RPL32 promoter had to be functionally intact, since the deletion of a small region upstream of the transcription start site demolished its positive action. We conclude that adding the RPL32 promoter to an expression cassette in cis may be a powerful tool to augment gene expression levels. PMID:21350661

Hoeksema, F.; Hamer, K.; Siep, M.; Verhees, J. A.; Otte, A. P.



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

PubMed Central

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

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



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


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

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



Comparative results between two groups of track-and-field athletes with or without the use of Master Amino acid Pattern® as protein substitute  

Microsoft Academic Search

Comparative results of this study have shown that athletes, by taking the Master Amino acid Pattern (MAP®) as a dietary protein\\u000a substitute and performing physical activity, have had (1) increased body muscle mass, strength, and endurance; (2) decreased\\u000a fat mass; (3) increased basal metabolism rate; (4) greater improvement in performance of the nonprevailing muscles compared\\u000a to the prevailing ones; and

M. Lucà-Moretti; A. Grandi; E. Lucà; E. Mariani; G. Vender; E. Arrigotti; M. Ferrario; E. Rovelli



A novel first exon of the Patched1 gene is upregulated by Hedgehog signaling resulting in a protein with pathway inhibitory functions  

Microsoft Academic Search

Patched homolog 1 (PTCH1) is a key component of the Hedgehog (HH) signaling pathway with three alternative first exons, but only exon 1B transcription depending on HH activation. Here, we show that in both human and mouse a novel PTCH1 first exon (1C) is expressed. Exon 1C transcription is upregulated by HH signaling, but the resulting PTCH1-1C protein has a

Takashi Shimokawa; Fahimeh Rahnama; Peter G. Zaphiropoulos



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

PubMed Central

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

Fernandez-Moya, Sandra M.; Carrington, Mark; Estevez, Antonio M.



Methotrexate treatment of FraX fibroblasts results in FMR1 transcription but not in detectable FMR1 protein levels  

PubMed Central

Background Fragile X syndrome is caused by the loss of FMRP expression due to methylation of the FMR1 promoter. Treatment of fragile X syndrome patients’ lymphoblastoid cells with 5-azadeoxycytidine results in demethylation of the promoter and reactivation of the gene. The aim of the study was to analyze if methotrexate, an agent which also reduces DNA methylation but with less toxicity than 5-azadeoxycytidine, has therapeutic potential in fragile X syndrome. Methods Fibroblasts of fragile X syndrome patients were treated with methotrexate in concentrations ranging from 1 to 4 ?g/ml for up to 14 days. FMR1 and FMRP expression were analyzed by quantitative PCR and western blotting. Results FMR1 mRNA was detected and levels correlated positively with methotrexate concentrations and time of treatment, but western blotting did not show detectable FMRP levels. Conclusions We show that it is possible to reactivate FMR1 transcription in fibroblasts of fragile X syndrome patients by treatment with methotrexate. However, we were not able to show FMRP expression, possibly due to the reduced translation efficacy caused by the triplet repeat extension. Unless FMR1 reactivation is more effective in vivo our results indicate that methotrexate has no role in the treatment of fragile X syndrome. PMID:24020679



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

SciTech Connect

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.

Luka, Zigmund; Pakhomova, Svetlana; Loukachevitch, Lioudmila V.; Newcomer, Marcia E.; Wagner, Conrad (Vanderbilt); (LSU)



Whey acidic protein extrinsically expressed from the mouse mammary tumor virus long terminal repeat results in hyperplasia of the coagulation gland epithelium and impaired mammary development.  


The whey acidic protein (WAP) is a milk protein that contains a cysteine-rich motif. This characteristic WAP signature has also been found in some protease inhibitors and certain proteins involved in tissue modeling. WAP is specifically synthesized in mammary tissue from late pregnant and lactating animals, and precocious synthesis results in impaired lobuloalveolar development of the gland in some transgenic lines. To determine whether growth modulatory effects of WAP are confined to mammary tissue, we expressed the WAP gene under the control of the mouse mammary tumor virus long terminal repeat in transgenic mice. The transgene was expressed at high levels in organs with exocrine function, such as mammary and salivary glands, prostate, seminal vesicle, and the coagulation gland. In addition to impaired mammary development, we observed hyperplasia and dysplasia of the coagulation gland epithelium. These findings suggest that WAP or a member of the WAP signature family can, in certain tissue contexts, function as an epithelial growth regulator. It appears from the present study that growth regulatory effects of WAP are restricted in the mouse to the mammary and coagulation gland epithelium. PMID:7522033

Hennighausen, L; McKnight, R; Burdon, T; Baik, M; Wall, R J; Smith, G H



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

SciTech Connect

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.

Corral, J.; Forster, A.; Thompson, S.; Rabbitts, T.H. (Medical Research Council Laboratory of Molecular Biology, Cambridge (United Kingdom)); Lampert, F. (Kinderklinik-Universitaet Giessen (Germany)); Kaneko, Y. (Saitama Cancer Centre, Saitama (Japan)); Slater, R.; Kroes, W.G. (Univ. of Amsterdam (Netherlands)); Van Der Schoot, C.E. (Central Laboratory of the Netherlands Red Cross, Amsterdam (Netherlands)); Ludwig, W.D. (Institut fur Humangenetik und Antrhopologie, Heidelberg (Germany)); Karpas, A. (Univ. of Cambridge (United Kingdom)); Pocock, C.; Cotter, F. (Institute of Child Health, London (United Kingdom))



Improving Docking Results via Reranking of Ensembles of Ligand Poses in Multiple X-ray Protein Conformations with MM-GBSA.  


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 Å 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

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



Structure of L-arabinose-binding protein from Escherichia coli at 5 A resolution and preliminary results at 3.5 A.  

PubMed Central

The three-dimensional crystal structure of the L-arabinose-binding protein from E. coli, an essential component in the active transport of L-arabinose, has been solved at 5 A resolution using the method of multiple isomorphous replacement. Five heavy atom derivatives were used. A preliminary 3.5 A electron density map has also been calculated. The results indicate that the molecule is ellipsoidal with approximate dimensions 68 A X 38 A X 30 A. Two similar domains within the molecule (which is a single polypeptide chain) are related by an approximate noncrystallographic rotation-translation axis. This relationship involves approximately 20% of the structure. Images PMID:781669

Phillips, G N; Mahajan, V K; Siu, A K; Quiocho, F A



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

PubMed Central

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

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



Factor VIII A3 domain substitution N1922S results in hemophilia A due to domain-specific misfolding and hyposecretion of functional protein.  


A point mutation leading to amino acid substitution N1922S in the A3 domain of factor VIII (fVIII) results in moderate to severe hemophilia A. A heterologous expression system comparing N1922S-fVIII and wild-type fVIII (wt-fVIII) demonstrated similar specific coagulant activities but poor secretion of N1922S-fVIII. Immunocytochemical analysis revealed that intracellular levels of N1922S-fVIII were similar to those of wt-fVIII. The specific activity of intracellular N1922S-fVIII was 10% of that of wt-fVIII, indicating the presence of large amounts of a nonfunctional N1922S-fVIII-folding intermediate. wt-fVIII colocalized with both endoplasmic reticulum (ER)- and Golgi-resident proteins. In contrast, N1922S-fVIII colocalized only with ER-resident proteins, indicating a block in transit from the ER to the Golgi. A panel of conformation-dependent monoclonal antibodies was used to determine native or nonnative folding of N1922S-fVIII. Intracellular N1922S-fVIII but not secreted N1922S-fVIII displayed abnormal folding in the A3 and C1 domains, indicating that the A1, A2, and C2 domains fold independently into antigenically intact tertiary structures, but that folding is stalled in the mutant A3 and its contiguous C1 domain. In summary, the N1922S substitution results in poor secretion of a functional protein, and the domain-specific defect in folding and intracellular trafficking of N1922S-fVIII is a novel mechanism for secretion defects leading to hemophilia A. PMID:21217077

Summers, Ryan J; Meeks, Shannon L; Healey, John F; Brown, Harrison C; Parker, Ernest T; Kempton, Christine L; Doering, Christopher B; Lollar, Pete



Factor VIII A3 domain substitution N1922S results in hemophilia A due to domain-specific misfolding and hyposecretion of functional protein  

PubMed Central

A point mutation leading to amino acid substitution N1922S in the A3 domain of factor VIII (fVIII) results in moderate to severe hemophilia A. A heterologous expression system comparing N1922S-fVIII and wild-type fVIII (wt-fVIII) demonstrated similar specific coagulant activities but poor secretion of N1922S-fVIII. Immunocytochemical analysis revealed that intracellular levels of N1922S-fVIII were similar to those of wt-fVIII. The specific activity of intracellular N1922S-fVIII was 10% of that of wt-fVIII, indicating the presence of large amounts of a nonfunctional N1922S-fVIII–folding intermediate. wt-fVIII colocalized with both endoplasmic reticulum (ER)– and Golgi-resident proteins. In contrast, N1922S-fVIII colocalized only with ER-resident proteins, indicating a block in transit from the ER to the Golgi. A panel of conformation-dependent monoclonal antibodies was used to determine native or nonnative folding of N1922S-fVIII. Intracellular N1922S-fVIII but not secreted N1922S-fVIII displayed abnormal folding in the A3 and C1 domains, indicating that the A1, A2, and C2 domains fold independently into antigenically intact tertiary structures, but that folding is stalled in the mutant A3 and its contiguous C1 domain. In summary, the N1922S substitution results in poor secretion of a functional protein, and the domain-specific defect in folding and intracellular trafficking of N1922S-fVIII is a novel mechanism for secretion defects leading to hemophilia A. PMID:21217077

Summers, Ryan J.; Meeks, Shannon L.; Healey, John F.; Brown, Harrison C.; Parker, Ernest T.; Kempton, Christine L.; Doering, Christopher B.



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


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

Cervantes-Rodríguez, M; Martínez-Gómez, M; Cuevas, E; Nicolás, L; Castelán, F; Nathanielsz, P W; Zambrano, E; Rodríguez-Antolín, J



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)

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) Å, b=11.052(2) Å, c=10.666(2) Å and ?=93.897(3)°), P1bar (a=7.051(2) Å, b=7.198(2) Å, c=8.314(2) Å, ?=107.897(3)°, ?=102.687(3)° and ?=100.564(3)°) and C2/c (a=17.862(4) Å, b=6.931(1) Å, c=20.133(4) Å 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.

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



Mutation of a single conserved tryptophan in multidrug resistance protein 1 (MRP1/ABCC1) results in loss of drug resistance and selective loss of organic anion transport.  


Multidrug resistance protein 1 (MRP1/ABCC1) belongs to the ATP-binding cassette transporter superfamily and is capable of conferring resistance to a broad range of chemotherapeutic agents and transporting structurally diverse conjugated organic anions. In this study, we found that substitution of a highly conserved tryptophan at position 1246 with cysteine (W1246C-MRP1) in the putative last transmembrane segment (TM17) of MRP1 eliminated 17beta-estradiol 17-(beta-d-glucuronide) (E(2)17betaG) transport by membrane vesicles prepared from transiently transfected human embryonic kidney cells while leaving the capacity for leukotriene C(4)- and verapamil-stimulated glutathione transport intact. In addition, in contrast to wild-type MRP1, leukotriene C(4) transport by the W1246C-MRP1 protein was no longer inhibitable by E(2)17betaG, indicating that the mutant protein had lost the ability to bind the glucuronide. A similar phenotype was observed when Trp(1246) was replaced with Ala, Phe, and Tyr. Confocal microscopy of cells expressing Trp(1246) mutant MRP1 molecules fused at the C terminus with green fluorescent protein showed that they were correctly routed to the plasma membrane. In addition to the loss of E(2)17betaG transport, HeLa cells stably transfected with W1246C-MRP1 cDNA were not resistant to the Vinca alkaloid vincristine and accumulated levels of [(3)H]vincristine comparable to those in vector control-transfected cells. Cells expressing W1246C-MRP1 were also not resistant to cationic anthracyclines (doxorubicin, daunorubicin) or the electroneutral epipodophyllotoxin VP-16. In contrast, resistance to sodium arsenite was only partially diminished, and resistance to potassium antimony tartrate remained comparable to that of cells expressing wild-type MRP1. This suggests that the structural determinants required for transport of heavy metal oxyanions differ from those for chemotherapeutic agents. Our results provide the first example of a tryptophan residue being so critically important for substrate specificity in a eukaryotic ATP-binding cassette transporter. PMID:11278867

Ito, K; Olsen, S L; Qiu, W; Deeley, R G; Cole, S P



Targeted disruption of Zfp36l2, encoding a CCCH tandem zinc finger RNA-binding protein, results in defective hematopoiesis  

PubMed Central

Members of the tristetraprolin family of tandem CCCH finger proteins can bind to AU-rich elements in the 3?-untranslated region of mRNAs, leading to their deadenylation and subsequent degradation. Partial deficiency of 1 of the 4 mouse tristetraprolin family members, Zfp36l2, resulted in complete female infertility because of early embryo death. We have now generated mice completely deficient in the ZFP36L2 protein. Homozygous Zfp36l2 knockout (KO) mice died within approximately 2 weeks of birth, apparently from intestinal or other hemorrhage. Analysis of peripheral blood from KO mice showed a decrease in red and white cells, hemoglobin, hematocrit, and platelets. Yolk sacs from embryonic day 11.5 (E11.5) Zfp36l2 KO mice and fetal livers from E14.5 KO mice gave rise to markedly reduced numbers of definitive multilineage and lineage-committed hematopoietic progenitors. Competitive reconstitution experiments demonstrated that Zfp36l2 KO fetal liver hematopoietic stem cells were unable to adequately reconstitute the hematopoietic system of lethally irradiated recipients. These data establish Zfp36l2 as a critical modulator of definitive hematopoiesis and suggest a novel regulatory pathway involving control of mRNA stability in the life cycle of hematopoietic stem and progenitor cells. PMID:19633199

Stumpo, Deborah J.; Broxmeyer, Hal E.; Ward, Toni; Cooper, Scott; Hangoc, Giao; Chung, Yang Jo; Shelley, William C.; Richfield, Eric K.; Ray, Manas K.; Yoder, Mervin C.; Aplan, Peter D.



TNF receptor I on human keratinocytes is a binding partner for staphylococcal protein A resulting in the activation of NF kappa B, AP-1, and downstream gene transcription.  


Primary human keratinocytes and immortalized HaCaT cells were analysed for their capacity to bind purified staphylococcal protein A (SpA). Co-incubation with FITC-labelled SpA led to a dose-depending attachment. Pull-down experiments with cellular extracts revealed the TNF? receptor I (TNF RI) as binding partner on keratinocytes. Thus, we next looked for expression of this receptor in human epidermis and cultured keratinocytes. TNF RI is strongly expressed on all keratinocytes analysed, both at the mRNA and protein level and activation by SpA at optimal doses of 50-100 ?g/ml resulted in the phosphorylation of the TNF RI downstream kinases MEK1/2, JNK1/2, and p38 subsequently leading to translocation of the p65 NF kappa B subunit and AP-1 into the nucleus. This translocation was then followed by increased expression of IL-8 and COX-2, two known NF kappa B-induced pro-inflammatory genes. To further test the relevance of our findings, we analysed in vitro production of over 100 strains isolated from atopic eczema showing that more than 85% of the tested strains produced extracellular SpA in substantial amounts. Thus, besides superantigens, haemolysins, and other cell wall components, Staphylococcus aureus exerts pro-inflammatory stimuli on human keratinocytes through the production of SpA signalling through TNF RI. PMID:20955203

Classen, Anna; Kalali, Behnam N; Schnopp, Christina; Andres, Christian; Aguilar-Pimentel, Juan A; Ring, Johannes; Ollert, Markus; Mempel, Martin



Inactivation of the alpha C Protein Antigen Gene, bca, by a Novel Shuttle\\/Suicide Vector Results in Attenuation of Virulence and Immunity in Group B Streptococcus  

Microsoft Academic Search

The alpha C protein of group B Streptococcus (GBS) is a major surface-associated antigen. Although its role in the biology and virulence of GBS has not been defined, it is opsonic and capable of eliciting protective immunity. The alpha C protein is widely distributed among clinical isolates and is a potential protein carrier and antigen in conjugate vaccines to prevent

Jing Li; Dennis L. Kasper; Frederick M. Ausubel; Bernard Rosner; James L. Michel



Disruption of the protein kinase N gene of Drosophila melanogaster Results in the Recessive delorean Allele (pkndln) With a Negative Impact on Wing Morphogenesis  

PubMed Central

We describe the delorean mutation of the Drosophila melanogaster protein kinase N gene (pkndln) with defects in wing morphology. Flies homozygous for the recessive pkndln allele have a composite wing phenotype that exhibits changes in relative position and shape of the wing blade as well as loss of specific vein and bristle structures. The pkndln allele is the result of a P-element insertion in the first intron of the pkn locus, and the delorean wing phenotype is contingent upon the interaction of insertion-bearing alleles in trans. The presence of the insertion results in production of a novel transcript that initiates from within the 3? end of the P-element. The delorean-specific transcript is predicted to produce a wild-type PKN protein. The delorean phenotype is not the result of a reduction in pkn expression, as it could not be recreated using a variety of wing-specific drivers of pkn-RNAi expression. Rather, it is the presence of the delorean-specific transcript that correlates with the mutant phenotype. We consider the delorean wing phenotype to be due to a pairing-dependent, recessive mutation that behaves as a dosage-sensitive, gain of function. Our analysis of genetic interactions with basket and nemo reflects an involvement of pkn and Jun-terminal kinase signaling in common processes during wing differentiation and places PKN as a potential effector of Rho1’s involvement in the Jun-terminal kinase pathway. The delorean phenotype, with its associated defects in wing morphology, provides evidence of a role for PKN in adult morphogenetic processes. PMID:24531729

Sass, Georgette L.; Ostrow, Bruce D.



Lipid raft- and protein kinase C-mediated synergism between glucocorticoid- and gonadotropin-releasing hormone signaling results in decreased cell proliferation.  


Cross-talk between the glucocorticoid receptor (GR) and other receptors is emerging as a mechanism for fine-tuning cellular responses. We have previously shown that gonadotropin-releasing hormone (GnRH) ligand-independently activates the GR and synergistically modulates glucocorticoid-induced transcription of an endogenous gene in L?T2 pituitary gonadotrope precursor cells. Here, we investigated GR and GnRH receptor (GnRHR) cross-talk that involves co-localization with lipid rafts in L?T2 cells. We report that the GnRHR and a small population of the GR co-localize with the lipid raft protein flotillin-1 (Flot-1) at the plasma membrane and that the GR is present in a complex with Flot-1, independent of the presence of ligands. We found that the SGK-1 gene is up-regulated by Dex and GnRH alone, whereas a combination of both ligands resulted in a synergistic increase in SGK-1 mRNA levels. Using siRNA-mediated knockdown and antagonist strategies, we show that the gene-specific synergistic transcriptional response requires the GR, GnRHR, and Flot-1 as well as the protein kinase C pathway. Interestingly, although several GR cofactors are differentially recruited to the SGK-1 promoter in the presence of Dex and GnRH, GR levels remain unchanged compared with Dex treatment alone, suggesting that lipid raft association of the GR has a role in enhancing its transcriptional output in the nucleus. Finally, we show that Dex plus GnRH synergistically inhibit cell proliferation in a manner dependent on SGK-1 and Flot-1. Collectively the results support a mechanism whereby GR and GnRHR cross-talk within Flot-1-containing lipid rafts modulates cell proliferation via PKC activation and SGK-1 up-regulation. PMID:24558046

Wehmeyer, Lancelot; Du Toit, Andrea; Lang, Dirk M; Hapgood, Janet P



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

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



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

SciTech Connect

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.

Congiu, A.M.; Casu, S.; Ugazio, G. (Istituto di Genetica (Italy))



Inhibition of Protein Geranylgeranylation Specifically Interferes with CD40-Dependent B Cell Activation, Resulting in a Reduced Capacity To Induce T Cell Immunity.  


Ab-independent effector functions of B cells, such as Ag presentation and cytokine production, have been shown to play an important role in a variety of immune-mediated conditions such as autoimmune diseases, transplant rejection, and graft-versus-host disease. Most current immunosuppressive treatments target T cells, are relatively unspecific, and result in profound immunosuppression that places patients at an increased risk of developing severe infections and cancer. Therapeutic strategies, which interfere with B cell activation, could therefore be a useful addition to the current immunosuppressive armamentarium. Using a transcriptomic approach, we identified upregulation of genes that belong to the mevalonate pathway as a key molecular event following CD40-mediated activation of B cells. Inhibition of 3-hydroxy-3-methylglutaryl CoA reductase, the rate-limiting enzyme of the mevalonate pathway, by lipophilic statins such as simvastatin and atorvastatin resulted in a specific inhibition of B cell activation via CD40 and impaired their ability to act as stimulatory APCs for allospecific T cells. Mechanistically, the inhibitory effect resulted from the inhibition of protein geranylgeranylation subsequent to the depletion of mevalonate, the metabolic precursor for geranylgeranyl. Thus, inhibition of geranylgeranylation either directly through geranylgeranyl transferase inhibitors or indirectly through statins represents a promising therapeutic approach for the treatment of diseases in which Ag presentation by B cells plays a role. PMID:25311809

Shimabukuro-Vornhagen, Alexander; Zoghi, Shahram; Liebig, Tanja M; Wennhold, Kerstin; Chemitz, Jens; Draube, Andreas; Kochanek, Matthias; Blaschke, Florian; Pallasch, Christian; Holtick, Udo; Scheid, Christof; Theurich, Sebastian; Hallek, Michael; von Bergwelt-Baildon, Michael S



Glucose deprivation results in the induction of glucose-regulated proteins and domes in MDCK monolayers in hormonally defined serum-free medium.  


Madin-Darby canine kidney (MDCK) cells spontaneously form dome-like structures in vitro, a phenomenon which has been proposed to be indicative of cellular differentiation. This study indicates the existence of a correlation between the induction of domes and of glucose-regulated proteins during glucose starvation. When MDCK monolayers were glucose deprived, domes appeared very rapidly. After only 3 h of glucose deprivation domes appeared in 69% of the microscope fields. The level of expression of glucose-regulated proteins (grps) as well as domes was examined over a 6-h time interval of glucose deprivation. Both grp 76 and 97 were induced over this time interval, with grp 76 being the more readily detectable. The level of induction of grp 76 as a function of time was quantitated by means of densitometry measurements. The induction of domes was examined in parallel with the induction of grp 76. The results indicated that the induction of grp 76 and domes occurs with a similar time course. The effect of glucose deprivation on the initial rate of ouabain-sensitive Rb+ uptake was also examined. Within the first 4 h of glucose deprivation, the initial rate of ouabain-sensitive Rb+ uptake did not differ significantly in glucose-deprived and control MDCK monolayers. These observations indicate that unlike the case with other methods of dome induction (e.g., treatment with either prostaglandin E1 (PGE1) or hexamethylene bis-acetamide (HMBA] glucose deprivation does not affect the Na+K+ ATPase activity of MDCK monolayers. These observations suggest that PGE1, HMBA, and glucose deprivation affect dome formation in MDCK monolayers by means of distinct mechanisms. PMID:2714397

Taub, M L; Syracuse, J A; Cai, J W; Fiorella, P; Subjeck, J R



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

PubMed Central

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

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



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


Staphylococcus aureus is the major pathogen among the staphylococci and the most common cause of bone infections. These infections are mainly characterized by bone destruction and inflammation, and are often debilitating and very difficult to treat. Previously we demonstrated that S. aureus protein A (SpA) can bind to osteoblasts, which results in inhibition of osteoblast proliferation and mineralization, apoptosis, and activation of osteoclasts. In this study we used small interfering RNA (siRNA) to demonstrate that osteoblast tumour necrosis factor receptor-1 (TNFR-1) is responsible for the recognition of and binding to SpA. TNFR-1 binding to SpA results in the activation of nuclear factor kappa B (NF?B). In turn, NF?B translocates to the nucleus of the osteoblast, which leads to release of interleukin 6 (IL-6). Silencing TNFR-1 in osteoblasts or disruption of the spa gene in S. aureus prevented both NF?B activation and IL-6 release. As well as playing a key role in proinflammatory reactions, IL-6 is also an important osteotropic factor. Release of IL-6 from osteoblasts results in the activation of the bone-resorbing cells, the osteoclasts. Consistent with our results described above, both silencing TNFR-1 in osteoblasts and disruption of spa in S. aureus prevented osteoclast activation. These studies are the first to demonstrate the importance of the TNFR-1-SpA interaction in bone infection, and may help explain the mechanism through which osteoclasts become overactivated, leading to bone destruction. Anti-inflammatory drug therapy could be used either alone or in conjunction with antibiotics to treat osteomyelitis or for prophylaxis in high-risk patients. PMID:23154968

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



Gene trap mutation of murine Outer dense fiber protein-2 gene can result in sperm tail abnormalities in mice with high percentage chimaerism  

PubMed Central

Background Outer dense fiber protein 2, Odf2, is a major component of the outer dense fibers, ODF, in the flagellum of spermatozoa. ODF are associated with microtubule doublets that form the axoneme. We recently demonstrated that tyrosine phosphorylation of Odf2 is important for sperm motility. In the course of a study of Odf2 using Odf2 mouse knockout lines we observed that males of a high percentage chimaerism, made using XL169 embryonic stem cells, were infertile, whereas mice of low-medium percentage chimaerism were fertile. Results XL169 ES cells have a ?-geo gene trap cassette inserted in the Odf2 gene. To determine possible underlying mechanisms resulting in infertility we analyzed epididymal sperm and observed that >50% displayed bent tails. We next performed ultrastructural analyses on testis of high percentage XL169 chimaeric mice. This analysis showed that high percentage XL169 chimaeric mice produce elongating spermatids that miss one or more entire outer dense fibers in their midpiece and principal piece. In addition, we observed elongating spermatids that show thinning of outer dense fibers. No other obvious abnormalities or defects are present in elongating spermatids. Spermatozoa from the caput and cauda epididymis of XL169 mice of high percentage chimaerism show additional tail defects, including absence of one or more axonemal microtubule doublets and bent tails. Sperm with bent tails display abnormal motility. Conclusions Our results document the possible impact of loss of one Odf2 allele on sperm tail structure and function, resulting in a novel sperm tail phenotype. PMID:20550699



A new chromogenic assay (HemosIL ThromboPath) is sensitive to major prothrombotic risk factors affecting the protein C pathway. Results of a multicenter study.  


The HemosIL ThromboPath assay (Instrumentation Laboratory) is a new chromogenic assay designed to globally evaluate the functionality of the protein C (PC) pathway. It is based on the ability of endogenous APC generated after activation of PC by a snake venom extract (Protac) to reduce the thrombin generation induced by a reagent containing tissue factor. The aim of this multicenter study involving three laboratories was to evaluate the test sensitivity to PC pathway abnormalities by retrospectively testing frozen plasma samples obtained in the different laboratories. Test results were significantly lower (p < 0.0001) in subjects who presented with any confirmed PC pathway abnormality than in those without. The cut-off value, defined in each participating center as the mean value minus one standard deviation of test results obtained in 30 normal samples, was found to provide a sensitivity-to-specificity ratio similar to that obtained using ROC-analysis. The assay performed well in carriers of the factor V Leiden mutation (n = 81), patients with PC deficiency (n = 40), combined defects (n = 55) or lupus anticoagulant (n = 44), with test results below the locally defined cut-off values in 97.5%, 95.0%, 100% and 100% of the tested subjects, respectively. The assay sensitivity for PS deficiency (n = 62) was 87.1%. Only 13.6% of the 272 subjects without any PC pathway abnormality had a decreased test result. So, using the locally defined cut-off values, the overall test sensitivity to all tested PC pathway abnormalities was 95.0% (95%CI = 91.8-97.3), its specificity 86.4% (95%CI = 81.8-90.2), its negative predictive value 94.4% (95%CI = 90.8-96.9) and its positive predictive value 87.9% (95%CI = 83.7-91.3). PMID:19157524

Toulon, Pierre; Smirnov, Mikhail; Triscott, Mark; Safa, Omid; Biguzzi, Eugenia; Bouziane, Kader; Tripodi, Armando



Differential and Inefficient Splicing of a Broadly Expressed Drosophila erect wing Transcript Results in Tissue-Specific Enrichment of the Vital EWG Protein Isoform  

PubMed Central

In this report, we document an unusual mode of tissue-enriched gene expression that is primarily mediated by alternative and inefficient splicing. We have analyzed posttranscriptional regulation of the Drosophila erect wing gene, which provides a vital neuronal function and is essential for the formation of certain muscles. Its predominant protein product, the 116-kDa EWG protein, a putative transcriptional regulator, can provide all known erect wing-associated functions. Moreover, consistent with its function, the 116-kDa protein is highly enriched in neurons and is also observed transiently in migrating myoblasts. In contrast to the protein distribution, we observed that erect wing transcripts are present in comparable levels in neuron-enriched heads and neuron-poor bodies of adult Drosophila. Our analyses shows that erect wing transcript consists of 10 exons and is alternatively spliced and that a subset of introns are inefficiently spliced. We also show that the 116-kDa EWG protein-encoding splice isoform is head enriched. In contrast, bodies have lower levels of transcripts that can encode the 116-kDa protein and greater amounts of unprocessed erect wing RNA. Thus, the enrichment of the 116-kDa protein in heads is ensured by tissue-specific alternative and inefficient splicing and not by transcriptional regulation. Furthermore, this regulation is biologically important, as an increased level of the 116-kDa protein outside the nervous system is lethal. PMID:10330140

Koushika, Sandhya P.; Soller, Matthias; DeSimone, Susan M.; Daub, Douglas M.; White, Kalpana


155 Arrive. Survive. Thrive.  

E-print Network

of university is a very exciting time as you meet new friends and engage in the social and scholarly life students are also encouraged to take advantage of our great athletic facilities. Carleton Complete includes, small-group activities, academic advising, and support with class registration will help every first

156 Arrive. Survive. Thrive.  

E-print Network

and learning, enhance the student experience, and contribute to the creation of an environment for success and community service learning opportunities that encourage student engagement. As you will see throughout. Likewise, connect with us on Facebook ( and Twitter (@Carleton

Dawson, Jeff W.


A Single Point Mutation in Nonstructural Protein NS2 of Bovine Viral Diarrhea Virus Results in Temperature-Sensitive Attenuation of Viral Cytopathogenicity ?  

PubMed Central

For Bovine viral diarrhea virus (BVDV), the type species of the genus Pestivirus in the family Flaviviridae, cytopathogenic (cp) and noncytopathogenic (ncp) viruses are distinguished according to their effect on cultured cells. It has been established that cytopathogenicity of BVDV correlates with efficient production of viral nonstructural protein NS3 and with enhanced viral RNA synthesis. Here, we describe generation and characterization of a temperature-sensitive (ts) mutant of cp BVDV strain CP7, termed TS2.7. Infection of bovine cells with TS2.7 and the parent CP7 at 33°C resulted in efficient viral replication and a cytopathic effect. In contrast, the ability of TS2.7 to cause cytopathogenicity at 39.5°C was drastically reduced despite production of high titers of infectious virus. Further experiments, including nucleotide sequencing of the TS2.7 genome and reverse genetics, showed that a Y1338H substitution at residue 193 of NS2 resulted in the temperature-dependent attenuation of cytopathogenicity despite high levels of infectious virus production. Interestingly, TS2.7 and the reconstructed mutant CP7-Y1338H produced NS3 in addition to NS2-3 throughout infection. Compared to the parent CP7, NS2-3 processing was slightly decreased at both temperatures. Quantification of viral RNAs that were accumulated at 10 h postinfection demonstrated that attenuation of the cytopathogenicity of the ts mutants at 39.5°C correlated with reduced amounts of viral RNA, while the efficiency of viral RNA synthesis at 33°C was not affected. Taken together, the results of this study show that a mutation in BVDV NS2 attenuates viral RNA replication and suppresses viral cytopathogenicity at high temperature without altering NS3 expression and infectious virus production in a temperature-dependent manner. PMID:19776121

Pankraz, Alexander; Preis, Simone; Thiel, Heinz-Jürgen; Gallei, Andreas; Becher, Paul



Deletion of RTS1, Encoding a Regulatory Subunit of Protein Phosphatase 2A, Results in Constitutive Amino Acid Signaling via Increased Stp1p Processing  

PubMed Central

In Saccharomyces cerevisiae, extracellular amino acids are sensed at the plasma membrane by the SPS sensor, consisting of the transporter homologue Ssy1p, Ptr3p, and the endoprotease Ssy5p. Amino acid sensing results in proteolytic truncation of the transcription factors Stp1p and Stp2p, followed by their relocation from the cytoplasm to the nucleus, where they activate transcription of amino acid permease genes. We screened a transposon mutant library for constitutively signaling mutants, with the aim of identifying down-regulating components of the SPS-mediated pathway. Three isolated mutants were carrying a transposon in the RTS1 gene, which encodes a regulatory subunit of protein phosphatase 2A. We investigated the basal activity of the AGP1 and BAP2 promoters in rts1? cells and found increased transcription from these promoters, as well as increased Stp1p processing, even in the absence of amino acids. Based on our findings we propose that the phosphatase complex containing Rts1p keeps the SPS-mediated pathway down-regulated in the absence of extracellular amino acids by dephosphorylating a component of the pathway. PMID:16400180

Eckert-Boulet, Nadine; Larsson, Katrin; Wu, Boqian; Poulsen, Peter; Regenberg, Birgitte; Nielsen, Jens; Kielland-Brandt, Morten C.



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

PubMed Central

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

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



Absence of myotonic dystrophy protein kinase (DMPK) mRNA as a result of a triplet repeat expansion in myotonic dystrophy  

SciTech Connect

Myotonic dystrophy is an autosomally dominant inherited disease in which system-wide abnormalities are caused by a triplet repeat expansion within the 3[prime] untranslated region of the myotonic dystrophy protein kinase (DMPK) gene. To determine the effect an expanded repeat region has on DMPK expression, the authors have separated the chromosome 19 homologues from a 36-year-old woman with myotonic dystrophy into different cell lines by way of somatic cell hybridization. Hybrid DM9101 contains the normal DMPK allele (13 repeats), whereas hybrid DM1115 harbors the mutant allele ([approximately]133 repeats). Reverse transcription/polymerase chain reaction (RT/PCR) amplification of coding sequences from the DMPK gene has shown both reduced levels of primary DMPK transcripts and impaired processing of these transcripts in hybrid cell line DM1115. These findings suggest that the presence of a large number of repeats in the 3[prime] untranslated region of the DMPK gene reduces both the synthesis and the processing of DMPK mRNA, resulting in undetectable levels of processed DMPK mRNA from the mutant allele. 41 refs., 6 figs., 1 tab.

Carango, P.; Noble, J.E.; Funanage, V.L.; Marks, H.G. (Alfred I. duPont Institute, Wilmington, DE (United States))



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

PubMed Central

A battery of thermodynamic, kinetic, and structural approaches has indicated that the small ?-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

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



Slow proton transfer coupled to unfolding explains the puzzling results of single-molecule experiments on BBL, a paradigmatic downhill folding protein.  


A battery of thermodynamic, kinetic, and structural approaches has indicated that the small ?-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

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



Association between serum insulin-like growth factor I or IGF-binding protein 3 and estimated glomerular filtration rate: results of a population-based sample  

PubMed Central

Background Insulin-like growth factor I (IGF-I), which is mostly carried in blood by IGF-binding protein 3 (IGFBP-3), was associated to the glomerular filtration rate and chronic kidney disease in a multiethnic study among US adults. The aim of the present study was to investigate whether serum IGF-I or IGFBP-3 are associated with estimated glomerular filtration rate (eGFR) in a population-based study of Caucasian adults. Methods Data from 4028 subjects (2048 women) aged 20 to 81 years from the Study of Health in Pomerania (SHIP) were analyzed. Total serum IGF-I and IGFBP-3 concentrations were determined by chemiluminescence immunoassays and categorized into sex- and age-specific quartiles. Results After adjusting for age, waist circumference and type 2 diabetes mellitus, analysis of variance (ANOVA) revealed inverse associations between serum IGF-I concentrations and eGFR in men as well as between serum IGFBP-3 concentrations and eGFR in men and women. Logistic regression analyses confirmed these findings and showed that high IGF-I or IGFBP-3 concentrations were associated with an increased risk of decreased eGFR (<60 mL/min/1.73 m2) in men or women. These relations became stronger when lower eGFR cut-offs were used for the analyses. Conclusion Our data revealed associations of increased serum IGF-I concentrations and decreased eGFR in men but not in women and an association of increased serum IGFBP-3 concentrations and decreased eGFR in both sexes. PMID:23237568



BBS7 is required for BBSome formation and its absence in mice results in Bardet-Biedl syndrome phenotypes and selective abnormalities in membrane protein trafficking  

PubMed Central

Summary Bardet-Biedl Syndrome (BBS) is a pleiotropic and genetically heterozygous disorder caused independently by numerous genes (BBS1–BBS17). Seven highly conserved BBS proteins (BBS1, 2, 4, 5, 7, 8 and 9) form a complex known as the BBSome, which functions in ciliary membrane biogenesis. BBS7 is both a unique subunit of the BBSome and displays direct physical interaction with a second BBS complex, the BBS chaperonin complex. To examine the in vivo function of BBS7, we generated Bbs7 knockout mice. Bbs7?/? mice show similar phenotypes to other BBS gene mutant mice including retinal degeneration, obesity, ventriculomegaly and male infertility characterized by abnormal spermatozoa flagellar axonemes. Using tissues from Bbs7?/? mice, we show that BBS7 is required for BBSome formation, and that BBS7 and BBS2 depend on each other for protein stability. Although the BBSome serves as a coat complex for ciliary membrane proteins, BBS7 is not required for the localization of ciliary membrane proteins polycystin-1, polycystin-2, or bitter taste receptors, but absence of BBS7 leads to abnormal accumulation of the dopamine D1 receptor to the ciliary membrane, indicating that BBS7 is involved in specific membrane protein localization to cilia. PMID:23572516

Zhang, Qihong; Nishimura, Darryl; Vogel, Tim; Shao, Jianqiang; Swiderski, Ruth; Yin, Terry; Searby, Charles; Carter, Calvin S.; Kim, GunHee; Bugge, Kevin; Stone, Edwin M.; Sheffield, Val C.



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

Microsoft Academic Search

Summary 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 inves- tigated if hsp70 can be used in a novel peptide vaccine for the induction of protective antiviral immunity and memory

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



Fetal Wastage Syndrome due to Blood Protein\\/Platelet Defects: Results of Prevalence Studies and Treatment Outcome with Low-Dose Heparin and Low-Dose Aspirin  

Microsoft Academic Search

Fetal wastage syndrome is characterized by recurrent spontaneous abortion. Many syndromes are associated with recurrent fetal loss, including anatomical anomalies, endocrine\\/hormonal abnormalities, and coagulation defects, with coagulation defects accounting for ?30% of cases. Most procoagulant factor defects are due to inadequate fibrin-mediated implantation of the fertilized ovum into the decidua. However, blood protein\\/ platelet defects leading to hypercoagulability and thrombosis

Rodger L. Bick; H. Robert Laughlin; Brian M. Cohen; A. Jay Staub; James Madden; Ali Toofanian



Muscle uncoupling protein 3 expression is unchanged by chronic ephedrine/caffeine treatment: results of a double blind, randomised clinical trial in morbidly obese females.  


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 (25-52 years of age, body-mass index 48.0±4.0 kg/m2, range 41.1-57.6) 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 5769±1105 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 -5.2±3.2%, p<0.0001) without significant differences between the treated groups. EC treatment increased the RMR (+9.2±6.8%, p?=?0.020), differently from placebo which was linked to a reduction of RMR (-7.6±6.5%, 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: NCT02048215. PMID:24905629

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



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

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 (25–52 years of age, body-mass index 48.0±4.0 kg/m2, range 41.1–57.6) 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 5769±1105 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 ?5.2±3.2%, p<0.0001) without significant differences between the treated groups. EC treatment increased the RMR (+9.2±6.8%, p?=?0.020), differently from placebo which was linked to a reduction of RMR (?7.6±6.5%, 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 NCT02048215 PMID:24905629

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



Cotranslational protein-RNA associations predict protein-protein interactions  

PubMed Central

Background Most cellular proteins function as part of stable protein complexes. We recently showed that around 38% of proteins associate with mRNAs that encode interacting proteins, reflecting the cotranslational formation of the complex between the bait protein and the nascent peptides encoded by the interacting mRNAs. Here we hypothesise that these cotranslational protein-mRNA associations can be used to predict protein-protein interactions. Results We found that the fission yeast Exo2 protein, which encodes an exonuclease of the XRN1 family, coimmunoprecipitates with the eti1 mRNA, which codes for a protein of unknown function and uninformative sequence. Based on this protein-mRNA association, we predicted that the Exo2 and Eti1 protein are part of the same complex, and confirmed this hypothesis by coimmunoprecipitation and colocalization of the proteins. Similarly, we show that the cotranslational interaction between the Sty1 MAP kinase and the cip2 mRNA, which encodes an RNA-binding protein, predicts a complex between Sty1 and Cip2. Conclusions Our results demonstrate that cotranslational protein-mRNA associations can be used to identify new components of protein complexes. PMID:24755092



Global Forebrain Ischemia Results in Differential Cellular Expression of Interleukin1? (IL1?) and its Receptor at mRNA and Protein Level  

Microsoft Academic Search

The mRNA expression of the proinflammatory cytokine interleukin-1? (IL-1?) has been shown to be induced in neural elements during ischemia. It is not clear which cells generate the IL-1? mRNA and eventually synthesize IL-1 protein and which cells respond to this signaling by producing IL-1 receptors during ischemia. To clarify this question, rats were subjected to global ischemia by bilateral

Tiina R. Sairanen; Perttu J. Lindsberg; Michael Brenner; Anna-Leena Sirén; Tiina R Vidman



Increased Atherosclerosis in ApoE and LDL Receptor Gene Knock-Out Mice as a Result of Human Cholesteryl Ester Transfer Protein Transgene Expression  

Microsoft Academic Search

Abstract—The plasma cholesteryl ester transfer protein (CETP) plays a major role in the catabolism of HDL cholesteryl ester (CE). CETP transgenic mice have decreased HDL cholesterol levels and have been reported to have either increased or decreased early atherosclerotic lesions. To evaluate the impact of CETP expression on more,advanced forms of atherosclerosis, we have cross-bred the human CETP transgene into

Andrew S. Plump; Lori Masucci-Magoulas; Can Bruce; Charles L. Bisgaier; Jan L. Breslow; Alan R. Tall



Protein annealing: Thermal treatment of met-hemoglobin bound to ?-zirconium phosphate\\/phosphonates results in initial denaturation followed by recovery of activity and structure  

Microsoft Academic Search

Thermal stability of met-hemoglobin (Hb) intercalated in three layered solid materials was investigated at 60°C. At this temperature, free Hb undergoes rapid thermal denaturation, which is accompanied by the loss of its peroxidase-like activity and protein secondary structure. Heating of Hb intercalated in the galleries of ?-zirconium(IV) phosphate (at 60°C) also indicated initial loss of peroxidase-like activity of bound Hb,

V. Jagannadham; Akhilesh Bhambhani; Challa V. Kumar



Absence of myotonic dystrophy protein kinase (DMPK) mRNA as a result of a triplet repeat expansion in myotonic dystrophy  

Microsoft Academic Search

Myotonic dystrophy is an autosomally dominant inherited disease in which system-wide abnormalities are caused by a triplet repeat expansion within the 3[prime] untranslated region of the myotonic dystrophy protein kinase (DMPK) gene. To determine the effect an expanded repeat region has on DMPK expression, the authors have separated the chromosome 19 homologues from a 36-year-old woman with myotonic dystrophy into

P. Carango; J. E. Noble; V. L. Funanage; H. G. Marks



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.  


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

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



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

PubMed Central

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

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



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

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

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



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


We previously reported that transgenic (TG) mice with cardiac-restricted alpha(1A)-adrenergic receptor (alpha(1A)-AR)-overexpression showed enhanced contractility, but no hypertrophy. Since chronic inotropic enhancement may be deleterious, we investigated if long-term, cardiac function and longevity are compromised. alpha(1A)-TG mice, but not their non-TG littermates (NTLs), showed progressive loss of left ventricular (LV) hypercontractility (dP/dt(max): 14,567+/-603 to 11,610+/-915 mmHg/s, P<0.05, A1A1 line: 170-fold overexpression; and 13,625+/-826 to 8322+/-682 mmHg/s, respectively, P<0.05, A1A4 line: 112-fold overexpression, at 2 and 6 months, respectively). Both TG lines developed LV fibrosis, but not LV dilatation or hypertrophy, despite activation of hypertrophic signaling pathways. Microarray and real time RT-PCR analyses revealed activation of matricellular protein genes, including those for thrombospondin 1, connective tissue growth factor and tenascin C, but not transforming growth factor beta1. Life-span was markedly shortened (mean age at death: 155 days, A1A1 line; 224 days, A1A4 line compared with NTLs: >300 days). Telemetric electrocardiography revealed that death in the alpha(1A)-AR TG mice was due to cardiac standstill preceded by a progressive diminution in QRS amplitude, but not by arrhythmias. The QRS changes and sudden death could be mimicked by alpha(1)-AR activation, and reversed preterminally by alpha(1)-AR blockade, suggesting a relationship to stress- or activity-associated catecholamine release. Thus, long-term augmentation of cardiac alpha(1A)-adrenergic drive leads to premature death and progressive LV fibrosis with reactivation of matricellular protein genes. To our knowledge this is the first evidence in vivo for a role of the alpha(1A)-AR in ventricular fibrosis and in pathological cardiac remodeling. PMID:16516910

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



Three-Dimensional Culture of an Ovine Pulmonary Adenocarcinoma-Derived Cell Line Results in Re-Expression of Surfactant Proteins and Jaagsiekte Sheep Retrovirus  

PubMed Central

Jaagsiekte sheep retrovirus (JSRV) is the causative agent of ovine pulmonary adenocarcinoma (OPA) in sheep. A major interest is elucidating the mechanism(s) of transformation by the viral envelope (Env) that functions as an oncogene. These studies would benefit from a cell line derived from type II pneumocytes that have maintained the differentiation state. In this study we used an OPA-derived cell line (JS7), which has lost structural and functional properties of type II pneumocytes, and no longer expresses JSRV when grown in 2-D monolayer culture. When JS7 cells were placed in 3-D culture using Matrigel, they grew as small spheres of polarized cells that re-expressed surfactant proteins characteristic of type II pneumocytes. Moreover, JS7 cells grown in 3-D re-expressed JSRV virus by several criteria. This study underscores the importance of the culture environment on maintaining the differentiation state of OPA tumor cells as well as expression of JSRV. PMID:21481432

Johnson, Chassidy; Fan, Hung



A 10-minute point-of-care assay for detection of blood protein adducts resulting from low level exposure to organophosphate nerve agents.  


The OrganoTox test is a rapid, point-of-care assay capable of detecting clinically relevant organophosphate (OP) poisoning after low-level exposure to sarin, soman, tabun, or VX chemical nerve agents. The test utilizes either a finger stick peripheral blood sample or plasma specimen. While high-level nerve agent exposure can quickly lead to death, low-level exposure produces vague, nondescript signs and symptoms that are not easily clinically differentiated from other conditions. In initial testing, the OrganoTox test was used to detect the presence of blood protein-nerve agent adducts in exposed blood samples. In order to mimic the in vivo exposure as closely as possible, nerve agents stored in organic solvents were spiked in minute quantities into whole blood samples. For performance testing, 40 plasma samples were spiked with sarin, soman, tabun, or VX and 10 normal plasma samples were used as the negative control. The 40 nerve agent-spiked plasma samples included 10 replicates of each agent. At the clinically relevant low-level exposure of 10 ng/ml, the OrganoTox test demonstrated 100% sensitivity for soman, tabun, and VX and 80% sensitivity for sarin. The OrganoTox test demonstrated greater than 97% specificity with 150 blood samples obtained from healthy adults. No cross-reactivity or interference from pesticide precursor compounds was found. A rapid test for nerve agent exposure will help identify affected patients earlier in the clinical course and trigger more appropriate medical management in a more timely manner. PMID:23200942

VanDine, Robert; Babu, Uma Mahesh; Condon, Peter; Mendez, Arlene; Sambursky, Robert



A bivalent Neisseria meningitidis recombinant lipidated factor H binding protein vaccine in young adults: results of a randomised, controlled, dose-escalation phase 1 trial.  


Neisseria meningitidis is a leading cause of meningitis and septicaemia, but a broadly-protective vaccine against endemic serogroup B disease is not licensed and available. The conserved, outer-membrane lipoprotein factor H binding protein (fHBP, also known as LP2086) is expressed as one of two subfamily variants in virtually all meningococci. This study investigated the safety, tolerability, and immunogenicity of a recombinant-expressed bivalent fHBP (r-fHBP) vaccine in healthy adults. Participants (N=103) aged 18-25 years were recruited into three ascending dose level cohorts of 20, 60, and 200?g of a bivalent r-fHBP vaccine formulation and randomised to receive vaccine or placebo at 0, 1, and 6 months. The vaccine was well tolerated. Geometric mean titres (GMTs) for r-fHBP subfamily-specific IgG antibodies increased 19-168-fold from pre-vaccination to post-dose 2 in a dose level-dependent manner. In addition, robust serum bactericidal assay using human complement (hSBA) responses for strains expressing both homologous and heterologous fHBP variants were observed. After three vaccinations, 16-52% of the placebo group and 47-90%, 75-100%, and 88-100%, of the 20, 60, and 200?g dose levels, respectively, had seroprotective (? 1:4) hSBA titres against six serogroup B strains. The bivalent r-fHBP vaccine was well tolerated and induced robust bactericidal activity against six diverse serogroup B strains in young adults at the 60 and 200?g dose levels. PMID:22871351

Richmond, P C; Nissen, M D; Marshall, H S; Lambert, S B; Roberton, D; Gruber, W C; Jones, T R; Arora, A



Lack of CD47 Impairs Bone Cell Differentiation and Results in an Osteopenic Phenotype in Vivo due to Impaired Signal Regulatory Protein ? (SIRP?) Signaling*  

PubMed Central

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

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



Reduced glutathione disrupts the intracellular trafficking of tyrosinase and tyrosinase-related protein-1 but not dopachrome tautomerase and Pmel17 to melanosomes, which results in the attenuation of melanization.  


We previously reported that treatment of B16 melanotic melanoma cells with reduced glutathione (GSH) converts them to amelanotic cells without any significant down-regulation of tyrosinase activity. To characterize the cellular mechanism(s) involved, we determined the intracellular distribution of melanocyte-specific proteins, especially in melanin synthesis-specific organelles, termed melanosomes by subcellular fractionation followed by Western blotting and confocal laser microscopy (CFLM). In the melanosome-rich large granule fraction and in highly purified melanosome fractions, while GSH-induced amelanotic B16 cells have significantly diminished levels of protein/activity of tyrosinase and tyrosinase-related protein-1 compared with control melanized B16 cells, there was substantially no difference in the distribution and levels of dopachrome tautomerase and the processed isoform of Pmel17 (HMB45) between control melanized and GSH-induced amelanotic B16 cells. Analysis of merged images obtained by CFLM revealed that whereas tyrosinase, Pmel17 and dopachrome tautomerase colocalize with each other in the control melanized B16 cells, tyrosinase does not colocalize with Pmel17 or its processed isoform and with dopachrome tautomerase in GSH-induced amelanotic B16 cells. The sum of these findings suggests that reduced glutathione selectively disrupts the intracellular trafficking of tyrosinase and tyrosinase-related protein-1 but not dopachrome tautomerase and Pmel17 to melanosomes, which results in the attenuation of melanization, probably serving as a putative model for oculocutaneous albinism type 4. PMID:23764898

Nakajima, Hiroaki; Nagata, Takeshi; Koga, Shihiro; Imokawa, Genji



Satellite cell proliferation in the adult rat trigeminal ganglion results from the release of a mitogenic protein from explanted sensory neurons [published erratum appears in J Cell Biol 1994 Jun;125(6):1429  

PubMed Central

Explant of trigeminal ganglia neurons in adult rats induces perineuronal glial proliferation of primarily satellite cells as opposed to Schwann cells. This proliferation begins at 15 h after explant culture and by 27 h there is a significant increase in glial proliferation as measured by scintillation counts of [3H]thymidine. Blocking protein synthesis between 0 and 3.5 h after explant culture (early) results in an enhanced proliferative response, while blocking protein synthesis between 3.5 and 7 h (late) causes a complete block of the proliferative response assessed at 27 h. Conditioned media experiments demonstrate that both the mitogenic and inhibitory signals are diffusible and heat labile. Finally, the addition of neurotrophic factors to rescue injured ganglionic neurons attenuates the proliferative glial response suggesting that injured neurons produce and release signals that induce glial proliferation. PMID:8132704



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  

Microsoft Academic Search

Cellulose synthase (CESA) is a critical catalytic subunit of the cellulose synthase complex responsible for glucan chain elongation.\\u000a Our knowledge about how CESA functions is still very limited. Here, we report the functional characterization of a rice mutant,\\u000a brittle culm11, that shows growth retardation and dramatically reduced plant strength. Map-based cloning revealed that all the mutant phenotypes\\u000a result from a

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



Silencing of BNIP3 Results from Promoter Methylation by DNA Methyltransferase 1 Induced by the Mitogen-Activated Protein Kinase Pathway  

PubMed Central

We have previously shown that Ras mediates NO-induced BNIP3 expression via the MEK-ERK-HIF-1 pathway in mouse macrophages, and that NO-induced death results at least in part from the induction of BNIP3. In the present study, we describe another aspect of Ras regulation of BNIP3 expression in pancreatic cancer cells. Human BNIP3 promoter-driven luciferase activity was efficiently induced by activated Ras in AsPC-1, Miapaca-2, PK-1 and PANC-1 cells. However, expression of endogenous BNIP3 was not induced, and BNIP3 up-regulation by hypoxia was also inhibited. Treatment of the cells with the DNMT inhibitor, 5- aza-2-deoxycytidine, restored BNIP3 induction, indicating that DNA methylation of the BNIP3 promoter was responsible for the inhibition of BNIP3 induction. Furthermore, inhibition of the MEK pathway with U0126 reduced DNMT1 expression, but not that of DNMT3a and 3b, and restored the hypoxia-inducibility of BNIP3, suggesting that the DNA methylation of the BNIP3 promoter was mediated by DNMT1 via the MEK pathway. PMID:21573703

An, Hyun-Jung; Lee, Hayyoung; Paik, Sang-Gi



ProteinProtein Interactions  

E-print Network

to nitrocellulose or PVDF membrane, and then incubating with a probe of interest. The probe is typically a protein, samples are run on SDS-PAGE gels, transferred to nitrocellulose or PVDF, and then overlaid with a soluble

Hall, Randy A


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


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

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



Resistance to nitrophenolic herbicides and metronidazole in the cyanobacterium Synechocystis sp. PCC 6803 as a result of the inactivation of a nitroreductase-like protein encoded by drgA gene.  


Dinoseb is a herbicide known to inhibit photosystem II electron transfer like DCMU, triazine and phenolic-type herbicides. The mutant Din7 of the cyanobacterium Synechocystis sp. PCC 6803, selected for resistance to dinoseb, and the mutant Ins2, constructed by the insertion of the kanamycin resistance cassette into the drgA gene, were cross-resistant to other nitrophenolic herbicides (DNOC, 2,4-dinitrophenol) and to the cell inhibitor metronidazole but not to the photosystem II inhibitors DCMU or ioxynil. The Din7 mutant had the same characteristics of photosystem II inhibition by dinoseb as the wild type. This result suggested the existence of another site for dinoseb inhibition. The wild type cells modified dinoseb to a non-toxic product that gave an absorption spectrum similar to that of dithionite treated dinoseb containing reduced nitro groups. In contrast, the Din7 mutant did not modify dinoseb. These phenomena were controlled by the drgA gene encoding a protein which showed similarity to several enzymes having nitroreductase activity. The addition of superoxide dismutase to the medium relieved the toxic effect of dinoseb in wild type cells but not in Din7. It is proposed that in wild type cells of Synechocystis sp. PCC 6803 the DrgA protein is involved in detoxification of dinoseb via the reduction of the nitro group(s) and this process is accompanied by the formation of toxic superoxide anions. Mutations blocking the activity of the DrgA protein lead to the development of resistance to nitrophenolic herbicides and metronidazole. PMID:9654132

Elanskaya, I V; Chesnavichene, E A; Vernotte, C; Astier, C



Protein Condensation  

NASA Astrophysics Data System (ADS)

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.

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



Protein Condensation  

NASA Astrophysics Data System (ADS)

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.

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



Deletion of mitochondrial associated ubiquitin fold modifier protein Ufm1 in Leishmania donovani results in loss of ?-oxidation of fatty acids and blocks cell division in the amastigote stage  

PubMed Central

Summary Recently, we described the existence of the ubiquitin fold modifier1 (Ufm1) and its conjugation pathway in Leishmania donovani. We demonstrated the conjugation of Ufm1 to proteins such as mitochondrial trifunctional protein (MTP) that catalyzes ?-oxidation of fatty acids in L. donovani. To elucidate the biological roles of the Ufm1-mediated modifications, we made an L. donovani Ufm1 null mutant (Ufm1?/?). Loss of Ufm1 and consequently absence of Ufm1 conjugation with MTP resulted in diminished acetyl-CoA, the end product of the ?-oxidation in the Ufm1?/? amastigote stage. The Ufm1?/? mutants showed reduced survival in the amastigote stage in vitro and ex vivo in human macrophages. This survival was restored by re-expression of wild type Ufm1 with concomitant induction of acetyl-CoA but not by re-expressing the non-conjugatable Ufm1, indicating the essential nature of Ufm1 conjugation and ?-oxidation. Both cell cycle analysis and ultrastructural studies of Ufm1?/? parasites confirmed the role of Ufm1 in amastigote growth. The defect in vitro growth of amstigotes in human macrophages was further substantiated by reduced survival. Therefore, these studies suggest the importance of Ufm1 in Leishmania pathogenesis with larger impact on other organisms and further provide an opportunity to test Ufm1?/? parasites as drug and vaccine targets. PMID:22897198

Gannavaram, Sreenivas; Connelly, Patricia S; Daniels, Mathew P; Duncan, Robert; Salotra, Poonam; Nakhasi, Hira L



Protein C  


Protein C is a normal substance in the body that prevents blood clotting. A blood test can be done ... forming (anticoagulants), such as warfarin (Coumadin), decrease protein C and protein S levels. Your doctor may ask you ...


Detection of IgM responses to bovine respiratory syncytial virus by indirect ELISA following experimental infection and reinfection of calves: abolition of false positive and false negative results by pre-treatment of sera with protein-G agarose.  


The IgM responses in three panels of sera generated by infection and reinfection of calves with bovine respiratory syncytial virus (BRSV) were measured by indirect ELISA (I-ELISA). The effect of depleting serum IgG by pre-treatment with protein G agarose (PGA) was evaluated. Following primary infection a weak IgM response was detected in the untreated sera of 3 out of 4 calves with maternally derived antibody (MDA). Both the magnitude and duration of the specific IgM responses in these calves were increased by pre-treatment with PGA. In addition, the fourth infected calf tested gave a single positive IgM result following PGA treatment. Transient or persistent IgM responses which were abolished by pre-treatment of sera with PGA were detected in 4/8 calves following reinfection. These were considered to be false positive results, consistent with the influence of IgM rheumatoid factor (IgM-RF). One of these calves and two additional calves showed transient increases in IgM which were resistant to PGA treatment. These were considered to represent specific IgM responses to reinfection. The results indicate the ability of PGA treatment to eliminate both false positive and false negative results and emphasise the necessity for controlling the influence of IgM-RF in IgM-specific indirect ELISAs. PMID:10522785

Graham, D A; Foster, J C; Mawhinney, K A; Elvander, M; Adair, B M; Merza, M



Effect of protein, unsaturated fat, and carbohydrate intakes on plasma apolipoprotein B and VLDL and LDL containing apolipoprotein C-III: results from the OmniHeart Trial2  

PubMed Central

Background Plasma apolipoprotein B (apo B) and VLDL and LDL with apolipoprotein C-III (apo C-III) are independent risk factors for cardiovascular disease (CVD). Dietary intake affects lipoprotein concentration and composition related to those apolipoproteins. Objective We studied differences in apo B lipoproteins with and without apo C-III after 3 healthy diets based on the Dietary Approaches to Stop Hypertension Trial diet. Design Healthy participants (n = 162) were fed each of 3 healthy diets for 6 wk in a crossover design. Diets differed by emphasis of either carbohydrate (Carb), unsaturated fat (Unsat), or protein (Prot). Blood was collected at baseline and after diets for analysis. Results Compared with the Carb diet, the Prot diet reduced plasma apo B and triglycerides in VLDL with apo C-III (16%, P = 0.07; 11%, P = 0.05, respectively) and apo B in LDL with apo C-III (16%, P = 0.04). Compared with the Unsat diet, the Prot diet reduced triglycerides in VLDL with apo C-III (16%, P = 0.02). Compared with baseline (subjects' usual diet was higher in saturated fat), the Prot diet reduced apo B in LDL with apo C-III (11%, P = 0.05), and all 3 diets reduced plasma total apo B (6?10%, P < 0.05) and apo B in the major type of LDL, LDL without apo C-III (8 ?10%, P < 0.01). All 3 diets reduced the ratio of apo C-III to apo E in VLDL. Conclusions Substituting protein for carbohydrate in the context of a healthy dietary pattern reduced atherogenic apo C-III–containing LDL and its precursor, apo C-III–containing VLDL, resulting in the most favorable profile of apo B lipoproteins. In addition, compared with a typical high-saturated fat diet, healthy diets that emphasize carbohydrate, protein, or unsaturated fat reduce plasma total and LDL apo B and produce a lower more metabolically favorable ratio of apo C-III to apo E. PMID:18541549

Furtado, Jeremy D; Campos, Hannia; Appel, Lawrence J; Miller, Edgar R; Laranjo, Nancy; Carey, Vincent J; Sacks, Frank M



Dramatic structural changes resulting from the loss of a crucial hydrogen bond in the hinge region involved in C-terminal helix swapping in SurE: a survival protein from Salmonella typhimurium.  


Domain swapping is an interesting feature of some oligomeric proteins in which each protomer of the oligomer provides an identical surface for exclusive interaction with a segment or domain belonging to another protomer. Here we report results of mutagenesis experiments on the structure of C-terminal helix swapped dimer of a stationary phase survival protein from Salmonella typhimurium (StSurE). Wild type StSurE is a dimer in which a large helical segment at the C-terminus and a tetramerization loop comprising two ? strands are swapped between the protomers. Key residues in StSurE that might promote C-terminal helix swapping were identified by sequence and structural comparisons. Three mutants in which the helix swapping is likely to be avoided were constructed and expressed in E. coli. Three-dimensional X-ray crystal structures of the mutants H234A and D230A/H234A could be determined at 2.1 Å and 2.35 Å resolutions, respectively. Contrary to expectations, helix swapping was mostly retained in both the mutants. The loss of the crucial D230 OD2- H234 NE2 hydrogen bond (2.89 Å in the wild type structure) in the hinge region was compensated by new inter and intra-chain interactions. However, the two fold molecular symmetry was lost and there were large conformational changes throughout the polypeptide. In spite of these changes, the dimeric structure and an approximate tetrameric organization were retained, probably due to the interactions involving the tetramerization loop. Mutants were mostly functionally inactive, highlighting the importance of precise inter-subunit interactions for the symmetry and function of StSurE. PMID:23409101

Mathiharan, Yamuna Kalyani; Pappachan, Anju; Savithri, H S; Murthy, Mathur R N



Protein solubility modeling  

NASA Technical Reports Server (NTRS)

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.

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



Delayed formation of zero-valent selenium nanoparticles by Bacillus mycoides SeITE01 as a consequence of selenite reduction under aerobic conditions  

PubMed Central

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



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

PubMed Central

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

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



Combined Results from Solution Studies on Intact Influenza Virus M1 Protein and from a New Crystal Form of Its N-Terminal Domain Show That M1 Is an Elongated Monomer  

Microsoft Academic Search

The amino-terminal domain of influenza A virus matrix protein (residues 1–164) was crystallized at pH 7 into a new crystal form in space group P1. This packing of the protein implies that M1(1–164) was monomeric in solution when it crystallized. Otherwise, the structure of the M1 fragment in the pH 7 crystals was the same as the monomers in crystals

Steffi Arzt; Florence Baudin; Annie Barge; Peter Timmins; Wim P. Burmeister; Rob W. H. Ruigrok



Protein folds and protein folding  

PubMed Central

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

Schaeffer, R. Dustin; Daggett, Valerie



Yoonjung Seo Phone: +1(347)287-7022  

E-print Network

of waste-to-energy (WTE) and the potential for improvement in the Republic of Korea · Contributed. Yoonjung, S. (2013), Current MSW Management and Waste-to-Energy Status in the Republic of Korea, WTERT and Assessment of Applicability of Waste- to-Energy Technologies in Korea. International Solid Waste Association


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.  


The insulin-like growth factor (IGF) axis plays an essential role in the development of the mammary gland. High circulating levels of IGF-I and of its major binding protein IGFBP3 have been related with increased mammographic density in Caucasian premenopausal women. Some common single nucleotide polymorphisms (SNPs) in genes of the IGF pathway have also been suggested to play a role in mammographic density. We conducted a cross-sectional study nested within the large Mexican ESMaestras cohort to investigate the relation between circulating levels of IGF-I, IGFBP-3, the IGF-I/IGFBP-3 ratio, five common SNPs in the IGF-1, IGFBP-3 and IGF-1R genes and mammographic density in 593 premenopausal Mexican women. Mean age at mammogram was 43.1 (standard deviation, SD = 3.7) years, and average body mass index (BMI) at recruitment was 28.5 kg/m(2). Mean percent mammographic density was 36.5% (SD: 17.1), with mean dense tissue area of 48.3 (SD: 33.3) cm(2) . Mean IGF-I and IGFBP-3 concentrations were 15.33 (SD: 5.52) nmol/l and 114.96 (SD: 21.34) nmol/l, respectively. No significant associations were seen between percent density and biomarker concentrations, but women with higher IGF-I and IGF-I/IGFBP-3 concentrations had lower absolute dense (p(trend) = 0.03 and 0.09, respectively) and nondense tissue areas (p(trend) < 0.001 for both parameters). However, these associations were null after adjustment by BMI. SNPs in specific genes were associated with circulating levels of growth factors, but not with mammographic density features. These results do not support the hypothesis of a strong association between circulating levels of growth hormones and mammographic density in Mexican premenopausal women. PMID:24037648

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



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.  


Body composition in HIV-infected individuals is subject to many influences. We conducted a pilot 6-month randomized trial of 68 women living with AIDS (WLA) from rural India. High protein intervention combined with education and supportive care delivered by HIV-trained village women (activated social health activist [Asha] 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

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



Biases in Drosophila melanogaster protein trap screens  

E-print Network

genome, the repertoire of genes that yield protein traps is still small. Results We analysed the collection of available protein trap lines in Drosophila melanogaster and identified potential biases that are likely to restrict genome coverage in protein...

Aleksic, Jelena; Lazic, Ranko; Muller, Ilka; Russell, Steven R; Adryan, Boris



Protein-Losing Gastroenteropathy  

PubMed Central

In the past 10 years with the development of improved methods, particularly radioisotope techniques, it has been demonstrated that a number of patients with gastrointestinal disease and depletion of plasma proteins become hypoproteinemic because of actual leakage of albumin and other plasma proteins into the lumen of the gastrointestinal tract. The site of protein leakage is variable depending on the underlying pathological state but the loss of protein-containing lymph through the gastrointestinal lymphatic channels seems to be the major mechanism for hypoproteinemia. It has become apparent that there exists a normal mechanism for secretion of plasma proteins into the gastrointestinal tract as part of the overall metabolism of the plasma proteins. When the process is exaggerated so that resynthesis of plasma protein cannot keep pace with its degradation, sometimes severe hypoproteinemia is the result. Such a pathological process has now been described in approximately 40 disease states. A review of all the techniques which can demonstrate gastroenteric protein loss reveals that there are no widely available quantitative tests but that accurate quantitation is not necessary for the diagnosis of protein losing gastroenteropathy. PMID:18730025

Pops, Martin A.



Protein Data Bank.  

National Technical Information Service (NTIS)

The Protein Data Bank is the international depository for the results of structural studies of biological macromolecules. At present, some 1200 biological macromolecules are reported to have been crystallized, of which approximately 350 have structures de...

E. E. Abola, F. C. Bernstein, T. F. Koetzle



Protein engineering of cellulases.  


This review covers the topic of protein engineering of cellulases, mostly after 2009. Two major trends that are identified in this work are: first, the increased importance of results from computational protein engineering to drive ideas in the field, as experimental ideas and results often are still scarce, and, second, the further development of helper proteins for cellulose hydrolysis, such as lytic polysaccharide monooxygenase (LPO). The discussion in this work focuses both on improved attributes of cellulases and on the domains of cellulase that have been improved. PMID:24794535

Bommarius, Andreas S; Sohn, Minjeong; Kang, Yuzhi; Lee, Jay H; Realff, Matthew J



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

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



Mutations in the yeast RNA14 and RNA15 genes result in an abnormal mRNA decay rate; sequence analysis reveals an RNA-binding domain in the RNA15 protein.  

PubMed Central

In Saccharomyces cerevisiae, temperature-sensitive mutations in the genes RNA14 and RNA15 correlate with a reduction of mRNA stability and poly(A) tail length. Although mRNA transcription is not abolished in these mutants, the transcripts are rapidly deadenylated as in a strain carrying an RNA polymerase B(II) temperature-sensitive mutation. This suggests that the primary defect could be in the control of the poly(A) status of the mRNAs and that the fast decay rate may be due to the loss of this control. By complementation of their temperature-sensitive phenotype, we have cloned the wild-type genes. They are essential for cell viability and are unique in the haploid genome. The RNA14 gene, located on chromosome H, is transcribed as three mRNAs, one major and two minor, which are 2.2, 1.5, and 1.1 kb in length. The RNA15 gene gives rise to a single 1.2-kb transcript and maps to chromosome XVI. Sequence analysis indicates that RNA14 encodes a 636-amino-acid protein with a calculated molecular weight of 75,295. No homology was found between RNA14 and RNA15 or between RNA14 and other proteins contained in data banks. The RNA15 DNA sequence predicts a protein of 296 amino acids with a molecular weight of 32,770. Sequence comparison reveals an N-terminal putative RNA-binding domain in the RNA15-encoded protein, followed by a glutamine and asparagine stretch similar to the opa sequences. Both RNA14 and RNA15 wild-type genes, when cloned on a multicopy plasmid, are able to suppress the temperature-sensitive phenotype of strains bearing either the rna14 or the rna15 mutation, suggesting that the encoded proteins could interact with each other. Images PMID:1674817

Minvielle-Sebastia, L; Winsor, B; Bonneaud, N; Lacroute, F



Quantification of the Influence of Protein-Protein Interactions on Adsorbed Protein Structure and Bioactivity  

PubMed Central

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 2 h to saturate the surface, followed by immersion in pure buffer solution for 15 h 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

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



HIV protein sequence hotspots for crosstalk with host hub proteins.  


HIV proteins target host hub proteins for transient binding interactions. The presence of viral proteins in the infected cell results in out-competition of host proteins in their interaction with hub proteins, drastically affecting cell physiology. Functional genomics and interactome datasets can be used to quantify the sequence hotspots on the HIV proteome mediating interactions with host hub proteins. In this study, we used the HIV and human interactome databases to identify HIV targeted host hub proteins and their host binding partners (H2). We developed a high throughput computational procedure utilizing motif discovery algorithms on sets of protein sequences, including sequences of HIV and H2 proteins. We identified as HIV sequence hotspots those linear motifs that are highly conserved on HIV sequences and at the same time have a statistically enriched presence on the sequences of H2 proteins. The HIV protein motifs discovered in this study are expressed by subsets of H2 host proteins potentially outcompeted by HIV proteins. A large subset of these motifs is involved in cleavage, nuclear localization, phosphorylation, and transcription factor binding events. Many such motifs are clustered on an HIV sequence in the form of hotspots. The sequential positions of these hotspots are consistent with the curated literature on phenotype altering residue mutations, as well as with existing binding site data. The hotspot map produced in this study is the first global portrayal of HIV motifs involved in altering the host protein network at highly connected hub nodes. PMID:21858059

Sarmady, Mahdi; Dampier, William; Tozeren, Aydin



Rab proteins  

Microsoft Academic Search

Rab proteins form the largest branch of the Ras superfamily of GTPases. They are localized to the cytoplasmic face of organelles and vesicles involved in the biosynthetic\\/secretory and endocytic pathways in eukaryotic cells. It is now well established that Rab proteins play an essential role in the processes that underlie the targeting and fusion of transport vesicles with their appropriate

Olivier Martinez; Bruno Goud



Interfacial protein-protein associations.  


While traditional models of protein adsorption focus primarily on direct protein-surface interactions, recent findings suggest that protein-protein interactions may play a central role. Using high-throughput intermolecular resonance energy transfer (RET) tracking, we directly observed dynamic, protein-protein associations of bovine serum albumin on polyethylene glycol modified surfaces. The associations were heterogeneous and reversible, and associating molecules resided on the surface for longer times. The appearance of three distinct RET states suggested a spatially heterogeneous surface - with areas of high protein density (i.e., strongly interacting clusters) coexisting with mobile monomers. Distinct association states exhibited characteristic behavior, i.e., partial-RET (monomer-monomer) associations were shorter-lived than complete-RET (protein-cluster) associations. While the fractional surface area covered by regions with high protein density (i.e., clusters) increased with increasing concentration, the distribution of contact times between monomers and clusters was independent of solution concentration, suggesting that associations were a local phenomenon, and independent of the global surface coverage. PMID:24274729

Langdon, Blake B; Kastantin, Mark; Walder, Robert; Schwartz, Daniel K



Protein disulfide engineering.  


Improving the stability of proteins is an important goal in many biomedical and industrial applications. A logical approach is to emulate stabilizing molecular interactions found in nature. Disulfide bonds are covalent interactions that provide substantial stability to many proteins and conform to well-defined geometric conformations, thus making them appealing candidates in protein engineering efforts. Disulfide engineering is the directed design of novel disulfide bonds into target proteins. This important biotechnological tool has achieved considerable success in a wide range of applications, yet the rules that govern the stabilizing effects of disulfide bonds are not fully characterized. Contrary to expectations, many designed disulfide bonds have resulted in decreased stability of the modified protein. We review progress in disulfide engineering, with an emphasis on the issue of stability and computational methods that facilitate engineering efforts. PMID:24291258

Dombkowski, Alan A; Sultana, Kazi Zakia; Craig, Douglas B



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


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

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



Green fluorescent protein.  


Several bioluminescent coelenterates use a secondary fluorescent protein, the green fluorescent protein (GFP), in an energy transfer reaction to produce green light. The most studied of these proteins have been the GFPs from the jellyfish Aequorea victoria and the sea pansy Renilla reniformis. Although the proteins from these organisms are not identical, they are thought to have the same chromophore, which is derived from the primary amino acid sequence of GFP. The differences are thought to be due to changes in the protein environment of the chromophore. Recent interest in these molecules has arisen from the cloning of the Aequorea gfp cDNA and the demonstration that its expression in the absence of other Aequorea proteins results in a fluorescent product. This demonstration indicated that GFP could be used as a marker of gene expression and protein localization in living and fixed tissues. Bacterial, plant and animal (including mammalian) cells all express GFP. The heterologous expression of the gfp cDNA has also meant that it could be mutated to produce proteins with different fluorescent properties. Variants with more intense fluorescence or alterations in the excitation and emission spectra have been produced. PMID:7480149

Chalfie, M



Comparative effects of selenite and selenate on nitrate assimilation in barley seedlings  

NASA Technical Reports Server (NTRS)

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.

Aslam, M.; Harbit, K. B.; Huffaker, R. C.



Strategies for the detection of potential beet necrotic yellow vein virus genome recombinations which might arise as a result of growing A type coat protein gene-expressing sugarbeets in soil containing B type virus.  


We have searched for beet necrotic yellow vein virus (BNYVV) populations with a recombined genome which could possibly arise when transgenic sugarbeets expressing the coat protein gene of A type BNYVV are grown in soil containing Polymyxa betae carrying B type BNYVV, in soil samples from previous field release experiments and in a greenhouse model experiment. In order to accelerate the potential evolution of virus populations with recombined genomes in the model experiment, eight successive crops of sugarbeet plantlets were grown in the same soil samples over a period of 3 years. For the sensitive detection of recombined BNYVV genomes, we used nested PCRs with sense primers that are preferentially extended on the A type BNYVV sequence in the region of the coat protein gene and antisense primers which are preferentially extended on the B type BNYVV sequence in a region downstream of the coat protein gene which is not present in the transgene. Controls with mixtures of sap from plants which were singly infected with A or with B type BNYVV only revealed that, unless proper precautions are taken, PCR-mediated recombination artifacts may readily be produced. A method was developed that is able to detect A type/B type recombinant RNA molecules up to dilutions of one to a million in pure B type RNA molecules. Inspite of this high sensitivity we failed to detect any BNYVV with a recombined genome in the transgenic plants of the model experiment or at the sites of the previous field release experiments. PMID:15070072

Koenig, R; Büttner, G



Electronic Transport via Proteins.  


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

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



Support vector machines for predicting protein-protein interactions using domains and hydrophobicity features  

Microsoft Academic Search

Since proteins work in the context of many other proteins and rarely work in isolation, it is highly important to study protein-protein interactions to understand proteins functions. The interactions data that have been identified by high-throughput technologies like the yeast two-hybrid system are known to yield many false positives. As a result, methods for computational prediction of protein-protein interactions based

Hany Alashwal; S. Deris; R. M. Othman



Protein Phosphatases  

NSDL National Science Digital Library

This Teaching Resource provides lecture notes and slides for a class covering the structure and function of protein phosphatases and is part of the course "Cell Signaling Systems: A Course for Graduate Students." The lecture begins with a discussion of the importance of phosphatases in physiology, recognized by the award of a Nobel Prize in 1992, and then proceeds to describe the two types of protein phosphatases: serine/threonine and tyrosine phosphatases. The information covered includes the structure, regulation, and substrate specificity of protein phosphatases, with an emphasis on their importance in disease and clinical settings.

Stephen R. Salton (Mount Sinai School of Medicine;Department of Neuroscience REV)



Transport Proteins  

NSDL National Science Digital Library

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

Jack D. Thatcher (Lewisburg;West Virginia School of Osteopathic Medicine REV)



EDITORIAL: Precision proteins Precision proteins  

NASA Astrophysics Data System (ADS)

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 gro

Demming, Anna



Protein Sequence Databases  

Microsoft Academic Search

\\u000a With the availability of almost 150 completed genome sequences from both eukaryotic and prokaryotic organisms, efforts are\\u000a now being focused on the identification and functional analysis of the proteins encoded by these genomes. The rapidly emerging\\u000a field of proteomics, the large-scale analysis of these proteins, has started to generate huge amounts of data as a result\\u000a of the new information

Michele Magrane; Maria Jesus Martin; Claire O’Donovan; Rolf Apweiler


PROTEIN DESIGN: Proteins from Scratch  

NSDL National Science Digital Library

Access to the article is free, however registration and sign-in are required. It is not easy to predict a protein's structure from its linear amino acid sequence, and the reverse problem--designing a protein that will assume a particular folded shape--is even harder. But on page 82 in this issue, Dahiyat and Mayo have succeeded in creating a zinc finger fold de novo, which forms the same shaped protein as the natural one but without the usual stabilizing zinc ion. In his Perspective, DeGrado explains why this problem has been so intractable and where we can go now that Dahiyat and Mayo have paved the way.

William F. DeGrado (University of Pennsylvania School of Medicine;Department of Biochemistry and Biophysics)



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

SciTech Connect

In the 8;21 translocation, the AML1 gene, located at chromosome band 21q22, is translocated to chromosome 8 (q22), where it is fused to the ETO gene and transcribed as a chimeric gene. AML1 is the human homolog of the recently cloned mouse gene pebp2[alpha]B, homologous to the DNA binding [alpha] subunit of the polyoma enhancer factor pebp2. AML1 is also involved in a translocation with chromosome 3 that is seen in patients with therapy-related acute myeloid leukemia and myelodysplastic syndrome and in chronic myelogenous leukemia in blast crisis. The authors have isolated a fusion cDNA clone from a t(3;21) library derived from a patient with therapy-related myelodysplastic syndrome; this clone contains sequences from AML1 and from EAP, which have now been localized to ban 3q26. EAP has previously been characterized as a highly expressed small nuclear protein of 128 residues (EBER 1) associated with Epstein-Barr virus small RNA. The fusion clone contains the DNA binding 5[prime] part of AML1 that is fused to ETO in the t(8;21) and, in addition, at least one other exon. The translocation replaces the last nine codons of AML1 with the last 96 codons of EAP. The fusion does not maintain the correct reading frame of EAP and may not lead to a functional chimeric protein. 23 refs., 6 figs.

Nucifora, G.; Begy, C.R.; Rowley, J.D. (Univ. of Chicago, IL (United States)); Erickson, P.; Drabkin, H.A. (Univ. of Colorado Health Sciences Center, Denver, CO (United States))



Direct polymerization of proteins.  


We report the synthesis of active polymers of superfolder green fluorescent protein (sfGFP) in one step using Click chemistry. Up to six copies of the non-natural amino acids (nnAAs) p-azido-l-phenylalanine (pAzF) or p-propargyloxy-l-phenylalanine (pPaF) were site-specifically inserted into sfGFP by cell-free protein synthesis (CFPS). sfGFP containing two or three copies of these nnAAs were coupled by copper-catalyzed azide-alkyne cycloaddition to synthesize linear or branched protein polymers, respectively. The protein polymers retained ?63% of their specific activity (i.e., fluorescence) after coupling. Polymerization of a concentrated solution of triply substituted sfGFP resulted in fluorescent macromolecular particles. Our method can be generalized to synthesize polymers of a protein or copolymers of any two or more proteins, and the conjugation sites can be determined exactly by standard genetic manipulation. Polymers of proteins and small molecules can also be created with this technology to make a new class of scaffolds or biomaterials. PMID:24200191

Albayrak, Cem; Swartz, James R



Studying protein-protein interactions using peptide arrays.  


Screening of arrays and libraries of compounds is well-established as a high-throughput method for detecting and analyzing interactions in both biological and chemical systems. Arrays and libraries can be composed from various types of molecules, ranging from small organic compounds to DNA, proteins and peptides. The applications of libraries for detecting and characterizing biological interactions are wide and diverse, including for example epitope mapping, carbohydrate arrays, enzyme binding and protein-protein interactions. Here, we will focus on the use of peptide arrays to study protein-protein interactions. Characterization of protein-protein interactions is crucial for understanding cell functionality. Using peptides, it is possible to map the precise binding sites in such complexes. Peptide array libraries usually contain partly overlapping peptides derived from the sequence of one protein from the complex of interest. The peptides are attached to a solid support using various techniques such as SPOT-synthesis and photolithography. Then, the array is incubated with the partner protein from the complex of interest. Finally, the detection of the protein-bound peptides is carried out by using immunodetection assays. Peptide array screening is semi-quantitative, and quantitative studies with selected peptides in solution are required to validate and complement the screening results. These studies can improve our fundamental understanding of cellular processes by characterizing amino acid patterns of protein-protein interactions, which may even develop into prediction algorithms. The binding peptides can then serve as a basis for the design of drugs that inhibit or activate the target protein-protein interactions. In the current review, we will introduce the recent work on this subject performed in our and in other laboratories. We will discuss the applications, advantages and disadvantages of using peptide arrays as a tool to study protein-protein interactions. PMID:21243154

Katz, Chen; Levy-Beladev, Liron; Rotem-Bamberger, Shahar; Rito, Tiago; Rüdiger, Stefan G D; Friedler, Assaf



Understanding and predicting protein structure  

SciTech Connect

Protein structure prediction from sequence remains a premiere computational problem for modern molecular biology. Just as protein structure prediction may be divided into sub-problems of main-chain and side-chain placement, so the protein structure prediction track this year has been divided into sub-tracks of protein threading (organized by Daniel Fischer and Adam Godzik) and side-chain packing (organized by Su Chung and S. Subbiah). The result is an unusually rich tour through different levels of protein structure prediction, from coarse-grained prediction of the tertiary fold to the fine-grained atomic detail of individual side-chains. 8 refs.

Fischer, D. [Univ. of California, Los Angeles, CA (United States); Godzik, A. [Scripps Research Institute, La Jolla, CA (United States); Chung, S. [Uniformed Services Univ. of the Health Sciences, Bethesda, MD (United States)] [and others



Protein Structure (Part 1) Proteins are heteropolymers  

E-print Network

Protein Structure (Part 1) #12;Proteins are heteropolymers · hetero ­ (from the "shape" of the protein #12;Protein Structure Heirarchy ...AGARTWLYLIPDNLDK... Primary structure Secondary structure helix sheet Ter=ary structure Quaternary structure

Summa, Christopher M.


Protein Conformation and Diagnostic Tests: The Prion Protein  

Microsoft Academic Search

Background: Many clinical diagnostic tests depend on the accurate detection and quantification of proteins and peptides and their functions. Alterations of protein structure, and the resulting consequences on dynamics, can affect the outcome of laboratory tests. These changes can be a result of mutations, other in vivo factors, or even the experimental conditions of the diagnostic test. Approach: The relationship

Brian J. Bennion; Valerie Daggett



Dielectric response of hydrated proteins  

NASA Astrophysics Data System (ADS)

We study dipolar susceptibility of hydrated proteins, representing the average dipole moment induced at the hydrated protein by a uniform external field. This parameter shows remarkable variation among proteins. We find a negative value of the dipolar susceptibility for some proteins, which implies a dia-electric dipolar response and negative dielectrophoresis. Such proteins, even though carrying significant permanent dipole moments, repel from the 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. We therefore suggest 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 approximately 2 nm from the protein surface into the bulk. A similar correlation length of about 1 nm is found for the electrostatic potential. The model is applied to the analysis of light absorption by protein solutions in the THz window of radiation. Here we also find significant deviations of the absorption coefficient from the predictions of traditional theories.

Matyushov, Dmitry



Membrane Proteins A membrane protein is a  

E-print Network

Membrane Proteins A membrane protein is a protein molecule that is attached to, or associated with the membrane of a cell or an organelle. More than half of all proteins interact with membranes. #12;Membrane Proteins - Func2on Biological membranes consist of a phospholipid bilayer and a variety of proteins

Cavanagh, John


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

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



Spatial propagation of protein polymerization.  


We consider the spatial dependence of filamentous protein self-assembly. Through studying the cases where the spreading of aggregated material is dominated either by diffusion or by growth, we derive analytical results for the spatial evolution of filamentous protein aggregation, which we validate against Monte Carlo simulations. Moreover, we compare the predictions of our theory with experimental measurements of two systems for which we identify the propagation as either growth or diffusion controlled. Our results connect the macroscopic observables that characterize the spatial propagation of protein self-assembly with the underlying microscopic processes and provide physical limits on spatial propagation and prionlike behavior associated with protein aggregation. PMID:24655282

Cohen, S I A; Rajah, L; Yoon, C H; Buell, A K; White, D A; Sperling, R A; Vendruscolo, M; Terentjev, E M; Dobson, C M; Weitz, D A; Knowles, T P J



The t(10;11)(p13;q14) in the U937 cell line results in the fusion of the AF10 gene and CALM, encoding a new member of the AP-3 clathrin assembly protein family.  

PubMed Central

The translocation t(10;11)(p13;q14) is a recurring chromosomal abnormality that has been observed in patients with acute lymphoblastic leukemia as well as acute myeloid leukemia. We have recently reported that the monocytic cell line U937 has a t(10;11)(p13;q14) translocation. Using a combination of positional cloning and candidate gene approach, we cloned the breakpoint and were able to show that AF10 is fused to a novel gene that we named CALM (Clathrin Assembly Lymphoid Myeloid leukemia gene) located at 11q14. AF10, a putative transcription factor, had recently been cloned as one of the fusion partners of MLL. CALM has a very high homology in its N-terminal third to the murine ap-3 gene which is one of the clathrin assembly proteins. The N-terminal region of ap-3 has been shown to bind to clathrin and to have a high-affinity binding site for phosphoinositols. The identification of the CALM/AF10 fusion gene in the widely used U937 cell line will contribute to our understanding of the malignant phenotype of this line. Images Fig. 1 Fig. 3 PMID:8643484

Dreyling, M H; Martinez-Climent, J A; Zheng, M; Mao, J; Rowley, J D; Bohlander, S K



Potential Survival Benefit of Anti-Apoptosis Protein: Survivin-Derived Peptide Vaccine with and without Interferon Alpha Therapy for Patients with Advanced or Recurrent Urothelial Cancer--Results from Phase I Clinical Trials  

PubMed Central

We previously identified a human leukocyte antigen (HLA)-A24-restricted antigenic peptide, survivin-2B80–88, a member of the inhibitor of apoptosis protein family, recognized by CD8+cytotoxic T lymphocytes (CTL). In a phase I clinical trial of survivin-2B80-88 vaccination for metastatic urothelial cancer (MUC), we achieved clinical and immunological responses with safety. Moreover, our previous study indicated that interferon alpha (IFN?) enhanced the effects of the vaccine for colorectal cancer. Therefore, we started a new phase I clinical trial of survivin-2B80–88 vaccination with IFN? for MUC patients. Twenty-one patients were enrolled and no severe adverse event was observed. HLA-A24/survivin-2B80–88 tetramer analysis and ELISPOT assay revealed a significant increase in the frequency of the peptide-specific CTLs after vaccination in nine patients. Six patients had stable disease. The effects of IFN? on the vaccination were unclear for MUC. Throughout two trials, 30 MUO patients received survivin-2B80–88 vaccination. Patients receiving the vaccination had significantly better overall survival than a comparable control group of MUO patients without vaccination (P = 0.0009). Survivin-2B80–88 vaccination may be a promising therapy for selected patients with MUC refractory to standard chemotherapy. This trial was registered with UMIN00005859. PMID:24363758

Kitamura, Hiroshi; Nishida, Sachiyo; Takahashi-Takaya, Akari; Kawami, Sachiyo; Torigoe, Toshihiko; Hirohashi, Yoshihiko; Tsukamoto, Taiji; Sato, Noriyuki; Masumori, Naoya



Cotton and Protein Interactions  

SciTech Connect

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.

Goheen, Steven C.; Edwards, J. V.; Rayburn, Alfred R.; Gaither, Kari A.; Castro, Nathan J.



Protein constituents of the eggshell: eggshell-specific matrix proteins  

Microsoft Academic Search

In this article, we review the results of recent proteomic and genomic analyses of eggshell matrix proteins and draw attention\\u000a to the impact of these data on current understanding of eggshell formation and function. Eggshell-specific matrix proteins\\u000a from avian (ovocleidins and ovocalyxins) and non-avian (paleovaterin) shells are discussed. Two possible roles for eggshell-specific\\u000a matrix proteins have been proposed; both reflect

Megan L. H. Rose; Maxwell T. Hincke



Predicting Protein Interactions by Brownian Dynamics Simulations  

PubMed Central

We present a newly adapted Brownian-Dynamics (BD)-based protein docking method for predicting native protein complexes. The approach includes global BD conformational sampling, compact complex selection, and local energy minimization. In order to reduce the computational costs for energy evaluations, a shell-based grid force field was developed to represent the receptor protein and solvation effects. The performance of this BD protein docking approach has been evaluated on a test set of 24 crystal protein complexes. Reproduction of experimental structures in the test set indicates the adequate conformational sampling and accurate scoring of this BD protein docking approach. Furthermore, we have developed an approach to account for the flexibility of proteins, which has been successfully applied to reproduce the experimental complex structure from the structure of two unbounded proteins. These results indicate that this adapted BD protein docking approach can be useful for the prediction of protein-protein interactions. PMID:22500075

Meng, Xuan-Yu; Xu, Yu; Zhang, Hong-Xing; Mezei, Mihaly; Cui, Meng



Predicting protein interactions by Brownian dynamics simulations.  


We present a newly adapted Brownian-Dynamics (BD)-based protein docking method for predicting native protein complexes. The approach includes global BD conformational sampling, compact complex selection, and local energy minimization. In order to reduce the computational costs for energy evaluations, a shell-based grid force field was developed to represent the receptor protein and solvation effects. The performance of this BD protein docking approach has been evaluated on a test set of 24 crystal protein complexes. Reproduction of experimental structures in the test set indicates the adequate conformational sampling and accurate scoring of this BD protein docking approach. Furthermore, we have developed an approach to account for the flexibility of proteins, which has been successfully applied to reproduce the experimental complex structure from the structure of two unbounded proteins. These results indicate that this adapted BD protein docking approach can be useful for the prediction of protein-protein interactions. PMID:22500075

Meng, Xuan-Yu; Xu, Yu; Zhang, Hong-Xing; Mezei, Mihaly; Cui, Meng



Prediction of thermodynamic instabilities of protein solutions from simple protein-protein interactions  

NASA Astrophysics Data System (ADS)

Statistical thermodynamics of protein solutions is often studied in terms of simple, microscopic models of particles interacting via pairwise potentials. Such modelling can reproduce the short range structure of protein solutions at equilibrium and predict thermodynamics instabilities of these systems. We introduce a square well model of effective protein-protein interaction that embeds the solvent’s action. We modify an existing model [45] by considering a well depth having an explicit dependence on temperature, i.e. an explicit free energy character, thus encompassing the statistically relevant configurations of solvent molecules around proteins. We choose protein solutions exhibiting demixing upon temperature decrease (lysozyme, enthalpy driven) and upon temperature increase (haemoglobin, entropy driven). We obtain satisfactory fits of spinodal curves for both the two proteins without adding any mean field term, thus extending the validity of the original model. Our results underline the solvent role in modulating or stretching the interaction potential.

D'Agostino, Tommaso; Solana, José Ramón; Emanuele, Antonio



Energy Landscape and Transition State of Protein-Protein Association  

PubMed Central

Formation of a stereospecific protein complex is favored by specific interactions between two proteins but disfavored by the loss of translational and rotational freedom. Echoing the protein folding process, we have previously proposed a transition state for protein-protein association. Here we clarify the specification of the transition state by working with two types of toy models for protein association. A “hemisphere” model consists of two matching hemispheres as associating proteins, and a “crater” model consists of a spherical protein with a crater to which another spherical protein fits snugly. Short-range pairwise interactions between sites across the interface hold together the bound complex. Small relative translation and rotation between the subunits quickly destroy the interactions, leading to a sharp transition between the bound state with numerous short-range interactions but restricted translation and rotational freedom and the unbound state with, at most, a small number of interactions but expanded configurational freedom. This transition sets the outer boundary of the bound state as well as the transition state for association. The energy landscape is funnel-like, with the deep well of the bound state surrounded by a broad shallow basin. Calculations with the toy models suggest that mutational change in the interaction energy in the x-ray structure of a protein-protein complex, commonly used to approximate the effect on the association constant, overestimates the effect of mutation by 10–20%. With an eye toward specifying the transition states of actual protein complexes, we find that the total number of contacts between the subunits serves as a good surrogate of the interaction energy. This formalism of protein-protein association is applied to the barnase-barstar complex, reproducing the experimental results for the association rate over a wide range of ionic strength. PMID:17142273

Alsallaq, Ramzi; Zhou, Huan-Xiang



Elevated levels of high-sensitivity C-reactive protein are associated with mild cognitive impairment and its subtypes: results of a population-based case-control study.  


As high-sensitivity C-reactive protein (hsCRP) seems to be associated with an increased risk of cognitive decline, this nested case-control study examined the relation of hsCRP and mild cognitive impairment (MCI) at different time points. 148 MCI cases (106 amnestic, 42 non-amnestic (aMCI/naMCI)) and 148 matched controls were identified from a prospective population based cohort study of 4,359 participants (aged 50-80). HsCRP levels were measured 5 years before (baseline) and at the time of neuropsychological testing (follow-up). Odds ratios (OR) for hsCRP quartiles serum levels were calculated for the two time points using logistic regression analyses and were adjusted for cardiovascular covariates. In the fully adjusted model, baseline hsCRP levels were significantly associated with both MCI and aMCI (OR = 2.29, 95% confidence interval (CI), 1.01-5.15, first versus fourth quartile, respective OR = 2.73, 95% CI, 1.09-6.84). At follow-up, the fourth hsCRP quartile was associated with MCI (OR = 3.60, 95% CI, 1.55-8.33), aMCI (OR = 3.73, 95% CI, 1.52-9.17) and naMCI (OR = 3.66, 95% CI, 1.00-13.77). Elevated hsCRP levels, even detected five years before diagnosis, are associated with an at least twofold increased probability of MCI. These findings suggest that inflammation plays an important role in the development and presence of cognitive impairment. PMID:22008268

Dlugaj, Martha; Gerwig, Marcus; Wege, Natalia; Siegrist, Johannes; Mann, Klaus; Bröcker-Preuss, Martina; Dragano, Nico; Moebus, Susanne; Jöckel, Karl-Heinz; Bokhof, Beate; Möhlenkamp, Stefan; Erbel, Raimund; Weimar, Christian



Breakfast Proteins  

NSDL National Science Digital Library

In this activity, learners construct a cereal chain as a model of how proteins are made in the cell. Learners build their chain off of an initial template which represents a single copy of DNA and hand-written notes, replicating the process of transcription and translation. In this model, the letters of the chain correspond to the colors of the cereal (not the amino acids).

Yu, Julie



Protein Kinases  

NSDL National Science Digital Library

This Teaching Resource provides lecture notes and slides for a class covering the structure and function of protein kinases and is part of the course "Cell Signaling Systems: A Course for Graduate Students." The lecture begins with a discussion of the genomics and evolutionary relationships among kinases and then proceeds to describe the structure-function relationships of specific kinases, the molecular mechanisms underlying substrate specificity, and selected issues in regulation of kinase activity.

Avrom Caplan (Mount Sinai School of Medicine;Department of Pharmacology and Biological Chemistry REV)



Protein Factory  

NSDL National Science Digital Library

In this activity, learners take on the role of various parts of the cell in order to model the process of protein synthesis. Each learner receives a card describing, step by step, what s/he should be doing. Learners play the roles of either RNA codons, an RNA polymerase, messenger RNA, a ribosome, or transfer RNA. This activity is designed for a large group of 30 learners, but could be used with smaller groups of at least 10 learners.

Salter, Irene



Regulation of protein intake in adult dogs.  


To determine whether adult dogs are able to regulate their intake of protein, 2-year-old female Beagles were allowed self-selection from 2 diets that differed in concentration of protein. Such a feeding regimen permitted the dogs to select for protein without affecting energy intake. Within the 1st week of the 4-week study, the dogs had established their pattern of protein intake. They selected approximately 30% of metabolizable energy from protein. Adult female rats offered the same diets also selected 30% of metabolizable energy from protein. The results indicated that adult dogs are able to regulate protein and energy intake. PMID:6822458

Romsos, D R; Ferguson, D



Frontal chromatography of proteins  

Microsoft Academic Search

A mathematical model describing the dynamic adsorption of proteins in columns packed with spherical porous adsorbent particles is used to study the effect of axial dispersion on the performance of chromatographic systems. The values of the axial dispersion coefficient, DL, are estimated from a correlation based on a model describing axial dispersion in packed beds that provides satisfactory results when

G. A. Heeter; A. I. Liapis



Native PAGE Protein Expression  

E-print Network

, mg/ml BA Bovine serum albumin Bovine g-globulin Standard curve generation using known standards. A, use Bio-Rad's bovine serum albumin or bovine g-globulin to make your standard curve. For best results and bovine serum albumin standard Catalog # Description 500-0121 RC DC Protein Assay Kit I, includes RC

Lebendiker, Mario


Proteins and protein\\/surfactant mixtures at interfaces in motion  

Microsoft Academic Search

The research described in this thesis covers a number of aspects of the relation between surface properties and foaming properties of proteins, low molecular surfactants and mixtures thereof. This work is the result of a question of the industrial partners if it is possible to understand the foaming properties of protein hydrolysates. As there are many aspects of the surface

F. J. G. Boerboom



Protein-Protein Interaction Detection: Methods and Analysis  

PubMed Central

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

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



DNA to Protein  

NSDL National Science Digital Library

Explore the relationship between the genetic code on the DNA strand and the resulting protein and rudimentary shape it forms. Through models of transcription and translation, you will discover this relationship and the resilience to mutations built into our genetic code. Start by exploring DNAÂs double helix with an interactive 3D model. Highlight base pairs, look at one or both strands, and turn hydrogen bonds on or off. Next, watch an animation of transcription, which creates RNA from DNA, and translation, which Âreads the RNA codons to create a protein.

Consortium, The C.



Improving protein function prediction using domain and protein complexes in PPI networks  

PubMed Central

Background Characterization of unknown proteins through computational approaches is one of the most challenging problems in silico biology, which has attracted world-wide interests and great efforts. There have been some computational methods proposed to address this problem, which are either based on homology mapping or in the context of protein interaction networks. Results In this paper, two algorithms are proposed by integrating the protein-protein interaction (PPI) network, proteins’ domain information and protein complexes. The one is domain combination similarity (DCS), which combines the domain compositions of both proteins and their neighbors. The other is domain combination similarity in context of protein complexes (DSCP), which extends the protein functional similarity definition of DCS by combining the domain compositions of both proteins and the complexes including them. The new algorithms are tested on networks of the model species of Saccharomyces cerevisiae to predict functions of unknown proteins using cross validations. Comparing with other several existing algorithms, the results have demonstrated the effectiveness of our proposed methods in protein function prediction. Furthermore, the algorithm DSCP using experimental determined complex data is robust when a large percentage of the proteins in the network is unknown, and it outperforms DCS and other several existing algorithms. Conclusions The accuracy of predicting protein function can be improved by integrating the protein-protein interaction (PPI) network, proteins’ domain information and protein complexes. PMID:24655481



Mobilization of seed protein reserves.  


The mobilization of seed storage proteins upon seed imbibition and germination is a crucial process in the establishment of the seedling. Storage proteins fold compactly, presenting only a few vulnerable regions for initial proteolytic digestion. Evolutionarily related storage proteins have similar three-dimensional structure, and thus tend to be initially cleaved at similar sites. The initial cleavage makes possible subsequent rapid and extensive breakdown catalyzed by endo- and exopeptidases. The proteolytic enzymes that degrade the storage proteins during mobilization identified so far are mostly cysteine proteases, but also include serine, aspartic and metalloproteases. Plants often ensure early initiation of storage protein mobilization by depositing active proteases during seed maturation, in the very compartments where storage proteins are sequestered. Various means are used in such cases to prevent proteolytic attack until after imbibition of the seed with water. This constraint, however, is not always enforced as the dry seeds of some plant species contain proteolytic intermediates as a result of limited proteolysis of some storage proteins. Besides addressing fundamental questions in plant protein metabolism, studies of the mobilization of storage proteins will point out proteolytic events to avoid in large-scale production of cloned products in seeds. Conversely, proteolytic enzymes may be applied toward reduction of food allergens, many of which are seed storage proteins. PMID:22017287

Tan-Wilson, Anna L; Wilson, Karl A



PAS/LOV proteins  

PubMed Central

The light, oxygen or voltage (LOV) domain belongs to the Per-ARNT-Sim (PAS) superfamily of domains, and functions with the flavin chromophore as a module for sensing blue light in plants and fungi. The Arabidopsis thaliana PAS/LOV proteins (PLPs), of unknown function, possess an N-terminal PAS domain and a C-terminal LOV domain. Our recent analysis using yeast two-hybrid and Escherichia coli protein production systems reveals that the interactions of Arabidopsis PLPs with several proteins diminish under blue light illumination and that the PLP LOV domain may bind to a flavin chromophore. These results suggest that PLP functions as a blue light receptor. Homologs of PLP exist in rice, tomato and moss. The LOV domains of these PLP homologs form a distinct group in phylogenetic analysis. These facts suggest that PLP belongs to a new class of plant blue light receptor. PMID:19704421

Ogura, Yasunobu; Tokutomi, Satoru; Wada, Masamitsu



Protein aggregation in Escherichia coli: role of proteases.  


Protein aggregation is involved in several human diseases, and presumed to be an important process in protein quality control. In bacteria, aggregation of proteins occurs during stress conditions, such as heat shock. We studied the protein aggregates of Escherichia coli during heat shock. Our results demonstrate that the concentration and diversity of proteins in the aggregates depend on the availability of proteases. Aggregates obtained from mutants in the Lon (La) protease contain three times more protein than wild-type aggregates and show the broadest protein diversity. The results support the assumption that protein aggregates are formed from partially unfolded proteins that were not refolded by chaperones or degraded by proteases. PMID:11886743

Rosen, Ran; Biran, Dvora; Gur, Eyal; Becher, Dörte; Hecker, Michael; Ron, Eliora Z



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

SciTech Connect

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.

Curtis, R.A.; Blanch, H.W. [Univ. of California, Berkeley, CA (United States). Dept. of Chemical Engineering] [Univ. of California, Berkeley, CA (United States). Dept. of Chemical Engineering; Prausnitz, J.M. [Lawrence Berkeley Lab., CA (United States)] [Lawrence Berkeley Lab., CA (United States)



Proteomic analysis of membrane proteins of vero cells: exploration of potential proteins responsible for virus entry.  


Vero cells are highly susceptible to many viruses in humans and animals, and its membrane proteins (MPs) are responsible for virus entry. In our study, the MP proteome of the Vero cells was investigated using a shotgun LC-MS/MS approach. Six hundred twenty-seven proteins, including a total of 1839 peptides, were identified in MP samples of the Vero cells. In 627 proteins, 307 proteins (48.96%) were annotated in terms of biological process of gene ontology (GO) categories; 356 proteins (56.78%) were annotated in terms of molecular function of GO categories; 414 proteins (66.03%) were annotated in terms of cellular components of GO categories. Of 627 identified proteins, seventeen proteins had been revealed to be virus receptor proteins. The resulting protein lists and highlighted proteins may provide valuable information to increase understanding of virus infection of Vero cells. PMID:24286161

Guo, Donghua; Zhu, Qinghe; Zhang, Hong; Sun, Dongbo



Protein-sparing therapy in the postoperative period  

Microsoft Academic Search

Surgical trauma leads to increased nitrogen loss which may result from increased protein breakdown, diminished protein synthesis, or both. Increased protein breakdown is predominant after surgical trauma, whereas protein synthesis has been reported to be either unchanged or increased. Attempts to improve nitrogen balance should be directed toward both mechanisms; protein breakdown is diminished by avoiding tissue damage, and by

Ulrich Keller; Didier Clerc; Marius Krfinzlin; M. Heberer



Reduced protein adsorption by osmolytes.  


Osmolytes are substances that affect osmosis and are used by cells to adapt to environmental stress. Here, we report a neutron reflectivity study on the influence of some osmolytes on protein adsorption at solid-liquid interfaces. Bovine ribonuclease A (RNase) and bovine insulin were used as model proteins adsorbing at a hydrophilic silica and at a hydrophobic polystyrene surface. From the neutron reflectivity data, the adsorbed protein layers were characterized in terms of layer thickness, protein packing density, and adsorbed protein mass in the absence and presence of urea, trehalose, sucrose, and glycerol. All data point to the clear effect of these nonionic cosolvents on the degree of protein adsorption. For example, 1 M sucrose leads to a reduction of the adsorbed amount of RNase by 39% on a silica surface and by 71% on a polystyrene surface. Trehalose was found to exhibit activity similar to that of sucrose. The changes in adsorbed protein mass can be attributed to a decreased packing density of the proteins in the adsorbed layers. Moreover, we investigated insulin adsorption at a hydrophobic surface in the absence and presence of glycerol. The degree of insulin adsorption is decreased by even 80% in the presence of 4 M of glycerol. The results of this study demonstrate that nonionic cosolvents can be used to tune and control nonspecific protein adsorption at aqueous-solid interfaces, which might be relevant for biomedical applications. PMID:21568286

Evers, Florian; Steitz, Roland; Tolan, Metin; Czeslik, Claus



Coulomb energies of protein-protein complexes with monopole-free charge distributions.  


From an analysis of Coulomb energy distributions of a large set of protein-protein complexes we show that the positive tail in the energy distribution disappears when the monopole-monopole term, the only energy term independent of inter-subunit orientations, is removed. This indicates that unfavorable Coulomb energies associated with subunit orientations are excluded in protein-protein complexes. The overall result remained unchanged when solvent effects were included. Our results have important bearing on the restriction of subunit orientations in protein-protein complexes and complement a recent work [K. Brock, K. Talley, K. Coley, P. Kundrotas, E. Alexov, Optimization of electrostatic interactions in protein-protein complexes, Biophys. J. 93 (2007) 3340-3352.] which showed that Coulomb energy of interaction in protein-protein complexes is sequence-optimized. PMID:19167253

Das, Madhurima; Basu, Gautam



Engineering protein farnesyltransferase for enzymatic protein labeling applications.  


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

Dozier, Jonathan K; Khatwani, Santoshkumar L; Wollack, James W; Wang, Yen-Chih; Schmidt-Dannert, Claudia; Distefano, Mark D



Discover protein sequence signatures from protein-protein interaction data  

E-print Network

Background: The development of high-throughput technologies such as yeast two- hybrid systems and mass spectrometry technologies has made it possible to generate large protein-protein interaction ( PPI) datasets. Mining ...

Fang, Jianwen; Haasl, R. J.; Dong, Yinghua; Lushington, Gerald H.



Dynamics of protein conformations  

NASA Astrophysics Data System (ADS)

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.

Stepanova, Maria



Protein Stability in Ice  

PubMed Central

This study presents an experimental approach, based on the change of Trp fluorescence between native and denatured states of proteins, which permits to monitor unfolding equilibria and the thermodynamic stability (?G°) of these macromolecules in frozen aqueous solutions. The results obtained by guanidinium chloride denaturation of the azurin mutant C112S from Pseudomonas aeruginosa, in the temperature range from ?8 to ?16°C, demonstrate that the stability of the native fold may be significantly perturbed in ice depending mainly on the size of the liquid water pool (VL) in equilibrium with the solid phase. The data establish a threshold, around VL = 1.5%, below which in ice ?G° decreases progressively relative to liquid state, up to 3 kcal/mole for VL = 0.285%. The sharp dependence of ?G° on VL is consistent with a mechanism based on adsorption of the protein to the ice surface. The reduction in ?G° is accompanied by a corresponding decrease in m-value indicating that protein-ice interactions increase the solvent accessible surface area of the native fold or reduce that of the denatured state, or both. The method opens the possibility for examining in a more quantitative fashion the influence of various experimental conditions on the ice perturbation and in particular to test the effectiveness of numerous additives used in formulations to preserve labile pharmaco proteins. PMID:17189314

Strambini, Giovanni B.; Gonnelli, Margherita



Protein Adsorption to Biomaterials  

Microsoft Academic Search

Within milliseconds after biomaterials come in contact with a biological fluid such as blood, proteins begin to adhere to\\u000a the surface through a process known as protein adsorption. Protein adsorption is initially strongly influenced by protein\\u000a diffusion, but protein affinity for the surface becomes critically important and, over time, higher-affinity proteins can\\u000a be replaced by lower-affinity proteins in a dynamic

David Richard Schmidt; Heather Waldeck; Weiyuan John Kao


SAMPI: Protein Identification with Mass Spectra Alignments  

Microsoft Academic Search

Background: Mass spectrometry based peptide mass fingerprints (PMFs) offer a fast, efficient, and robust method for protein identification. A protein is digested (usually by trypsin) and its mass spectrum is compared to simulated spectra for protein sequences in a database. However, existing tools for analyzing PMFs often suffer from missing or heuristic analysis of the significance of search results and

Hans-michael Kaltenbach; Andreas Wilke; Sebastian Böcker



Preservation of protein clefts in comparative models  

Microsoft Academic Search

BACKGROUND: Comparative, or homology, modelling of protein structures is the most widely used prediction method when the target protein has homologues of known structure. Given that the quality of a model may vary greatly, several studies have been devoted to identifying the factors that influence modelling results. These studies usually consider the protein as a whole, and only a few

David Piedra; Sergi Lois; Xavier de la Cruz



On Hydrophobicity and Conformational Specificity in Proteins  

E-print Network

On Hydrophobicity and Conformational Specificity in Proteins Erik Sandelin1 2 Stockholm of monomeric globular single domain proteins. We find that the total fraction of hydrophobic residues is roughly constant and has no discernible dependence on protein size. This results in a decrease

Sandelin, Erik


Study of the Assembly of Vesicular Stomatitis Virus N Protein: Role of the P Protein  

PubMed Central

To derive structural information about the vesicular stomatitis virus (VSV) nucleocapsid (N) protein, the N protein and the VSV phosphoprotein (P protein) were expressed together in Escherichia coli. The N and P proteins formed soluble protein complexes of various molar ratios when coexpressed. The major N/P protein complex was composed of 10 molecules of the N protein, 5 molecules of the P protein, and an RNA. A soluble N protein-RNA oligomer free of the P protein was isolated from the N/P protein-RNA complex using conditions of lowered pH. The molecular weight of the N protein-RNA oligomer, 513,879, as determined by analytical ultracentrifugation, showed that it was composed of 10 molecules of the N protein and an RNA of approximately 90 nucleotides. The N protein-RNA oligomer had the appearance of a disk with outer diameter, inner diameter, and thickness of 148 ± 10 ?, 78 ± 9 ?, and 83 ± 8 ?, respectively, as determined by electron microscopy. RNA in the complexes was protected from RNase digestion and was stable at pH 11. This verified that N/P protein complexes expressed in E. coli were competent for encapsidation. In addition to coexpression with the full-length P protein, the N protein was expressed with the C-terminal 72 amino acids of the P protein. This portion of the P protein was sufficient for binding to the N protein, maintaining it in a soluble state, and for assembly of N protein-RNA oligomers. With the results provided in this report, we propose a model for the assembly of an N/P protein-RNA oligomer. PMID:11000221

Green, Todd J.; Macpherson, Silvia; Qiu, Shihong; Lebowitz, Jacob; Wertz, Gail W.; Luo, Ming



Protein Aggregation and Protein Instability Govern Familial Amyotrophic Lateral Sclerosis Patient Survival  

Microsoft Academic Search

The nature of the “toxic gain of function” that results from amyotrophic lateral sclerosis (ALS)-, Parkinson-, and Alzheimer-related mutations is a matter of debate. As a result no adequate model of any neurodegenerative disease etiology exists. We demonstrate that two synergistic properties, namely, increased protein aggregation propensity (increased likelihood that an unfolded protein will aggregate) and decreased protein stability (increased

Qi Wang; Joshua L Johnson; Nathalie Y. R Agar; Jeffrey N Agar



Dietary protein to support muscle hypertrophy.  


Intact protein, protein hydrolysates, and free amino acids are popular ingredients in contemporary sports nutrition, and have been suggested to augment post-exercise recovery. Protein and/or amino acid ingestion stimulates skeletal muscle protein synthesis, inhibits protein breakdown and, as such, stimulates muscle protein accretion following resistance and endurance type exercise. This has been suggested to lead to a greater adaptive response to each successive exercise bout, resulting in more effective muscle reconditioning. Despite limited evidence, some basic guidelines can be defined regarding the preferred type, amount, and timing of dietary protein that should be ingested to maximize post-exercise muscle protein accretion. Whey protein seems most effective in stimulating muscle protein synthesis during acute post-exercise recovery. This is likely attributable to its rapid digestion and absorption kinetics and specific amino acid composition. Ingestion of approximately 20 g protein during and/or immediately after exercise is sufficient to maximize post-exercise muscle protein synthesis rates. Coingestion of a large amount of carbohydrate or free leucine is not warranted to further augment post- exercise muscle protein synthesis when ample protein is already ingested. Future research should focus on the relevance of the acute anabolic response following exercise to optimize the skeletal muscle adaptive response to exercise training. PMID:22301837

van Loon, Luc J C; Gibala, Martin J



Protein Quality Control in the Nucleus  

PubMed Central

In their natural environment, cells are regularly exposed to various stress conditions that may lead to protein misfolding, but also in the absence of stress, misfolded proteins occur as the result of mutations or failures during protein synthesis. Since such partially denatured proteins are prone to aggregate, cells have evolved several elaborate quality control systems to deal with these potentially toxic proteins. First, various molecular chaperones will seize the misfolded protein and either attempt to refold the protein or target it for degradation via the ubiquitin-proteasome system. The degradation of misfolded proteins is clearly compartmentalized, so unique degradation pathways exist for misfolded proteins depending on whether their subcellular localization is ER/secretory, mitochondrial, cytosolic or nuclear. Recent studies, mainly in yeast, have shown that the nucleus appears to be particularly active in protein quality control. Thus, specific ubiquitin-protein ligases located in the nucleus, target not only misfolded nuclear proteins, but also various misfolded cytosolic proteins which are transported to the nucleus prior to their degradation. In comparison, much less is known about these mechanisms in mammalian cells. Here we highlight recent advances in our understanding of nuclear protein quality control, in particular regarding substrate recognition and proteasomal degradation. PMID:25010148

Nielsen, Sofie V.; Poulsen, Esben G.; Rebula, Caio A.; Hartmann-Petersen, Rasmus



Quantitative study of protein-protein interactions by quartz nanopipettes.  


In this report, protein-modified quartz nanopipettes were used to quantitatively study protein-protein interactions in attoliter sensing volumes. As shown by numerical simulations, the ionic current through the conical-shaped nanopipette is very sensitive to the surface charge variation near the pore mouth. With the appropriate modification of negatively charged human neuroglobin (hNgb) onto the inner surface of a nanopipette, we were able to detect concentration-dependent current change when the hNgb-modified nanopipette tip was exposed to positively charged cytochrome c (Cyt c) with a series of concentrations in the bath solution. Such current change is due to the adsorption of Cyt c to the inner surface of the nanopipette through specific interactions with hNgb. In contrast, a smaller current change with weak concentration dependence was observed when Cyt c was replaced with lysozyme, which does not specifically bind to hNgb. The equilibrium dissociation constant (KD) for the Cyt c-hNgb complex formation was derived and the value matched very well with the result from surface plasmon resonance measurement. This is the first quantitative study of protein-protein interactions by a conical-shaped nanopore based on charge sensing. Our results demonstrate that nanopipettes can potentially be used as a label-free analytical tool to quantitatively characterize protein-protein interactions. PMID:25060094

Tiwari, Purushottam Babu; Astudillo, Luisana; Miksovska, Jaroslava; Wang, Xuewen; Li, Wenzhi; Darici, Yesim; He, Jin



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


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

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



Dual targeting of peroxisomal proteins  

PubMed Central

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

Ast, Julia; Stiebler, Alina C.; Freitag, Johannes; Bolker, Michael



Water at interface with proteins  

E-print Network

Water is essential for the activity of proteins. However, the effect of the properties of water on the behavior of proteins is only partially understood. Recently, several experiments have investigated the relation between the dynamics of the hydration water and the dynamics of protein. These works have generated a large amount of data whose interpretation is debated. New experiments measure the dynamics of water at low temperature on the surface of proteins, finding a qualitative change (crossover) that might be related to the slowing down and stop of the protein's activity (protein glass transition), possibly relevant for the safe preservation of organic material at low temperature. To better understand the experimental data several scenarios have been discussed. Here, we review these experiments and discuss their interpretations in relation with the anomalous properties of water. We summarize the results for the thermodynamics and dynamics of supercooled water at an interface. We consider also the effect of water on protein stability, making a step in the direction of understanding, by means of Monte Carlo simulations and theoretical calculations, how the interplay of water cooperativity and hydrogen bonds interfacial strengthening affects the protein cold denaturation.

Giancarlo Franzese; Valentino Bianco; Svilen Iskrov



Protein microarrays to detect protein-protein interactions using red and green fluorescent proteins.  


Proteomics, the study of protein function on a global scale, will play an important role in furthering our understanding of gene functions, complex biological pathways, and discovery of novel drug targets. A number of techniques have been developed for proteomic studies to identify and analyze proteins, compare protein expression levels, and study protein-protein interactions. Recent developments have applied a DNA array-type approach to immobilize proteins on a surface for high-throughput analysis. Here we report the development and construction of protein chips using derivatized glass and nitrocellulose-coated slides and the employment of recombinant proteins fused with green and red fluorescent proteins for detection. Fluorescent signals were found to be proportional to the amount of arrayed proteins and could be readily detected with a conventional fluorescence slide scanner. This technique allows the investigation of protein-protein interactions without the need for additional labeling steps of probe proteins. PMID:12069413

Kukar, Thomas; Eckenrode, Sarah; Gu, Yunrong; Lian, Wei; Megginson, Mike; She, Jin-Xiong; Wu, Donghai



Bioactive proteins from mushrooms  

Microsoft Academic Search

Mushrooms have been used as food or medicine for thousands of years. Due to low-fat content and absence of cholesterol, many mushrooms are excellent sources of protein. There are various mushroom proteins with interesting biological activities, such as lectins, fungal immunomodulatory proteins (FIP), ribosome inactivating proteins (RIP), ribonucleases, laccases, and other proteins, which have become popular sources of natural antitumor,

Xiaofei Xu; Huidan Yan; Jian Chen; Xuewu Zhang



Electrostatic design of protein-protein association rates.  


De novo design and redesign of proteins and protein complexes have made promising progress in recent years. Here, we give an overview of how to use available computer-based tools to design proteins to bind faster and tighter to their protein-complex partner by electrostatic optimization between the two proteins. Electrostatic optimization is possible because of the simple relation between the Debye-Huckel energy of interaction between a pair of proteins and their rate of association. This can be used for rapid, structure-based calculations of the electrostatic attraction between the two proteins in the complex. Using these principles, we developed two computer programs that predict the change in k(on), and as such the affinity, on introducing charged mutations. The two programs have a web interface that is available at and When mutations leading to charge optimization are introduced outside the physical binding site, the rate of dissociation is unchanged and therefore the change in k(on) parallels that of the affinity. This design method was evaluated on a number of different protein complexes resulting in binding rates and affinities of hundreds of fold faster and tighter compared to wild type. In this chapter, we demonstrate the procedure and go step by step over the methodology of using these programs for protein-association design. Finally, the way to easily implement the principle of electrostatic design for any protein complex of choice is shown. PMID:16957340

Schreiber, Gideon; Shaul, Yossi; Gottschalk, Kay E



Benchmarking protein-protein interface predictions: why you should care about protein size.  


A number of predictive methods have been developed to predict protein-protein binding sites. Each new method is traditionally benchmarked using sets of protein structures of various sizes, and global statistics are used to assess the quality of the prediction. Little attention has been paid to the potential bias due to protein size on these statistics. Indeed, small proteins involve proportionally more residues at interfaces than large ones. If a predictive method is biased toward small proteins, this can lead to an over-estimation of its performance. Here, we investigate the bias due to the size effect when benchmarking protein-protein interface prediction on the widely used docking benchmark 4.0. First, we simulate random scores that favor small proteins over large ones. Instead of the 0.5 AUC (Area Under the Curve) value expected by chance, these biased scores result in an AUC equal to 0.6 using hypergeometric distributions, and up to 0.65 using constant scores. We then use real prediction results to illustrate how to detect the size bias by shuffling, and subsequently correct it using a simple conversion of the scores into normalized ranks. In addition, we investigate the scores produced by eight published methods and show that they are all affected by the size effect, which can change their relative ranking. The size effect also has an impact on linear combination scores by modifying the relative contributions of each method. In the future, systematic corrections should be applied when benchmarking predictive methods using data sets with mixed protein sizes. PMID:24420747

Martin, Juliette



Effect of the quality of the interaction data on predicting protein function from protein-protein interactions.  


Protein function prediction is an important issue in the post-genomic era. When protein function is deduced from protein interaction data, the traditional methods treat each interaction sample equally, where the qualities of the interaction samples are seldom taken into account. In this paper, we investigate the effect of the quality of protein-protein interaction data on predicting protein function. Moreover, two improved methods, weight neighbour counting method (WNC) and weight chi-square method (WCHI), are proposed by considering the quality of interaction samples with the neighbour counting method (NC) and chi-square method (CHI). Experimental results have shown that the qualities of interaction samples affect the performances of protein function prediction methods seriously. It is also demonstrated that WNC and WCHI methods outperform NC and CHI methods in protein function prediction when example weights are chosen properly. PMID:20640817

Ni, Qing-Shan; Wang, Zheng-Zhi; Li, Gang-Guo; Wang, Guang-Yun; Zhao, Ying-Jie



The N and C Termini of ZO-1 Are Surrounded by Distinct Proteins and Functional Protein Networks*  

PubMed Central

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

Van Itallie, Christina M.; Aponte, Angel; Tietgens, Amber Jean; Gucek, Marjan; Fredriksson, Karin; Anderson, James Melvin



Genetic disorders of surfactant proteins.  


Inherited disorders of pulmonary surfactant-associated proteins are rare but provide important insights into unique mechanisms of surfactant dysfunction. Recessive loss-of-function mutations in the surfactant protein-B and the ATP-binding cassette family member A3 (ABCA3) genes present as lethal surfactant deficiency in the newborn, whereas other recessive mutations in ABCA3 and dominant mutations in the surfactant protein-C gene result in interstitial lung disease in older infants and children. The molecular basis and the genetic and tissue-based approaches to the evaluation of children suspected of having one of these disorders are discussed. PMID:17575475

Hamvas, Aaron; Cole, F Sessions; Nogee, Lawrence M



Protein Phosphatase 1? Mediates Ceramide-induced ERM Protein Dephosphorylation  

PubMed Central

ERM (ezrin, radixin, and moesin) proteins are cytoskeletal interacting proteins that bind cortical actin, the plasma membrane, and membrane proteins, which are found in specialized plasma membrane structures such as microvilli and filopodia. ERM proteins are regulated by phosphatidylinositol 4, 5-biphosphate (PIP2) and by phosphorylation of a C-terminal threonine, and its inactivation involves PIP2 hydrolysis and/or myosin phosphatase (MP). Recently, we demonstrated that ERM proteins are also subject to counter regulation by the bioactive sphingolipids ceramide and sphingosine 1-phosphate. Plasma membrane ceramide induces ERM dephosphorylation whereas sphingosine 1-phosphate induces their phosphorylation. In this work, we pursue the mechanisms by which ceramide regulates dephosphorylation. We found that this dephosphorylation was independent of hydrolysis and localization of PIP2 and MP. However, the results show that ERM dephosphorylation was blocked by treatment with protein phosphatase 1 (PP1) pharmacological inhibitors and specifically by siRNA to PP1?, whereas okadaic acid, a PP2A inhibitor, failed. Moreover, a catalytic inactive mutant of PP1? acted as dominant negative of the endogenous PP1?. Additional results showed that the ceramide mechanism of PP1? activation is largely independent of PIP2 hydrolysis and MP. Taken together, these results demonstrate a novel, acute mechanism of ERM regulation dependent on PP1? and plasma membrane ceramide. PMID:22311981

Canals, Daniel; Roddy, Patrick; Hannun, Yusuf A.



Split-protein systems: beyond binary protein-protein interactions.  


It has been estimated that 650,000 protein-protein interactions exist in the human interactome (Stumpf et al., 2008), a subset of all possible macromolecular partnerships that dictate life. Thus there is a continued need for the development of sensitive and user-friendly methods for cataloguing biomacromolecules in complex environments and for detecting their interactions, modifications, and cellular location. Such methods also allow for establishing differences in the interactome between a normal and diseased cellular state and for quantifying the outcome of therapeutic intervention. A promising approach for deconvoluting the role of macromolecular partnerships is split-protein reassembly, also called protein fragment complementation. This approach relies on the appropriate fragmentation of protein reporters, such as the green fluorescent protein or firefly luciferase, which when attached to possible interacting partners can reassemble and regain function, thereby confirming the partnership. Split-protein methods have been effectively utilized for detecting protein-protein interactions in cell-free systems, Escherichia coli, yeast, mammalian cells, plants, and live animals. Herein, we present recent advances in engineering split-protein systems that allow for the rapid detection of ternary protein complexes, small molecule inhibitors, as well as a variety of macromolecules including nucleic acids, poly(ADP) ribose, and iron sulfur clusters. We also present advances that combine split-protein systems with chemical inducers of dimerization strategies that allow for regulating the activity of orthogonal split-proteases as well as aid in identifying enzyme inhibitors. Finally, we discuss autoinhibition strategies leading to turn-on sensors as well as future directions in split-protein methodology including possible therapeutic approaches. PMID:22070901

Shekhawat, Sujan S; Ghosh, Indraneel



Protein purification and preparation.  

E-print Network

) Protein Extraction with Inhibitors ...........................................................12 Protease such as activity assays, protein microarrays, SDS-PAGE, immunoblotting, ELISA, two-dimensional gel electrophoresis and phosphatase inhibitor cocktails to prevent proteolysis and dephosphorylation of your proteins Affinity

Lebendiker, Mario


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

PubMed Central

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

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



The Geobiochemistry of Methanogen Proteins  

NASA Astrophysics Data System (ADS)

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.

Prasad, A.; Shock, E.



Intramesoporous silica structure differentiating protein loading density  

PubMed Central

We report that hydrothermal aging temperature had a critical effect on intramesoporous structure of mesoporous silica and thus the intramesoporous structure affected protein loading in the mesoporous silica significantly. For a neutral protein Immunoglobulin G with a Y-like molecular shape, the larger desorption pore size allowed the larger protein loading. For a charged protein glucose oxidase with an elliptical molecular shape, the larger surface area resulted in the larger protein loading. Fluorescence emission spectra from tyrosinyl and tryptophanyl residues of the proteins in mesoporous silicas indicated that the charged protein was electrostatically attached inside the mesopores in a way of monolayer, while the neutral protein IgG could continue to aggregate after the monolayer occupancy. PMID:22745517

Qi, Wen; Li, Xiaolin; Chen, Baowei; Yao, Pei; Lei, Chenghong; Liu, Jun



Local Network Patterns in Protein-Protein Interfaces  

PubMed Central

Protein-protein interfaces hold the key to understanding protein-protein interactions. In this paper we investigated local interaction network patterns beyond pair-wise contact sites by considering interfaces as contact networks among residues. A contact site was defined as any residue on the surface of one protein which was in contact with a residue on the surface of another protein. We labeled the sub-graphs of these contact networks by their amino acid types. The observed distributions of these labeled sub-graphs were compared with the corresponding background distributions and the results suggested that there were preferred chemical patterns of closely packed residues at the interface. These preferred patterns point to biological constraints on physical proximity between those residues on one protein which were involved in binding to residues which were close on the interacting partner. Interaction interfaces were far from random and contain information beyond pairs and triangles. To illustrate the possible application of the local network patterns observed, we introduced a signature method, called iScore, based on these local patterns to assess interface predictions. On our data sets iScore achieved 83.6% specificity with 82% sensitivity. PMID:23520460

Luo, Qiang; Hamer, Rebecca; Reinert, Gesine; Deane, Charlotte M.



Do REITs Manipulate Their Financial Results Around Seasoned Equity Offerings? Evidence from US Equity REITs  

Microsoft Academic Search

This study addresses two questions: Is there earnings management in the REIT industry around seasoned equity offerings (SEO)?\\u000a How is earnings management affected by financial and governance factors? Discretionary accruals methods are used to measure\\u000a earnings management. In addition, the difference between actual and calculated FFO is used to capture the potential FFO manipulation.\\u000a We examine how these manipulation measures

Yuan Wei Zhu; Seow Eng Ong; Wee Yong Yeo



Engineering novel fluorescent proteins.  

E-print Network

??Fluorescent proteins are intrinsically fluorescent, genetically encodable tags that can be expressed in many heterologous organisms. Originally cloned from jellyfish and corals, these proteins and… (more)

Shaner, Nathan Christopher



Non-classical protein secretion in bacteria  

PubMed Central

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

Bendtsen, Jannick D; Kiemer, Lars; Fausb?ll, Anders; Brunak, S?ren



iAlign: a method for the structural comparison of protein-protein interfaces  

PubMed Central

Motivation: Protein–protein interactions play an essential role in many cellular processes. The rapid accumulation of protein–protein complex structures provides an unprecedented opportunity for comparative studies of protein–protein interactions. To facilitate such studies, it is necessary to develop an accurate and efficient computational algorithm for the comparison of protein–protein interaction modes. While there are many structural comparison approaches developed for individual proteins, very few methods are available for protein–protein complexes. Results: We present a novel interface alignment method, iAlign, for the structural alignment of protein–protein interfaces. New scoring schemes for measuring interface similarity are introduced, and an iterative dynamic programming algorithm is implemented. We find that the similarity scores follow extreme value distributions. Using statistical models, we empirically estimate their statistical significance, which is in good agreement with manual classifications by human experts. Large-scale tests of iAlign were conducted on both artificial docking models and experimental structures. In a benchmark test on 1517 dimers, iAlign successfully detects biologically related, structurally similar protein–protein interfaces at a coverage percentage of 90% and an error per query of 0.05. When compared against previously published methods, iAlign is substantially more accurate and efficient. Availability: The iAlign software package is freely available at Contact: Supplementary information: Supplementary data are available at Bioinformatics online. PMID:20624782

Gao, Mu; Skolnick, Jeffrey



Protein sensing with engineered protein nanopores*  

PubMed Central

The use of nanopores is a powerful new frontier in single-molecule sciences. Nanopores have been used effectively in exploring various biophysical features of small polypeptides and proteins, such as their folding state and structure, ligand interactions, and enzymatic activity. In particular, the ?-hemolysin protein pore (?HL) has been used extensively for the detection, characterization and analysis of polypeptides, because this protein nanopore is highly robust, versatile and tractable under various experimental conditions. Inspired by the mechanisms of protein translocation across the outer membrane translocases of mitochondria, we have shown the ability to use nanopore-probe techniques in controlling a single protein using engineered ?HL pores. Here, we provide a detailed protocol for the preparation of ?HL protein nanopores. Moreover, we demonstrate that placing attractive electrostatic traps is instrumental in tackling single-molecule stochastic sensing of folded proteins. PMID:22528256

Mohammad, Mohammad M.; Movileanu, Liviu



Protein designs in HP models  

NASA Astrophysics Data System (ADS)

The inverse protein folding problem is that of designing an amino acid sequence which folds into a prescribed shape. This problem arises in drug design where a particular structure is necessary to ensure proper protein-protein interactions and could have applications in nanotechnology. A major challenge in designing proteins with native folds that attain a specific shape is to avoid proteins that have multiple native folds (unstable proteins). In this technical note we present our results on protein designs in the variant of Hydrophobic-Polar (HP) model introduced by Dill [6] on 2D square lattice. The HP model distinguishes only polar and hydrophobic monomers and only counts the number of hydrophobic contacts in the energy function. To achieve better stability of our designs we use the Hydrophobic-Polar-Cysteine (HPC) model which distinguishes the third type of monomers called "cysteines" and incorporates also the disulfid bridges (SS-bridges) into the energy function. We present stable designs in 2D square lattice and 3D hexagonal prism lattice in the HPC model.

Gupta, Arvind; Khodabakhshi, Alireza Hadj; Ma?uch, Ján; Rafiey, Arash; Stacho, Ladislav



Encounter complexes and dimensionality reduction in protein-protein association  

PubMed Central

An outstanding challenge has been to understand the mechanism whereby proteins associate. We report here the results of exhaustively sampling the conformational space in protein–protein association using a physics-based energy function. The agreement between experimental intermolecular paramagnetic relaxation enhancement (PRE) data and the PRE profiles calculated from the docked structures shows that the method captures both specific and non-specific encounter complexes. To explore the energy landscape in the vicinity of the native structure, the nonlinear manifold describing the relative orientation of two solid bodies is projected onto a Euclidean space in which the shape of low energy regions is studied by principal component analysis. Results show that the energy surface is canyon-like, with a smooth funnel within a two dimensional subspace capturing over 75% of the total motion. Thus, proteins tend to associate along preferred pathways, similar to sliding of a protein along DNA in the process of protein-DNA recognition. DOI: PMID:24714491

Kozakov, Dima; Li, Keyong; Hall, David R; Beglov, Dmitri; Zheng, Jiefu; Vakili, Pirooz; Schueler-Furman, Ora; Paschalidis, Ioannis Ch; Clore, G Marius; Vajda, Sandor



Hash subgraph pairwise kernel for protein-protein interaction extraction.  


Extracting protein-protein interaction (PPI) from biomedical literature is an important task in biomedical text mining (BioTM). In this paper, we propose a hash subgraph pairwise (HSP) kernel-based approach for this task. The key to the novel kernel is to use the hierarchical hash labels to express the structural information of subgraphs in a linear time. We apply the graph kernel to compute dependency graphs representing the sentence structure for protein-protein interaction extraction task, which can efficiently make use of full graph structural information, and particularly capture the contiguous topological and label information ignored before. We evaluate the proposed approach on five publicly available PPI corpora. The experimental results show that our approach significantly outperforms all-path kernel approach on all five corpora and achieves state-of-the-art performance. PMID:22595237

Zhang, Yijia; Lin, Hongfei; Yang, Zhihao; Wang, Jian; Li, Yanpeng



Protein surface-distribution and protein-protein interactions in the binding of peripheral proteins to charged lipid membranes.  

PubMed Central

The binding of native cytochrome c to negatively charged lipid dispersions of dioleoyl phosphatidylglycerol has been studied over a wide range of ionic strengths. Not only is the strength of protein binding found to decrease rapidly with increasing ionic strength, but also the binding curves reach an apparent saturation level that decreases rapidly with increasing ionic strength. Analysis of the binding isotherms with a general statistical thermodynamic model that takes into account not only the free energy of the electrostatic double layer, but also the free energy of the surface distribution of the protein, demonstrates that the apparent saturation effects could arise from a competition between the out-of-plane binding reaction and the lateral in-plane interactions between proteins at the surface. It is found that association with nonlocalized sites results in binding isotherms that display the apparent saturation effect to a much more pronounced extent than does the Langmuir adsorption isotherm for binding to localized sites. With the model for nonlocalized sites, the binding isotherms of native cytochrome c can be described adequately by taking into account only the entropy of the surface distribution of the protein, without appreciable enthalpic interactions between the bound proteins. The binding of cytochrome c to dioleoyl phosphatidylglycerol dispersions at a temperature at which the bound protein is denatured on the lipid surface, but is nondenatured when free in solution, has also been studied. The binding curves for the surface-denatured protein differ from those for the native protein in that the apparent saturation at high ionic strength is less pronounced. This indicates the tendency of the denatured protein to aggregate on the lipid surface, and can be described by the binding isotherms for nonlocalized sites only if attractive interactions between the surface-bound proteins are included in addition to the distributional entropic terms. Additionally, it is found that the binding capacity for the native protein is increased at low ionic strength to a value that is greater than that for complete surface coverage, and that corresponds more closely to neutralization of the effective charge (determined from the ionic strength dependence), rather than of the total net charge, on the protein. Electron spin resonance experiments with spin-labeled lipids indicate that this different mode of binding arises from a penetration or disturbance of the bilayer surface by the protein that may alleviate the effects of in-plane interactions under conditions of strong binding. PMID:7696507

Heimburg, T; Marsh, D



Methylation of ribosomal proteins in Bacillus subtilis.  

PubMed Central

We measured the methylation of ribosomal proteins from the 30S and 50S subunits of Bacillus subtilis after growing the cells in the presence of [1-14C]methionine and [methyl-3H]methionine. Two-dimensional polyacrylamide gel electrophoretic analysis revealed a preferential methylation of the 50S ribosomal proteins. Proteins L11 and L16, and possibly L9, L10, L18, and L20, were methylated. On the other hand, only two possibly methylated proteins were found on the 30S subunit. A comparison of these results with those for Escherichia coli suggests a common methylation pattern for the bacterial ribosomal proteins. Images PMID:6768716

Mardones, E; Amaro, A M; Jerez, C A



Prion protein and aging  

PubMed Central

The cellular prion protein (PrPC) has been widely investigated ever since its conformational isoform, the prion (or PrPSc), was identified as the etiological agent of prion disorders. The high homology shared by the PrPC-encoding gene among mammals, its high turnover rate and expression in every tissue strongly suggest that PrPC may possess key physiological functions. Therefore, defining PrPC roles, properties and fate in the physiology of mammalian cells would be fundamental to understand its pathological involvement in prion diseases. Since the incidence of these neurodegenerative disorders is enhanced in aging, understanding PrPC functions in this life phase may be of crucial importance. Indeed, a large body of evidence suggests that PrPC plays a neuroprotective and antioxidant role. Moreover, it has been suggested that PrPC is involved in Alzheimer disease, another neurodegenerative pathology that develops predominantly in the aging population. In prion diseases, PrPC function is likely lost upon protein aggregation occurring in the course of the disease. Additionally, the aging process may alter PrPC biochemical properties, thus influencing its propensity to convert into PrPSc. Both phenomena may contribute to the disease development and progression. In Alzheimer disease, PrPC has a controversial role because its presence seems to mediate ?-amyloid toxicity, while its down-regulation correlates with neuronal death. The role of PrPC in aging has been investigated from different perspectives, often leading to contrasting results. The putative protein functions in aging have been studied in relation to memory, behavior and myelin maintenance. In aging mice, PrPC changes in subcellular localization and post-translational modifications have been explored in an attempt to relate them to different protein roles and propensity to convert into PrPSc. Here we provide an overview of the most relevant studies attempting to delineate PrPC functions and fate in aging. PMID:25364751

Gasperini, Lisa; Legname, Giuseppe



Protein Structure Prediction by Protein Threading  

NASA Astrophysics Data System (ADS)

The seminal work of Bowie, Lüthy, and Eisenberg (Bowie et al., 1991) on "the inverse protein folding problem" laid the foundation of protein structure prediction by protein threading. By using simple measures for fitness of different amino acid types to local structural environments defined in terms of solvent accessibility and protein secondary structure, the authors derived a simple and yet profoundly novel approach to assessing if a protein sequence fits well with a given protein structural fold. Their follow-up work (Elofsson et al., 1996; Fischer and Eisenberg, 1996; Fischer et al., 1996a,b) and the work by Jones, Taylor, and Thornton (Jones et al., 1992) on protein fold recognition led to the development of a new brand of powerful tools for protein structure prediction, which we now term "protein threading." These computational tools have played a key role in extending the utility of all the experimentally solved structures by X-ray crystallography and nuclear magnetic resonance (NMR), providing structural models and functional predictions for many of the proteins encoded in the hundreds of genomes that have been sequenced up to now.

Xu, Ying; Liu, Zhijie; Cai, Liming; Xu, Dong


A Least Square Method Based Model for Identifying Protein Complexes in Protein-Protein Interaction Network  

PubMed Central

Protein complex formed by a group of physical interacting proteins plays a crucial role in cell activities. Great effort has been made to computationally identify protein complexes from protein-protein interaction (PPI) network. However, the accuracy of the prediction is still far from being satisfactory, because the topological structures of protein complexes in the PPI network are too complicated. This paper proposes a novel optimization framework to detect complexes from PPI network, named PLSMC. The method is on the basis of the fact that if two proteins are in a common complex, they are likely to be interacting. PLSMC employs this relation to determine complexes by a penalized least squares method. PLSMC is applied to several public yeast PPI networks, and compared with several state-of-the-art methods. The results indicate that PLSMC outperforms other methods. In particular, complexes predicted by PLSMC can match known complexes with a higher accuracy than other methods. Furthermore, the predicted complexes have high functional homogeneity.

Dai, Qiguo; Guo, Maozu; Guo, Yingjie; Liu, Xiaoyan; Liu, Yang; Teng, Zhixia



When is protein binding important?  


The present paper is an ode to a classic citation by Benet and Hoener (2002. Clin Pharm Ther 71(3):115-121). The now classic paper had a huge impact on drug development and the way the issue of protein binding is perceived and interpreted. Although the authors very clearly pointed out the limitations and underlying assumptions for their delineations, these are too often overlooked and the classic paper's message is misinterpreted by broadening to cases that were not intended. Some members of the scientific community concluded from the paper that protein binding is not important. This was clearly not intended by the authors, as they finished their paper with a paragraph entitled: "When is protein binding important?" Misinterpretation of the underlying assumptions in the classic work can result in major pitfalls in drug development. Therefore, we revisit the topic of protein binding with the intention of clarifying when clinically relevant changes should be considered during drug development. PMID:23650013

Heuberger, Jules; Schmidt, Stephan; Derendorf, Hartmut



Protein denaturation and protein:drugs interactions from intrinsic protein  

E-print Network

. Here we present a quantitative and accurate method for the determination of protein stability engineering and drug screening within life science research. Current techniques either consume too much labeling, and that takes advantage of the intrinsic tryptophan fluorescence of most proteins. Coupled

Jackson, Sophie


Protein crystallization in vivo  

E-print Network

Protein crystallization in vivo provides some fascinating examples of biological self-assembly. Here, we provide a selective survey to show the diversity of functions for which protein crystals are used, and the physical properties of the crystals thatare exploited. Where known, we emphasize how the nature of the protein-protein interactions leads to control of the crystallization behaviour.

Jonathan P. K. Doye; Wilson C. K. Poon



Origins of Myc Proteins - Using Intrinsic Protein Disorder to Trace Distant Relatives  

PubMed Central

Mammalian Myc proteins are important determinants of cell proliferation as well as the undifferentiated state of stem cells and their activity is frequently deregulated in cancer. Based mainly on conservation in the C-terminal DNA-binding and dimerization domain, Myc-like proteins have been reported in many simpler organisms within and outside the Metazoa but they have not been found in fungi or plants. Several important signature motifs defining mammalian Myc proteins are found in the N-terminal domain but the extent to which these are found in the Myc-like proteins from simpler organisms is not well established. The extent of N-terminal signature sequence conservation would give important insights about the evolution of Myc proteins and their current function in mammalian physiology and disease. In a systematic study of Myc-like proteins we show that N-terminal signature motifs are not readily detectable in individual Myc-like proteins from invertebrates but that weak similarities to Myc boxes 1 and 2 can be found in the N-termini of the simplest Metazoa as well as the unicellular choanoflagellate, Monosiga brevicollis, using multiple protein alignments. Phylogenetic support for the connections of these proteins to established Myc proteins is however poor. We show that the pattern of predicted protein disorder along the length of Myc proteins can be used as a complementary approach to making dendrograms of Myc proteins that aids the classification of Myc proteins. This suggests that the pattern of disorder within Myc proteins is more conserved through evolution than their amino acid sequence. In the disorder-based dendrograms the Myc-like proteins from simpler organisms, including M. brevicollis, are connected to established Myc proteins with a higher degree of certainty. Our results suggest that protein disorder based dendrograms may be of general significance for studying distant relationships between proteins, such as transcription factors, that have high levels of intrinsic disorder. PMID:24086436

Mahani, Amir; Henriksson, Johan; Wright, Anthony P. H.



PDZ Domain Proteins: Plug and Play!  

NSDL National Science Digital Library

PDZ domains (an acronym from PSD-95, Dlg, and ZO-1) are protein modules found in many cytoplasmic proteins (more than 400 in humans); they are discussed in this STKE Review. They are associated with a wide range of other protein-protein interaction domains (for example, WW, PTB, LRR, SH3) or with domains that exhibit particular enzymatic activities (such as guanosine triphosphatases, serine-threonine kinases, phosphatases), and they participate in various intracellular protein networks. PDZ domains bind to very diverse carboxyl-termini of protein partners in a specific (and sometimes reversible) manner, which enables the formation of supramolecular networks. Scaffolding of proteins by PDZ domain proteins usually occurs at specific sites within the cell (such as the plasma membrane or the Golgi apparatus) and is frequently involved in localizing proteins to specialized subcellular compartments of polarized cells, such as the presynaptic terminals and postsynaptic densities of neurons and the basolateral or apical membranes of epithelial cells. Genetic models in invertebrates and vertebrates that are now available for some PDZ proteins illuminate the large set of biological processes in which this protein family is involved, from the establishment and maintenance of the cytoarchitecture to signaling events. Accordingly, defects of PDZ proteins that play a central role in tissue homeostasis result in pathological conditions including cancer and developmental abnormalities. The simplicity of PDZ domain interactions has enabled the design of pharmacological inhibitors of potential therapeutic interest.

Claire Nourry (Marseille;Laboratory of Molecular Pharmacology REV); Seth G. N. Grant (University of Edinburgh;Centre for Neuroscience Research REV); Jean-Paul Borg (Marseille;Laboratory of Molecular Pharmacology REV)



Cross-talk unfolded: MARCKS proteins.  

PubMed Central

The proteins of the MARCKS (myristoylated alanine-rich C kinase substrate) family were first identified as prominent substrates of protein kinase C (PKC). Since then, these proteins have been implicated in the regulation of brain development and postnatal survival, cellular migration and adhesion, as well as endo-, exo- and phago-cytosis, and neurosecretion. The effector domain of MARCKS proteins is phosphorylated by PKC, binds to calmodulin and contributes to membrane binding. This multitude of mutually exclusive interactions allows cross-talk between the signal transduction pathways involving PKC and calmodulin. This review focuses on recent, mostly biophysical and biochemical results renewing interest in this protein family. MARCKS membrane binding is now understood at the molecular level. From a structural point of view, there is a consensus emerging that MARCKS proteins are "natively unfolded". Interestingly, domains similar to the effector domain have been discovered in other proteins. Furthermore, since the effector domain enhances the polymerization of actin in vitro, MARCKS proteins have been proposed to mediate regulation of the actin cytoskeleton. However, the recent observations that MARCKS might serve to sequester phosphatidylinositol 4,5-bisphosphate in the plasma membrane of unstimulated cells suggest an alternative model for the control of the actin cytoskeleton. While myristoylation is classically considered to be a co-translational, irreversible event, new reports on MARCKS proteins suggest a more dynamic picture of this protein modification. Finally, studies with mice lacking MARCKS proteins have investigated the functions of these proteins during embryonic development in the intact organism. PMID:11829734

Arbuzova, Anna; Schmitz, Arndt A P; Vergeres, Guy



Origins of Protein Functions in Cells  

NASA Technical Reports Server (NTRS)

In modern organisms proteins perform a majority of cellular functions, such as chemical catalysis, energy transduction and transport of material across cell walls. Although great strides have been made towards understanding protein evolution, a meaningful extrapolation from contemporary proteins to their earliest ancestors is virtually impossible. In an alternative approach, the origin of water-soluble proteins was probed through the synthesis and in vitro evolution of very large libraries of random amino acid sequences. In combination with computer modeling and simulations, these experiments allow us to address a number of fundamental questions about the origins of proteins. Can functionality emerge from random sequences of proteins? How did the initial repertoire of functional proteins diversify to facilitate new functions? Did this diversification proceed primarily through drawing novel functionalities from random sequences or through evolution of already existing proto-enzymes? Did protein evolution start from a pool of proteins defined by a frozen accident and other collections of proteins could start a different evolutionary pathway? Although we do not have definitive answers to these questions yet, important clues have been uncovered. In one example (Keefe and Szostak, 2001), novel ATP binding proteins were identified that appear to be unrelated in both sequence and structure to any known ATP binding proteins. One of these proteins was subsequently redesigned computationally to bind GTP through introducing several mutations that introduce targeted structural changes to the protein, improve its binding to guanine and prevent water from accessing the active center. This study facilitates further investigations of individual evolutionary steps that lead to a change of function in primordial proteins. In a second study (Seelig and Szostak, 2007), novel enzymes were generated that can join two pieces of RNA in a reaction for which no natural enzymes are known. Recently it was found that, as in the previous case, the proteins have a structure unknown among modern enzymes. In this case, in vitro evolution started from a small, non-enzymatic protein. A similar selection process initiated from a library of random polypeptides is in progress. These results not only allow for estimating the occurrence of function in random protein assemblies but also provide evidence for the possibility of alternative protein worlds. Extant proteins might simply represent a frozen accident in the world of possible proteins. Alternative collections of proteins, even with similar functions, could originate alternative evolutionary paths.

Seelig, Burchard; Pohorille, Andrzej



Large-scale mapping of human protein-protein interactions by mass spectrometry  

PubMed Central

Mapping protein–protein interactions is an invaluable tool for understanding protein function. Here, we report the first large-scale study of protein–protein interactions in human cells using a mass spectrometry-based approach. The study maps protein interactions for 338 bait proteins that were selected based on known or suspected disease and functional associations. Large-scale immunoprecipitation of Flag-tagged versions of these proteins followed by LC-ESI-MS/MS analysis resulted in the identification of 24 540 potential protein interactions. False positives and redundant hits were filtered out using empirical criteria and a calculated interaction confidence score, producing a data set of 6463 interactions between 2235 distinct proteins. This data set was further cross-validated using previously published and predicted human protein interactions. In-depth mining of the data set shows that it represents a valuable source of novel protein–protein interactions with relevance to human diseases. In addition, via our preliminary analysis, we report many novel protein interactions and pathway associations. PMID:17353931

Ewing, Rob M; Chu, Peter; Elisma, Fred; Li, Hongyan; Taylor, Paul; Climie, Shane; McBroom-Cerajewski, Linda; Robinson, Mark D; O'Connor, Liam; Li, Michael; Taylor, Rod; Dharsee, Moyez; Ho, Yuen; Heilbut, Adrian; Moore, Lynda; Zhang, Shudong; Ornatsky, Olga; Bukhman, Yury V; Ethier, Martin; Sheng, Yinglun; Vasilescu, Julian; Abu-Farha, Mohamed; Lambert, Jean-Philippe; Duewel, Henry S; Stewart, Ian I; Kuehl, Bonnie; Hogue, Kelly; Colwill, Karen; Gladwish, Katharine; Muskat, Brenda; Kinach, Robert; Adams, Sally-Lin; Moran, Michael F; Morin, Gregg B; Topaloglou, Thodoros; Figeys, Daniel



PPLook: an automated data mining tool for protein-protein interaction  

PubMed Central

Background Extracting and visualizing of protein-protein interaction (PPI) from text literatures are a meaningful topic in protein science. It assists the identification of interactions among proteins. There is a lack of tools to extract PPI, visualize and classify the results. Results We developed a PPI search system, termed PPLook, which automatically extracts and visualizes protein-protein interaction (PPI) from text. Given a query protein name, PPLook can search a dataset for other proteins interacting with it by using a keywords dictionary pattern-matching algorithm, and display the topological parameters, such as the number of nodes, edges, and connected components. The visualization component of PPLook enables us to view the interaction relationship among the proteins in a three-dimensional space based on the OpenGL graphics interface technology. PPLook can also provide the functions of selecting protein semantic class, counting the number of semantic class proteins which interact with query protein, counting the literature number of articles appearing the interaction relationship about the query protein. Moreover, PPLook provides heterogeneous search and a user-friendly graphical interface. Conclusions PPLook is an effective tool for biologists and biosystem developers who need to access PPI information from the literature. PPLook is freely available for non-commercial users at PMID:20550717



Isofocusing and immunological investigations on cephalopod lens proteins  

Microsoft Academic Search

Soluble lens proteins from Octopus vulgaris, Sepia officinalis, and Loligo vulgaris were analyzed by thin-layer isoelectric focusing and compared by various immunochemical methods using antibodies directed against total soluble lens protein antigens from the said three species. The results show close similarity between Sepia and Loligo lens proteins. Antibodies made against Sepia and Loligo lens proteins did not react against

S. K. Brahma; M. Lancieri



Mechanism of covalent adduct formation of aucubin to proteins  

Microsoft Academic Search

The iridoid glucoside aucubin can irreversibly bind to proteins through the formation of its aglycone. In view of a possible involvement of these protein adducts in the toxicity of aucubin, we investigated the mechanism of binding of aucubin to proteins. [3H]aucubin in itself did not result in binding to protein whereas it covalently bound to rat serum albumin as a

Dong-Hyun Kim; Bok-Ryang Kim; Ji-Yeon Kim; Yo-Chan Jeong



Protein-protein binding affinities by pulse proteolysis: Application to TEM-1/BLIP protein complexes  

PubMed Central

Efficient methods for quantifying dissociation constants have become increasingly important for high-throughput mutagenesis studies in the postgenomic era. However, experimentally determining binding affinity is often laborious, requires large amounts of purified protein, and utilizes specialized equipment. Recently, pulse proteolysis has been shown to be a robust and simple method to determine the dissociation constants for a protein–ligand pair based on the increase in thermodynamic stability upon ligand binding. Here, we extend this technique to determine binding affinities for a protein–protein complex involving the ?-lactamase TEM-1 and various ?-lactamase inhibitor protein (BLIP) mutants. Interaction with BLIP results in an increase in the denaturation curve midpoint, Cm, of TEM-1, which correlates with the rank order of binding affinities for several BLIP mutants. Hence, pulse proteolysis is a simple, effective method to assay for mutations that modulate binding affinity in protein–protein complexes. From a small set (n = 4) of TEM-1/BLIP mutant complexes, a linear relationship between energy of stabilization (dissociation constant) and ?Cm was observed. From this “calibration curve,” accurate dissociation constants for two additional BLIP mutants were calculated directly from proteolysis-derived ?Cm values. Therefore, in addition to qualitative information, armed with knowledge of the dissociation constants from the WT protein and a limited number of mutants, accurate quantitation of binding affinities can be determined for additional mutants from pulse proteolysis. Minimal sample requirements and the suitability of impure protein preparations are important advantages that make pulse proteolysis a powerful tool for high-throughput mutagenesis binding studies. PMID:20669180

Hanes, Melinda S; Ratcliff, Kathleen; Marqusee, Susan; Handel, Tracy M



Cluster-based assessment of protein-protein interaction confidence  

PubMed Central

Background Protein-protein interaction networks are key to a systems-level understanding of cellular biology. However, interaction data can contain a considerable fraction of false positives. Several methods have been proposed to assess the confidence of individual interactions. Most of them require the integration of additional data like protein expression and interaction homology information. While being certainly useful, such additional data are not always available and may introduce additional bias and ambiguity. Results We propose a novel, network topology based interaction confidence assessment method called CAPPIC (cluster-based assessment of protein-protein interaction confidence). It exploits the network’s inherent modular architecture for assessing the confidence of individual interactions. Our method determines algorithmic parameters intrinsically and does not require any parameter input or reference sets for confidence scoring. Conclusions On the basis of five yeast and two human physical interactome maps inferred using different techniques, we show that CAPPIC reliably assesses interaction confidence and its performance compares well to other approaches that are also based on network topology. The confidence score correlates with the agreement in localization and biological process annotations of interacting proteins. Moreover, it corroborates experimental evidence of physical interactions. Our method is not limited to physical interactome maps as we exemplify with a large yeast genetic interaction network. An implementation of CAPPIC is available at PMID:23050565



Why do proteins aggregate? "Intrinsically insoluble proteins" and "dark mediators" revealed by studies on "insoluble proteins" solubilized in pure water  

PubMed Central

In 2008, I reviewed and proposed a model for our discovery in 2005 that unrefoldable and insoluble proteins could in fact be solubilized in unsalted water. Since then, this discovery has offered us and other groups a powerful tool to characterize insoluble proteins, and we have further addressed several fundamental and disease-relevant issues associated with this discovery. Here I review these results, which are conceptualized into several novel scenarios. 1) Unlike 'misfolded proteins', which still retain the capacity to fold into well-defined structures but are misled to 'off-pathway' aggregation, unrefoldable and insoluble proteins completely lack this ability and will unavoidably aggregate in vivo with ~150 mM ions, thus designated as 'intrinsically insoluble proteins (IIPs)' here. IIPs may largely account for the 'wastefully synthesized' DRiPs identified in human cells. 2) The fact that IIPs including membrane proteins are all soluble in unsalted water, but get aggregated upon being exposed to ions, logically suggests that ions existing in the background play a central role in mediating protein aggregation, thus acting as 'dark mediators'. Our study with 14 salts confirms that IIPs lack the capacity to fold into any well-defined structures. We uncover that salts modulate protein dynamics and anions bind proteins with high selectivity and affinity, which is surprisingly masked by pre-existing ions. Accordingly, I modified my previous model. 3) Insoluble proteins interact with lipids to different degrees. Remarkably, an ALS-causing P56S mutation transforms the ?-sandwich MSP domain into a helical integral membrane protein. Consequently, the number of membrane-interacting proteins might be much larger than currently recognized. To attack biological membranes may represent a common mechanism by which aggregated proteins initiate human diseases. 4) Our discovery also implies a solution to the 'chicken-and-egg paradox' for the origin of primitive membranes embedded with integral membrane proteins, if proteins originally emerged in unsalted prebiotic media. PMID:24555050

Song, Jianxing



Replacement of the V3 domain in the surface subunit of the feline immunodeficiency virus envelope glycoprotein with the equivalent region of a T cell-tropic human immunodeficiency virus type 1 results in a chimeric surface protein that efficiently binds to CXCR4.  


Feline immunodeficiency virus (FIV) and the T cell-tropic strains of human immunodeficiency virus type 1 (HIV-1) share the use of the chemokine receptor CXCR4 for cell entry. To study this process further we developed a cell surface binding assay based on the expression of a soluble version of the FIV SU C-terminally tagged with the influenza virus hemagglutinin epitope (HA). The specificity of the assay was demonstrated by the following evidence: (1) the SU-HA protein bound to HeLa cells that express CXCR4 but not to MDCK cells that lack this chemokine receptor; and (2) binding of the SU-HA to HeLa cells was blocked by incubation with the CXCR4 antagonist AMD3100 as well as with the anti-CXCR4 monoclonal antibody (MAb) 12G5. Deletion of the V3 region from the FIV SU glycoprotein abolished its ability to bind CXCR4-expressing cells. Remarkably, substitution of the V3 domain of the FIV SU by the equivalent region of the HIV-1 NL4-3 isolate resulted in efficient cell surface binding of the chimeric SU protein to CXCR4. Moreover, transfection of MDCK cells with a plasmid encoding human CXCR4 allowed the association of the chimeric SU-HA glycoprotein to the transfected cells. Interestingly, while cell binding of the chimeric FIV-HIV SU was inhibited by an anti-HIV-1 V3 MAb, its association with CXCR4 was found to be resistant to AMD3100. Of note, the chimeric FIV-HIV Env glycoprotein was capable of promoting CXCR4-dependent cell-to-cell fusion. PMID:24148007

González, Silvia A; Falcón, Juan I; Affranchino, José L



Structural similarity of loops in protein families: toward the understanding of protein evolution  

PubMed Central

Background Protein evolution and protein classification are usually inferred by comparing protein cores in their conserved aligned parts. Structurally aligned protein regions are separated by less conserved loop regions, where sequence and structure locally deviate from each other and do not superimpose well. Results Our results indicate that even longer protein loops can not be viewed as "random coils" and for the majority of protein families in our test set there exists a linear correlation between the measures of sequence similarity and loop structural similarity. Results suggest that distance matrices derived from the loop (dis)similarity measure may produce in some cases more reliable cluster trees compared to the distance matrices based on the conventional measures of sequence and structural (dis)similarity. Conclusions We show that by considering "dissimilar" loop regions rather than only conserved core regions it is possible to improve our understanding of protein evolution. PMID:15691378

Panchenko, Anna R; Madej, Thomas



Protein Structure Analysis Iosif Vaisman  

E-print Network

Protein Structure Analysis Iosif Vaisman 2009 BINF 731 Protein Engineering Protein Engineering stability Increase proteins resistance to proteases Change codon composition Computational Mutagenesis Assumption: the structural differences between each mutant and the wild-type protein are usually minor and

Vaisman, Iosif


Modelling protein-protein interaction networks via a stickiness index  

PubMed Central

What type of connectivity structure are we seeing in protein–protein interaction networks? A number of random graph models have been mooted. After fitting model parameters to real data, the models can be judged by their success in reproducing key network properties. Here, we propose a very simple random graph model that inserts a connection according to the degree, or ‘stickiness’, of the two proteins involved. This model can be regarded as a testable distillation of more sophisticated versions that attempt to account for the presence of interaction surfaces or binding domains. By computing a range of network similarity measures, including relative graphlet frequency distance, we find that our model outperforms other random graph classes. In particular, we show that given the underlying degree information, fitting a stickiness model produces better results than simply choosing a degree-matching graph uniformly at random. Therefore, the results lend support to the basic modelling methodology. PMID:16971339

Przulj, Natasa; Higham, Desmond J



A combinatorial perspective of the protein inference problem.  


In a shotgun proteomics experiment, proteins are the most biologically meaningful output. The success of proteomics studies depends on the ability to accurately and efficiently identify proteins. Many methods have been proposed to facilitate the identification of proteins from peptide identification results. However, the relationship between protein identification and peptide identification has not been thoroughly explained before. In this paper, we devote ourselves to a combinatorial perspective of the protein inference problem. We employ combinatorial mathematics to calculate the conditional protein probabilities (protein probability means the probability that a protein is correctly identified) under three assumptions, which lead to a lower bound, an upper bound, and an empirical estimation of protein probabilities, respectively. The combinatorial perspective enables us to obtain an analytical expression for protein inference. Our method achieves comparable results with ProteinProphet in a more efficient manner in experiments on two data sets of standard protein mixtures and two data sets of real samples. Based on our model, we study the impact of unique peptides and degenerate peptides (degenerate peptides are peptides shared by at least two proteins) on protein probabilities. Meanwhile, we also study the relationship between our model and ProteinProphet. We name our program ProteinInfer. Its Java source code, our supplementary document and experimental results are available at: > PMID:24407311

Yang, Chao; He, Zengyou; Yu, Weichuan



Protein-protein interaction Functional Genomics  

E-print Network

., (2007) Molecular Systems Biology ­ ~300 purifications · E. coli ­ Butland G et al., (2005) Nature ­ 857 ­ Krogan et al., (2006) · 547 complexes, 2600 bait protein, 4000 prey proteins · Human ­ Ewing RB et al of the effects of the individual strains · Highly functional interaction ­ Collins SR et al. (2007) Nature test

Spang, Rainer


Protein Sequence, Structure, Stability and Functionality  

E-print Network

Protein-protein interactions (protein functionalities) are mediated by water, which compacts individual proteins and promotes close and temporarily stable large-area protein-protein interfaces. Proteins are peptide chains decorated by amino acids, and protein scientists have long described protein-water interactions in terms of qualitative amino acid hydrophobicity scales. Here we examine several recent scales and argue plausibly (in terms of self-organized criticality) that one of them should be regarded as an absolute scale (within the protein universe), analogous to the dielectric scale of bond ionicity in inorganic octet compounds. Applications to repeat proteins (containing upwards of 900 amino acids) are successful, far beyond reasonable expectations, in all cases studied so far. While some of the results are obvious and can be obtained from the ex vitro spatial structures alone, many are hidden from plain view, and can be called phantom relations. As a byproduct, the network theory explains the exceptional functionality of leucine in zippers, heptads, and repeat consensus sites.

J. C. Phillips



Multilabel learning for protein subcellular location prediction.  


Protein subcellular localization aims at predicting the location of a protein within a cell using computational methods. Knowledge of subcellular localization of proteins indicates protein functions and helps in identifying drug targets. Prediction of protein subcellular localization is an important but challenging problem, particularly when proteins may simultaneously exist at, or move between, two or more different subcellular location sites. Most of the existing protein subcellular localization methods are only used to deal with the single-location proteins. To better reflect the characteristics of multiplex proteins, we formulate prediction of subcellular localization of multiplex proteins as a multilabel learning problem. We present and compare two multilabel learning approaches, which exploit correlations between labels and leverage label-specific features, respectively, to induce a high quality prediction model. Experimental results on six protein data sets under various organisms show that our described methods achieve significantly higher performance than any of the existing methods. Among the different multilabel learning methods, we find that methods exploiting label correlations performs better than those leveraging label-specific features. PMID:22987129

Li, Guo-Zheng; Wang, Xiao; Hu, Xiaohua; Liu, Jia-Ming; Zhao, Rui-Wei



Protein Folding and Misfolding on Surfaces  

PubMed Central

Protein folding, misfolding and aggregation, as well as the way misfolded and aggregated proteins affects cell viability are emerging as key themes in molecular and structural biology and in molecular medicine. Recent advances in the knowledge of the biophysical basis of protein folding have led to propose the energy landscape theory which provides a consistent framework to better understand how a protein folds rapidly and efficiently to the compact, biologically active structure. The increased knowledge on protein folding has highlighted its strict relation to protein misfolding and aggregation, either process being in close competition with the other, both relying on the same physicochemical basis. The theory has also provided information to better understand the structural and environmental factors affecting protein folding resulting in protein misfolding and aggregation into ordered or disordered polymeric assemblies. Among these, particular importance is given to the effects of surfaces. The latter, in some cases make possible rapid and efficient protein folding but most often recruit proteins/peptides increasing their local concentration thus favouring misfolding and accelerating the rate of nucleation. It is also emerging that surfaces can modify the path of protein misfolding and aggregation generating oligomers and polymers structurally different from those arising in the bulk solution and endowed with different physical properties and cytotoxicities. PMID:19330090

Stefani, Massimo



Cementum attachment protein/protein-tyrosine phosphotase-like member A is not expressed in teeth.  


Cementum is a highly specialized connective tissue that covers tooth roots. The only cementum-specific protein described to date is the cementum attachment protein (CAP). A putative sequence for CAP was established from a cDNA clone isolated from a human cementifying fibroma cDNA library. This sequence overlaps with a phosphatase-like protein in muscle termed the protein-tyrosine phosphatase-like member A (PTPLA). To clarify the nature of CAP/PTPLA, we cloned the homologous rat protein and determined its sequence. The rat protein shared 94% sequence identity with the human protein. On Northern blots containing RNA from various rat tissues of different developmental stages, the cDNA hybridized to an mRNA expressed in heart and skeletal muscle but not in teeth. These results were confirmed by real-time PCR. Thus, the sequence deposited in public databanks under the name 'cementum attachment protein' does not represent genuine CAP. PMID:19148556

Schild, Christof; Beyeler, Michael; Lang, Niklaus P; Trueb, Beat



Cold Chemical Oxidation of Proteins  

PubMed Central

Various methods of protein footprinting use hydrogen peroxide as oxidant. Its removal by various solid-phase desalting methods, catalase treatment, or freeze-drying after the footprinting is critical to insure no uncontrolled oxidation. Although catalase treatment removes hydrogen peroxide with little loss of protein or additional protein oxidation, we discovered that freeze-drying or freezing of the protein in peroxide solution does lead to protein oxidation. Interestingly, the oxidation is not a result of freeze or thaw processes but depends on the temperature and length of time for incubation. After 2 h, apomyoglobin undergoes nearly complete single oxidation at ?80 °C and double oxidation at ?15 °C. The oxidation extents are in addition to the number of methionine residues. Minimal oxidation is observed at 4 °C and 22 °C compared to oxidation at ?80 °C or ?20 °C. The concentration of hydrogen peroxide is critical; 75 mM (0.2%) is required to oxidize > 50% of the protein at ?15 °C and 100-mM (0.3%) at ?80 °C. In addition to Met, ~ 5% of the tryptophan and tyrosine residues are oxidized as well as lower amounts of His and Phe. Oxidation of Val 68 and Val 17 (a buried residue) also occurs, with the oxidation of Val 17 likely occurring by electron transfer from one of two of the oxidized aromatic residues contacting Val 17. We describe here the need to remove the hydrogen peroxide prior to cold storage of proteins, and we also report some preliminary results pertaining to the mechanism of cold, solid-state oxidation. PMID:19715356

Hambly, David M.; Gross, Michael L.



Low-homology protein threading  

PubMed Central

Motivation: The challenge of template-based modeling lies in the recognition of correct templates and generation of accurate sequence-template alignments. Homologous information has proved to be very powerful in detecting remote homologs, as demonstrated by the state-of-the-art profile-based method HHpred. However, HHpred does not fare well when proteins under consideration are low-homology. A protein is low-homology if we cannot obtain sufficient amount of homologous information for it from existing protein sequence databases. Results: We present a profile-entropy dependent scoring function for low-homology protein threading. This method will model correlation among various protein features and determine their relative importance according to the amount of homologous information available. When proteins under consideration are low-homology, our method will rely more on structure information; otherwise, homologous information. Experimental results indicate that our threading method greatly outperforms the best profile-based method HHpred and all the top CASP8 servers on low-homology proteins. Tested on the CASP8 hard targets, our threading method is also better than all the top CASP8 servers but slightly worse than Zhang-Server. This is significant considering that Zhang-Server and other top CASP8 servers use a combination of multiple structure-prediction techniques including consensus method, multiple-template modeling, template-free modeling and model refinement while our method is a classical single-template-based threading method without any post-threading refinement. Contact: PMID:20529920

Peng, Jian; Xu, Jinbo



Ensemble learning prediction of protein-protein interactions using proteins functional annotations.  


Protein-protein interactions are important for the majority of biological processes. A significant number of computational methods have been developed to predict protein-protein interactions using protein sequence, structural and genomic data. Vast experimental data is publicly available on the Internet, but it is scattered across numerous databases. This fact motivated us to create and evaluate new high-throughput datasets of interacting proteins. We extracted interaction data from DIP, MINT, BioGRID and IntAct databases. Then we constructed descriptive features for machine learning purposes based on data from Gene Ontology and DOMINE. Thereafter, four well-established machine learning methods: Support Vector Machine, Random Forest, Decision Tree and Naïve Bayes, were used on these datasets to build an Ensemble Learning method based on majority voting. In cross-validation experiment, sensitivity exceeded 80% and classification/prediction accuracy reached 90% for the Ensemble Learning method. We extended the experiment to a bigger and more realistic dataset maintaining sensitivity over 70%. These results confirmed that our datasets are suitable for performing PPI prediction and Ensemble Learning method is well suited for this task. Both the processed PPI datasets and the software are available at . PMID:24469380

Saha, Indrajit; Zubek, Julian; Klingström, Tomas; Forsberg, Simon; Wikander, Johan; Kierczak, Marcin; Maulik, Ujjwal; Plewczynski, Dariusz



Analysis of protein-protein interaction between late cornified envelope proteins and corneodesmosin.  


Deletion of two members of the late cornified envelope (LCE) family, LCE3B and LCE3C (LCE3C_LCE3B-del), has been identified as risk factor for psoriasis with a possible role in skin barrier function. Moreover, genetic interaction between LCE3C_LCE3B-del and HLA-C*06, located in the psoriasis susceptibility regions 4 and 1 (PSORS4 and 1), has been reported in several populations. Because of high linkage disequilibrium between the PSORS1 genes HLA-C*06 and corneodesmosin (CDSN), both genes are potentially involved in psoriasis. As corneodesmosin and LCE proteins are both constituents of the stratum corneum, we investigated potential direct protein-protein interactions between six LCE proteins and two corneodesmosin sequence variants. Partial colocalization of LCE2 and CDSN was observed in normal and psoriasis skin using immunofluorescence microscopy. Co-expression of eCFP-LCE and mRFP-CDSN proteins in COS-1 cells and human adult keratinocytes, and GST pull-down results did not provide evidence for direct interactions between LCE proteins and CDSN variants. PMID:25078048

Bergboer, Judith G M; Dulak, Maria G; van Vlijmen-Willems, Ivonne M J J; Jonca, Nathalie; van Wijk, Erwin; Hendriks, Wiljan J A J; Zeeuwen, Patrick L J M; Schalkwijk, Joost



Protein structure prediction using hybrid AI methods  

SciTech Connect

This paper describes a new approach for predicting protein structures based on Artificial Intelligence methods and genetic algorithms. We combine nearest neighbor searching algorithms, neural networks, heuristic rules and genetic algorithms to form an integrated system to predict protein structures from their primary amino acid sequences. First we describe our methods and how they are integrated, and then apply our methods to several protein sequences. The results are very close to the real structures obtained by crystallography. Parallel genetic algorithms are also implemented.

Guan, X.; Mural, R.J.; Uberbacher, E.C.



Protein misfolding disorders: Pathogenesis and intervention  

Microsoft Academic Search

Summary  Newly synthesized proteins in the living cell must go through a folding process to attain their functional structure. To achieve\\u000a this in an efficient fashion, all organisms, including humans, have evolved a large set of molecular chaperones that assist\\u000a the folding as well as the maintenance of the functional structure of cellular proteins. Aberrant proteins, the result of\\u000a production errors,

N. Gregersen



Protein Aggregation as a Cause for Disease  

Microsoft Academic Search

The ability of proteins to fold into a defined and functional conformation is one of the most fundamental processes in biology.\\u000a Certain conditions, however, initiate misfolding or unfolding of proteins. This leads to the loss of functional protein or\\u000a it can result in a wide range of diseases. One group of diseases, which includes Alzheimer’s, Parkinson’s, Huntington’s disease,\\u000a and the

T. Scheibel; J. Buchner


Enhancement of rabbit protein S anticoagulant cofactor activity in vivo by modulation of the protein S C4B binding protein interaction.  

PubMed Central

The carboxy-terminal region of protein S has been recently been observed to be involved in the interaction between protein S and C4b-binding protein (Walker, F. J. 1989. J. Biol. Chem. 264:17645-17658). A synthetic peptide, GVQLDLDEAI, corresponding to that region of protein S has been used to investigate the protein S/C4b-binding protein interaction in vitro and in vivo. Rabbit activated protein C possesses species-specific anticoagulant activity for which rabbit protein S functions as a cofactor. In plasma, rabbit protein S is found in complex with C4b-binding protein. GVQLDLDEAI can inhibit this interaction, resulting in enhancement of the anticoagulant activity of rabbit activated protein C. The effect of the peptide can be blocked by the concurrent addition of human or rabbit C4b-binding protein. When infused into rabbits, GVQLDLDEAI was cleared from the circulation with a half-life of 80 min. This is significantly less rapid than the clearance of similarly sized control peptides (half-life of 15 min), but much more than that of bovine protein S, a much larger protein (half-life of 15 h). Plasma samples removed from the rabbits after infusion with GVQLDLDEAI were found to have increased concentrations of free protein S and to show enhanced anticoagulation by rabbit activated protein C ex vivo in a dose-dependent manner. The concentration for half-maximal effect (5 microM) was very similar to that observed in vitro. These results suggest that the formation of a complex between protein S and C4b-binding protein is important in the regulation of protein S activity in vivo, and that modulation of this interaction allows one to influence the anticoagulant activity of the protein C pathway. Images PMID:2147696

Weinstein, R E; Walker, F J



SoyProLow: A protein database enriched in low abundant soybean proteins  

PubMed Central

Soybeans are an important legume crop that contain 2 major storage proteins, ?-conglycinin and glycinin, which account about 70- 80% of total seed proteins. These abundant proteins hinder the isolation and characterization of several low abundant proteins in soybean seeds. Several protein extraction methodologies were developed in our laboratory to decrease these abundant storage proteins in seed extracts and to also decrease the amount of ribulose-1, 5-bisphosphate carboxylase/oxygenase (RuBisCO), which is normally very abundant in leaf extracts. One of the extraction methodologies used 40% isopropanol and was more effective in depleting soybean storage proteins and enhancing low abundant seed proteins than similar methods using 10-80% isopropanol. Extractions performed with 40% isopropanol decreased the amount of storage proteins and revealed 107 low abundant proteins when using the combined approaches of two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and Mass Spectrometry (MS). The separation of proteins was achieved by iso-electric focusing (IEF) and 2D-PAGE. The proteins were analyzed with MS techniques to provide amino acid sequence. The proteins were identified by comparing their amino acid sequences with those in different databases including NCBI-non redundant, UniprotKB and MSDB databases. In this investigation, previously published results on low abundant soybean seed proteins were used to create an online database (SoyProLow) to provide a data repository that can be used as a reference to identify and characterize low abundance proteins. This database is freely accessible to individuals using similar techniques and can be for the subsequent genetic manipulation to produce value added soybean traits. An intuitive user interface based on dynamic HTML enables users to browse the network and the profiles of the low abundant proteins. Availability PMID:25352730

Tavakolan, Mona; Alkharouf, Nadim W; Matthews, Benjamin F; Natarajan, Savithiry S



Characterizing Protein Conformation Space  

E-print Network

In this work, we propose a radical approach for exploring the space of all possible protein structures. We present techniques to explore the clash-free conformation space, which comprises all protein structures whose atoms ...

Nigham, Anshul


Determination of immunoreactive proteins of Babesia ovis.  


Babesia ovis, an intraerythrocytic protozoan parasite transmitted by ticks, causes severe infections in sheep in tropical and subtropical regions of the world. Parasite-specific immunoreactive proteins have been used as antigen in the serological diagnosis of babesiosis. There is no study about determination of B. ovis-specific proteins in sheep. This study was planned to determine the immunoreactive proteins of B. ovis. In this study, two splenectomized lambs, and twelve seropositive sheep and five seronegative lambs for anti-B. ovis antibodies were used as materials. Infected blood samples at 5% of parasitemia from the two splenectomized lambs experimentally infected with a virulent B. ovis field strain were analyzed for B. ovis-specific proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting (WB). B. ovis-specific five major proteins were recognized by anti-B. ovis serum but not by healthy sheep serum. They were of approximate molecular weights 154, 109, 77, 58, and 38 kDa. As the control samples, protein profiles of the blood extracts of two lambs before splenectomy operation were also blotted with the immune sera, but none of the five proteins was detected. These proteins were also immunoblotted with heterologous positive and negative sheep sera. All of twelve positive sera recognized the 109 kDa protein with 100 percent sensitivity. The 77 kDa protein reacted in 11 of 12 sera (91.6%). The sensitivities of the other 3 proteins ranged between 83.3% and 25%. The five protein bands immunoblotted with sera of the 5 negative lambs did not give any positive reaction. The results of this study revealed the presence of proteins recognized by the serum antibodies of experimentally and naturally infected sheep with B. ovis. Additional studies on the purification of these proteins and on subsequently their utilization in a serodiagnostic method are required to improve the serological diagnosis of ovine babesiosis. PMID:24209710

Sevinc, Ferda; Guler, Leyla; Sevinc, Mutlu; Ekici, Ozlem Derinbay; Isik, Nermin



Proteins as "dopable" bio-electronic materials  

NASA Astrophysics Data System (ADS)

Proteins are surprisingly good solid-state electronic conductors. This holds also for proteins without any known biological electron transfer function. How do they do it? To answer this question we measure solid-state electron transport (ETp) across proteins that are "dry" (only tightly bound water, to retain the conformation, still present). We compare results for the electron transfer (ET) protein, Azurin (Az), the proton-pumping membrane protein Bacteriorhodopsin (bR), and for Human and Bovine Serum Albumin (HSA and BSA). Clear differences between these proteins are seen, which preserve their structure in the solid state measurement configuration. Importantly for future bioelectronics, the results are sensitive to protein modification, e.g., removing or disconnecting the retinal in bR and removing or replacing the Cu redox centre in Az. These cofactors can thus be viewed as natural dopants for proteins. Insight in the ETp mechanism comes from temperature-dependent studies. Az shows 40-360K temperature-independent ETp across its 3.5 nm long axis, until its denaturation temperature, indicative of tunneling. Cu removal, replacement (by Zn) or deuteration changes this to thermally activated ETp. This suggests hopping and involvement of the amide backbone in the ETp. The latter, which rhymes with indications from ETp experiments on oligopeptide and simulations of ET in proteins, opens the way for modeling what otherwise is an awfully complex system. Below 200K all proteins and their variants show temperature-independent ETp. We can furthermore make a totally electrically inactive protein, HSA, into an efficient ETp medium by doping it with natural poly-ene. Putting our data in perspective by comparing them to all known protein ETp data in the literature, we conclude that, in general, proteins are well described as dopable molecular wires.

Cahen, David



Protein Structure Analysis Iosif Vaisman  

E-print Network

Protein Structure Analysis Iosif Vaisman 2010 BINF 731 Protein sequence (helices, beta-sheets, turns) ·Tertiary - the three-dimensional fold of a protein subunit prediction Protein structure classification Protein representations Protein representations Protein

Vaisman, Iosif


Protein Structure Analysis Iosif Vaisman  

E-print Network

Protein Structure Analysis Iosif Vaisman 2009 BINF 731 Protein sequence (helices, beta-sheets, turns) ·Tertiary - the three-dimensional fold of a protein subunit prediction Protein structure classification Protein representations Protein representations Protein

Vaisman, Iosif


Protein Explorer Tutorial  

NSDL National Science Digital Library

Protein Explorer is free software for visualizing the three-dimensional structures of protein, DNA, and RNA macromolecules, and their interactions and binding of ligands, inhibitors, and drugs. It is arguably the easiest-to-use software of its kind. It is suitable for high school and college students (ages 16 years and older), yet it is also widely used by graduate students and researchers. This site provides instruction in the use of Protein Explorer and simplifies queries of the Protein Data Bank.


Protein Clustering and Classification  

Microsoft Academic Search

Proteins are the building blocks of all organisms. The name protein is derived from the Greek word ‘protos’ which means first or primal. Indeed proteins are the most fundamental substance of\\u000a life, as they are the key component of the protoplasm of all cells. In addition to their role as the building blocks of cells\\u000a and tissue, proteins also play

Ori Sasson; Michal Linial



Protein-spanning water networks and implications for prediction of protein-protein interactions mediated through hydrophobic effects.  


Hydrophobic effects, often conflated with hydrophobic forces, are implicated as major determinants in biological association and self-assembly processes. Protein-protein interactions involved in signaling pathways in living systems are a prime example where hydrophobic effects have profound implications. In the context of protein-protein interactions, a priori knowledge of relevant binding interfaces (i.e., clusters of residues involved directly with binding interactions) is difficult. In the case of hydrophobically mediated interactions, use of hydropathy-based methods relying on single residue hydrophobicity properties are routinely and widely used to predict propensities for such residues to be present in hydrophobic interfaces. However, recent studies suggest that consideration of hydrophobicity for single residues on a protein surface require accounting of the local environment dictated by neighboring residues and local water. In this study, we use a method derived from percolation theory to evaluate spanning water networks in the first hydration shells of a series of small proteins. We use residue-based water density and single-linkage clustering methods to predict hydrophobic regions of proteins; these regions are putatively involved in binding interactions. We find that this simple method is able to predict with sufficient accuracy and coverage the binding interface residues of a series of proteins. The approach is competitive with automated servers. The results of this study highlight the importance of accounting of local environment in determining the hydrophobic nature of individual residues on protein surfaces. Proteins 2014; 82:3312-3326. © 2014 Wiley Periodicals, Inc. PMID:25204743

Cui, Di; Ou, Shuching; Patel, Sandeep



Whey Protein Concentrate Market Enhancement.  

National Technical Information Service (NTIS)

The report describes results of an investigation into the marketability and potential uses for whey protein concentrate (WPC), a co-product formed in the process of converting cheese whey to ethanol. With more than four billion pounds of whey produced ann...

R. R. Zall, A. H. Teixeira, J. H. Chen



Protein Engineering of Penicillin Acylase  

PubMed Central

Penicillin acylases (PA) are widely used for the production of semi-synthetic ?-lactam antibiotics and chiral compounds. In this review, the latest achievements in the production of recombinant enzymes are discussed, as well as the results of PA type G protein engineering. PMID:22649651

Tishkov, V.I.; Savin, S.S.; Yasnaya, A.S.



A disulfide polymerized protein crystal.  


The vDED coiled coil domain from human BAP29 was crystallized in dimeric and tetrameric forms. For the dimer, a disulfide bond was unexpectedly found to bridge a crystal contact, resulting in complete cross-linking along the c-axis. This indicates that it is in principle possible to design spontaneously polymerizing protein crystals. PMID:25327138

Quistgaard, Esben M



Amino-acid site variability among natural and designed proteins  

PubMed Central

Computational protein design attempts to create protein sequences that fold stably into pre-specified structures. Here we compare alignments of designed proteins to alignments of natural proteins and assess how closely designed sequences recapitulate patterns of sequence variation found in natural protein sequences. We design proteins using RosettaDesign, and we evaluate both fixed-backbone designs and variable-backbone designs with different amounts of backbone flexibility. We find that proteins designed with a fixed backbone tend to underestimate the amount of site variability observed in natural proteins while proteins designed with an intermediate amount of backbone flexibility result in more realistic site variability. Further, the correlation between solvent exposure and site variability in designed proteins is lower than that in natural proteins. This finding suggests that site variability is too uniform across different solvent exposure states (i.e., buried residues are too variable or exposed residues too conserved). When comparing the amino acid frequencies in the designed proteins with those in natural proteins we find that in the designed proteins hydrophobic residues are underrepresented in the core. From these results we conclude that intermediate backbone flexibility during design results in more accurate protein design and that either scoring functions or backbone sampling methods require further improvement to accurately replicate structural constraints on site variability. PMID:24255821

Jackson, Eleisha L.; Ollikainen, Noah; Covert, Arthur W.; Kortemme, Tanja



Amino-acid site variability among natural and designed proteins.  


Computational protein design attempts to create protein sequences that fold stably into pre-specified structures. Here we compare alignments of designed proteins to alignments of natural proteins and assess how closely designed sequences recapitulate patterns of sequence variation found in natural protein sequences. We design proteins using RosettaDesign, and we evaluate both fixed-backbone designs and variable-backbone designs with different amounts of backbone flexibility. We find that proteins designed with a fixed backbone tend to underestimate the amount of site variability observed in natural proteins while proteins designed with an intermediate amount of backbone flexibility result in more realistic site variability. Further, the correlation between solvent exposure and site variability in designed proteins is lower than that in natural proteins. This finding suggests that site variability is too uniform across different solvent exposure states (i.e., buried residues are too variable or exposed residues too conserved). When comparing the amino acid frequencies in the designed proteins with those in natural proteins we find that in the designed proteins hydrophobic residues are underrepresented in the core. From these results we conclude that intermediate backbone flexibility during design results in more accurate protein design and that either scoring functions or backbone sampling methods require further improvement to accurately replicate structural constraints on site variability. PMID:24255821

Jackson, Eleisha L; Ollikainen, Noah; Covert, Arthur W; Kortemme, Tanja; Wilke, Claus O



Structural classification of proteins and structural genomics: new insights into protein folding and evolution  

PubMed Central

During the past decade, the Protein Structure Initiative (PSI) centres have become major contributors of new families, superfamilies and folds to the Structural Classification of Proteins (SCOP) database. The PSI results have increased the diversity of protein structural space and accelerated our understanding of it. This review article surveys a selection of protein structures determined by the Joint Center for Structural Genomics (JCSG). It presents previously undescribed ?-sheet architectures such as the double barrel and spiral ?-roll and discusses new examples of unusual topologies and peculiar structural features observed in proteins characterized by the JCSG and other Structural Genomics centres. PMID:20944210

Andreeva, Antonina; Murzin, Alexey G.



Towards a detailed atlas of protein-protein interactions.  


Protein interaction maps are the key to understand the complex world of biological processes inside the cell. Public protein databases have already catalogued hundreds of thousands of experimentally discovered interactions, and struggle to curate all the existing information dispersed through the literature. However, to be most efficient, standard protocols need to be implemented for direct submission of new interaction sets directly into databases. At the same time, great efforts are invested to expand the coverage of the interaction space and unveil the molecular details of such interactions up to the atomistic level. The net result will be the definition of a detailed atlas spanning the universe of protein interactions to guide the everyday work of the biologist. PMID:23896349

Mosca, Roberto; Pons, Tirso; Céol, Arnaud; Valencia, Alfonso; Aloy, Patrick



Imaging Protein-protein Interactions in vivo  

PubMed Central

Protein-protein interactions are a hallmark of all essential cellular processes. However, many of these interactions are transient, or energetically weak, preventing their identification and analysis through traditional biochemical methods such as co-immunoprecipitation. In this regard, the genetically encodable fluorescent proteins (GFP, RFP, etc.) and their associated overlapping fluorescence spectrum have revolutionized our ability to monitor weak interactions in vivo using Förster resonance energy transfer (FRET)1-3. Here, we detail our use of a FRET-based proximity assay for monitoring receptor-receptor interactions on the endothelial cell surface. PMID:20972411

Seegar, Tom; Barton, William



General protein-protein cross-linking.  


This protocol describes a general protein-to-protein cross-linking procedure using the water-soluble amine-reactive homobifunctional BS(3) (bis[sulfosuccinimidyl] suberate); however, the protocol can be easily adapted using other cross-linkers of similar properties. BS(3) is composed of two sulfo-NHS ester groups and an 11.4 Å linker. Sulfo-NHS ester groups react with primary amines in slightly alkaline conditions (pH 7.2-8.5) and yield stable amide bonds. The reaction releases N-hydroxysuccinimide (see an application of NHS esters on Labeling a protein with fluorophores using NHS ester derivitization). PMID:24581440

Alegria-Schaffer, Alice



Palmitoylation of Hedgehog Proteins  

PubMed Central

Hedgehog (Hh) proteins are secreted signaling proteins that contain amide-linked palmitate at the N-terminus and cholesterol at the C-terminus. Palmitoylation of Hh proteins is critical for effective long- and short-range signaling. The palmitoylation reaction occurs during transit of Hh through the secretory pathway, most likely in the lumen of the ER. Attachment of palmitate to Hh proteins is independent of cholesterol modification and autoprocessing and is catalyzed by Hhat (Hedgehog acyltransferase). Hhat is a member of the membrane bound O-acyltransferase (MBOAT) family, a subgroup of multipass membrane proteins that catalyze transfer of fatty acyl groups to lipids and proteins. Several classes of secreted proteins have recently been shown to be substrates for MBOAT acyltransferases, including Hh proteins and Spitz (palmitoylated by Hhat), Wg/Wnt proteins (modified with palmitate and/or palmitoleate by Porcupine) and ghrelin (octanoylated by ghrelin O-acyltransferase). These findings highlight protein fatty acylation as a mechanism that not only influences membrane binding of intracellular proteins but also regulates the signaling range and efficacy of secreted proteins. PMID:22391306

Buglino, John A.; Resh, Marilyn D.



Intrinsically disordered protein  

Microsoft Academic Search

Proteins can exist in a trinity of structures: the ordered state, the molten globule, and the random coil. The five following examples suggest that native protein structure can correspond to any of the three states (not just the ordered state) and that protein function can arise from any of the three states and their transitions. (1) In a process that

A. Keith Dunker; J. David Lawson; Celeste J Brown; Ryan M Williams; Pedro Romero; Jeong S Oh; Christopher J Oldfield; Andrew M Campen; Catherine M Ratliff; Kerry W Hipps; Juan Ausio; Mark S Nissen; Raymond Reeves; ChulHee Kang; Charles R Kissinger; Robert W Bailey; Michael D Griswold; Wah Chiu; Ethan C Garner; Zoran Obradovic



Protein Design Zhilei Chen  

E-print Network

and materials in the pharmaceutical, chemical, food and agriculture industries. However, the number was limited to chemical modification methods in which specific residues in a protein are modified has greatly changed the landscape of protein design. Numerous powerful protein design techniques have

Zhao, Huimin


Modeling Protein Self Assembly  

ERIC Educational Resources Information Center

Understanding the structure and function of proteins is an important part of the standards-based science curriculum. Proteins serve vital roles within the cell and malfunctions in protein self assembly are implicated in degenerative diseases. Experience indicates that this topic is a difficult one for many students. We have found that the concept…

Baker, William P.; Jones, Carleton Buck; Hull, Elizabeth



The E5 Proteins  

PubMed Central

The E5 proteins are short transmembrane proteins encoded by many animal and human papillomaviruses. These proteins display transforming activity in cultured cells and animals, and they presumably also play a role in the productive virus life cycle. The E5 proteins are thought to act by modulating the activity of cellular proteins. Here, we describe the biological activities of the best-studied E5 proteins and discuss the evidence implicating specific protein targets and pathways in mediating these activities. The primary target of the 44-amino acid BPV1 E5 is the PDGF ? receptor, whereas the EGF receptor appears to be an important target of the 83-amino acid HPV16 E5 protein. Both E5 proteins also bind to the vacuolar ATPase and affect MHC class I expression and cell-cell communication. Continued studies of the E5 proteins will elucidate important aspects of transmembrane protein-protein interactions, cellular signal transduction, cell biology, virus replication, and tumorigenesis. PMID:23731971

DiMaio, Daniel; Petti, Lisa



Protein Preservation BIOMATERIALS  

E-print Network

Protein Preservation BIOMATERIALS Our goal is to develop measurements for characterizing sugar-based glasses with respect to their ability to serve as preservation media for therapeutic proteins to the stability of proteins in stabilizing sugar-based glass. This property had been completely overlooked


Protein thermostability in extremophiles  

Microsoft Academic Search

Thermostability of a protein is a property which cannot be attributed to the presence of a particular amino acid or to a post synthetic modification. Thermostability seems to be a property acquired by a protein through many small structural modifications obtained with the exchange of some amino acids and the modulation of the canonical forces found in all proteins such

Roberto Scandurra; Valerio Consalvi; Roberta Chiaraluce; Laura Politi; Paul C. Engel



A Stevedore's Protein Knot  

Microsoft Academic Search

Protein knots, mostly regarded as intriguing oddities, are gradually being recognized as significant structural motifs. Seven distinctly knotted folds have already been identified. It is by and large unclear how these exceptional structures actually fold, and only recently, experiments and simulations have begun to shed some light on this issue. In checking the new protein structures submitted to the Protein

Daniel Bölinger; Joanna I. Su?kowska; Hsiao-Ping Hsu; Leonid A. Mirny; Mehran Kardar; José N. Onuchic; Peter Virnau



Protein detection Proteomics  

E-print Network

Keywords Protein detection Proteomics Peptide Synthesis Alzheimer´s Disease Huntington of new techniques, which allow the specific detec- tion of proteins in the zeptomole range (1021 mole). This means to specifically detect a few thousand molecules of a single protein pre- sent in a complex mixture

Schüler, Axel


Measurement of solvent accessibility at protein-protein interfaces.  


Methods are presented for monitoring solvent accessibility of protein-ligand and protein-protein interfaces. The kinetics of solvent accessibility at the protein-protein interface is monitored by amide hydrogen/deuterium (H/2H) exchange detected by matrix assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS). A straightforward theoretical analysis is presented for determining the concentration of a weakly binding ligand that is required for achieving a situation in which the receptor is essentially 100% bound, and this is verified by control experiments. We show that when the receptor is essentially 100% bound it is possible to distinguish amide exchange as a result of solvent accessibility at the interface from amide exchange caused by complex dissociation. Methods are also presented for the measurement of tightly bound complexes of large interactions such as antibody-antigen complexes. Quantitation of the number of amides sequestered at the interface can be related to the number of H2O molecules excluded from the interface. PMID:15939994

Mandell, Jeffrey G; Baerga-Ortiz, Abel; Falick, Arnold M; Komives, Elizabeth A



RAFT polymers for protein recognition  

PubMed Central

Summary A new family of linear polymers with pronounced affinity for arginine- and lysine-rich proteins has been created. To this end, N-isopropylacrylamide (NIPAM) was copolymerized in water with a binding monomer and a hydrophobic comonomer using a living radical polymerization (RAFT). The resulting copolymers were water-soluble and displayed narrow polydispersities. They formed tight complexes with basic proteins depending on the nature and amount of the binding monomer as well as on the choice of the added hydrophobic comonomer. PMID:20703378

Tominey, Alan F; Liese, Julia; Wei, Sun; Kowski, Klaus



The 58-kDa microspherule protein (MSP58), a nucleolar protein, interacts with nucleolar protein p120.  


Protein p120 is a proliferation-related nucleolar protein which is detectable early in the G1 phase of the cell cycle and peaks early in the S phase. Most human malignant tumors contain much higher levels of protein p120 than normal resting cells. To identify p120-associated protein(s), a yeast two-hybrid screen was carried out using protein p120 as the bait. Two positive clones encoded portions of a novel protein, designated microspherule protein 58 kDa (MSP58). MSP58 mRNA is 1.9 kb and encodes an approximately 58-kDa polypeptide of 462 amino acids as shown by Western blotting of HeLa nucleolar proteins. The mouse MSP58 homolog has 97% amino acid similarity to human MSP58, but no MSP58 homolog was found in the yeast genome. The MSP58 N-terminal region contains serine-rich clusters and its C-terminal region has a coiled-coil domain. In insect Sf9 cells, recombinant p120 and MSP58 proteins associated with each other, confirming the results of the yeast two-hybrid assay. Deletion mutations revealed that the binding of MSP58 to p120 required a previously unrecognized coiled-coil domain within the N-terminal region of p120 and the C-terminal region of MSP58 protein. Immunofluorescence indicated that the MSP58 protein is localized in microspherules in the nucleolus. Anti-MSP58 Ig labeled nucleolar 'caps' when HeLa cells were treated with actinomycin D. When the MSP58 protein was overexpressed in COS-7 cells, the nucleolus became irregularly enlarged, which suggests that MSP58 may affect the size and shape of the nucleolus. PMID:9654073

Ren, Y; Busch, R K; Perlaky, L; Busch, H



Protein Binding: Do We Ever Learn??  

PubMed Central

Although the influence of protein binding (PB) on antibacterial activity has been reported for many antibiotics and over many years, there is currently no standardization for pharmacodynamic models that account for the impact of protein binding of antimicrobial agents in vitro. This might explain the somewhat contradictory results obtained from different studies. Simple in vitro models which compare the MIC obtained in protein-free standard medium versus a protein-rich medium are prone to methodological pitfalls and may lead to flawed conclusions. Within in vitro test systems, a range of test conditions, including source of protein, concentration of the tested antibiotic, temperature, pH, electrolytes, and supplements may influence the impact of protein binding. As new antibiotics with a high degree of protein binding are in clinical development, attention and action directed toward the optimization and standardization of testing the impact of protein binding on the activity of antibiotics in vitro become even more urgent. In addition, the quantitative relationship between the effects of protein binding in vitro and in vivo needs to be established, since the physiological conditions differ. General recommendations for testing the impact of protein binding in vitro are suggested. PMID:21537013

Zeitlinger, Markus A.; Derendorf, Hartmut; Mouton, Johan W.; Cars, Otto; Craig, William A.; Andes, David; Theuretzbacher, Ursula



Engineering cells to improve protein expression.  


Cellular engineering of bacteria, fungi, insect cells and mammalian cells is a promising methodology to improve recombinant protein production for structural, biochemical, and commercial applications. Increased understanding of the host organism biology has suggested engineering strategies targeting bottlenecks in transcription, translation, protein processing and secretory pathways, as well as cell growth and survival. A combination of metabolic engineering and synthetic biology has been used to improve the properties of cells for protein production, which has resulted in enhanced yields of multiple protein classes. PMID:24704806

Xiao, Su; Shiloach, Joseph; Betenbaugh, Michael J



The SALAMI protein structure search server  

PubMed Central

Protein structures often show similarities to another which would not be seen at the sequence level. Given the coordinates of a protein chain, the SALAMI server at will search the protein data bank and return a set of similar structures without using sequence information. The results page lists the related proteins, details of the sequence and structure similarity and implied sequence alignments. Via a simple structure viewer, one can view superpositions of query and library structures and finally download superimposed coordinates. The alignment method is very tolerant of large gaps and insertions, and tends to produce slightly longer alignments than other similar programs. PMID:19465380

Margraf, Thomas; Schenk, Gundolf; Torda, Andrew E.



Quantitative thermodynamic model for globular protein folding  

NASA Astrophysics Data System (ADS)

We present a statistical mechanics formalism for theoretical description of the process of protein folding ? unfolding transition in water environment. The formalism is based on the construction of the partition function of a protein obeying two-stage-like folding kinetics. Using the statistical mechanics model of solvation of hydrophobic hydrocarbons we obtain the partition function of infinitely diluted solution of proteins in water environment. The calculated dependencies of the protein heat capacities upon temperature are compared with the corresponding results of experimental measurements for staphylococcal nuclease and metmyoglobin.

Yakubovich, Alexander V.; Solov'yov, Andrey V.



Origins of the protein synthesis cycle  

NASA Technical Reports Server (NTRS)

Largely derived from experiments in molecular evolution, a theory of protein synthesis cycles has been constructed. The sequence begins with ordered thermal proteins resulting from the self-sequencing of mixed amino acids. Ordered thermal proteins then aggregate to cell-like structures. When they contained proteinoids sufficiently rich in lysine, the structures were able to synthesize offspring peptides. Since lysine-rich proteinoid (LRP) also catalyzes the polymerization of nucleoside triphosphate to polynucleotides, the same microspheres containing LRP could have synthesized both original cellular proteins and cellular nucleic acids. The LRP within protocells would have provided proximity advantageous for the origin and evolution of the genetic code.

Fox, S. W.



Thioredoxin-interacting protein regulates protein disulfide isomerases and endoplasmic reticulum stress  

PubMed Central

The endoplasmic reticulum (ER) is responsible for protein folding, modification, and trafficking. Accumulation of unfolded or misfolded proteins represents the condition of ER stress and triggers the unfolded protein response (UPR), a key mechanism linking supply of excess nutrients to insulin resistance and type 2 diabetes in obesity. The ER harbors proteins that participate in protein folding including protein disulfide isomerases (PDIs). Changes in PDI activity are associated with protein misfolding and ER stress. Here, we show that thioredoxin-interacting protein (Txnip), a member of the arrestin protein superfamily and one of the most strongly induced proteins in diabetic patients, regulates PDI activity and UPR signaling. We found that Txnip binds to PDIs and increases their enzymatic activity. Genetic deletion of Txnip in cells and mice led to increased protein ubiquitination and splicing of the UPR regulated transcription factor X-box-binding protein 1 (Xbp1s) at baseline as well as under ER stress. Our results reveal Txnip as a novel direct regulator of PDI activity and a feedback mechanism of UPR signaling to decrease ER stress. PMID:24843047

Lee, Samuel; Min Kim, Soo; Dotimas, James; Li, Letitia; Feener, Edward P; Baldus, Stephan; Myers, Ronald B; Chutkow, William A; Patwari, Parth; Yoshioka, Jun; Lee, Richard T



Thioredoxin-interacting protein regulates protein disulfide isomerases and endoplasmic reticulum stress.  


The endoplasmic reticulum (ER) is responsible for protein folding, modification, and trafficking. Accumulation of unfolded or misfolded proteins represents the condition of ER stress and triggers the unfolded protein response (UPR), a key mechanism linking supply of excess nutrients to insulin resistance and type 2 diabetes in obesity. The ER harbors proteins that participate in protein folding including protein disulfide isomerases (PDIs). Changes in PDI activity are associated with protein misfolding and ER stress. Here, we show that thioredoxin-interacting protein (Txnip), a member of the arrestin protein superfamily and one of the most strongly induced proteins in diabetic patients, regulates PDI activity and UPR signaling. We found that Txnip binds to PDIs and increases their enzymatic activity. Genetic deletion of Txnip in cells and mice led to increased protein ubiquitination and splicing of the UPR regulated transcription factor X-box-binding protein 1 (Xbp1s) at baseline as well as under ER stress. Our results reveal Txnip as a novel direct regulator of PDI activity and a feedback mechanism of UPR signaling to decrease ER stress. PMID:24843047

Lee, Samuel; Min Kim, Soo; Dotimas, James; Li, Letitia; Feener, Edward P; Baldus, Stephan; Myers, Ronald B; Chutkow, William A; Patwari, Parth; Yoshioka, Jun; Lee, Richard T



Evolution and function of CAG/polyglutamine repeats in protein-protein interaction networks  

PubMed Central

Expanded runs of consecutive trinucleotide CAG repeats encoding polyglutamine (polyQ) stretches are observed in the genes of a large number of patients with different genetic diseases such as Huntington's and several Ataxias. Protein aggregation, which is a key feature of most of these diseases, is thought to be triggered by these expanded polyQ sequences in disease-related proteins. However, polyQ tracts are a normal feature of many human proteins, suggesting that they have an important cellular function. To clarify the potential function of polyQ repeats in biological systems, we systematically analyzed available information stored in sequence and protein interaction databases. By integrating genomic, phylogenetic, protein interaction network and functional information, we obtained evidence that polyQ tracts in proteins stabilize protein interactions. This happens most likely through structural changes whereby the polyQ sequence extends a neighboring coiled-coil region to facilitate its interaction with a coiled-coil region in another protein. Alteration of this important biological function due to polyQ expansion results in gain of abnormal interactions, leading to pathological effects like protein aggregation. Our analyses suggest that research on polyQ proteins should shift focus from expanded polyQ proteins into the characterization of the influence of the wild-type polyQ on protein interactions. PMID:22287626

Schaefer, Martin H.; Wanker, Erich E.; Andrade-Navarro, Miguel A.



Role of partial protein unfolding in alcohol-induced protein aggregation  

PubMed Central

Proteins aggregate in response to various stresses including changes in solvent conditions. Addition of alcohols has been recently shown to induce aggregation of disease-related as well as non-disease-related proteins. Here we probed the biophysical mechanisms underlying alcohol-induced protein aggregation, in particular the role of partial protein unfolding in aggregation. We have studied aggregation mechanisms due to benzyl alcohol which is used in numerous biochemical and biotechnological applications. We chose cytochrome c as a model protein, for the reason that various optical and structural probes are available to monitor its global and partial unfolding reactions. Benzyl alcohol induced the aggregation of cytochrome c in isothermal conditions and decreased the temperature at which the protein aggregates. However, benzyl alcohol did not perturb the overall native conformation of cytochrome c. Instead, it caused partial unfolding of a local protein region around the methionine residue at position 80. Site-specific optical probes, two-dimensional NMR titrations, and hydrogen exchange all support this conclusion. The protein aggregation temperature varied linearly with the melting temperature of the Met80 region. Stabilizing the Met80 region by heme iron reduction drastically decreased protein aggregation, which confirmed that the local unfolding of this region causes protein aggregation. These results indicate that a possible mechanism by which alcohols induce protein aggregation is through partial rather than complete unfolding of native proteins. PMID:20597088

Singh, Surinder M.; Cabello-Villegas, Javier; Hutchings, Regina L.; Mallela, Krishna M.G.



Protein toxins: intracellular trafficking for targeted therapy  

Microsoft Academic Search

The immunotoxin approach is based on the use of tumor-targeting ligands or antibodies that are linked to the catalytic (toxic) moieties of bacterial or plant protein toxins. In this review, we first discuss the current state of clinical development of immunotoxin approaches describing the results obtained with the two toxins most frequently used: diphtheria and Pseudomonas toxin-derived proteins. In the

L Johannes; D Decaudin



Multiscale Simulation of Protein Mediated Membrane Remodeling  

PubMed Central

Proteins interacting with membranes can result in substantial membrane deformations and curvatures. This effect is known in its broadest terms as membrane remodeling. This review article will survey current multiscale simulation methodologies that have been employed to examine protein-mediated membrane remodeling. PMID:19922811

Ayton, Gary S.; Voth, Gregory A.



Targeting proteins for degradation  

PubMed Central

Protein degradation plays a central role in many cellular functions. Misfolded and damaged proteins are removed from the cell to avoid toxicity. The concentrations of regulatory proteins are adjusted by degradation at the appropriate time. Both foreign and native proteins are digested into small peptides as part of the adaptive immune response. In eukaryotic cells, an ATP-dependent protease called the proteasome is responsible for much of this proteolysis. Proteins are targeted for proteasomal degradation by a two-part degron, which consists of a proteasome binding signal and a degradation initiation site. Here we describe how both components contribute to the specificity of degradation. PMID:19841631

Schrader, Erin K; Harstad, Kristine G; Matouschek, Andreas



Highly thermostable fluorescent proteins  


Thermostable fluorescent proteins (TSFPs), methods for generating these and other stability-enhanced proteins, polynucleotides encoding such proteins, and assays and method for using the TSFPs and TSFP-encoding nucleic acid molecules are provided. The TSFPs of the invention show extremely enhanced levels of stability and thermotolerance. In one case, for example, a TSFP of the invention is so stable it can be heated to C. for short periods of time without denaturing, and retains 85% of its fluorescence when heated to C. for several minutes. The invention also provides a method for generating stability-enhanced variants of a protein, including but not limited to fluorescent proteins.

Bradbury, Andrew M. (Santa Fe, NM); Waldo, Geoffrey S. (Santa Fe, NM); Kiss, Csaba (Los Alamos, NM)



A Rapid and Efficient Immunoenzymatic Assay to Detect Receptor Protein Interactions: G Protein-Coupled Receptors  

PubMed Central

G protein-coupled receptors (GPCRs) represent one of the largest families of cell surface receptors, and are the target of at least one-third of the current therapeutic drugs on the market. Along their life cycle, GPCRs are accompanied by a range of specialized GPCR-interacting proteins (GIPs), which take part in receptor proper folding, targeting to the appropriate subcellular compartments and in receptor signaling tasks, and also in receptor regulation processes, such as desensitization and internalization. The direction of protein-protein interactions and multi-protein complexes formation is crucial in understanding protein function and their implication in pathological events. Although several methods have been already developed to assay protein complexes, some of them are quite laborious, expensive, and, more important, they do not generate fully quantitative results. Herein, we show a rapid immunoenzymatic assay to quantify GPCR interactionswith its signaling proteins. The recently de-orphanized GPCR, GPR17, was chosen as a GPCR prototype to optimize the assay. In a GPR17 transfected cell line and primary oligodendrocyte precursor cells, GPR17 interaction with proteins involved in the typical GPCR regulation, such as desensitization and internalization machinery, was investigated. The obtained results were validated by co-immunoprecipitation experiments, confirming this new method as a rapid and quantitative assay to study protein-protein interactions. PMID:24733071

Zappelli, Elisa; Daniele, Simona; Abbracchio, Maria P.; Martini, Claudia; Trincavelli, Maria Letizia



Material characterization of porcine lenticular soluble proteins.  


The soluble proteins present in the ocular lens impart important optical and dynamic mechanical properties on the lens. The short-range order of crystallin proteins grants transparency to a very concentrated protein solution. This unique protein system directly enables proper visual function of the eye. These proteins were investigated in steady and oscillatory shear. Steady shear data were fitted with a modified Herschel-Bulkley yield stress model that allows for a Newtonian plateau at low shear rates. The Cox-Merz rule was used in conjunction with large amplitude oscillatory shear to give insight into the degradation of the fluid structure with increasing strain. The shear thinning viscoelastic behavior of these proteins gives rise to beneficial mechanical properties and results from the same short-range order granting optical transparency. PMID:18471013

Reilly, Matthew A; Rapp, Brian; Hamilton, Paul D; Shen, Amy Q; Ravi, Nathan



Microbial production of spider silk proteins.  


The remarkable properties of spider dragline silk and related protein polymers will find many applications if the materials can be produced economically. We have demonstrated the production of high molecular weight spider dragline silk analog proteins encoded by synthetic genes in several microbial systems, including Escherichia coli and Pichia pastoris. In E. coli, proteins of up to 1000 amino acids in length could be produced efficiently, but the yield and homogeneity of higher molecular weight silk proteins were found to be limited by truncated synthesis, probably as a result of ribosome termination errors. No such phenomenon was observed in the yeast P. pastoris, where higher molecular weight silk proteins could be produced without heterogeneity due to truncated synthesis. Spider dragline silk analog proteins could be secreted by P. pastoris when fused to both the signal sequence and N-terminal pro-sequence of the Saccharomyces cerevisiae alpha-mating factor gene. PMID:11763501

Fahnestock, S R; Yao, Z; Bedzyk, L A



Protein patterning in polycarbonate microfluidic channels  

NASA Astrophysics Data System (ADS)

In this work protein patterning has been achieved within a polycarbonate microfluidic device. Channel structures were first coated with plasma polymerized allylamine (ALAPP) followed by the "cloud point" deposition of polyethylene oxide (PEO), a protein repellent molecule. Excimer laser micromachining was used to pattern the PEO to control protein localization. Subsequent removal of a sacrificial layer of polycarbonate resulted in the patterned polymer coating only in the channels of a simple fluidic device. Following a final diffusion bonding fabrication step the devices were filled with a buffer containing Streptavidin conjugated with fluorescein, and visualized under a confocal fluorescent microscope. This confirmed that protein adhesion occurred only in laser patterned areas. The ability to control protein adhesion in microfludic channels leads to the possibility of generating arrays of proteins or cells within polymer microfludics for cheap automated biosensors and synthesis systems.

Thomson, David A.; Hayes, Jason P.; Thissen, Helmut



Localization of peroxisomal matrix proteins by photobleaching  

SciTech Connect

The distribution of some enzymes between peroxisomes and cytosol, or a dual localization in both these compartments, can be difficult to reconcile. We have used photobleaching in live cells expressing green fluorescent protein (GFP)-fusion proteins to show that imported bona fide peroxisomal matrix proteins are retained in the peroxisome. The high mobility of the GFP-fusion proteins in the cytosol and absence of peroxisomal escape makes it possible to eliminate the cytosolic fluorescence by photobleaching, to distinguish between exclusively cytosolic proteins and proteins that are also present at low levels in peroxisomes. Using this technique we found that GFP tagged bile acid-CoA:amino acid N-acyltransferase (BAAT) was exclusively localized in the cytosol in HeLa cells. We conclude that the cytosolic localization was due to its carboxyterminal non-consensus peroxisomal targeting signal (-SQL) since mutation of the -SQL to -SKL resulted in BAAT being efficiently imported into peroxisomes.

Buch, Charlotta [Department of Biosciences and Nutrition, Karolinska Institutet, SE-141 57 Huddinge (Sweden) [Department of Biosciences and Nutrition, Karolinska Institutet, SE-141 57 Huddinge (Sweden); Soedertoerns University, Life Sciences, SE-141 89 Huddinge (Sweden); Hunt, Mary C. [Department of Genetics, Microbiology and Toxicology, Stockholm University, Arrhenius Laboratory, Svante Arrhenius vaeg 16F, SE-106 91 Stockholm (Sweden)] [Department of Genetics, Microbiology and Toxicology, Stockholm University, Arrhenius Laboratory, Svante Arrhenius vaeg 16F, SE-106 91 Stockholm (Sweden); Alexson, Stefan E.H. [Karolinska Institutet, Department of Laboratory Medicine, Division of Clinical Chemistry C1-74, Karolinska University Hospital at Huddinge, SE-141 86 Stockholm (Sweden)] [Karolinska Institutet, Department of Laboratory Medicine, Division of Clinical Chemistry C1-74, Karolinska University Hospital at Huddinge, SE-141 86 Stockholm (Sweden); Hallberg, Einar, E-mail: [Soedertoerns University, Life Sciences, SE-141 89 Huddinge (Sweden)] [Soedertoerns University, Life Sciences, SE-141 89 Huddinge (Sweden)



Protein biosynthesis with conformationally restricted amino acids  

SciTech Connect

The incorporation of conformationally constrained amino acids into peptides is a powerful approach for generating structurally defined peptides as conformational probes and bioactive agents. The ability to site-specifically introduce constrained amino acids into large polypeptide chains would provide a similar opportunity to probe the flexibility, conformation, folding and stability of proteins. To this end, we have examined the competence of the Escherichia coli protein biosynthetic machinery to incorporate a number of these unnatural amino acids into the 164 residue protein T4 lysozyme (T4L). Results clearly demonstrate that the protein biosynthetic machinery can accommodate a wide variety of conformationally constrained amino acids. The expansion of structural motifs that can be biosynthetically incorporated into proteins to include a large number of conformationally constrained amino acids significantly increases the power of mutagenesis methods as probes of protein structure and function and provides additional insights into the steric requirements of the translational machinery. 13 refs., 2 figs.

Mendel, D. (Univ. of California, Berkeley (United States) Lawrence Berkeley Lab., CA (United States)); Ellman, J.; Schultz, P.G. (Univ. of California, Berkeley (United States))



Protein self-diffusion in crowded solutions  

PubMed Central

Macromolecular crowding in biological media is an essential factor for cellular function. The interplay of intermolecular interactions at multiple time and length scales governs a fine-tuned system of reaction and transport processes, including particularly protein diffusion as a limiting or driving factor. Using quasielastic neutron backscattering, we probe the protein self-diffusion in crowded aqueous solutions of bovine serum albumin on nanosecond time and nanometer length scales employing the same protein as crowding agent. The measured diffusion coefficient D(?) strongly decreases with increasing protein volume fraction ? explored within 7% ? ? ? 30%. With an ellipsoidal protein model and an analytical framework involving colloid diffusion theory, we separate the rotational Dr(?) and translational Dt(?) contributions to D(?). The resulting Dt(?) is described by short-time self-diffusion of effective spheres. Protein self-diffusion at biological volume fractions is found to be slowed down to 20% of the dilute limit solely due to hydrodynamic interactions. PMID:21730176

Roosen-Runge, Felix; Hennig, Marcus; Zhang, Fajun; Jacobs, Robert M. J.; Sztucki, Michael; Schober, Helmut; Seydel, Tilo; Schreiber, Frank



Librational fluctuations in protein glasses.  


Librational motions in the region of the protein "glass" (or dynamic) transition are analysed for spin-labelled haemoglobin, serum albumin and ?-lactoglobulin by EPR spectroscopy. A discontinuity in the temperature dependence of the mean-square librational amplitude, , occurs in the region of 200K as found for the mean-square atomic displacement, , at the protein dynamic transition by Mössbauer spectroscopy and neutron scattering. The discontinuity in vs. T can be described by the Vogel-Tammann-Fulcher equation, implying a finite glass transition temperature. Above the dynamic transition, vs. 1/T can be approximated by the Arrhenius law with activation energies similar to those usually found for , and relaxation processes in glass-forming media and the hydration shells of proteins. Similar results are found for librational fluctuations of membranous Na,K-ATPase spin-labelled either on superficial SH groups or on those essential to activity. PMID:23669570

Marsh, Derek; Bartucci, Rosa; Guzzi, Rita; Sportelli, Luigi; Esmann, Mikael



Sequence repeats and protein structure  

NASA Astrophysics Data System (ADS)

Repeats are frequently found in known protein sequences. The level of sequence conservation in tandem repeats correlates with their propensities to be intrinsically disordered. We employ a coarse-grained model of a protein with a two-letter amino acid alphabet, hydrophobic (H) and polar (P), to examine the sequence-structure relationship in the realm of repeated sequences. A fraction of repeated sequences comprises a distinct class of bad folders, whose folding temperatures are much lower than those of random sequences. Imperfection in sequence repetition improves the folding properties of the bad folders while deteriorating those of the good folders. Our results may explain