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Sample records for additional protein factors

  1. Transcriptional Regulation of Zein Gene Expression in Maize through the Additive and Synergistic Action of opaque2, Prolamine-Box Binding Factor, and O2 Heterodimerizing Proteins

    PubMed Central

    Zhang, Zhiyong; Yang, Jun; Wu, Yongrui

    2015-01-01

    Maize (Zea mays) zeins are some of the most abundant cereal seed storage proteins (SSPs). Their abundance influences kernel hardness but compromises its nutritional quality. Transcription factors regulating the expression of zein and other SSP genes in cereals are endosperm-specific and homologs of maize opaque2 (O2) and prolamine-box binding factor (PBF). This study demonstrates that the ubiquitously expressed transcription factors, O2 heterodimerizing proteins (OHPs), specifically regulate 27-kD γ-zein gene expression (through binding to an O2-like box in its promoter) and interact with PBF. The zein content of double mutants OhpRNAi;o2 and PbfRNAi;o2 and the triple mutant PbfRNAi;OhpRNAi;o2 is reduced by 83, 89, and 90%, respectively, compared with the wild type. The triple mutant developed the smallest zein protein bodies, which were merely one-tenth the wild type’s size. Total protein levels in these mutants were maintained in a relatively constant range through proteome rebalancing. These data show that OHPs, O2, and PBF are master regulators of zein storage protein synthesis, acting in an additive and synergistic mode. The differential expression patterns of OHP and O2 genes may cause the slight differences in the timing of 27-kD γ-zein and 22-kD α-zein accumulation during protein body formation. PMID:25901087

  2. High levels of acute phase proteins and soluble 70 kDa heat shock proteins are independent and additive risk factors for mortality in colorectal cancer

    PubMed Central

    Kocsis, Judit; Mészáros, Tamás; Madaras, Balázs; Tóth, Éva Katalin; Kamondi, Szilárd; Gál, Péter; Varga, Lilian; Prohászka, Zoltán

    2010-01-01

    Recently, we reported that high soluble Hsp70 (sHsp70) level was a significant predictor of mortality during an almost 3-year-long follow-up period in patients with colorectal cancer. This association was the strongest in the group of <70-year-old female patients as well as in those who were in a less advanced stage of the disease at baseline. According to these observations, measurement of the serum level of sHsp70 is a useful, stage-independent prognostic marker in colorectal cancer, especially in patients without distant metastasis. Since many literature data indicated that measurement of C-reactive protein (CRP) and other acute phase proteins (APPs) may also be suitable for predicting the mortality of patients with colorectal cancer, it seemed reasonable to study whether the effect of sHsp70 and other APPs are related or independent. In order to answer this question, we measured the concentrations of CRP as well as of other complement-related APPs (C1 inhibitor, C3, and C9) along with that of the MASP-2 complement component in the sera of 175 patients with colorectal cancer and known levels of sHsp70, which have been used in our previous study. High (above median) levels of CRP, C1 esterase inhibitor (C1-INH), and sHsp70 were found to be independently associated with poor patient survival, whereas no such association was observed with the other proteins tested. According to the adjusted Cox proportional hazards analysis, the additive effect of high sHsp70, CRP, and C1-INH levels on the survival of patients exceeded that of high sHsp70 alone, with a hazard ratio (HR) of 2.83 (1.13–70.9). In some subgroups of patients, such as in females [HR 4.80 (1.07–21.60)] or in ≤70-year-old patients [HR 11.53 (2.78–47.70)], even greater differences were obtained. These findings indicate that the clinical mortality–prediction value of combined measurements of sHsp70, CRP, and C1-INH with inexpensive methods can be very high, especially in specific subgroups of

  3. Effect of additives on protein aggregation.

    PubMed

    Hamada, Hiroyuki; Arakawa, Tsutomu; Shiraki, Kentaro

    2009-06-01

    This paper overviews solution additives that affect protein stability and aggregation during refolding, heating, and freezing processes. Solution additives are mainly grouped into two classes, i.e., protein denaturants and stabilizers. The former includes guanidine, urea, strong ionic detergents, and certain chaotropic salts; the latter includes certain amino acids, sugars, polyhydric alcohols, osmolytes, and kosmotropic salts. However, there are solution additives that are not unambiguously placed into these two classes, including arginine, certain divalent cation salts (e.g., MgCl(2)) and certain polyhydric alcohols (e.g., ethylene glycol). Certain non-ionic or non-detergent surfactants, ionic liquids, amino acid derivatives, polyamines, and certain amphiphilic polymers may belong to this class. They have marginal effects on protein structure and stability, but are able to disrupt protein interactions. Information on additives that do not catalyze chemical reactions nor affect protein functions helps us to design protein solutions for increased stability or reduced aggregation. PMID:19519415

  4. A comparison of human prothrombin, factor IX (Christmas factor), factor X (Stuart factor), and protein S.

    PubMed

    Di Scipio, R G; Hermodson, M A; Yates, S G; Davie, E W

    1977-02-22

    Human prothrombin, factor IX, and factor X have been idolated in high yield and characterized as the their amino-terminal sequence, molecular weight, amino acid composition, and migration in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. An additional human plasma protein, called protein S, has also been purified and its properties have been compared with those of prothrombin, factor IX, and factor X. Prothrombin (mol wt 72 000), factor IX (mol wt 57 000), and protein S (mol wt 69 000) are single-chain glycoproteins, while factor X (mol wt 59 000) is a glycoprotein composed of two polypeptide chains held together by a disulfide bond(s). The amino-terminal sequence of the light chain of human factor X is homologous with prothrombin, factor IX, and protein S. The heavy chain of human factor X is slightly larger than the heavy chain of bovine factor X and differs from bovine factor X in its amino-terminal sequence.

  5. Activity of the upstream TATA-less promoter of the p21(Waf1/Cip1) gene depends on transcription factor IIA (TFIIA) in addition to TFIIA-reactive TBP-like protein.

    PubMed

    Suzuki, Hidefumi; Maeda, Ryo; Nakadai, Tomoyoshi; Tamura, Taka-aki

    2014-07-01

    TATA-binding protein-like protein (TLP) binds to transcription factor IIA (TFIIA) with high affinity, although the significance of this binding is poorly understood. In this study, we investigated the role of TFIIA in transcriptional regulation of the p21(Waf1/Cip1) (p21) gene. It has been shown that TLP is indispensable for p53-activated transcription from an upstream TATA-less promoter of the p21 gene. We found that mutant TLPs having decreased TFIIA-binding ability exhibited weakened transcriptional activation function for the upstream promoter. Activity of the upstream promoter was enhanced considerably by an increased amount of TFIIA in a p53-dependent manner, whereas activity of the TATA-containing downstream promoter was enhanced only slightly. TFIIA potentiated the upstream promoter additively with TLP. Although TFIIA is recruited to both promoters, activity of the upstream promoter was much more dependent on TFIIA. Recruitment of TFIIA and TLP to the upstream promoter was augmented in etoposide-treated cells, in which the amount of TFIIA-TLP complex is increased, and TFIIA-reactive TLP was required for the recruitment of both factors. It was confirmed that etoposide-stimulated transcription depends on TLP. We also found that TFIIA-reactive TLP acts to decrease cell growth rate, which can be explained by interaction of the p21 promoter with the transcription factors that we examined. The results of the present study suggest that the upstream TATA-less promoter of p21 needs TFIIA and TFIIA-reactive TLP for p53-dependent transcriptional enhancement.

  6. 14 CFR 1203.406 - Additional classification factors.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 14 Aeronautics and Space 5 2010-01-01 2010-01-01 false Additional classification factors. 1203.406... PROGRAM Guides for Original Classification § 1203.406 Additional classification factors. In determining the appropriate classification category, the following additional factors should be considered:...

  7. Oxygen additions in serial femtosecond crystallographic protein structures.

    PubMed

    Wang, Jimin

    2016-10-01

    In principle, serial femtosecond crystallography (SFX) could yield data sets that are completely free of the effects caused by slow, radiation-induced chemical reactions, for example, oxygen additions, responsible for radiation damage. However, experimental evidence is presented here that SFX data sets obtained by techniques that expose different parts of the same specimen to single pulses of radiation do not have this property, even if the specimen in question is frozen. The diffraction image of each such crystal obtained with the first pulse of radiation is certain to represent the structure of a protein that has not been modified chemically, but all of the images obtained subsequently from the same crystal will represent structures that have been modified to a lesser or greater extent by oxygen additions because of the rapid diffusion of oxygenic free radicals through the specimen. The higher the level of oxygen additions a crystal suffers during data collection, the poorer the statistical quality of data set obtained from it will, and the higher the free R-factors of the resulting structural model. PMID:27438534

  8. 14 CFR 1203.406 - Additional classification factors.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 14 Aeronautics and Space 5 2013-01-01 2013-01-01 false Additional classification factors. 1203.406 Section 1203.406 Aeronautics and Space NATIONAL AERONAUTICS AND SPACE ADMINISTRATION INFORMATION SECURITY PROGRAM Guides for Original Classification § 1203.406 Additional classification factors. In...

  9. 14 CFR 1203.406 - Additional classification factors.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 14 Aeronautics and Space 5 2011-01-01 2010-01-01 true Additional classification factors. 1203.406 Section 1203.406 Aeronautics and Space NATIONAL AERONAUTICS AND SPACE ADMINISTRATION INFORMATION SECURITY PROGRAM Guides for Original Classification § 1203.406 Additional classification factors. In...

  10. 14 CFR 1203.406 - Additional classification factors.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 14 Aeronautics and Space 5 2012-01-01 2012-01-01 false Additional classification factors. 1203.406 Section 1203.406 Aeronautics and Space NATIONAL AERONAUTICS AND SPACE ADMINISTRATION INFORMATION SECURITY PROGRAM Guides for Original Classification § 1203.406 Additional classification factors. In...

  11. Protein Synthesis Initiation Factors: Phosphorylation and Regulation

    SciTech Connect

    Karen S. Browning

    2009-06-15

    The initiation of the synthesis of proteins is a fundamental process shared by all living organisms. Each organism has both shared and unique mechanisms for regulation of this vital process. Higher plants provide for a major amount of fixation of carbon from the environment and turn this carbon into food and fuel sources for our use. However, we have very little understanding of how plants regulate the synthesis of the proteins necessary for these metabolic processes. The research carried out during the grant period sought to address some of these unknowns in the regulation of protein synthesis initiation. Our first goal was to determine if phosphorylation plays a significant role in plant initiation of protein synthesis. The role of phosphorylation, although well documented in mammalian protein synthesis regulation, is not well studied in plants. We showed that several of the factors necessary for the initiation of protein synthesis were targets of plant casein kinase and showed differential phosphorylation by the plant specific isoforms of this kinase. In addition, we identified and confirmed the phosphorylation sites in five of the plant initiation factors. Further, we showed that phosphorylation of one of these factors, eIF5, affected the ability of the factor to participate in the initiation process. Our second goal was to develop a method to make initiation factor 3 (eIF3) using recombinant methods. To date, we successfully cloned and expressed 13/13 subunits of wheat eIF3 in E. coli using de novo gene construction methods. The final step in this process is to place the subunits into three different plasmid operons for co-expression. Successful completion of expression of eIF3 will be an invaluable tool to the plant translation community.

  12. 14 CFR § 1203.406 - Additional classification factors.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 14 Aeronautics and Space 5 2014-01-01 2014-01-01 false Additional classification factors. § 1203.406 Section § 1203.406 Aeronautics and Space NATIONAL AERONAUTICS AND SPACE ADMINISTRATION INFORMATION SECURITY PROGRAM Guides for Original Classification § 1203.406 Additional classification...

  13. Tuning protein-protein interactions using cosolvents: specific effects of ionic and non-ionic additives on protein phase behavior.

    PubMed

    Hansen, Jan; Platten, Florian; Wagner, Dana; Egelhaaf, Stefan U

    2016-04-21

    Cosolvents are routinely used to modulate the (thermal) stability of proteins and, hence, their interactions with proteins have been studied intensely. However, less is known about their specific effects on protein-protein interactions, which we characterize in terms of the protein phase behavior. We analyze the phase behavior of lysozyme solutions in the presence of sodium chloride (NaCl), guanidine hydrochloride (GuHCl), glycerol, and dimethyl sulfoxide (DMSO). We experimentally determined the crystallization boundary (XB) and, in combination with data on the cloud-point temperatures (CPTs), the crystallization gap. In agreement with other studies, our data indicate that the additives might affect the protein phase behavior through electrostatic screening and additive-specific contributions. At high salt concentrations, where electrostatic interactions are screened, both the CPT and the XB are found to be linear functions of the additive concentration. Their slopes quantify the additive-specific changes of the phase behavior and thus of the protein-protein interactions. While the specific effect of NaCl is to induce attractions between proteins, DMSO, glycerol and GuHCl (with increasing strength) weaken attractions and/or induce repulsions. Except for DMSO, changes of the CPT are stronger than those of the XB. Furthermore, the crystallization gap widens in the case of GuHCl and glycerol and narrows in the case of NaCl. We relate these changes to colloidal interaction models, namely square-well and patchy interactions. PMID:27020538

  14. Colloidal graphenes as heterogeneous additives to enhance protein crystal yield.

    PubMed

    Gully, Benjamin S; Zou, Jianli; Cadby, Gemma; Passon, Daniel M; Iyer, K Swaminathan; Bond, Charles S

    2012-09-01

    In the structural analysis of proteins via X-ray diffraction, a rate-limiting step is in favourable nucleation, a problematic obstacle in successful generation of protein crystals. Here graphene and graphene oxide were applied to protein crystallisation trials, offering improvements in crystalline output and nucleation.

  15. Additives

    NASA Technical Reports Server (NTRS)

    Smalheer, C. V.

    1973-01-01

    The chemistry of lubricant additives is discussed to show what the additives are chemically and what functions they perform in the lubrication of various kinds of equipment. Current theories regarding the mode of action of lubricant additives are presented. The additive groups discussed include the following: (1) detergents and dispersants, (2) corrosion inhibitors, (3) antioxidants, (4) viscosity index improvers, (5) pour point depressants, and (6) antifouling agents.

  16. Factors influencing the inhibition of protein kinases.

    PubMed

    Brockhoff, Marielle; Hau, Jean-Christophe; Fontana, Patrizia; Zimmermann, Catherine; Pover, Alain De; Erdmann, Dirk; Chène, Patrick

    2012-04-01

    The protein kinase field is a very active research area in the pharmaceutical industry and many activities are ongoing to identify inhibitors of these proteins. The design of new chemical entities with improved pharmacological properties requires a deeper understanding of the factors that modulate inhibitor-kinase interactions. In this report, we studied the effect of two of these factors--the magnesium ion cofactor and the protein substrate--on inhibitors of the type I insulin-like growth factor receptor. Our results show that the concentration of magnesium ion influences the potency of adenosine triphosphate (ATP) competitive inhibitors, suggesting an explanation for the observation that such compounds retain their nanomolar potency in cells despite the presence of millimolar levels of ATP. We also showed that the peptidic substrate affects the potency of these inhibitors in a different manner, suggesting that the influence of this substrate on compound potency should be taken into consideration during drug discovery.

  17. EKylation: Addition of an Alternating-Charge Peptide Stabilizes Proteins.

    PubMed

    Liu, Erik J; Sinclair, Andrew; Keefe, Andrew J; Nannenga, Brent L; Coyle, Brandon L; Baneyx, François; Jiang, Shaoyi

    2015-10-12

    For nearly 40 years, therapeutic proteins have been stabilized by chemical conjugation of polyethylene glycol (PEG), but recently zwitterionic materials have proved to be a more effective substitute. In this work, we demonstrate that genetic fusion of alternating-charge extensions consisting of anionic glutamic acid (E) and cationic lysine (K) is an effective strategy for protein stabilization. This bioinspired "EKylation" method not only confers the stabilizing benefits of poly(zwitterions) but also allows for rapid biosynthesis of target constructs. Poly(EK) peptides of different predetermined lengths were appended to the C-terminus of a native β-lactamase and its destabilized TEM-19 mutant. The EK-modified enzymes retained biological activity and exhibited increased stability to environmental stressors such as high temperature and high-salt solutions. This one-step strategy provides a broadly applicable alternative to synthetic polymer conjugation that is biocompatible and degradable. PMID:26407134

  18. AP-42 ADDITIONS AND REVISIONS - TRANSPORTABILITY FACTORS FOR FUGITIVE DUST

    EPA Science Inventory

    The product is a table of factors, one for each county in the US, reflecting the portion of fugitive dust removed very close to the source via impaction on vegetation and similar mechanisms. Factors were based on land cover in area (county or grid cell) A praft final product was...

  19. Protein-Protein Interaction Analysis Highlights Additional Loci of Interest for Multiple Sclerosis

    PubMed Central

    Ragnedda, Giammario; Disanto, Giulio; Giovannoni, Gavin; Ebers, George C.; Sotgiu, Stefano; Ramagopalan, Sreeram V.

    2012-01-01

    Genetic factors play an important role in determining the risk of multiple sclerosis (MS). The strongest genetic association in MS is located within the major histocompatibility complex class II region (MHC), but more than 50 MS loci of modest effect located outside the MHC have now been identified. However, the relative candidate genes that underlie these associations and their functions are largely unknown. We conducted a protein-protein interaction (PPI) analysis of gene products coded in loci recently reported to be MS associated at the genome-wide significance level and in loci suggestive of MS association. Our aim was to identify which suggestive regions are more likely to be truly associated, which genes are mostly implicated in the PPI network and their expression profile. From three recent independent association studies, SNPs were considered and divided into significant and suggestive depending on the strength of the statistical association. Using the Disease Association Protein-Protein Link Evaluator tool we found that direct interactions among genetic products were significantly higher than expected by chance when considering both significant regions alone (p<0.0002) and significant plus suggestive (p<0.007). The number of genes involved in the network was 43. Of these, 23 were located within suggestive regions and many of them directly interacted with proteins coded within significant regions. These included genes such as SYK, IL-6, CSF2RB, FCLR3, EIF4EBP2 and CHST12. Using the gene portal BioGPS, we tested the expression of these genes in 24 different tissues and found the highest values among immune-related cells as compared to non-immune tissues (p<0.001). A gene ontology analysis confirmed the immune-related functions of these genes. In conclusion, loci currently suggestive of MS association interact with and have similar expression profiles and function as those significantly associated, highlighting the fact that more common variants remain to be

  20. Backbone Additivity in the Transfer Model of Protein Solvation

    SciTech Connect

    Hu, Char Y.; Kokubo, Hironori; Lynch, Gillian C.; Bolen, D Wayne; Pettitt, Bernard M.

    2010-05-01

    The transfer model implying additivity of the peptide backbone free energy of transfer is computationally tested. Molecular dynamics simulations are used to determine the extent of change in transfer free energy (ΔGtr) with increase in chain length of oligoglycine with capped end groups. Solvation free energies of oligoglycine models of varying lengths in pure water and in the osmolyte solutions, 2M urea and 2M trimethylamine N-oxide (TMAO), were calculated from simulations of all atom models, and ΔGtr values for peptide backbone transfer from water to the osmolyte solutions were determined. The results show that the transfer free energies change linearly with increasing chain length, demonstrating the principle of additivity, and provide values in reasonable agreement with experiment. The peptide backbone transfer free energy contributions arise from van der Waals interactions in the case of transfer to urea, but from electrostatics on transfer to TMAO solution. The simulations used here allow for the calculation of the solvation and transfer free energy of longer oligoglycine models to be evaluated than is currently possible through experiment. The peptide backbone unit computed transfer free energy of –54 cal/mol/Mcompares quite favorably with –43 cal/mol/M determined experimentally.

  1. Buffer additives other than the surfactant sodium dodecyl sulfate for protein separations by capillary electrophoresis.

    PubMed

    Corradini, D

    1997-10-10

    The different compounds utilized as additives to the electrolyte solutions employed in protein capillary zone electrophoresis (CZE) for minimizing protein-capillary wall interactions, for improving selectivity and resolution and for controlling the electroosmotic flow are reviewed. The dependence of the electroosmotic flow on the different variables that can be affected by the incorporation of an additive into the electrolytic solution is discussed. A list of the most effective additives employed for protein separations by CZE is reported in Appendix A.

  2. R Factor Proteins Synthesized in Escherichia coli Minicells: Incorporation Studies with Different R Factors and Detection of Deoxyribonucleic Acid-Binding Proteins1

    PubMed Central

    Levy, Stuart B.

    1974-01-01

    Analysis of the protein synthesized by Escherichia coli minicells containing R factors demonstrated a variety of low- and high-molecular-weight polypeptides in sodium dodecyl sulfate (SDS)-polyacrylamide gels. Only half of this protein was released into a soluble fraction on lysis of these minicells. The other half remained associated with the minicell envelope. The efficiency of precursor incorporation into protein and the kinds of proteins synthesized changed with the age of the minicells at the time of harvest. About 1 to 2% of the soluble R factor-coded protein bound to calf thymus, E. coli, or R factor DNA-cellulose. Although most of these proteins were excluded from Sephadex G-100 columns, they migrated chiefly as low-molecular-weight-polypeptides (13,000 to 15,000) in SDS-polyacrylamide gels. Additional DNA-binding proteins that appeared to be higher-molecular-weight peptides were noted in extracts from younger minicells. At least one protein, identified as an SDS band, appeared to bind selectively to R factor DNA-cellulose. Minicells with R factors also contained DNA-binding proteins of cell origin, including the core RNA polymerase. No such binding proteins were found in R− minicells. These studies suggest that: (i) R factors code for proteins that may be involved in their own DNA metabolism; (ii) R factor DNA-binding proteins may be associated with larger host cell DNA-binding proteins or subunits of larger R factor proteins; and (iii) the age of the minicell influences the extent of protein synthesis and the kinds of proteins synthesized by R factors in minicells. Images PMID:4612023

  3. An atomic view of additive mutational effects in a protein structure

    SciTech Connect

    Skinner, M.M.; Terwilliger, T.C.

    1996-04-01

    Substitution of a single amino acid in a protein will often lead to substantial changes in properties. If these properties could be altered in a rational way then proteins could be readily generated with functions tailored to specific uses. When amino acid substitutions are made at well-separated locations in a single protein, their effects are generally additive. Additivity of effects of amino acid substitutions is very useful because the properties of proteins with any combination of substitutions can be inferred directly from those of the proteins with single changes. It would therefore be of considerable interest to have a means of knowing whether substitutions at a particular pair of sites in a protein are likely to lead to additive effects. The structural basis for additivity of effects of mutations on protein function was examined by determining crystal structures of single and double mutants in the hydrophobic core of gene V protein. Structural effects of mutations were found to be cumulative when two mutations were made in a single protein. Additivity occurs in this case because the regions structurally affected by mutations at the two sites do not overlap even though the sites are separated by only 9 {angstrom}. Structural distortions induced by mutations in gene V protein decrease rapidly, but not isotropically, with distance from the site of mutation. It is anticipated that cases where structural and functional effects of mutations will be additive could be identified simply by examining whether the regions structurally affected by each component mutation overlap.

  4. A protein factor essential for microtubule assembly.

    PubMed Central

    Weingarten, M D; Lockwood, A H; Hwo, S Y; Kirschner, M W

    1975-01-01

    A heat stable protein essentail for microtubule assembly has been isolated. This protein, which we designate tau (tau), is present in association with tubulin purified from porcine brain by repeated cycles of polymerization. Tau is separated from tubulin by ion exchange chromatography on phosphocellulose. In the absence of tau, tubulin exists entirely as a 6S dimer of two polypeptide chains (alpha and beta tubulin) with a molecular weight of 120,000, which will not assemble into microtubules in vitro. Addition of tau completely restores tubule-forming capacity. Under nonpolymerizing conditions, tau converts 6S dimers to 36S rings-structures which have been implicated as intermediates in tubule formation. Hence, tau appears to act on the 6S tubulin dimer, activating it for polymerization. The unique ability of tau to restore the normal features of in vitro microtubule assembly makes it likely that tau is a major regulator of microtubule formation in cells. Images PMID:1057175

  5. Rational design of solution additives for the prevention of protein aggregation.

    PubMed

    Baynes, Brian M; Trout, Bernhardt L

    2004-09-01

    We have developed a statistical-mechanical model of the effect of solution additives on protein association reactions. This model incorporates solvent radial distribution functions obtained from all-atom molecular dynamics simulations of particular proteins into simple models of protein interactions. In this way, the effects of additives can be computed along the entire association/dissociation reaction coordinate. We used the model to test our hypothesis that a class of large solution additives, which we term "neutral crowders," can slow protein association and dissociation by being preferentially excluded from protein-protein encounter complexes, in a manner analogous to osmotic stress. The magnitude of this proposed "gap effect" was probed for two simple model systems: the association of two spheres and the association of two planes. Our results suggest that for a protein of 20 A radius, an 8 A additive can increase the free energy barrier for association and dissociation by as much as 3-6 kcal/mol. Because the proposed gap effect is present only for reactions involving multiple molecules, it can be exploited to develop novel additives that affect protein association reactions although having little or no effect on unimolecular reactions such as protein folding. This idea has many potential applications in areas such as the stabilization of proteins against aggregation during folding and in pharmaceutical formulations.

  6. Structural changes in gluten protein structure after addition of emulsifier. A Raman spectroscopy study

    NASA Astrophysics Data System (ADS)

    Ferrer, Evelina G.; Gómez, Analía V.; Añón, María C.; Puppo, María C.

    2011-06-01

    Food protein product, gluten protein, was chemically modified by varying levels of sodium stearoyl lactylate (SSL); and the extent of modifications (secondary and tertiary structures) of this protein was analyzed by using Raman spectroscopy. Analysis of the Amide I band showed an increase in its intensity mainly after the addition of the 0.25% of SSL to wheat flour to produced modified gluten protein, pointing the formation of a more ordered structure. Side chain vibrations also confirmed the observed changes.

  7. Pathogenesis-Related Proteins Limit the Retention of Condensed Tannin Additions to Red Wines.

    PubMed

    Springer, Lindsay F; Sherwood, Robert W; Sacks, Gavin L

    2016-02-17

    Exogenous additions of condensed tannin (CT) to must or wine are a common winemaking practice, but many studies have reported inexplicably low and variable retention of added CT. We observed that additions of purified CT to red wines can result in the formation of an insoluble precipitate with high nitrogen content. Proteomic analysis of the precipitant identified several classes of pathogenesis-related proteins. Proteins in juices and red wines were quantitated by SDS-PAGE and were highest in native Vitis spp., followed by interspecific hybrids and Vitis vinifera. Wine protein was positively correlated with the ratio of juice protein to the quantity of tannin derived from fruit. The binding of added CT by wine protein could be well modeled by the Freundlich equation. These observations may explain the poor CT retention in previous studies, particularly for interspecific hybrids, and also indicate that protein removal during winemaking may improve exogenous CT retention.

  8. Three WRKY transcription factors additively repress abscisic acid and gibberellin signaling in aleurone cells.

    PubMed

    Zhang, Liyuan; Gu, Lingkun; Ringler, Patricia; Smith, Stanley; Rushton, Paul J; Shen, Qingxi J

    2015-07-01

    Members of the WRKY transcription factor superfamily are essential for the regulation of many plant pathways. Functional redundancy due to duplications of WRKY transcription factors, however, complicates genetic analysis by allowing single-mutant plants to maintain wild-type phenotypes. Our analyses indicate that three group I WRKY genes, OsWRKY24, -53, and -70, act in a partially redundant manner. All three showed characteristics of typical WRKY transcription factors: each localized to nuclei and yeast one-hybrid assays indicated that they all bind to W-boxes, including those present in their own promoters. Quantitative real time-PCR (qRT-PCR) analyses indicated that the expression levels of the three WRKY genes varied in the different tissues tested. Particle bombardment-mediated transient expression analyses indicated that all three genes repress the GA and ABA signaling in a dosage-dependent manner. Combination of all three WRKY genes showed additive antagonism of ABA and GA signaling. These results suggest that these WRKY proteins function as negative transcriptional regulators of GA and ABA signaling. However, different combinations of these WRKY genes can lead to varied strengths in suppression of their targets.

  9. Additional Protein Fortification Is Necessary in Extremely Low-Birth-Weight Infants Fed Human Milk.

    PubMed

    Picaud, Jean-Charles; Houeto, Nellie; Buffin, Rachel; Loys, Claire-Marie; Godbert, Isabelle; Haÿs, Stephane

    2016-07-01

    In the present study, approximately one in three (49/152, 32.2%) extremely low-birth-weight infants were demonstrated to require additional protein intake to supplement the standard fortification to achieve satisfactory weight gain. This additional protein fortification also resulted in a rapid increase in length-for-age (P < 0.001) and head circumference-for-age (P = 0.02) z scores.

  10. Cellular factors modulating the mechanism of tau protein aggregation

    PubMed Central

    Fontaine, Sarah N.; Sabbagh, Jonathan J.; Baker, Jeremy; Martinez-Licha, Carlos R.; Darling, April

    2015-01-01

    Pathological accumulation of the microtubule-associated protein tau, in the form of neurofibrillary tangles, is a major hallmark of Alzheimer’s disease, the most prevalent neurodegenerative condition worldwide. In addition to Alzheimer’s disease, a number of neurodegenerative diseases, called tauopathies, are characterized by the accumulation of aggregated tau in a variety of brain regions. While tau normally plays an important role in stabilizing the microtubule network of the cytoskeleton, its dissociation from microtubules and eventual aggregation into pathological deposits is an area of intense focus for therapeutic development. Here we discuss the known cellular factors that affect tau aggregation, from post-translational modifications to molecular chaperones. PMID:25666877

  11. Effect of cleaning agents and additives on Protein A ligand degradation and chromatography performance.

    PubMed

    Yang, Lihua; Harding, Jason D; Ivanov, Alexander V; Ramasubramanyan, Natarajan; Dong, Diane D

    2015-03-13

    Protein A chromatography, employing the recombinant Protein A ligand, is widely used as a capture step for antibody and Fc-fusion proteins manufacture. Protein A ligands in these matrices are susceptible to degradation/loss when exposed to cleaning agents such as sodium hydroxide, resulting in loss of capacity on reuse. In this study, MabSelect Protein A ligand and MabSelect SuRe Protein A ligand were chosen to evaluate the impact of alkaline cleaning solutions on the ligands and the packed columns. The Protein A ligands alone and the Protein A columns were incubated or cycled in different concentrations of sodium hydroxide solutions with and without additives, respectively. Ligand integrity (degradation) and ligand function (binding affinity) were studied using SDS-PAGE and customized Biacore technology, surface plasma resonance (SPR) and were successfully correlated with column performance measurement in terms of static binding capacity (SBC), dynamic binding capacity (DBC) and recovery as a function of exposure to cleaning agents with and without additives. The findings and the methodology presented in this study are not only able to determine appropriate cleaning conditions for Protein A chromatography, but also provided tools to enable systematic and rapid study of the cleaning solutions and conditions. PMID:25680549

  12. [Research progress in HIV auxiliary proteins counteracting host restriction factors].

    PubMed

    Chen, Qian-Qian; Xu, Qing-Gang; Zhang, Chi-Yu

    2014-01-01

    Identification and functional analyses of antiviral restriction factors in hosts have become hot research topics. Four HIV restriction factors, APOBEC3G, Trim5alpha, Tetherin, and SAMHD1, have been identified in recent years. By encoding auxiliary proteins, lentiviruses can counteract host restriction factors. For example, the auxiliary proteins Vif, Vpu, and Vpx of HIV antagonize APOBEC3G, Tetherin, and SAMHD1, respectively. Furthermore, these auxiliary proteins enable the entry of HIV into host cells and influence the replication and pathogenicity of HIV. In this paper, we review the research progress in the functions of the three HIV auxiliary proteins that can antagonize the host restriction factors.

  13. Addition of interleukin 1 (IL1) and IL17 soluble receptors to a tumour necrosis factor α soluble receptor more effectively reduces the production of IL6 and macrophage inhibitory protein-3α and increases that of collagen in an in vitro model of rheumatoid synoviocyte activation

    PubMed Central

    Chevrel, G; Garnero, P; Miossec, P

    2002-01-01

    Methods: A simplified model was set up to evaluate the effect of tumour necrosis factor α (TNFα) soluble receptors (sTNFR) used alone and in combination with soluble interleukin 1 receptor (sIL1R) and sIL17R on the production of markers of inflammation (IL6), of migration of dendritic cells (macrophage inhibitory protein-3α (MIP-3α)), and of matrix synthesis (C-propeptide of type 1 collagen (P1CP)). Synoviocytes were stimulated with supernatants of activated peripheral blood mononuclear cells (PBMC) from patients with rheumatoid arthritis (RA). Soluble receptors (sR) were preincubated at 1 γg/ml alone or in combination with the supernatants before addition to RA synoviocytes. IL6, MIP-3α, and P1CP production was measured by enzyme linked immunosorbent assay (ELISA) in 48 hour synoviocyte supernatants. Results: IL6 production decreased by 16% with sTNFR alone compared with no sTNFR (p<0.001) and by 41% with the combination of the three sR (p<0.001). MIP-3α production decreased by 77% with sTNFR alone compared with no sTNFR (p<0.001) and by 98% with the combination of the three sR (p<0.001). In the presence of sTNFR alone, P1CP production increased by 25% compared with no sR (p<0.01). The combination of the three sR increased P1CP production by 48% (p<0.01). Conclusion: The effect of sTNFR on IL6, MIP-3α, and P1CP production by RA synoviocytes stimulated by activated PBMC supernatants was further enhanced when combined with sIL1R and sIL17R. PMID:12117682

  14. Effect of the addition of CMC on the aggregation behaviour of proteins

    NASA Astrophysics Data System (ADS)

    Yu, H.; Sabato, S. F.; D'Aprano, G.; Lacroix, M.

    2004-09-01

    The effect of carboxymethylcellulose (CMC) on the aggregation of formulation based on calcium caseinate, commercial whey protein (WPC), and a 1:1 mixture of soy protein isolate (SPI) and whey protein isolate (WPI) was investigated. Protein aggregation could be observed upon addition of CMC, as demonstrated by size-exclusion chromatography. This aggregation behaviour was enhanced by means of physical treatments, such as heating at 90°C for 30 min or gamma-irradiation at 32 kGy. A synergy resulted from the combination of CMC to gamma-irradiation in Caseinate/CMC and SPI/WPI/CMC formulations. Furthermore, CMC prevented precipitation in irradiated protein solutions for a period of more than 3 months at 4°C.

  15. Precipitation of sword bean proteins by heating and addition of magnesium chloride in a crude extract.

    PubMed

    Nishizawa, Kaho; Masuda, Tetsuya; Takenaka, Yasuyuki; Masui, Hironori; Tani, Fumito; Arii, Yasuhiro

    2016-08-01

    Sword bean (Canavalia gladiata) seeds are a traditional food in Asian countries. In this study, we aimed to determine the optimal methods for the precipitation of sword bean proteins useful for the food development. The soaking time for sword beans was determined by comparing it with that for soybeans. Sword bean proteins were extracted from dried seeds in distilled water using novel methods. We found that most proteins could be precipitated by heating the extract at more than 90 °C. Interestingly, adding magnesium chloride to the extract at lower temperatures induced specific precipitation of a single protein with a molecular weight of approximately 48 kDa. The molecular weight and N-terminal sequence of the precipitated protein was identical to that of canavalin. These data suggested that canavalin was precipitated by the addition of magnesium chloride to the extract. Our results provide important insights into the production of processed foods from sword bean.

  16. Shrimp laminin receptor binds with capsid proteins of two additional shrimp RNA viruses YHV and IMNV.

    PubMed

    Busayarat, Nattaphon; Senapin, Saengchan; Tonganunt, Moltira; Phiwsaiya, Kornsunee; Meemetta, Watcharachai; Unajak, Sasimanas; Jitrapakdee, Sarawut; Lo, Chu-Fang; Phongdara, Amornrat

    2011-07-01

    Laminin receptor (Lamr) in shrimp was previously proposed to be a potential receptor protein for Taura syndrome virus (TSV) based on yeast two-hybrid assays. Since shrimp Lamr bound to the VP1 capsid protein of TSV, we were interested to know whether capsid/envelope proteins from other shrimp viruses would also bind to Lamr. Thus, capsid/envelope encoding genes from 5 additional shrimp viruses were examined. These were Penaeus stylirostris densovirus (PstDNV), white spot syndrome virus (WSSV), infectious myonecrosis virus (IMNV), Macrobrachium rosenbergii nodavirus (MrNV), and yellow head virus (YHV). Protein interaction analysis using yeast two-hybrid assay revealed that Lamr specifically interacted with capsid/envelope proteins of RNA viruses IMNV and YHV but not MrNV and not with the capsid/envelope proteins of DNA viruses PstDNV and WSSV. In vitro pull-down assay also confirmed the interaction between Lamr and YHV gp116 envelope protein, and injection of recombinant Lamr (rLamr) protein produced in yeast cells protected shrimp against YHV in laboratory challenge tests. PMID:21414409

  17. Effect of additives on the tensile performance and protein solubility of industrial oilseed residual based plastics.

    PubMed

    Newson, William R; Kuktaite, Ramune; Hedenqvist, Mikael S; Gällstedt, Mikael; Johansson, Eva

    2014-07-16

    Ten chemical additives were selected from the literature for their proposed modifying activity in protein-protein interactions. These consisted of acids, bases, reducing agents, and denaturants and were added to residual deoiled meals of Crambe abyssinica (crambe) and Brassica carinata (carinata) to modify the properties of plastics produced through hot compression molding at 130 °C. The films produced were examined for tensile properties, protein solubility, molecular weight distribution, and water absorption. Of the additives tested, NaOH had the greatest positive effect on tensile properties, with increases of 105% in maximum stress and 200% in strain at maximum stress for crambe and a 70% increase in strain at maximum stress for carinata. Stiffness was not increased by any of the applied additives. Changes in tensile strength and elongation for crambe and elongation for carinata were related to changes in protein solubility. Increased pH was the most successful in improving the protein aggregation and mechanical properties within the complex chemistry of residual oilseed meals.

  18. Effect of additives on the tensile performance and protein solubility of industrial oilseed residual based plastics.

    PubMed

    Newson, William R; Kuktaite, Ramune; Hedenqvist, Mikael S; Gällstedt, Mikael; Johansson, Eva

    2014-07-16

    Ten chemical additives were selected from the literature for their proposed modifying activity in protein-protein interactions. These consisted of acids, bases, reducing agents, and denaturants and were added to residual deoiled meals of Crambe abyssinica (crambe) and Brassica carinata (carinata) to modify the properties of plastics produced through hot compression molding at 130 °C. The films produced were examined for tensile properties, protein solubility, molecular weight distribution, and water absorption. Of the additives tested, NaOH had the greatest positive effect on tensile properties, with increases of 105% in maximum stress and 200% in strain at maximum stress for crambe and a 70% increase in strain at maximum stress for carinata. Stiffness was not increased by any of the applied additives. Changes in tensile strength and elongation for crambe and elongation for carinata were related to changes in protein solubility. Increased pH was the most successful in improving the protein aggregation and mechanical properties within the complex chemistry of residual oilseed meals. PMID:24971658

  19. 34 CFR 377.22 - What additional factors does the Secretary consider in making grants?

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... PROJECTS TO INCREASE CLIENT CHOICE PROGRAM How Does the Secretary Make an Award? § 377.22 What additional factors does the Secretary consider in making grants? In addition to the criteria in § 377.21, the... strategies to increase client choice, in order to ensure that a variety of approaches are demonstrated...

  20. Nonlinearly Additive Forces in Multivalent Ligand Binding to a Single Protein Revealed with Force Spectroscopy

    SciTech Connect

    Ratto, T V; Rudd, R E; Langry, K C; Balhorn, R L; McElfresh, M W

    2005-07-15

    We present evidence of multivalent interactions between a single protein molecule and multiple carbohydrates at a pH where the protein can bind four ligands. The evidence is based not only on measurements of the force required to rupture the bonds formed between ConcanavalinA (ConA) and {alpha}-D-mannose, but also on an analysis of the polymer-extension force curves to infer the polymer architecture that binds the protein to the cantilever and the ligands to the substrate. We find that although the rupture forces for multiple carbohydrate connections to a single protein are larger than the rupture force for a single connection, they do not scale additively with increasing number. Specifically, the most common rupture forces are approximately 46, 66, and 85 pN, which we argue corresponds to 1, 2, and 3 ligands being pulled simultaneously from a single protein as corroborated by an analysis of the linkage architecture. As in our previous work polymer tethers allow us to discriminate between specific and non-specific binding. We analyze the binding configuration (i.e. serial versus parallel connections) through fitting the polymer stretching data with modified Worm-Like Chain (WLC) models that predict how the effective stiffness of the tethers is affected by multiple connections. This analysis establishes that the forces we measure are due to single proteins interacting with multiple ligands, the first force spectroscopy study that establishes single-molecule multivalent binding unambiguously.

  1. Effect of salt addition on the thermal behavior of proteins of bovine meat from Argentina.

    PubMed

    Pighin, D G; Sancho, A M; Gonzalez, C B

    2008-07-01

    Research was undertaken to investigate how the addition of sodium chloride (NaCl) and/or sodium tripolyphosphate (TPP) to sous vide cooked meat pieces produces an increase in water holding capacity (WHC). Semitendinosus muscles were injected to obtain tissue final concentrations of 0.70% NaCl, 0.25% TPP, 0.70% NaCl+0.25% TPP, and 1.20% NaCl+0.25% TPP. SDS-PAGE analysis showed increased protein solubilization in those treatments which included NaCl. Thermal analysis of whole muscles and isolated myofibrils showed the destabilizing effect of NaCl and a global stabilizing effect of TPP. Both salts together induced a destabilizing global effect, where TPP assisted NaCl in breaking the meat structure. It is suggested that the WHC increments are related to conformational changes in myofibrillar proteins and to the weakening of myofibrillar structure by the removal of myofibrillar proteins. PMID:22062916

  2. Are major behavioral and sociodemographic risk factors for mortality additive or multiplicative in their effects?

    PubMed

    Mehta, Neil; Preston, Samuel

    2016-04-01

    All individuals are subject to multiple risk factors for mortality. In this paper, we consider the nature of interactions between certain major sociodemographic and behavioral risk factors associated with all-cause mortality in the United States. We develop the formal logic pertaining to two forms of interaction between risk factors, additive and multiplicative relations. We then consider the general circumstances in which additive or multiplicative relations might be expected. We argue that expectations about interactions among socio-demographic variables, and their relation to behavioral variables, have been stated in terms of additivity. However, the statistical models typically used to estimate the relation between risk factors and mortality assume that risk factors act multiplicatively. We examine empirically the nature of interactions among five major risk factors associated with all-cause mortality: smoking, obesity, race, sex, and educational attainment. Data were drawn from the cross-sectional NHANES III (1988-1994) and NHANES 1999-2010 surveys, linked to death records through December 31, 2011. Our analytic sample comprised 35,604 respondents and 5369 deaths. We find that obesity is additive with each of the remaining four variables. We speculate that its additivity is a reflection of the fact that obese status is generally achieved later in life. For all pairings of socio-demographic variables, risks are multiplicative. For survival chances, it is much more dangerous to be poorly educated if you are black or if you are male. And it is much riskier to be a male if you are black. These traits, established at birth or during childhood, literally result in deadly combinations. We conclude that the identification of interactions among risk factors can cast valuable light on the nature of the process being studied. It also has public health implications by identifying especially vulnerable groups and by properly identifying the proportion of deaths

  3. Effect of salts and organic additives on the solubility of proteins in aqueous solutions.

    PubMed

    Ruckenstein, Eli; Shulgin, Ivan L

    2006-11-16

    The goal of this review is to examine the effect of salts and organic additives on the solubility of proteins in aqueous mixed solvents. The focus is on the correlation between the aqueous protein solubility and the osmotic second virial coefficient or the preferential binding parameter. First, several approaches which connect the solubility and the osmotic second virial coefficient are presented. Most of the experimental and theoretical results correlate the solubility and the osmotic second virial coefficient in the presence of salts. The correlation of the aqueous protein solubility with the osmotic second virial coefficient when the cosolvent is an organic component requires additional research. Second, the aqueous protein solubility is correlated with the preferential binding parameter on the basis of a theory developed by the authors of the present review. This theory can predict (i) the salting-in or -out effect of a cosolvent and (ii) the initial slope of the solubility curve. Good agreement was obtained between theoretical predictions and experimental results.

  4. Controlling for gene expression changes in transcription factor protein networks.

    PubMed

    Banks, Charles A S; Lee, Zachary T; Boanca, Gina; Lakshminarasimhan, Mahadevan; Groppe, Brad D; Wen, Zhihui; Hattem, Gaye L; Seidel, Chris W; Florens, Laurence; Washburn, Michael P

    2014-06-01

    The development of affinity purification technologies combined with mass spectrometric analysis of purified protein mixtures has been used both to identify new protein-protein interactions and to define the subunit composition of protein complexes. Transcription factor protein interactions, however, have not been systematically analyzed using these approaches. Here, we investigated whether ectopic expression of an affinity tagged transcription factor as bait in affinity purification mass spectrometry experiments perturbs gene expression in cells, resulting in the false positive identification of bait-associated proteins when typical experimental controls are used. Using quantitative proteomics and RNA sequencing, we determined that the increase in the abundance of a set of proteins caused by overexpression of the transcription factor RelA is not sufficient for these proteins to then co-purify non-specifically and be misidentified as bait-associated proteins. Therefore, typical controls should be sufficient, and a number of different baits can be compared with a common set of controls. This is of practical interest when identifying bait interactors from a large number of different baits. As expected, we found several known RelA interactors enriched in our RelA purifications (NFκB1, NFκB2, Rel, RelB, IκBα, IκBβ, and IκBε). We also found several proteins not previously described in association with RelA, including the small mitochondrial chaperone Tim13. Using a variety of biochemical approaches, we further investigated the nature of the association between Tim13 and NFκB family transcription factors. This work therefore provides a conceptual and experimental framework for analyzing transcription factor protein interactions.

  5. The latent transforming growth factor beta binding protein (LTBP) family.

    PubMed Central

    Oklü, R; Hesketh, R

    2000-01-01

    The transforming growth factor beta (TGFbeta) cytokines are a multi-functional family that exert a wide variety of effects on both normal and transformed mammalian cells. The secretion and activation of TGFbetas is regulated by their association with latency-associated proteins and latent TGFbeta binding proteins (LTBPs). Over the past few years, three members of the LTBP family have been identified, in addition to the protoype LTBP1 first sequenced in 1990. Three of the LTBP family are expressed in a variety of isoforms as a consequence of alternative splicing. This review summarizes the differences between the isoforms in terms of the effects on domain structure and hence possible function. The close identity between LTBPs and members of the fibrillin family, mutations in which have been linked directly to Marfan's syndrome, suggests that anomalous expression of LTBPs may be associated with disease. Recent data indicating that differential expression of LTBP1 isoforms occurs during the development of coronary heart disease is considered, together with evidence that modulation of LTBP function, and hence of TGFbeta activity, is associated with a variety of cancers. PMID:11104663

  6. A Recommendation for Naming Transcription Factor Proteins in the Grasses

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Transcription factors are central for the exquisite temporal and spatial expression patterns of many genes. These proteins are characterized by their ability to be tethered to particular regulatory sequences in the genes that they control. While many other proteins participate in the regulation of g...

  7. Effect of stabilizing additives on the structure and hydration of proteins: a study involving monoclinic lysozyme.

    PubMed

    Saraswathi, N T; Sankaranarayanan, R; Vijayan, M

    2002-07-01

    In pursuance of a long-range programme on the hydration, mobility and action of proteins, the structural basis of the stabilizing effect of sugars and polyols is being investigated. With two crystallographically independent molecules with slightly different packing environments in the crystal, monoclinic lysozyme constitutes an ideal system for exploring the problem. The differences in the structure and hydration of the two molecules provide a framework for examining the changes caused by stabilizing additives. Monoclinic crystals were grown under native conditions and also in the presence of 10% sucrose, 15% trehalose, 10% trehalose, 10% sorbitol and 5% glycerol. The crystal structures were refined at resolutions ranging from 1.8 to 2.1 A. The average B values, and hence the mobility of the structure, are lower in the presence of additives than in the native crystals. However, a comparison of the structures indicates that the effect of the additives on the structure and the hydration shell around the protein molecule is considerably less than that caused by differences in packing. It is also less than that caused by the replacement of NaNO(3) by NaCl as the precipitant in the crystallization experiments. This result is not in conformity with the commonly held belief that additives exert their stabilizing effect through the reorganization of the hydration shell, at least as far as the ordered water molecules are concerned.

  8. Molecular cloning and expression of an additional epidermal growth factor receptor-related gene.

    PubMed Central

    Plowman, G D; Whitney, G S; Neubauer, M G; Green, J M; McDonald, V L; Todaro, G J; Shoyab, M

    1990-01-01

    Epidermal growth factor (EGF), transforming growth factor alpha (TGF-alpha), and amphiregulin are structurally and functionally related growth regulatory proteins. These secreted polypeptides all bind to the 170-kDa cell-surface EGF receptor, activating its intrinsic kinase activity. However, amphiregulin exhibits different activities than EGF and TGF-alpha in a number of biological assays. Amphiregulin only partially competes with EGF for binding EGF receptor, and amphiregulin does not induce anchorage-independent growth of normal rat kidney cells (NRK) in the presence of TGF-beta. Amphiregulin also appears to abrogate the stimulatory effect of TGF-alpha on the growth of several aggressive epithelial carcinomas that overexpress EGF receptor. These findings suggest that amphiregulin may interact with a separate receptor in certain cell types. Here we report the cloning of another member of the human EGF receptor (HER) family of receptor tyrosine kinases, which we have named "HER3/ERRB3." The cDNA was isolated from a human carcinoma cell line, and its 6-kilobase transcript was identified in various human tissues. We have generated peptide-specific antisera that recognizes the 160-kDa HER3 protein when transiently expressed in COS cells. These reagents will allow us to determine whether HER3 binds amphiregulin or other growth regulatory proteins and what role HER3 protein plays in the regulation of cell growth. Images PMID:2164210

  9. 34 CFR 648.32 - What additional factors does the Secretary consider?

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 34 Education 3 2010-07-01 2010-07-01 false What additional factors does the Secretary consider? 648.32 Section 648.32 Education Regulations of the Offices of the Department of Education (Continued) OFFICE OF POSTSECONDARY EDUCATION, DEPARTMENT OF EDUCATION GRADUATE ASSISTANCE IN AREAS OF NATIONAL...

  10. 34 CFR 491.22 - What additional factor does the Secretary consider?

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 34 Education 3 2013-07-01 2013-07-01 false What additional factor does the Secretary consider? 491.22 Section 491.22 Education Regulations of the Offices of the Department of Education (Continued) OFFICE OF VOCATIONAL AND ADULT EDUCATION, DEPARTMENT OF EDUCATION ADULT EDUCATION FOR THE...

  11. 34 CFR 491.22 - What additional factor does the Secretary consider?

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 34 Education 3 2012-07-01 2012-07-01 false What additional factor does the Secretary consider? 491.22 Section 491.22 Education Regulations of the Offices of the Department of Education (Continued) OFFICE OF VOCATIONAL AND ADULT EDUCATION, DEPARTMENT OF EDUCATION ADULT EDUCATION FOR THE...

  12. 34 CFR 491.22 - What additional factor does the Secretary consider?

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 34 Education 3 2014-07-01 2014-07-01 false What additional factor does the Secretary consider? 491.22 Section 491.22 Education Regulations of the Offices of the Department of Education (Continued) OFFICE OF VOCATIONAL AND ADULT EDUCATION, DEPARTMENT OF EDUCATION ADULT EDUCATION FOR THE...

  13. 34 CFR 491.22 - What additional factor does the Secretary consider?

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 34 Education 3 2011-07-01 2011-07-01 false What additional factor does the Secretary consider? 491.22 Section 491.22 Education Regulations of the Offices of the Department of Education (Continued) OFFICE OF VOCATIONAL AND ADULT EDUCATION, DEPARTMENT OF EDUCATION ADULT EDUCATION FOR THE...

  14. 34 CFR 491.22 - What additional factor does the Secretary consider?

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 34 Education 3 2010-07-01 2010-07-01 false What additional factor does the Secretary consider? 491.22 Section 491.22 Education Regulations of the Offices of the Department of Education (Continued) OFFICE OF VOCATIONAL AND ADULT EDUCATION, DEPARTMENT OF EDUCATION ADULT EDUCATION FOR THE...

  15. 34 CFR 636.22 - What additional factors does the Secretary consider?

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 34 Education 3 2010-07-01 2010-07-01 false What additional factors does the Secretary consider? 636.22 Section 636.22 Education Regulations of the Offices of the Department of Education (Continued) OFFICE OF POSTSECONDARY EDUCATION, DEPARTMENT OF EDUCATION URBAN COMMUNITY SERVICE PROGRAM How Does...

  16. 34 CFR 636.22 - What additional factors does the Secretary consider?

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 34 Education 3 2014-07-01 2014-07-01 false What additional factors does the Secretary consider? 636.22 Section 636.22 Education Regulations of the Offices of the Department of Education (Continued) OFFICE OF POSTSECONDARY EDUCATION, DEPARTMENT OF EDUCATION URBAN COMMUNITY SERVICE PROGRAM How Does...

  17. 34 CFR 636.22 - What additional factors does the Secretary consider?

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 34 Education 3 2012-07-01 2012-07-01 false What additional factors does the Secretary consider? 636.22 Section 636.22 Education Regulations of the Offices of the Department of Education (Continued) OFFICE OF POSTSECONDARY EDUCATION, DEPARTMENT OF EDUCATION URBAN COMMUNITY SERVICE PROGRAM How Does...

  18. 34 CFR 636.22 - What additional factors does the Secretary consider?

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 34 Education 3 2013-07-01 2013-07-01 false What additional factors does the Secretary consider? 636.22 Section 636.22 Education Regulations of the Offices of the Department of Education (Continued) OFFICE OF POSTSECONDARY EDUCATION, DEPARTMENT OF EDUCATION URBAN COMMUNITY SERVICE PROGRAM How Does...

  19. 34 CFR 636.22 - What additional factors does the Secretary consider?

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 34 Education 3 2011-07-01 2011-07-01 false What additional factors does the Secretary consider? 636.22 Section 636.22 Education Regulations of the Offices of the Department of Education (Continued) OFFICE OF POSTSECONDARY EDUCATION, DEPARTMENT OF EDUCATION URBAN COMMUNITY SERVICE PROGRAM How Does...

  20. 21 CFR 1311.115 - Additional requirements for two-factor authentication.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 9 2013-04-01 2013-04-01 false Additional requirements for two-factor authentication. 1311.115 Section 1311.115 Food and Drugs DRUG ENFORCEMENT ADMINISTRATION, DEPARTMENT OF JUSTICE... criteria of FIPS 140-2 Security Level 1, as incorporated by reference in § 1311.08, for...

  1. 21 CFR 1311.115 - Additional requirements for two-factor authentication.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 9 2012-04-01 2012-04-01 false Additional requirements for two-factor authentication. 1311.115 Section 1311.115 Food and Drugs DRUG ENFORCEMENT ADMINISTRATION, DEPARTMENT OF JUSTICE... criteria of FIPS 140-2 Security Level 1, as incorporated by reference in § 1311.08, for...

  2. 34 CFR 425.22 - What additional factors does the Secretary consider?

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 34 Education 3 2010-07-01 2010-07-01 false What additional factors does the Secretary consider? 425.22 Section 425.22 Education Regulations of the Offices of the Department of Education (Continued) OFFICE OF VOCATIONAL AND ADULT EDUCATION, DEPARTMENT OF EDUCATION DEMONSTRATION PROJECTS FOR...

  3. 34 CFR 648.32 - What additional factors does the Secretary consider?

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... private institutions of higher education. (Authority: 20 U.S.C. 1135-1135c) ... 34 Education 3 2011-07-01 2011-07-01 false What additional factors does the Secretary consider? 648.32 Section 648.32 Education Regulations of the Offices of the Department of Education...

  4. 34 CFR 472.23 - What additional factor does the Secretary consider?

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 34 Education 3 2010-07-01 2010-07-01 false What additional factor does the Secretary consider? 472.23 Section 472.23 Education Regulations of the Offices of the Department of Education (Continued) OFFICE OF VOCATIONAL AND ADULT EDUCATION, DEPARTMENT OF EDUCATION NATIONAL WORKPLACE LITERACY PROGRAM...

  5. 34 CFR 472.23 - What additional factor does the Secretary consider?

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 34 Education 3 2012-07-01 2012-07-01 false What additional factor does the Secretary consider? 472.23 Section 472.23 Education Regulations of the Offices of the Department of Education (Continued) OFFICE OF VOCATIONAL AND ADULT EDUCATION, DEPARTMENT OF EDUCATION NATIONAL WORKPLACE LITERACY PROGRAM...

  6. 34 CFR 472.23 - What additional factor does the Secretary consider?

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 34 Education 3 2014-07-01 2014-07-01 false What additional factor does the Secretary consider? 472.23 Section 472.23 Education Regulations of the Offices of the Department of Education (Continued) OFFICE OF VOCATIONAL AND ADULT EDUCATION, DEPARTMENT OF EDUCATION NATIONAL WORKPLACE LITERACY PROGRAM...

  7. 34 CFR 472.23 - What additional factor does the Secretary consider?

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 34 Education 3 2011-07-01 2011-07-01 false What additional factor does the Secretary consider? 472.23 Section 472.23 Education Regulations of the Offices of the Department of Education (Continued) OFFICE OF VOCATIONAL AND ADULT EDUCATION, DEPARTMENT OF EDUCATION NATIONAL WORKPLACE LITERACY PROGRAM...

  8. 34 CFR 472.23 - What additional factor does the Secretary consider?

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 34 Education 3 2013-07-01 2013-07-01 false What additional factor does the Secretary consider? 472.23 Section 472.23 Education Regulations of the Offices of the Department of Education (Continued) OFFICE OF VOCATIONAL AND ADULT EDUCATION, DEPARTMENT OF EDUCATION NATIONAL WORKPLACE LITERACY PROGRAM...

  9. Nitrogen balancing and xylose addition enhances growth capacity and protein content in Chlorella minutissima cultures.

    PubMed

    Freitas, B C B; Esquível, M G; Matos, R G; Arraiano, C M; Morais, M G; Costa, J A V

    2016-10-01

    This study aimed to examine the metabolic changes in Chlorella minutissima cells grown under nitrogen-deficient conditions and with the addition of xylose. The cell density, maximum photochemical efficiency, and chlorophyll and lipid levels were measured. The expression of two photosynthetic proteins, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and the beta subunit (AtpB) of adenosine triphosphate synthase, were measured. Comparison of cells grown in medium with a 50% reduction in the nitrogen concentration versus the traditional medium solution revealed that the cells grown under nitrogen-deficient conditions exhibited an increased growth rate, higher maximum cell density (12.7×10(6)cellsmL(-1)), optimal PSII efficiency (0.69) and decreased lipid level (25.08%). This study has taken the first steps toward protein detection in Chlorella minutissima, and the results can be used to optimize the culturing of other microalgae. PMID:27359061

  10. Drugs for 'protein clouds': targeting intrinsically disordered transcription factors.

    PubMed

    Dunker, A Keith; Uversky, Vladimir N

    2010-12-01

    Transcription factors (TFs) are very attractive but difficult drug targets. The difficulties come from several directions including the binding promiscuity of TFs and the intrinsically disordered nature of their binding sites, which often resemble 'protein clouds'. For a long time the targeting of proteins without defined structures was considered infeasible. Data have now emerged showing that selective blocking of specific interactions of intrinsically disordered TFs with their protein binding partners is possible. Initial hits have been optimized to increase their specificity and affinity. Several strategies have been elaborated for elucidating the mechanisms of blocking of intrinsic disorder-based protein-protein interactions. However, challenges remain in the field of drug development for 'protein clouds'; such development is still in its earliest stage.

  11. Cloning, expression and purification of the factor H binding protein and its interaction with factor H

    PubMed Central

    Yarian, Fatemeh; Bandehpour, Mojgan; Seyed, Negar; Kazemi, Bahram

    2016-01-01

    Background and Objective: Neisseria meningitidis is a leading cause of meningitis and sepsis worldwide. The factor H binding protein (fHBP) is a key virulence factor of Neisseria meningitidis that is able to selectively bind to human factor H, the key regulator of the alternative complement pathway, which it has important implications for meningococcal pathogenesis and vaccine design. The aims of present research were cloning, expression, purification of fHbp and confirmation of the interaction between serum factor H (fH) and produced factor H binding protein. Materials and Methods: A 820 base pairs fhbp gene fragment was amplified by PCR and cloned into expression vector pET28a (+) in Bam HI and SalI restriction enzymes sites. Recombinant DNA was expressed in BL21 (DE3) cell. fHBP protein was purified by Ni-NTA agarose resin. Coupling of recombinant protein into CNBr activated Sepharose 4B resin was carried out for application in serum fH protein purification. (fH-fHBP) interaction was confirmed by SDS-PAGE and far-western blotting. Results and Conclusions: SDS-PAGE results showed a 35 kDa protein band. 150 kDa fH protein was purified by designed Sepharose 4B resin. Far-western blotting confirmed (fH-fHBP) interaction and proper folding of factor H binding protein. PMID:27092222

  12. Isolation of an additional member of the fibroblast growth factor receptor family, FGFR-3.

    PubMed Central

    Keegan, K; Johnson, D E; Williams, L T; Hayman, M J

    1991-01-01

    The fibroblast growth factors are a family of polypeptide growth factors involved in a variety of activities including mitogenesis, angiogenesis, and wound healing. Fibroblast growth factor receptors (FGFRs) have previously been identified in chicken, mouse, and human and have been shown to contain an extracellular domain with either two or three immunoglobulin-like domains, a transmembrane domain, and a cytoplasmic tyrosine kinase domain. We have isolated a human cDNA for another tyrosine kinase receptor that is highly homologous to the previously described FGFR. Expression of this receptor cDNA in COS cells directs the expression of a 125-kDa glycoprotein. We demonstrate that this cDNA encodes a biologically active receptor by showing that human acidic and basic fibroblast growth factors activate this receptor as measured by 45Ca2+ efflux assays. These data establish the existence of an additional member of the FGFR family that we have named FGFR-3. Images PMID:1847508

  13. Stimulation of protein phosphatase activity by insulin and growth factors in 3T3 cells

    SciTech Connect

    Chan, C.P.; McNall, S.J.; Krebs, E.G.; Fischer, E.H. )

    1988-09-01

    Incubation of Swiss mouse 3T3-D1 cells with physiological concentrations of insulin resulted in a rapid and transient activation of protein phosphatase activity as measure by using ({sup 32}P)phosphorylase {alpha} as substrate. Activation reached a maximum level (140% of control value) within 5 min of addition and returned to control levels within 20 min. The effect of insulin was dose-dependent with half-maximal activation occurring at {approx}5 nM insulin. This activity could be completely inhibited by addition of the heat-stable protein inhibitor 2, which suggests the presence of an activated type-1 phosphatase. Similar effects on phosphatase activity were seen when epidermal growth factor and platelet-derived growth factor were tested. These results suggest that some of the intracellular effects caused by insulin and growth factors are mediated through the activation of a protein phosphatase.

  14. Modeling protein density of states: additive hydrophobic effects are insufficient for calorimetric two-state cooperativity.

    PubMed

    Chan, H S

    2000-09-01

    A well-established experimental criterion for two-state thermodynamic cooperativity in protein folding is that the van't Hoff enthalpy DeltaH(vH) around the transition midpoint is equal, or very nearly so, to the calorimetric enthalpy DeltaH(cal) of the entire transition. This condition is satisfied by many small proteins. We use simple lattice models to provide a statistical mechanical framework to elucidate how this calorimetric two-state picture may be reconciled with the hierarchical multistate scenario emerging from recent hydrogen exchange experiments. We investigate the feasibility of using inverse Laplace transforms to recover the underlying density of states (i.e., enthalpy distribution) from calorimetric data. We find that the constraint imposed by DeltaH(vH)/DeltaH(cal) approximately 1 on densities of states of proteins is often more stringent than other "two-state" criteria proposed in recent theoretical studies. In conjunction with reasonable assumptions, the calorimetric two-state condition implies a narrow distribution of denatured-state enthalpies relative to the overall enthalpy difference between the native and the denatured conformations. This requirement does not always correlate with simple definitions of "sharpness" of a transition and has important ramifications for theoretical modeling. We find that protein models that assume capillarity cooperativity can exhibit overall calorimetric two-state-like behaviors. However, common heteropolymer models based on additive hydrophobic-like interactions, including highly specific two-dimensional Gō models, fail to produce proteinlike DeltaH(vH)/DeltaH(cal) approximately 1. A simple model is constructed to illustrate a proposed scenario in which physically plausible local and nonlocal cooperative terms, which mimic helical cooperativity and environment-dependent hydrogen bonding strength, can lead to thermodynamic behaviors closer to experiment. Our results suggest that proteinlike thermodynamic

  15. A Dominant Factor for Structural Classification of Protein Crystals.

    PubMed

    Qi, Fei; Fudo, Satoshi; Neya, Saburo; Hoshino, Tyuji

    2015-08-24

    With the increasing number of solved protein crystal structures, much information on protein shape and atom geometry has become available. It is of great interest to know the structural diversity for a single kind of protein. Our preliminary study suggested that multiple crystal structures of a single kind of protein can be classified into several groups from the viewpoint of structural similarity. In order to broadly examine this finding, cluster analysis was applied to the crystal structures of hemoglobin (Hb), myoglobin (Mb), human serum albumin (HSA), hen egg-white lysozyme (HEWL), and human immunodeficiency virus type 1 protease (HIV-1 PR), downloaded from the Protein Data Bank (PDB). As a result of classification by cluster analysis, 146 crystal structures of Hb were separated into five groups. The crystal structures of Mb (n = 284), HEWL (n = 336), HSA (n = 63), and HIV-1 PR (n = 488) were separated into six, five, three, and six groups, respectively. It was found that a major factor causing these structural separations is the space group of crystals and that crystallizing agents have an influence on the crystal structures. Amino acid mutation is a minor factor for the separation because no obvious point mutation making a specific cluster group was observed for the five kinds of proteins. In the classification of Hb and Mb, the species of protein source such as humans, rabbits, and mice is another significant factor. When the difference in amino sequence is large among species, the species of protein source is the primary factor causing cluster separation in the classification of crystal structures. PMID:26230289

  16. Sodium Benzoate, a Metabolite of Cinnamon and a Food Additive, Upregulates Ciliary Neurotrophic Factor in Astrocytes and Oligodendrocytes.

    PubMed

    Modi, Khushbu K; Jana, Malabendu; Mondal, Susanta; Pahan, Kalipada

    2015-11-01

    Ciliary neurotrophic factor (CNTF) is a promyelinating trophic factor that plays an important role in multiple sclerosis (MS). However, mechanisms by which CNTF expression could be increased in the brain are poorly understood. Recently we have discovered anti-inflammatory and immunomodulatory activities of sodium benzoate (NaB), a metabolite of cinnamon and a widely-used food additive. Here, we delineate that NaB is also capable of increasing the mRNA and protein expression of CNTF in primary mouse astrocytes and oligodendrocytes and primary human astrocytes. Accordingly, oral administration of NaB and cinnamon led to the upregulation of astroglial and oligodendroglial CNTF in vivo in mouse brain. Induction of experimental allergic encephalomyelitis, an animal model of MS, reduced the level of CNTF in the brain, which was restored by oral administration of cinnamon. While investigating underlying mechanisms, we observed that NaB induced the activation of protein kinase A (PKA) and H-89, an inhibitor of PKA, abrogated NaB-induced expression of CNTF. The activation of cAMP response element binding (CREB) protein by NaB, the recruitment of CREB and CREB-binding protein to the CNTF promoter by NaB and the abrogation of NaB-induced expression of CNTF in astrocytes by siRNA knockdown of CREB suggest that NaB increases the expression of CNTF via the activation of CREB. These results highlight a novel myelinogenic property of NaB and cinnamon, which may be of benefit for MS and other demyelinating disorders.

  17. The Neuroprotective Functions of Transforming Growth Factor Beta Proteins

    PubMed Central

    Dobolyi, Arpád; Vincze, Csilla; Pál, Gabriella; Lovas, Gábor

    2012-01-01

    Transforming growth factor beta (TGF-β) proteins are multifunctional cytokines whose neural functions are increasingly recognized. The machinery of TGF-β signaling, including the serine kinase type transmembrane receptors, is present in the central nervous system. However, the 3 mammalian TGF-β subtypes have distinct distributions in the brain suggesting different neural functions. Evidence of their involvement in the development and plasticity of the nervous system as well as their functions in peripheral organs suggested that they also exhibit neuroprotective functions. Indeed, TGF-β expression is induced following a variety of types of brain tissue injury. The neuroprotective function of TGF-βs is most established following brain ischemia. Damage in experimental animal models of global and focal ischemia was shown to be attenuated by TGF-βs. In addition, support for their neuroprotective actions following trauma, sclerosis multiplex, neurodegenerative diseases, infections, and brain tumors is also accumulating. The review will also describe the potential mechanisms of neuroprotection exerted by TGF-βs including anti-inflammatory, -apoptotic, -excitotoxic actions as well as the promotion of scar formation, angiogenesis, and neuroregeneration. The participation of these mechanisms in the neuroprotective effects of TGF-βs during different brain lesions will also be discussed. PMID:22942700

  18. Factors influencing dietary protein sources in the PREMIER trial population.

    PubMed

    Lin, Pao-Hwa; Miwa, Saki; Li, Yi-Ju; Wang, Yanfang; Levy, Erma; Lastor, Katherine; Champagne, Catherine

    2010-02-01

    Previous research suggests that protein intake, particularly plant protein, may benefit blood pressure control. However, very little has been published regarding protein sources in diets of US adults and factors influencing these choices. The purpose of this report is to describe specific sources of animal and plant proteins in diets of PREMIER clinical trial participants at baseline and how the PREMIER intervention, along with participant demographics, affected protein sources. Adult participants (n=809) who completed the 18-month PREMIER lifestyle intervention trial and had at least one diet recall at each of three study visits were included. Participants were recruited from four clinical centers in the Eastern, Southern, and Northeastern regions of United States. The PREMIER trial, conducted from 1999 to 2002, compared the impact on blood pressure of two structured behavioral interventions focusing on the traditional lifestyle modifications for blood pressure control with or without the Dietary Approaches to Stop Hypertension dietary pattern. Protein sources were assessed by two unannounced 24-hour recalls at each of three study visits. Differences in protein sources were mainly related to participant demographics, with relatively moderate impact of the intervention. The top four protein sources for all the study participants were poultry, dairy, refined grains and beef, each contributing approximately 10% to 17% in descending order to the total protein intake at baseline. Animal and plant protein each comprised approximately 66% and 34%, respectively, to the total daily protein intake at baseline, and such overall contribution pattern remained relatively constant over time. However, sex, race, age, and body weight status all influenced contribution patterns from different food groups significantly. These influences significantly impact choice and are essential elements to consider when designing intervention programs to alter protein contributions from animal

  19. Extracellular matrix protein in calcified endoskeleton: a potential additive for crystal growth and design

    NASA Astrophysics Data System (ADS)

    Azizur Rahman, M.; Fujimura, Hiroyuki; Shinjo, Ryuichi; Oomori, Tamotsu

    2011-06-01

    In this study, we demonstrate a key function of extracellular matrix proteins (ECMPs) on seed crystals, which are isolated from calcified endoskeletons of soft coral and contain only CaCO 3 without any living cells. This is the first report that an ECMP protein extracted from a marine organism could potentially influence in modifying the surface of a substrate for designing materials via crystallization. We previously studied with the ECMPs from a different type of soft coral ( Sinularia polydactyla) without introducing any seed crystals in the process , which showed different results. Thus, crystallization on the seed in the presence of ECMPs of present species is an important first step toward linking function to individual proteins from soft coral. For understanding this interesting phenomenon, in vitro crystallization was initiated in a supersaturated solution on seed particles of calcite (1 0 4) with and without ECMPs. No change in the crystal growth shape occurred without ECMPs present during the crystallization process. However, with ECMPs, the morphology and phase of the crystals in the crystallization process changed dramatically. Upon completion of crystallization with ECMPs, an attractive crystal morphology was found. Scanning electron microscopy (SEM) was utilized to observe the crystal morphologies on the seeds surface. The mineral phases of crystals nucleated by ECMPs on the seeds surface were examined by Raman spectroscopy. Although 50 mM Mg 2+ is influential in making aragonite in the crystallization process, the ECMPs significantly made calcite crystals even when 50 mM Mg 2+ was present in the process. Crystallization with the ECMP additive seems to be a technically attractive strategy to generate assembled micro crystals that could be used in crystals growth and design in the Pharmaceutical and biotechnology industries.

  20. Titration of the bacteriorhodopsin Schiff base involves titration of an additional protein residue.

    PubMed

    Zadok, Uri; Asato, Alfred E; Sheves, Mordechai

    2005-06-14

    The retinal protein protonated Schiff base linkage plays a key role in the function of bacteriorhodopsin (bR) as a light-driven proton pump. In the unphotolyzed pigment, the Schiff base (SB) is titrated with a pK(a) of approximately 13, but following light absorption, it experiences a decrease in the pK(a) and undergoes several alterations, including a deprotonation process. We have studied the SB titration using retinal analogues which have intrinsically lower pK(a)'s which allow for SB titrations over a much lower pH range. We found that above pH 9 the channel for the SB titration is perturbed, and the titration rate is considerably reduced. On the basis of studies with several mutants, it is suggested that the protonation state of residue Glu204 is responsible for the channel perturbation. We suggest that above pH 12 a channel for the SB titration is restored probably due to titration of an additional protein residue. The observations may imply that during the bR photocycle and M photointermediate formation the rate of Schiff base protonation from the bulk is decreased. This rate decrease may be due to the deprotonation process of the "proton-releasing complex" which includes Glu204. In contrast, during the lifetime of the O intermediate, the protonated SB is exposed to the bulk. Possible implications for the switch mechanism, and the directionality of the proton movement, are discussed.

  1. Medium modification with bone morphogenetic protein 2 addition for odontogenic differentiation.

    PubMed

    Atalayin, Cigdem; Tezel, Huseyin; Dagci, Taner; Yavasoglu, Nefise Ulku Karabay; Oktem, Gulperi

    2016-01-01

    The aim of this study was to evaluate whether medium modification improves the odontogenic differentiation of human dental pulp stem cells (DPSC) in vitro and in vivo. DPSC isolated from human impacted third molar teeth were analysed for clusters of differentiation with flow cytometry. Odontogenic differentiation was stimulated by medium modification with the addition of bone morphogenetic protein 2 (BMP2). The expression of dentin sialophosphoprotein, dentin matrix protein 1, enamelysin/matrix metalloproteinase 20 and the phosphate-regulating gene with homologies to endopeptidases on the X chromosome of the cells were analysed with RT-PCR at 7, 14 and 21 days. Then, DPSC were transplanted on the back of immunocompromised mice via a hydroxyapatite tricalcium phosphate scaffold, and the structure of the formed tissue was investigated. The cells were identified as mesenchymal stem cells with a 98.3% CD73 and CD90 double-positive cell rate. The increase in mineralization capacity and expression of human enamel-dentin specific transcripts proportional to the culture period were determined after differentiation. Six weeks after transplantation, an osteo-dentin matrix was formed in the group in which odontogenic differentiation was stimulated, and the odontogenic characteristics of the matrix were confirmed by histological examination and RT-PCR analysis. Odontogenic differentiation of the isolated and characterized human DPSC was improved with medium modification by the addition of BMP2 in vitro and in vivo. The defined medium and applied technique have a potential use for forming reparative dentin in the future, but the effects of the method should be investigated in long-term studies. PMID:26981753

  2. Latent transforming growth factor binding protein 4 regulates transforming growth factor beta receptor stability.

    PubMed

    Su, Chi-Ting; Huang, Jenq-Wen; Chiang, Chih-Kang; Lawrence, Elizabeth C; Levine, Kara L; Dabovic, Branka; Jung, Christine; Davis, Elaine C; Madan-Khetarpal, Suneeta; Urban, Zsolt

    2015-07-15

    Mutations in the gene for the latent transforming growth factor beta binding protein 4 (LTBP4) cause autosomal recessive cutis laxa type 1C. To understand the molecular disease mechanisms of this disease, we investigated the impact of LTBP4 loss on transforming growth factor beta (TGFβ) signaling. Despite elevated extracellular TGFβ activity, downstream signaling molecules of the TGFβ pathway, including pSMAD2 and pERK, were down-regulated in LTBP4 mutant human dermal fibroblasts. In addition, TGFβ receptors 1 and 2 (TGFBR1 and TGFBR2) were reduced at the protein but not at the ribonucleic acid level. Treatment with exogenous TGFβ1 led to an initially rapid increase in SMAD2 phosphorylation followed by a sustained depression of phosphorylation and receptor abundance. In mutant cells TGFBR1 was co-localized with lysosomes. Treatment with a TGFBR1 kinase inhibitor, endocytosis inhibitors or a lysosome inhibitor, normalized the levels of TGFBR1 and TGFBR2. Co-immunoprecipitation demonstrated a molecular interaction between LTBP4 and TGFBR2. Knockdown of LTBP4 reduced TGFβ receptor abundance and signaling in normal cells and supplementation of recombinant LTBP4 enhanced these measures in mutant cells. In a mouse model of Ltbp4 deficiency, reduced TGFβ signaling and receptor levels were normalized upon TGFBR1 kinase inhibitor treatment. Our results show that LTBP4 interacts with TGFBR2 and stabilizes TGFβ receptors by preventing their endocytosis and lysosomal degradation in a ligand-dependent and receptor kinase activity-dependent manner. These findings identify LTBP4 as a key molecule required for the stability of the TGFβ receptor complex, and a new mechanism by which the extracellular matrix regulates cytokine receptor signaling.

  3. Latent transforming growth factor binding protein 4 regulates transforming growth factor beta receptor stability

    PubMed Central

    Su, Chi-Ting; Huang, Jenq-Wen; Chiang, Chih-Kang; Lawrence, Elizabeth C.; Levine, Kara L.; Dabovic, Branka; Jung, Christine; Davis, Elaine C.; Madan-Khetarpal, Suneeta; Urban, Zsolt

    2015-01-01

    Mutations in the gene for the latent transforming growth factor beta binding protein 4 (LTBP4) cause autosomal recessive cutis laxa type 1C. To understand the molecular disease mechanisms of this disease, we investigated the impact of LTBP4 loss on transforming growth factor beta (TGFβ) signaling. Despite elevated extracellular TGFβ activity, downstream signaling molecules of the TGFβ pathway, including pSMAD2 and pERK, were down-regulated in LTBP4 mutant human dermal fibroblasts. In addition, TGFβ receptors 1 and 2 (TGFBR1 and TGFBR2) were reduced at the protein but not at the ribonucleic acid level. Treatment with exogenous TGFβ1 led to an initially rapid increase in SMAD2 phosphorylation followed by a sustained depression of phosphorylation and receptor abundance. In mutant cells TGFBR1 was co-localized with lysosomes. Treatment with a TGFBR1 kinase inhibitor, endocytosis inhibitors or a lysosome inhibitor, normalized the levels of TGFBR1 and TGFBR2. Co-immunoprecipitation demonstrated a molecular interaction between LTBP4 and TGFBR2. Knockdown of LTBP4 reduced TGFβ receptor abundance and signaling in normal cells and supplementation of recombinant LTBP4 enhanced these measures in mutant cells. In a mouse model of Ltbp4 deficiency, reduced TGFβ signaling and receptor levels were normalized upon TGFBR1 kinase inhibitor treatment. Our results show that LTBP4 interacts with TGFBR2 and stabilizes TGFβ receptors by preventing their endocytosis and lysosomal degradation in a ligand-dependent and receptor kinase activity-dependent manner. These findings identify LTBP4 as a key molecule required for the stability of the TGFβ receptor complex, and a new mechanism by which the extracellular matrix regulates cytokine receptor signaling. PMID:25882708

  4. Polydom: a secreted protein with pentraxin, complement control protein, epidermal growth factor and von Willebrand factor A domains.

    PubMed Central

    Gilgès, D; Vinit, M A; Callebaut, I; Coulombel, L; Cacheux, V; Romeo, P H; Vigon, I

    2000-01-01

    To identify extracellular proteins with epidermal growth factor (EGF) domains that are potentially involved in the control of haemopoiesis, we performed degenerate reverse-transcriptase-mediated PCR on the murine bone-marrow stromal cell line MS-5 and isolated a new partial cDNA encoding EGF-like domains related to those in the Notch proteins. Cloning and sequencing of the full-length cDNA showed that it encoded a new extracellular multi-domain protein that we named polydom. This 387 kDa mosaic protein contained a signal peptide followed by a new association of eight different protein domains, including a pentraxin domain and a von Willebrand factor type A domain, ten EGF domains, and 34 complement control protein modules. The human polydom mRNA is strongly expressed in placenta, its expression in the other tissues being weak or undetectable. The particular multidomain structure of the encoded protein suggests an important biological role in cellular adhesion and/or in the immune system. PMID:11062057

  5. Effect of salt additives on protein partition in polyethylene glycol-sodium sulfate aqueous two-phase systems.

    PubMed

    Ferreira, Luisa; Madeira, Pedro P; Mikheeva, Larissa; Uversky, Vladimir N; Zaslavsky, Boris

    2013-12-01

    Partitioning of 15 proteins in polyethylene glycol (PEG)-sodium sulfate aqueous two-phase systems (ATPS) formed by PEG of two different molecular weights, PEG-600 and PEG-8000 in the presence of different buffers at pH7.4 was studied. The effect of two salt additives (NaCl and NaSCN) on the protein partition behavior was examined. The salt effects on protein partitioning were analyzed by using the Collander solvent regression relationship between the proteins partition coefficients in ATPS with and without salt additives. The results obtained show that the concentration of buffer as well as the presence and concentration of salt additives affects the protein partition behavior. Analysis of ATPS in terms of the differences between the relative hydrophobicity and electrostatic properties of the phases does not explain the protein partition behavior. The differences between protein partitioning in PEG-600-salt and PEG-8000-salt ATPS cannot be explained by the protein size or polymer excluded volume effect. It is suggested that the protein-ion and protein-solvent interactions in the phases of ATPS are primarily important for protein partitioning.

  6. Factor H-related proteins determine complement-activating surfaces.

    PubMed

    Józsi, Mihály; Tortajada, Agustin; Uzonyi, Barbara; Goicoechea de Jorge, Elena; Rodríguez de Córdoba, Santiago

    2015-06-01

    Complement factor H-related proteins (FHRs) are strongly associated with different diseases involving complement dysregulation, which suggests a major role for these proteins regulating complement activation. Because FHRs are evolutionarily and structurally related to complement inhibitor factor H (FH), the initial assumption was that the FHRs are also negative complement regulators. Whereas weak complement inhibiting activities were originally reported for these molecules, recent developments indicate that FHRs may enhance complement activation, with important implications for the role of these proteins in health and disease. We review these findings here, and propose that FHRs represent a complex set of surface recognition molecules that, by competing with FH, provide improved discrimination of self and non-self surfaces and play a central role in determining appropriate activation of the complement pathway.

  7. Assembly of Neuronal Connectivity by Neurotrophic Factors and Leucine-Rich Repeat Proteins

    PubMed Central

    Ledda, Fernanda; Paratcha, Gustavo

    2016-01-01

    Proper function of the nervous system critically relies on sophisticated neuronal networks interconnected in a highly specific pattern. The architecture of these connections arises from sequential developmental steps such as axonal growth and guidance, dendrite development, target determination, synapse formation and plasticity. Leucine-rich repeat (LRR) transmembrane proteins have been involved in cell-type specific signaling pathways that underlie these developmental processes. The members of this superfamily of proteins execute their functions acting as trans-synaptic cell adhesion molecules involved in target specificity and synapse formation or working in cis as cell-intrinsic modulators of neurotrophic factor receptor trafficking and signaling. In this review, we will focus on novel physiological mechanisms through which LRR proteins regulate neurotrophic factor receptor signaling, highlighting the importance of these modulatory events for proper axonal extension and guidance, tissue innervation and dendrite morphogenesis. Additionally, we discuss few examples linking this set of LRR proteins to neurodevelopmental and psychiatric disorders. PMID:27555809

  8. Assembly of Neuronal Connectivity by Neurotrophic Factors and Leucine-Rich Repeat Proteins.

    PubMed

    Ledda, Fernanda; Paratcha, Gustavo

    2016-01-01

    Proper function of the nervous system critically relies on sophisticated neuronal networks interconnected in a highly specific pattern. The architecture of these connections arises from sequential developmental steps such as axonal growth and guidance, dendrite development, target determination, synapse formation and plasticity. Leucine-rich repeat (LRR) transmembrane proteins have been involved in cell-type specific signaling pathways that underlie these developmental processes. The members of this superfamily of proteins execute their functions acting as trans-synaptic cell adhesion molecules involved in target specificity and synapse formation or working in cis as cell-intrinsic modulators of neurotrophic factor receptor trafficking and signaling. In this review, we will focus on novel physiological mechanisms through which LRR proteins regulate neurotrophic factor receptor signaling, highlighting the importance of these modulatory events for proper axonal extension and guidance, tissue innervation and dendrite morphogenesis. Additionally, we discuss few examples linking this set of LRR proteins to neurodevelopmental and psychiatric disorders. PMID:27555809

  9. Two RNA recognition motif-containing proteins are plant mitochondrial editing factors

    PubMed Central

    Shi, Xiaowen; Hanson, Maureen R.; Bentolila, Stéphane

    2015-01-01

    Post-transcriptional C-to-U RNA editing occurs in plant plastid and mitochondrial transcripts. Members of the Arabidopsis RNA-editing factor interacting protein (RIP) family and ORRM1 (Organelle RNA Recognition Motif-containing protein 1) have been recently characterized as essential components of the chloroplast RNA editing apparatus. ORRM1 belongs to a distinct clade of RNA Recognition Motif (RRM)-containing proteins, most of which are predicted to be organelle-targeted. Here we report the identification of two proteins, ORRM2 (organelle RRM protein 2) and ORRM3 (organelle RRM protein 3), as the first members of the ORRM clade to be identified as mitochondrial editing factors. Transient silencing of ORRM2 and ORRM3 resulted in reduced editing efficiency at ∼6% of the mitochondrial C targets. In addition to an RRM domain at the N terminus, ORRM3 carries a glycine-rich domain at the C terminus. The N-terminal RRM domain by itself provides the editing activity of ORRM3. In yeast-two hybrid assays, ORRM3 interacts with RIP1, ORRM2 and with itself. Transient silencing of ORRM2 in the orrm3 mutant further impairs the editing activity at sites controlled by both ORRM2 and ORRM3. Identification of the effect of ORRM2 and ORRM3 on RNA editing reveals a previously undescribed role of RRM-containing proteins as mitochondrial RNA editing factors. PMID:25800738

  10. Monitoring Wnt Protein Acylation Using an In Vitro Cyclo-Addition Reaction

    PubMed Central

    Tuladhar, Rubina; Yarravarapu, Nageswari; Lum, Lawrence

    2016-01-01

    We describe here a technique for visualizing the lipidation status of Wnt proteins using azide-alkyne cycloaddition chemistry (click chemistry) and SDS-PAGE. This protocol incorporates in vivo labeling of a Wnt-IgG Fc fusion protein using an alkynylated palmitate probe but departs from a traditional approach by incorporating a secondary cycloaddition reaction performed on single-step purified Wnt protein immobilized on protein A resin. This approach mitigates experimental noise by decreasing the contribution of labeling from other palmitoylated proteins and by providing a robust method for normalizing labeling efficiency based on protein abundance. PMID:27590147

  11. Monitoring Wnt Protein Acylation Using an In Vitro Cyclo-Addition Reaction.

    PubMed

    Tuladhar, Rubina; Yarravarapu, Nageswari; Lum, Lawrence

    2016-01-01

    We describe here a technique for visualizing the lipidation status of Wnt proteins using azide-alkyne cycloaddition chemistry (click chemistry) and SDS-PAGE. This protocol incorporates in vivo labeling of a Wnt-IgG Fc fusion protein using an alkynylated palmitate probe but departs from a traditional approach by incorporating a secondary cycloaddition reaction performed on single-step purified Wnt protein immobilized on protein A resin. This approach mitigates experimental noise by decreasing the contribution of labeling from other palmitoylated proteins and by providing a robust method for normalizing labeling efficiency based on protein abundance. PMID:27590147

  12. TALE factors poise promoters for activation by Hox proteins.

    PubMed

    Choe, Seong-Kyu; Ladam, Franck; Sagerström, Charles G

    2014-01-27

    Hox proteins form complexes with TALE cofactors from the Pbx and Prep/Meis families to control transcription, but it remains unclear how Hox:TALE complexes function. Examining a Hoxb1b:TALE complex that regulates zebrafish hoxb1a transcription, we find maternally deposited TALE proteins at the hoxb1a promoter already during blastula stages. These TALE factors recruit histone-modifying enzymes to promote an active chromatin profile at the hoxb1a promoter and also recruit RNA polymerase II (RNAPII) and P-TEFb. However, in the presence of TALE factors, RNAPII remains phosphorylated on serine 5 and hoxb1a transcription is inefficient. By gastrula stages, Hoxb1b binds together with TALE factors to the hoxb1a promoter. This triggers P-TEFb-mediated transitioning of RNAPII to the serine 2-phosphorylated form and efficient hoxb1a transcription. We conclude that TALE factors access promoters during early embryogenesis to poise them for activation but that Hox proteins are required to trigger efficient transcription.

  13. Insulin-like growth factor (IGF) binding protein enhances the biologic response to IGF-I

    SciTech Connect

    Elgin, R.G.; Busby, H.W. Jr.; Clemmons, D.R.

    1987-05-01

    The insulin-like growth factors IGF-I and IGF-II circulate in blood bound to carrier proteins. The higher molecular mass IGF-binding protein complex (150 kDa) is composed of subunits, and one subunits that forms this complex is growth hormone dependent. In addition, many cell types and tissues secrete another form of IGF binding protein that is not growth hormone dependent. Both forms of the IGF binding protein are believed to inactivate the IGFs and to function as delivery systems to tissues. This conclusion was based on studies that determined the effects of impure preparations of these binding proteins or that examined the effect of these proteins only on the insulin-like actions of the IGFs. The authors report here that a pure preparation of the extracellular form of the IGF binding protein (purified from human amniotic fluid) markedly potentiated replication of several cell types in response to human IGF-I. Secondary cultures of human, mouse, and chicken embryo fibroblasts as well as porcine aortic smooth muscle cells showed marked enhancement of their DNA synthesis response to IGF-I in the presence of this protein. The binding protein not only potentiated the DNA synthesis response but also enhanced the increase in cell number in response to IGF-I. This stimulation is specific for growth factors that bind to the binding protein since incubation with insulin, which binds to the type I IGF receptor but not to the binding protein, did not result in potentiation of this response. They conclude that a form of IGF binding protein that is present in extracellular fluids and is secreted by many types of cells can markedly potentiate the cellular response to IGF-I.

  14. Interactions of signaling proteins, growth factors and other proteins with heparan sulfate: mechanisms and mysteries.

    PubMed

    Billings, Paul C; Pacifici, Maurizio

    2015-01-01

    Heparan sulfate (HS) is a component of cell surface and matrix-associated proteoglycans (HSPGs) that, collectively, play crucial roles in many physiologic processes including cell differentiation, organ morphogenesis and cancer. A key function of HS is to bind and interact with signaling proteins, growth factors, plasma proteins, immune-modulators and other factors. In doing so, the HS chains and HSPGs are able to regulate protein distribution, bio-availability and action on target cells and can also serve as cell surface co-receptors, facilitating ligand-receptor interactions. These proteins contain an HS/heparin-binding domain (HBD) that mediates their association and contacts with HS. HBDs are highly diverse in sequence and predicted structure, contain clusters of basic amino acids (Lys and Arg) and possess an overall net positive charge, most often within a consensus Cardin-Weintraub (CW) motif. Interestingly, other domains and residues are now known to influence protein-HS interactions, as well as interactions with other glycosaminoglycans, such as chondroitin sulfate. In this review, we provide a description and analysis of HBDs in proteins including amphiregulin, fibroblast growth factor family members, heparanase, sclerostin and hedgehog protein family members. We discuss HBD structural and functional features and important roles carried out by other protein domains, and also provide novel conformational insights into the diversity of CW motifs present in Sonic, Indian and Desert hedgehogs. Finally, we review progress in understanding the pathogenesis of a rare pediatric skeletal disorder, Hereditary Multiple Exostoses (HME), characterized by HS deficiency and cartilage tumor formation. Advances in understanding protein-HS interactions will have broad implications for basic biology and translational medicine as well as for the development of HS-based therapeutics.

  15. Eukaryotic damaged DNA-binding proteins: DNA repair proteins or transcription factors?

    SciTech Connect

    Protic, M.

    1994-12-31

    Recognition and removal of structural defects in the genome, caused by diverse physical and chemical agents, are among the most important cell functions. Proteins that recognize and bind to modified DNA, and thereby initiate damage-induced recovery processes, have been identified in prokaryotic and eukaryotic cells. Damaged DNA-binding (DDB) proteins from prokaryotes are either DNA repair enzymes or noncatalytic subunits of larger DNA repair complexes that participate in excision repair, or in recombinational repair and SOS-mutagenesis. Although the methods employed may not have allowed detection of all eukaryotic DDB proteins and identification of their functions, it appears that during evolution cells have developed a wide array of DDB proteins that can discriminate among the diversity of DNA conformations found in the eukaryotic nucleus, as well as a gene-sharing feature found in DDB proteins that also act as transcription factors.

  16. Use of additives to enhance the properties of cottonseed protein as wood adhesives

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Soy protein is currently being used commercially as a “green” wood adhesive. Previous work in this laboratory has shown that cottonseed protein isolate, tested on maple wood veneer, produced higher adhesive strength and hot water resistance relative to soy protein. In the present study, cottonseed...

  17. Soy Protein Isolate As Fluid Loss Additive in Bentonite-Water-Based Drilling Fluids.

    PubMed

    Li, Mei-Chun; Wu, Qinglin; Song, Kunlin; Lee, Sunyoung; Jin, Chunde; Ren, Suxia; Lei, Tingzhou

    2015-11-11

    Wellbore instability and formation collapse caused by lost circulation are vital issues during well excavation in the oil industry. This study reports the novel utilization of soy protein isolate (SPI) as fluid loss additive in bentonite-water based drilling fluids (BT-WDFs) and describes how its particle size and concentration influence on the filtration property of SPI/BT-WDFs. It was found that high pressure homogenization (HPH)-treated SPI had superior filtration property over that of native SPI due to the improved ability for the plugging pore throat. HPH treatment also caused a significant change in the surface characteristic of SPI, leading to a considerable surface interaction with BT in aqueous solution. The concentration of SPI had a significant impact on the dispersion state of SPI/BT mixtures in aquesous solution. At low SPI concentrations, strong aggregations were created, resulting in the formation of thick, loose, high-porosity and high-permeability filter cakes and high fluid loss. At high SPI concentrations, intercatlated/exfoliated structures were generated, resulting in the formation of thin, compact, low-porosity and low-permeability filter cakes and low fluid loss. The SPI/BT-WDFs exhibited superior filtration property than pure BT-WDFs at the same solid concentraion, demonstrating the potential utilization of SPI as an effective, renewable, and biodegradable fluid loss reducer in well excavation applications. PMID:26492498

  18. Soy Protein Isolate As Fluid Loss Additive in Bentonite-Water-Based Drilling Fluids.

    PubMed

    Li, Mei-Chun; Wu, Qinglin; Song, Kunlin; Lee, Sunyoung; Jin, Chunde; Ren, Suxia; Lei, Tingzhou

    2015-11-11

    Wellbore instability and formation collapse caused by lost circulation are vital issues during well excavation in the oil industry. This study reports the novel utilization of soy protein isolate (SPI) as fluid loss additive in bentonite-water based drilling fluids (BT-WDFs) and describes how its particle size and concentration influence on the filtration property of SPI/BT-WDFs. It was found that high pressure homogenization (HPH)-treated SPI had superior filtration property over that of native SPI due to the improved ability for the plugging pore throat. HPH treatment also caused a significant change in the surface characteristic of SPI, leading to a considerable surface interaction with BT in aqueous solution. The concentration of SPI had a significant impact on the dispersion state of SPI/BT mixtures in aquesous solution. At low SPI concentrations, strong aggregations were created, resulting in the formation of thick, loose, high-porosity and high-permeability filter cakes and high fluid loss. At high SPI concentrations, intercatlated/exfoliated structures were generated, resulting in the formation of thin, compact, low-porosity and low-permeability filter cakes and low fluid loss. The SPI/BT-WDFs exhibited superior filtration property than pure BT-WDFs at the same solid concentraion, demonstrating the potential utilization of SPI as an effective, renewable, and biodegradable fluid loss reducer in well excavation applications.

  19. Interplay between trigger factor and other protein biogenesis factors on the ribosome

    NASA Astrophysics Data System (ADS)

    Bornemann, Thomas; Holtkamp, Wolf; Wintermeyer, Wolfgang

    2014-06-01

    Nascent proteins emerging from translating ribosomes in bacteria are screened by a number of ribosome-associated protein biogenesis factors, among them the chaperone trigger factor (TF), the signal recognition particle (SRP) that targets ribosomes synthesizing membrane proteins to the membrane and the modifying enzymes, peptide deformylase (PDF) and methionine aminopeptidase (MAP). Here, we examine the interplay between these factors both kinetically and at equilibrium. TF rapidly scans the ribosomes until it is stabilized on ribosomes presenting TF-specific nascent chains. SRP binding to those complexes is strongly impaired. Thus, TF in effect prevents SRP binding to the majority of ribosomes, except those presenting SRP-specific signal sequences, explaining how the small amount of SRP in the cell can be effective in membrane targeting. PDF and MAP do not interfere with TF or SRP binding to translating ribosomes, indicating that nascent-chain processing can take place before or in parallel with TF or SRP binding.

  20. A diverse family of proteins containing tumor necrosis factor receptor-associated factor domains.

    PubMed

    Zapata, J M; Pawlowski, K; Haas, E; Ware, C F; Godzik, A; Reed, J C

    2001-06-29

    We have identified three new tumor necrosis factor-receptor associated factor (TRAF) domain-containing proteins in humans using bioinformatics approaches, including: MUL, the product of the causative gene in Mulibrey Nanism syndrome; USP7 (HAUSP), an ubiquitin protease; and SPOP, a POZ domain-containing protein. Unlike classical TRAF family proteins involved in TNF family receptor (TNFR) signaling, the TRAF domains (TDs) of MUL, USP7, and SPOP are located near the NH(2) termini or central region of these proteins, rather than carboxyl end. MUL and USP7 are capable of binding in vitro via their TDs to all of the previously identified TRAF family proteins (TRAF1, TRAF2, TRAF3, TRAF4, TRAF5, and TRAF6), whereas the TD of SPOP interacts weakly with TRAF1 and TRAF6 only. The TD of MUL also interacted with itself, whereas the TDs of USP7 and SPOP did not self-associate. Analysis of various MUL and USP7 mutants by transient transfection assays indicated that the TDs of these proteins are necessary and sufficient for suppressing NF-kappaB induction by TRAF2 and TRAF6 as well as certain TRAF-binding TNF family receptors. In contrast, the TD of SPOP did not inhibit NF-kappaB induction. Immunofluorescence confocal microscopy indicated that MUL localizes to cytosolic bodies, with targeting to these structures mediated by a RBCC tripartite domain within the MUL protein. USP7 localized predominantly to the nucleus, in a TD-dependent manner. Data base searches revealed multiple proteins containing TDs homologous to those found in MUL, USP7, and SPOP throughout eukaryotes, including yeast, protists, plants, invertebrates, and mammals, suggesting that this branch of the TD family arose from an ancient gene. We propose the moniker TEFs (TD-encompassing factors) for this large family of proteins.

  1. Factors which Limit the Value of Additional Redundancy in Human Rated Launch Vehicle Systems

    NASA Technical Reports Server (NTRS)

    Anderson, Joel M.; Stott, James E.; Ring, Robert W.; Hatfield, Spencer; Kaltz, Gregory M.

    2008-01-01

    The National Aeronautics and Space Administration (NASA) has embarked on an ambitious program to return humans to the moon and beyond. As NASA moves forward in the development and design of new launch vehicles for future space exploration, it must fully consider the implications that rule-based requirements of redundancy or fault tolerance have on system reliability/risk. These considerations include common cause failure, increased system complexity, combined serial and parallel configurations, and the impact of design features implemented to control premature activation. These factors and others must be considered in trade studies to support design decisions that balance safety, reliability, performance and system complexity to achieve a relatively simple, operable system that provides the safest and most reliable system within the specified performance requirements. This paper describes conditions under which additional functional redundancy can impede improved system reliability. Examples from current NASA programs including the Ares I Upper Stage will be shown.

  2. Development and molecular characterization of wheat--Aegilops kotschyi addition and substitution lines with high grain protein, iron, and zinc.

    PubMed

    Rawat, Nidhi; Neelam, Kumari; Tiwari, Vijay K; Randhawa, Gursharn S; Friebe, Bernd; Gill, Bikram S; Dhaliwal, Harcharan S

    2011-11-01

    Over two billion people, depending largely on staple foods, suffer from deficiencies in protein and some micronutrients such as iron and zinc. Among various approaches to overcome protein and micronutrient deficiencies, biofortification through a combination of conventional and molecular breeding methods is the most feasible, cheapest, and sustainable approach. An interspecific cross was made between the wheat cultivar 'Chinese Spring' and Aegilops kotschyi Boiss. accession 396, which has a threefold higher grain iron and zinc concentrations and about 33% higher protein concentration than wheat cultivars. Recurrent backcrossing and selection for the micronutrient content was performed at each generation. Thirteen derivatives with high grain iron and zinc concentrations and contents, ash and ash micronutrients, and protein were analyzed for alien introgression. Morphological markers, high molecular weight glutenin subunit profiles, anchored wheat microsatellite markers, and GISH showed that addition and substitution of homoeologous groups 1, 2, and 7 chromosomes of Ae. kotschyi possess gene(s) for high grain micronutrients. The addition of 1U/1S had high molecular weight glutenin subunits with higher molecular weight than those of wheat, and the addition of 2S in most of the derivatives also enhanced grain protein content by over 20%. Low grain protein content in a derivative with a 2S-wheat translocation, waxy leaves, and absence of the gdm148 marker strongly suggests that the gene for higher grain protein content on chromosome 2S is orthologous to the grain protein QTL on the short arm of group 2 chromosomes.

  3. Characterization of insulin-like growth factor-binding proteins from sheep thyroid cells.

    PubMed

    Bachrach, L K; Liu, F R; Burrow, G N; Eggo, M C

    1989-12-01

    The insulin-like growth factors (IGFs) are bound by specific, high affinity binding proteins. Distinct classes of IGF-binding proteins have been described in human serum, amniotic fluid, cerebrospinal fluid, and conditioned medium from cultured cells. Sheep thyroid cells produce IGF-binding proteins under hormonal regulation. Cells grown without or with standard medium supplements (transferrin, glycyl-histidyl-lysine, hydrocortisone, somatostatin, insulin, and TSH) released binding proteins with apparent mol wt of 23, 29, and 32 kDa on Western ligand blot (nonreduced). Binding proteins from these cells appeared as 21, 26, 34, 36, and 41 kDa bands when cross-linked to [125I]IGF-I under reducing conditions. The addition of epidermal growth factor (EGF) or phorbol esters, thyroid cell mitogens stimulated the production of larger binding proteins with mol wt of 40-44 and 48-52 by ligand blot and cross-linking methods, respectively. Deglycosylation of conditioned medium cross-linked to [125I]IGF-I with endoglycosidase-F did not alter the size of the smaller binding proteins, but reduced EGF-stimulated binding proteins to 36-40 kDa. Similarly, tunicamycin treatment, which inhibits glycosylation, reduced only the size of this larger binding protein species. Polyclonal antisera directed against the human amniotic fluid binding protein (BP-28) immunoprecipitated the 32 kDa sheep thyroid binding protein seen on ligand blot and the cross-linked binding protein at 36-38 kDa. Antibody against the major human serum binding protein (BP-53) recognized only the larger EGF-stimulated binding proteins. In contrast to sheep thyroid cells, rat FRTL5 thyroid cells produced no detectable IGF-binding proteins. We conclude that the predominant binding proteins produced by sheep thyroid cells under standard culture conditions are non-glycosylated and immunoreact with antiserum directed against BP-28. EGF and phorbol esters stimulate production of larger glycosylated binding proteins

  4. Minimalistic predictor of protein binding energy: contribution of solvation factor to protein binding.

    PubMed

    Choi, Jeong-Mo; Serohijos, Adrian W R; Murphy, Sean; Lucarelli, Dennis; Lofranco, Leo L; Feldman, Andrew; Shakhnovich, Eugene I

    2015-02-17

    It has long been known that solvation plays an important role in protein-protein interactions. Here, we use a minimalistic solvation-based model for predicting protein binding energy to estimate quantitatively the contribution of the solvation factor in protein binding. The factor is described by a simple linear combination of buried surface areas according to amino-acid types. Even without structural optimization, our minimalistic model demonstrates a predictive power comparable to more complex methods, making the proposed approach the basis for high throughput applications. Application of the model to a proteomic database shows that receptor-substrate complexes involved in signaling have lower affinities than enzyme-inhibitor and antibody-antigen complexes, and they differ by chemical compositions on interfaces. Also, we found that protein complexes with components that come from the same genes generally have lower affinities than complexes formed by proteins from different genes, but in this case the difference originates from different interface areas. The model was implemented in the software PYTHON, and the source code can be found on the Shakhnovich group webpage: http://faculty.chemistry.harvard.edu/shakhnovich/software.

  5. Additive Factors Do Not Imply Discrete Processing Stages: A Worked Example Using Models of the Stroop Task

    PubMed Central

    Stafford, Tom; Gurney, Kevin N.

    2011-01-01

    Previously, it has been shown experimentally that the psychophysical law known as Piéron’s Law holds for color intensity and that the size of the effect is additive with that of Stroop condition (Stafford et al., 2011). According to the additive factors method (Donders, 1868–1869/1969; Sternberg, 1998), additivity is assumed to indicate independent and discrete processing stages. We present computational modeling work, using an existing Parallel Distributed Processing model of the Stroop task (Cohen et al., 1990) and a standard model of decision making (Ratcliff, 1978). This demonstrates that additive factors can be successfully accounted for by existing single stage models of the Stroop effect. Consequently, it is not valid to infer either discrete stages or separate loci of effects from additive factors. Further, our modeling work suggests that information binding may be a more important architectural property for producing additive factors than discrete stages. PMID:22102842

  6. Bacillus licheniformis Contains Two More PerR-Like Proteins in Addition to PerR, Fur, and Zur Orthologues

    PubMed Central

    Ju, Shin-Yeong; Yang, Yoon-Mo; Ryu, Su-Hyun; Kwon, Yumi; Won, Young-Bin; Lee, Yeh-Eun; Youn, Hwan; Lee, Jin-Won

    2016-01-01

    The ferric uptake regulator (Fur) family proteins include sensors of Fe (Fur), Zn (Zur), and peroxide (PerR). Among Fur family proteins, Fur and Zur are ubiquitous in most prokaryotic organisms, whereas PerR exists mainly in Gram positive bacteria as a functional homologue of OxyR. Gram positive bacteria such as Bacillus subtilis, Listeria monocytogenes and Staphylococcus aureus encode three Fur family proteins: Fur, Zur, and PerR. In this study, we identified five Fur family proteins from B. licheniformis: two novel PerR-like proteins (BL00690 and BL00950) in addition to Fur (BL05249), Zur (BL03703), and PerR (BL00075) homologues. Our data indicate that all of the five B. licheniformis Fur homologues contain a structural Zn2+ site composed of four cysteine residues like many other Fur family proteins. Furthermore, we provide evidence that the PerR-like proteins (BL00690 and BL00950) as well as PerRBL (BL00075), but not FurBL (BL05249) and ZurBL (BL03703), can sense H2O2 by histidine oxidation with different sensitivity. We also show that PerR2 (BL00690) has a PerR-like repressor activity for PerR-regulated genes in vivo. Taken together, our results suggest that B. licheniformis contains three PerR subfamily proteins which can sense H2O2 by histidine oxidation not by cysteine oxidation, in addition to Fur and Zur. PMID:27176811

  7. Bacillus licheniformis Contains Two More PerR-Like Proteins in Addition to PerR, Fur, and Zur Orthologues.

    PubMed

    Kim, Jung-Hoon; Ji, Chang-Jun; Ju, Shin-Yeong; Yang, Yoon-Mo; Ryu, Su-Hyun; Kwon, Yumi; Won, Young-Bin; Lee, Yeh-Eun; Youn, Hwan; Lee, Jin-Won

    2016-01-01

    The ferric uptake regulator (Fur) family proteins include sensors of Fe (Fur), Zn (Zur), and peroxide (PerR). Among Fur family proteins, Fur and Zur are ubiquitous in most prokaryotic organisms, whereas PerR exists mainly in Gram positive bacteria as a functional homologue of OxyR. Gram positive bacteria such as Bacillus subtilis, Listeria monocytogenes and Staphylococcus aureus encode three Fur family proteins: Fur, Zur, and PerR. In this study, we identified five Fur family proteins from B. licheniformis: two novel PerR-like proteins (BL00690 and BL00950) in addition to Fur (BL05249), Zur (BL03703), and PerR (BL00075) homologues. Our data indicate that all of the five B. licheniformis Fur homologues contain a structural Zn2+ site composed of four cysteine residues like many other Fur family proteins. Furthermore, we provide evidence that the PerR-like proteins (BL00690 and BL00950) as well as PerRBL (BL00075), but not FurBL (BL05249) and ZurBL (BL03703), can sense H2O2 by histidine oxidation with different sensitivity. We also show that PerR2 (BL00690) has a PerR-like repressor activity for PerR-regulated genes in vivo. Taken together, our results suggest that B. licheniformis contains three PerR subfamily proteins which can sense H2O2 by histidine oxidation not by cysteine oxidation, in addition to Fur and Zur.

  8. Additive cytotoxicity of different monoclonal antibody-cobra venom factor conjugates for human neuroblastoma cells.

    PubMed

    Juhl, H; Petrella, E C; Cheung, N K; Bredehorst, R; Vogel, C W

    1997-11-01

    Insufficient numbers of antigen molecules and heterogeneity of antigen expression on tumor cells are major factors limiting the immunotherapeutic potential of the few clinically useful monoclonal antibodies capable of mediating complement cytotoxicity and antibody-dependent cellular cytotoxicity. To overcome this limitation, we converted two non-cytotoxic monoclonal anti-neuroblastoma antibodies, designated 3E7 (IgG2b) and 8H9 (IgG1), and the non-cytotoxic F(ab')2 fragment of the cytotoxic monoclonal anti-GD2 antibody 3F8 (IgG3) into cytotoxic antibody conjugates by covalent attachment of cobra venom factor (CVF), a structural and functional homologue of the activated third component of complement. Competitive binding experiments confirmed the different specificities of the three antibodies. In the presence of human complement, all three antibody-CVF conjugates mediated selective complement-dependent lysis of human neuroblastoma cells. Consistent with the kinetics of the alternative pathway of complement, approximately seven hours incubation were required to reach maximum cytotoxicity of up to 25% for the 3E7-CVF conjugate, up to 60% for the 8H9-CVF conjugate, and up to 95% for the 3F8 F(ab')2-CVF conjugate. The different extent of maximal cytotoxic activity of the three conjugates was reflected by corresponding differences in the extent of binding of both unconjugated antibodies and the respective conjugates. Any combination of the three antibody-CVF conjugates caused an additive effect in complement-mediated lysis. Using a cocktail of all three conjugates, the extent of complement-mediated killing could be increased up to 100%. These data demonstrate that by coupling of CVF the relative large number of non-cytotoxic monoclonal anti-tumor antibodies of interesting specificity can be used to design cocktails of cytotoxic conjugates and, thereby, to overcome the problem of insufficient and heterogeneous antigen expression on tumor cells for immunotherapy.

  9. How protein chemists learned about the hydrophobic factor.

    PubMed Central

    Tanford, C.

    1997-01-01

    It is generally accepted today that the hydrophobic force is the dominant energetic factor that leads to the folding of polypeptide chains into compact globular entities. This principle was first explicitly introduced to protein chemists in 1938 by Irving Langmuir, past master in the application of hydrophobicity to other problems, and was enthusiastically endorsed by J.D. Bernal. But both proposal and endorsement came in the course of a debate about a quite different structural principle, the so-called "cyclol hypothesis" proposed by D. Wrinch, which soon proved to be theoretically and experimentally unsupportable. Being a more tangible idea, directly expressed in structural terms, the cyclol hypothesis received more attention than the hydrophobic principle and the latter never actually entered the mainstream of protein science until 1959, when it was thrust into the limelight in a lucid review by W. Kauzmann. A theoretical paper by H.S. Frank and M. Evans, not itself related to protein folding, probably played a major role in the acceptance of the hydrophobicity concept by protein chemists because it provided a crude but tangible picture of the origin of hydrophobicity per se in terms of water structure. PMID:9194199

  10. The NS1 protein: a multitasking virulence factor.

    PubMed

    Ayllon, Juan; García-Sastre, Adolfo

    2015-01-01

    The non-structural protein 1 of influenza virus (NS1) is a relatively small polypeptide with an outstanding number of ascribed functions. NS1 is the main viral antagonist of the innate immune response during influenza virus infection, chiefly by inhibiting the type I interferon system at multiple steps. As such, its role is critical to overcome the first barrier the host presents to halt the viral infection. However, the pro-viral activities of this well-studied protein go far beyond and include regulation of viral RNA and protein synthesis, and disruption of the host cell homeostasis by dramatically affecting general gene expression while tweaking the PI3K signaling network. Because of all of this, NS1 is a key virulence factor that impacts influenza pathogenesis, and adaptation to new hosts, making it an attractive target for control strategies. Here, we will overview the many roles that have been ascribed to the NS1 protein, and give insights into the sequence features and structural properties that make them possible, highlighting the need to understand how NS1 can actually perform all of these functions during viral infection. PMID:25007846

  11. How protein chemists learned about the hydrophobic factor.

    PubMed

    Tanford, C

    1997-06-01

    It is generally accepted today that the hydrophobic force is the dominant energetic factor that leads to the folding of polypeptide chains into compact globular entities. This principle was first explicitly introduced to protein chemists in 1938 by Irving Langmuir, past master in the application of hydrophobicity to other problems, and was enthusiastically endorsed by J.D. Bernal. But both proposal and endorsement came in the course of a debate about a quite different structural principle, the so-called "cyclol hypothesis" proposed by D. Wrinch, which soon proved to be theoretically and experimentally unsupportable. Being a more tangible idea, directly expressed in structural terms, the cyclol hypothesis received more attention than the hydrophobic principle and the latter never actually entered the mainstream of protein science until 1959, when it was thrust into the limelight in a lucid review by W. Kauzmann. A theoretical paper by H.S. Frank and M. Evans, not itself related to protein folding, probably played a major role in the acceptance of the hydrophobicity concept by protein chemists because it provided a crude but tangible picture of the origin of hydrophobicity per se in terms of water structure. PMID:9194199

  12. The NS1 protein: a multitasking virulence factor.

    PubMed

    Ayllon, Juan; García-Sastre, Adolfo

    2015-01-01

    The non-structural protein 1 of influenza virus (NS1) is a relatively small polypeptide with an outstanding number of ascribed functions. NS1 is the main viral antagonist of the innate immune response during influenza virus infection, chiefly by inhibiting the type I interferon system at multiple steps. As such, its role is critical to overcome the first barrier the host presents to halt the viral infection. However, the pro-viral activities of this well-studied protein go far beyond and include regulation of viral RNA and protein synthesis, and disruption of the host cell homeostasis by dramatically affecting general gene expression while tweaking the PI3K signaling network. Because of all of this, NS1 is a key virulence factor that impacts influenza pathogenesis, and adaptation to new hosts, making it an attractive target for control strategies. Here, we will overview the many roles that have been ascribed to the NS1 protein, and give insights into the sequence features and structural properties that make them possible, highlighting the need to understand how NS1 can actually perform all of these functions during viral infection.

  13. Effect of the addition of conventional additives and whey proteins concentrates on technological parameters, physicochemical properties, microstructure and sensory attributes of sous vide cooked beef muscles.

    PubMed

    Szerman, N; Gonzalez, C B; Sancho, A M; Grigioni, G; Carduza, F; Vaudagna, S R

    2012-03-01

    Beef muscles submitted to four enhancement treatments (1.88% whey protein concentrate (WPC)+1.25% sodium chloride (NaCl); 1.88% modified whey protein concentrate (MWPC)+1.25%NaCl; 0.25% sodium tripolyphosphate (STPP)+1.25%NaCl; 1.25%NaCl) and a control treatment (non-injected muscles) were sous vide cooked. Muscles with STPP+NaCl presented a significantly higher total yield (106.5%) in comparison to those with WPC/MWPC+NaCl (94.7% and 92.9%, respectively), NaCl alone (84.8%) or controls (72.1%). Muscles with STPP+NaCl presented significantly lower shear force values than control ones; also, WPC/MWPC+NaCl added muscles presented similar values than those from the other treatments. After cooking, muscles with STPP+NaCl or WPC/MWPC+NaCl depicted compacted and uniform microstructures. Muscles with STPP+NaCl showed a pink colour, meanwhile other treatment muscles presented colours between pinkish-grey and grey-brown. STPP+NaCl added samples presented the highest values of global tenderness and juiciness. The addition of STPP+NaCl had a better performance than WPC/MWPC+NaCl. However, the addition of WPC/MWPC+NaCl improved total yield in comparison to NaCl added or control ones. PMID:22112522

  14. Effect of the addition of conventional additives and whey proteins concentrates on technological parameters, physicochemical properties, microstructure and sensory attributes of sous vide cooked beef muscles.

    PubMed

    Szerman, N; Gonzalez, C B; Sancho, A M; Grigioni, G; Carduza, F; Vaudagna, S R

    2012-03-01

    Beef muscles submitted to four enhancement treatments (1.88% whey protein concentrate (WPC)+1.25% sodium chloride (NaCl); 1.88% modified whey protein concentrate (MWPC)+1.25%NaCl; 0.25% sodium tripolyphosphate (STPP)+1.25%NaCl; 1.25%NaCl) and a control treatment (non-injected muscles) were sous vide cooked. Muscles with STPP+NaCl presented a significantly higher total yield (106.5%) in comparison to those with WPC/MWPC+NaCl (94.7% and 92.9%, respectively), NaCl alone (84.8%) or controls (72.1%). Muscles with STPP+NaCl presented significantly lower shear force values than control ones; also, WPC/MWPC+NaCl added muscles presented similar values than those from the other treatments. After cooking, muscles with STPP+NaCl or WPC/MWPC+NaCl depicted compacted and uniform microstructures. Muscles with STPP+NaCl showed a pink colour, meanwhile other treatment muscles presented colours between pinkish-grey and grey-brown. STPP+NaCl added samples presented the highest values of global tenderness and juiciness. The addition of STPP+NaCl had a better performance than WPC/MWPC+NaCl. However, the addition of WPC/MWPC+NaCl improved total yield in comparison to NaCl added or control ones.

  15. Electrical inhibition of lens epithelial cell proliferation: an additional factor in secondary cataract?

    PubMed Central

    Wang, Entong; Reid, Brian; Lois, Noemi; Forrester, John V.; McCaig, Colin D.; Zhao, Min

    2005-01-01

    Cataract is the most common cause of blindness but is at least curable by surgery. Unfortunately, many patients gradually develop the complication of posterior capsule opacification (PCO) or secondary cataract. This arises from stimulated cell growth within the lens capsule and can greatly impair vision. It is not fully understood why residual lens epithelial cell growth occurs after surgery. We propose and show that cataract surgery might remove an important inhibitory factor for lens cell growth, namely electric fields. The lens generates a unique pattern of electric currents constantly flowing out from the equator and entering the anterior and posterior poles. We show here that cutting and removing part of the anterior capsule as in cataract surgery significantly decreases the equatorial outward electric currents. Application of electric fields in culture inhibits proliferation of human lens epithelial cells. This inhibitory effect is likely to be mediated through a cell cycle control mechanism that decreases entry of cells into S phase from G1 phase by decreasing the G1-specific cell cycle protein cyclin E and increasing the cyclin-Cdk complex inhibitor p27kip1. Capsulorrhexis in vivo, which reduced endogenous lens electric fields, significantly increased LEC growth. This, together with our previous findings that electric fields have significant effects on the direction of lens cell migration, points to a controlling mechanism for the aberrant cell growth in posterior capsule opacification. A novel approach to control growth of lens epithelial cells using electric fields combined with other controlling mechanisms may be more effective in the prevention and treatment of this common complication of cataract surgery. PMID:15764648

  16. Insulin resistance: an additional risk factor in the pathogenesis of cardiovascular disease in type 2 diabetes.

    PubMed

    Patel, Tushar P; Rawal, Komal; Bagchi, Ashim K; Akolkar, Gauri; Bernardes, Nathalia; Dias, Danielle da Silva; Gupta, Sarita; Singal, Pawan K

    2016-01-01

    Sedentary life style and high calorie dietary habits are prominent leading cause of metabolic syndrome in modern world. Obesity plays a central role in occurrence of various diseases like hyperinsulinemia, hyperglycemia and hyperlipidemia, which lead to insulin resistance and metabolic derangements like cardiovascular diseases (CVDs) mediated by oxidative stress. The mortality rate due to CVDs is on the rise in developing countries. Insulin resistance (IR) leads to micro or macro angiopathy, peripheral arterial dysfunction, hampered blood flow, hypertension, as well as the cardiomyocyte and the endothelial cell dysfunctions, thus increasing risk factors for coronary artery blockage, stroke and heart failure suggesting that there is a strong association between IR and CVDs. The plausible linkages between these two pathophysiological conditions are altered levels of insulin signaling proteins such as IR-β, IRS-1, PI3K, Akt, Glut4 and PGC-1α that hamper insulin-mediated glucose uptake as well as other functions of insulin in the cardiomyocytes and the endothelial cells of the heart. Reduced AMPK, PFK-2 and elevated levels of NADP(H)-dependent oxidases produced by activated M1 macrophages of the adipose tissue and elevated levels of circulating angiotensin are also cause of CVD in diabetes mellitus condition. Insulin sensitizers, angiotensin blockers, superoxide scavengers are used as therapeutics in the amelioration of CVD. It evidently becomes important to unravel the mechanisms of the association between IR and CVDs in order to formulate novel efficient drugs to treat patients suffering from insulin resistance-mediated cardiovascular diseases. The possible associations between insulin resistance and cardiovascular diseases are reviewed here. PMID:26542377

  17. Integrating products of Bessel functions with an additional exponential or rational factor

    NASA Astrophysics Data System (ADS)

    Van Deun, Joris; Cools, Ronald

    2008-04-01

    We provide two MATLAB programs to compute integrals of the form ex∏i=1kJν_i(ax)dxand 0∞xr+x∏i=1kJν_i(ax)dx with Jν_i(x) the Bessel function of the first kind and (real) order ν. The parameter m is a real number such that ∑ν+m>-1 (to assure integrability near zero), r is real and the numbers c and a are all strictly positive. The program can deliver accurate error estimates. Program summaryProgram title: BESSELINTR, BESSELINTC Catalogue identifier: AEAH_v1_0 Program summary URL:http://cpc.cs.qub.ac.uk/summaries/AEAH_v1_0.html Program obtainable from: CPC Program Library, Queen's University, Belfast, N. Ireland Licensing provisions: Standard CPC licence, http://cpc.cs.qub.ac.uk/licence/licence.html No. of lines in distributed program, including test data, etc.: 1601 No. of bytes in distributed program, including test data, etc.: 13 161 Distribution format: tar.gz Programming language: Matlab (version ⩾6.5), Octave (version ⩾2.1.69) Computer: All supporting Matlab or Octave Operating system: All supporting Matlab or Octave RAM: For k Bessel functions our program needs approximately ( 500+140k) double precision variables Classification: 4.11 Nature of problem: The problem consists in integrating an arbitrary product of Bessel functions with an additional rational or exponential factor over a semi-infinite interval. Difficulties arise from the irregular oscillatory behaviour and the possible slow decay of the integrand, which prevents truncation at a finite point. Solution method: The interval of integration is split into a finite and infinite part. The integral over the finite part is computed using Gauss-Legendre quadrature. The integrand on the infinite part is approximated using asymptotic expansions and this approximation is integrated exactly with the aid of the upper incomplete gamma function. In the case where a rational factor is present, this factor is first expanded in a Taylor series around infinity. Restrictions: Some (and eventually all

  18. Regulation of RE1 protein silencing transcription factor (REST) expression by HIP1 protein interactor (HIPPI).

    PubMed

    Datta, Moumita; Bhattacharyya, Nitai P

    2011-09-30

    Earlier we have shown that the proapoptotic protein HIPPI (huntingtin interacting protein 1 (HIP1) protein interactor) along with its molecular partner HIP1 could regulate transcription of the caspase-1 gene. Here we report that RE1-silencing transcription factor/neuron-restrictive silencer factor (REST/NRSF) is a new transcriptional target of HIPPI. HIPPI could bind to the promoter of REST and increased its expression in neuronal as well as non-neuronal cells. Such activation of REST down-regulated expression of REST target genes, such as brain-derived neurotrophic factor (BDNF) or proenkephalin (PENK). The ability of HIPPI to activate REST gene transcription was dependent on HIP1, the nuclear transporter of HIPPI. Using a Huntington disease cell model, we have demonstrated that feeble interaction of HIP1 with mutant huntingtin protein resulted in increased nuclear accumulation of HIPPI and HIP1, leading to higher occupancy of HIPPI at the REST promoter, triggering its transcriptional activation and consequent repression of REST target genes. This novel transcription regulatory mechanism of REST by HIPPI may contribute to the deregulation of transcription observed in the cell model of Huntington disease. PMID:21832040

  19. The sequential addition of ribosomal proteins during the formation of the small ribosomal subunit in Friend erythroleukemia cells.

    PubMed

    Todorov, I T; Noll, F; Hadjiolov, A A

    1983-03-15

    Nucleolar '80-S' and '40-S' preribosomes (containing 45-S and 21-S pre-rRNA, respectively), as well as cytoplasmic ribosomes, were isolated from Friend erythroleukemia cells. The presence of structural ribosomal proteins in the isolated particles was studied by using antisera against individual rat liver small ribosomal subunit proteins. The analysis is based on the established crossreactivity between rat and mouse ribosomes [F. Noll and H. Bielka (1970) Mol. Gen. Genet. 106, 106-113]. The identification of the proteins was achieved by two independent immunological techniques: the passive haemagglutination test and the enzyme immunoassay of electrophoretically fractionated proteins, blotted on nitrocellulose. All 17 proteins tested are present in cytoplasmic ribosomes. A large number of proteins (S3a, S6, S7, S8, S11, S14, S18, S20, S23/24 and S25) are present in the '80-S' preribosome. Only two proteins (S3 and S21) are added during the formation of the '40-S' preribosome in the nucleolus. Four proteins (S2, S19, S26 and S29) are added at later, possibly extranucleolar, stages of ribosome formation. The results obtained provide evidence for the sequential addition of proteins during the formation of the small ribosomal subunit in Friend erythroleukemia cells.

  20. Telomere Capping Proteins are Structurally Related to RPA with an additional Telomere-Specific Domain

    SciTech Connect

    Gelinas, A.; Paschini, M; Reyes, F; Heroux, A; Batey, R; Lundblad, V; Wuttke, D

    2009-01-01

    Telomeres must be capped to preserve chromosomal stability. The conserved Stn1 and Ten1 proteins are required for proper capping of the telomere, although the mechanistic details of how they contribute to telomere maintenance are unclear. Here, we report the crystal structures of the C-terminal domain of the Saccharomyces cerevisiae Stn1 and the Schizosaccharomyces pombe Ten1 proteins. These structures reveal striking similarities to corresponding subunits in the replication protein A complex, further supporting an evolutionary link between telomere maintenance proteins and DNA repair complexes. Our structural and in vivo data of Stn1 identify a new domain that has evolved to support a telomere-specific role in chromosome maintenance. These findings endorse a model of an evolutionarily conserved mechanism of DNA maintenance that has developed as a result of increased chromosomal structural complexity.

  1. Effective protein-protein interaction from structure factor data of a lysozyme solution

    SciTech Connect

    Abramo, M. C.; Caccamo, C.; Costa, D.; Ruberto, R.; Wanderlingh, U.; Cavero, M.; Pellicane, G.

    2013-08-07

    We report the determination of an effective protein-protein central potential for a lysozyme solution, obtained from the direct inversion of the total structure factor of the system, as extracted from small angle neutron scattering. The inversion scheme rests on a hypernetted-chain relationship between the effective potential and the structural functions, and is preliminarily tested for the case of a Lennard-Jones interaction. The characteristics of our potential are discussed in comparison with current models of effective interactions in complex fluids. The phase behavior predictions are also investigated.

  2. Effective protein-protein interaction from structure factor data of a lysozyme solution

    NASA Astrophysics Data System (ADS)

    Abramo, M. C.; Caccamo, C.; Cavero, M.; Costa, D.; Pellicane, G.; Ruberto, R.; Wanderlingh, U.

    2013-08-01

    We report the determination of an effective protein-protein central potential for a lysozyme solution, obtained from the direct inversion of the total structure factor of the system, as extracted from small angle neutron scattering. The inversion scheme rests on a hypernetted-chain relationship between the effective potential and the structural functions, and is preliminarily tested for the case of a Lennard-Jones interaction. The characteristics of our potential are discussed in comparison with current models of effective interactions in complex fluids. The phase behavior predictions are also investigated.

  3. ProFold: Protein Fold Classification with Additional Structural Features and a Novel Ensemble Classifier

    PubMed Central

    2016-01-01

    Protein fold classification plays an important role in both protein functional analysis and drug design. The number of proteins in PDB is very large, but only a very small part is categorized and stored in the SCOPe database. Therefore, it is necessary to develop an efficient method for protein fold classification. In recent years, a variety of classification methods have been used in many protein fold classification studies. In this study, we propose a novel classification method called proFold. We import protein tertiary structure in the period of feature extraction and employ a novel ensemble strategy in the period of classifier training. Compared with existing similar ensemble classifiers using the same widely used dataset (DD-dataset), proFold achieves 76.2% overall accuracy. Another two commonly used datasets, EDD-dataset and TG-dataset, are also tested, of which the accuracies are 93.2% and 94.3%, higher than the existing methods. ProFold is available to the public as a web-server.

  4. ProFold: Protein Fold Classification with Additional Structural Features and a Novel Ensemble Classifier

    PubMed Central

    2016-01-01

    Protein fold classification plays an important role in both protein functional analysis and drug design. The number of proteins in PDB is very large, but only a very small part is categorized and stored in the SCOPe database. Therefore, it is necessary to develop an efficient method for protein fold classification. In recent years, a variety of classification methods have been used in many protein fold classification studies. In this study, we propose a novel classification method called proFold. We import protein tertiary structure in the period of feature extraction and employ a novel ensemble strategy in the period of classifier training. Compared with existing similar ensemble classifiers using the same widely used dataset (DD-dataset), proFold achieves 76.2% overall accuracy. Another two commonly used datasets, EDD-dataset and TG-dataset, are also tested, of which the accuracies are 93.2% and 94.3%, higher than the existing methods. ProFold is available to the public as a web-server. PMID:27660761

  5. Factor H-binding protein, a unique meningococcal vaccine antigen.

    PubMed

    Pizza, Mariagrazia; Donnelly, John; Rappuoli, Rino

    2008-12-30

    GNA1870, also named factor H-binding protein (fHbp) or rLP-2086, is a genome-derived antigen and one of the components of a rationally designed vaccine against Neisseria meningitidis serogroup B, which has entered phase III clinical trials. It has been classified into three main non-cross-protective variant groups. GNA1870 has also been termed fHbp because of its ability to bind factor H, a key regulatory component of the alternative complement pathway. fHbp is important for survival in human blood, human sera, and in presence of antimicrobial peptides, independently of its expression level. All these properties make fHbp a unique vaccine antigen.

  6. Recombinant Expression, Purification, and Functional Characterisation of Connective Tissue Growth Factor and Nephroblastoma-Overexpressed Protein

    PubMed Central

    Bohr, Wilhelm; Kupper, Michael; Hoffmann, Kurt; Weiskirchen, Ralf

    2010-01-01

    The CCN family of proteins, especially its prominent member, the Connective tissue growth factor (CTGF/CCN2) has been identified as a possible biomarker for the diagnosis of fibrotic diseases. As a downstream mediator of TGF-β1 signalling, it is involved in tissue scarring, stimulates interstitial deposition of extracellular matrix proteins, and promotes proliferation of several cell types. Another member of this family, the Nephroblastoma-Overexpressed protein (NOV/CCN3), has growth-inhibiting properties. First reports further suggest that these two CCN family members act opposite to each other in regulating extracellular matrix protein expression and reciprocally influence their own expression when over-expressed. We have established stable HEK and Flp-In-293 clones as productive sources for recombinant human CCN2/CTGF. In addition, we generated an adenoviral vector for recombinant expression of rat NOV and established protocols to purify large quantities of these CCN proteins. The identity of purified human CCN2/CTGF and rat CCN3/NOV was proven by In-gel digest followed by ESI-TOF/MS mass spectrometry. The biological activity of purified proteins was demonstrated using a Smad3-sensitive reporter gene and BrdU proliferation assay in permanent cell line EA•hy 926 cells. We further demonstrate for the first time that both recombinant CCN proteins are N-glycosylated. PMID:21209863

  7. Macrophage Stimulating Protein Is a Novel Neurotrophic Factor

    PubMed Central

    Stella, Maria Cristina; Vercelli, Alessandro; Repici, Mariaelena; Follenzi, Antonia; Comoglio, Paolo M.

    2001-01-01

    Macrophage stimulating protein (MSP), also known as hepatocyte growth factor-like, is a soluble cytokine that belongs to the family of the plasminogen-related growth factors (PRGFs). PRGFs are α/β heterodimers that bind to transmembrane tyrosine kinase receptors. MSP was originally isolated as a chemotactic factor for peritoneal macrophages. Through binding to its receptor, encoded by the RON gene, it stimulates dissociation of epithelia and works as an inflammatory mediator by repressing the production of nitric oxide (NO). Here, we identify a novel role for MSP in the central nervous system. As a paradigm to analyze this function we chose the hypoglossal system of adult mice. We demonstrate in vivo that either administration of exogenous MSP or transplantation of MSP-producing cells at the proximal stump of the resected nerve is sufficient to prevent motoneuron atrophy upon axotomy. We also show that the MSP gene is expressed in the tongue, the target of the hypoglossal nerve, and that MSP induces biosynthesis of Ron receptor in the motoneuron somata. Finally, we show that MSP suppresses NO production in the injured hypoglossal nuclei. Together, these data suggest that MSP is a novel neurotrophic factor for cranial motoneurons and, by regulating the production of NO, may have a role in brain plasticity and regeneration. PMID:11359926

  8. Lengths of Orthologous Prokaryotic Proteins Are Affected by Evolutionary Factors

    PubMed Central

    Tatarinova, Tatiana; Dien Bard, Jennifer; Cohen, Irit

    2015-01-01

    Proteins of the same functional family (for example, kinases) may have significantly different lengths. It is an open question whether such variation in length is random or it appears as a response to some unknown evolutionary driving factors. The main purpose of this paper is to demonstrate existence of factors affecting prokaryotic gene lengths. We believe that the ranking of genomes according to lengths of their genes, followed by the calculation of coefficients of association between genome rank and genome property, is a reasonable approach in revealing such evolutionary driving factors. As we demonstrated earlier, our chosen approach, Bubble-sort, combines stability, accuracy, and computational efficiency as compared to other ranking methods. Application of Bubble Sort to the set of 1390 prokaryotic genomes confirmed that genes of Archaeal species are generally shorter than Bacterial ones. We observed that gene lengths are affected by various factors: within each domain, different phyla have preferences for short or long genes; thermophiles tend to have shorter genes than the soil-dwellers; halophiles tend to have longer genes. We also found that species with overrepresentation of cytosines and guanines in the third position of the codon (GC3 content) tend to have longer genes than species with low GC3 content. PMID:26114113

  9. Salivary protein factors are elevated in breast cancer patients

    PubMed Central

    BROOKS, MAI N.; WANG, JIANGHUA; LI, YANG; ZHANG, RONG; ELASHOFF, DAVID; WONG, DAVID T.

    2009-01-01

    While saliva is a source of easily accessible bodily fluids, there has been little effort to study its value in cancer diagnosis. We hypothesized that certain proteins would be elevated in the saliva of patients with breast cancer. Our study included 49 healthy individuals and 49 breast cancer patients. The levels of vascular endothelial growth factor (VEGF), epidermal growth factor (EGF) and carcinoembryonic antigen (CEA) in the saliva were measured with enzyme-linked immunosorbent assay (ELISA). We observed that salivary fluid protein levels were significantly elevated in cancer patients as follows: i) VEGF, 3.7±1.6 in cancer versus 2.1±1.2 ng/ml in control (p<0.0001); ii) EGF, 3.7±1.7 versus 2.1±1.3 ng/ml (p<0.0001); and iii) CEA, 83±31 versus 66.1±27.1 ng/ml (p=0.0106). The areas under the receiver operating characteristic (ROC) curve (AUC) were 80, 77 and 65%, respectively. The best prediction was from the combination of salivary VEGF and EGF with a sensitivity of 83%, specificity of 74% and AUC of 84%. We conclude that saliva is a novel avenue for tumor marker research and deserves further studies. Saliva may potentially be useful in supplementing current methods of breast cancer detection. PMID:19844594

  10. Towards the synthesis of hydroxyapatite/protein scaffolds with controlled porosities: bulk and interfacial shear rheology of a hydroxyapatite suspension with protein additives.

    PubMed

    Maas, Michael; Bodnar, Pedro Marcus; Hess, Ulrike; Treccani, Laura; Rezwan, Kurosch

    2013-10-01

    The synthesis of porous hydroxyapatite scaffolds is essential for biomedical applications such as bone tissue engineering and replacement. One way to induce macroporosity, which is needed to support bone in-growth, is to use protein additives as foaming agents. Another reason to use protein additives is the potential to introduce a specific biofunctionality to the synthesized scaffolds. In this work, we study the rheological properties of a hydroxyapatite suspension system with additions of the proteins bovine serum albumin (BSA), lysozyme (LSZ) and fibrinogen (FIB). Both the rheology of the bulk phase as well as the interfacial shear rheology are studied. The bulk rheological data provides important information on the setting behavior of the thixotropic suspension, which we find to be faster with the addition of FIB and LSZ and much slower with BSA. Foam bubble stabilization mechanisms can be rationalized via interfacial shear rheology and we show that it depends on the growth of interfacial films at the suspension/air interface. These interfacial films support the stabilization of bubbles within the ceramic matrix and thereby introduce macropores. Due to the weak interaction of the protein molecules with the hydroxyapatite particles of the suspension, we find that BSA forms the most stable interfacial films, followed by FIB. LSZ strongly interacts with the hydroxyapatite particles and thus only forms thin films with very low elastic moduli. In summary, our study provides fundamental rheological insights which are essential for tailoring hydroxyapatite/protein suspensions in order to synthesize scaffolds with controlled porosities.

  11. 34 CFR 359.32 - What additional factors does the Secretary consider in making a grant under this program?

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... EDUCATION DISABILITY AND REHABILITATION RESEARCH: SPECIAL PROJECTS AND DEMONSTRATIONS FOR SPINAL CORD INJURIES How Does the Secretary Make a Grant? § 359.32 What additional factors does the Secretary...

  12. 34 CFR 359.32 - What additional factors does the Secretary consider in making a grant under this program?

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... EDUCATION DISABILITY AND REHABILITATION RESEARCH: SPECIAL PROJECTS AND DEMONSTRATIONS FOR SPINAL CORD INJURIES How Does the Secretary Make a Grant? § 359.32 What additional factors does the Secretary...

  13. 34 CFR 359.32 - What additional factors does the Secretary consider in making a grant under this program?

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... EDUCATION DISABILITY AND REHABILITATION RESEARCH: SPECIAL PROJECTS AND DEMONSTRATIONS FOR SPINAL CORD INJURIES How Does the Secretary Make a Grant? § 359.32 What additional factors does the Secretary...

  14. 34 CFR 359.32 - What additional factors does the Secretary consider in making a grant under this program?

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... EDUCATION DISABILITY AND REHABILITATION RESEARCH: SPECIAL PROJECTS AND DEMONSTRATIONS FOR SPINAL CORD INJURIES How Does the Secretary Make a Grant? § 359.32 What additional factors does the Secretary...

  15. 34 CFR 359.32 - What additional factors does the Secretary consider in making a grant under this program?

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... EDUCATION DISABILITY AND REHABILITATION RESEARCH: SPECIAL PROJECTS AND DEMONSTRATIONS FOR SPINAL CORD INJURIES How Does the Secretary Make a Grant? § 359.32 What additional factors does the Secretary...

  16. Differential protein phosphorylation in induction of thyroid cell proliferation by thyrotropin, epidermal growth factor, or phorbol ester.

    PubMed Central

    Contor, L; Lamy, F; Lecocq, R; Roger, P P; Dumont, J E

    1988-01-01

    Protein phosphorylation was studied in primary cultures of thyroid epithelial cells after the addition of different mitogens: thyrotropin (TSH) acting through cyclic AMP, epidermal growth factor (EGF), or 12-O-tetradecanoylphorbol-13-acetate (TPA). EGF or TPA increased the phosphorylation of five common polypeptides. Among these, two 42-kilodalton proteins contained phosphotyrosine and phosphoserine with or without phosphothreonine. Their characteristics suggested that they are similar to the two 42-kilodalton target proteins for tyrosine protein phosphorylation demonstrated in fibroblasts in response to mitogens. No common phosphorylated proteins were detected in TSH-treated cells and in EGF- or TPA-treated cells. The differences in the protein phosphorylation patterns in response to TSH, EGF, and TPA suggested that the newly emerging cyclic AMP-mediated mitogenic pathway is distinct from the better known growth factor- and tumor promoter-induced pathways. Images PMID:3261388

  17. Coagulation factor V mediates inhibition of tissue factor signaling by activated protein C in mice.

    PubMed

    Liang, Hai Po H; Kerschen, Edward J; Basu, Sreemanti; Hernandez, Irene; Zogg, Mark; Jia, Shuang; Hessner, Martin J; Toso, Raffaella; Rezaie, Alireza R; Fernández, José A; Camire, Rodney M; Ruf, Wolfram; Griffin, John H; Weiler, Hartmut

    2015-11-19

    The key effector molecule of the natural protein C pathway, activated protein C (aPC), exerts pleiotropic effects on coagulation, fibrinolysis, and inflammation. Coagulation-independent cell signaling by aPC appears to be the predominant mechanism underlying its highly reproducible therapeutic efficacy in most animal models of injury and infection. In this study, using a mouse model of Staphylococcus aureus sepsis, we demonstrate marked disease stage-specific effects of the anticoagulant and cell signaling functions of aPC. aPC resistance of factor (f)V due to the R506Q Leiden mutation protected against detrimental anticoagulant effects of aPC therapy but also abrogated the anti-inflammatory and mortality-reducing effects of the signaling-selective 5A-aPC variant that has minimal anticoagulant function. We found that procofactor V (cleaved by aPC at R506) and protein S were necessary cofactors for the aPC-mediated inhibition of inflammatory tissue-factor signaling. The anti-inflammatory cofactor function of fV involved the same structural features that govern its cofactor function for the anticoagulant effects of aPC, yet its anti-inflammatory activities did not involve proteolysis of activated coagulation factors Va and VIIIa. These findings reveal a novel biological function and mechanism of the protein C pathway in which protein S and the aPC-cleaved form of fV are cofactors for anti-inflammatory cell signaling by aPC in the context of endotoxemia and infection.

  18. Arabidopsis sigma factor binding proteins are activators of the WRKY33 transcription factor in plant defense.

    PubMed

    Lai, Zhibing; Li, Ying; Wang, Fei; Cheng, Yuan; Fan, Baofang; Yu, Jing-Quan; Chen, Zhixiang

    2011-10-01

    Necrotrophic pathogens are important plant pathogens that cause many devastating plant diseases. Despite their impact, our understanding of the plant defense response to necrotrophic pathogens is limited. The WRKY33 transcription factor is important for plant resistance to necrotrophic pathogens; therefore, elucidation of its functions will enhance our understanding of plant immunity to necrotrophic pathogens. Here, we report the identification of two WRKY33-interacting proteins, nuclear-encoded SIGMA FACTOR BINDING PROTEIN1 (SIB1) and SIB2, which also interact with plastid-encoded plastid RNA polymerase SIGMA FACTOR1. Both SIB1 and SIB2 contain an N-terminal chloroplast targeting signal and a putative nuclear localization signal, suggesting that they are dual targeted. Bimolecular fluorescence complementation indicates that WRKY33 interacts with SIBs in the nucleus of plant cells. Both SIB1 and SIB2 contain a short VQ motif that is important for interaction with WRKY33. The two VQ motif-containing proteins recognize the C-terminal WRKY domain and stimulate the DNA binding activity of WRKY33. Like WRKY33, both SIB1 and SIB2 are rapidly and strongly induced by the necrotrophic pathogen Botrytis cinerea. Resistance to B. cinerea is compromised in the sib1 and sib2 mutants but enhanced in SIB1-overexpressing transgenic plants. These results suggest that dual-targeted SIB1 and SIB2 function as activators of WRKY33 in plant defense against necrotrophic pathogens.

  19. Addition of magnesium chloride to enhance mono-dispersity of a coiled-coil recombinant mouse macrophage protein.

    PubMed

    Pahuja, Parveen; Srinivasan, Alagiri; Puri, Munish

    2014-04-01

    X-ray crystallography for the determination of three-dimensional structures of protein macromolecules represents an important tool in function assignment of uncharacterized proteins. However, crystallisation is often difficult to achieve. A protein sample fully characterized in terms of dispersity may increase the likelihood of successful crystallisation by improving the predictability of the crystallisation process. To maximize the probability of crystallisation of a novel mouse macrophage protein (rMMP), target molecule was characterized and refined to improve monodispersity. Addition of MgCl2 at low concentrations resolves the rMMP into a monodisperse solution, and finally successful crystallization of rMMP was achieved. The effect of MgCl2 was studied using gel filtration chromatography and dynamic light scattering.

  20. Differentiation inducing factor-1 (DIF-1) induces gene and protein expression of the Dictyostelium nuclear calmodulin-binding protein nucleomorphin.

    PubMed

    O'Day, Danton H; Poloz, Yekaterina; Myre, Michael A

    2009-02-01

    The nucleomorphin gene numA1 from Dictyostelium codes for a multi-domain, calmodulin binding protein that regulates nuclear number. To gain insight into the regulation of numA, we assessed the effects of the stalk cell differentiation inducing factor-1 (DIF-1), an extracellular signalling molecule, on the expression of numA1 RNA and protein. For comparison, the extracellular signalling molecules cAMP (mediates chemotaxis, prestalk and prespore differentiation) and ammonia (NH(3)/NH(4)(+); antagonizes DIF) were also studied. Starvation, which is a signal for multicellular development, results in a greater than 80% decrease in numA1 mRNA expression within 4 h. Treatment with ammonium chloride led to a greater than 90% inhibition of numA1 RNA expression within 2 h. In contrast, the addition of DIF-1 completely blocked the decrease in numA1 gene expression caused by starvation. Treatment of vegetative cells with cAMP led to decreases in numA1 RNA expression that were equivalent to those seen with starvation. Western blotting after various morphogen treatments showed that the maintenance of vegetative levels of numA1 RNA by DIF-1 in starved cells was reflected in significantly increased numA1 protein levels. Treatment with cAMP and/or ammonia led to decreased protein expression and each of these morphogens suppressed the stimulatory effects of DIF-1. Protein expression levels of CBP4a, a calcium-dependent binding partner of numA1, were regulated in the same manner as numA1 suggesting this potential co-regulation may be related to their functional relationship. NumA1 is the first calmodulin binding protein shown to be regulated by developmental morphogens in Dictyostelium being upregulated by DIF-1 and down-regulated by cAMP and ammonia. PMID:19000924

  1. Relative Importance and Additive Effects of Maternal and Infant Risk Factors on Childhood Asthma

    PubMed Central

    Rosas-Salazar, Christian; James, Kristina; Escobar, Gabriel; Gebretsadik, Tebeb; Li, Sherian Xu; Carroll, Kecia N.; Walsh, Eileen; Mitchel, Edward; Das, Suman; Kumar, Rajesh; Yu, Chang; Dupont, William D.; Hartert, Tina V.

    2016-01-01

    Background Environmental exposures that occur in utero and during early life may contribute to the development of childhood asthma through alteration of the human microbiome. The objectives of this study were to estimate the cumulative effect and relative importance of environmental exposures on the risk of childhood asthma. Methods We conducted a population-based birth cohort study of mother-child dyads who were born between 1995 and 2003 and were continuously enrolled in the PRIMA (Prevention of RSV: Impact on Morbidity and Asthma) cohort. The individual and cumulative impact of maternal urinary tract infections (UTI) during pregnancy, maternal colonization with group B streptococcus (GBS), mode of delivery, infant antibiotic use, and older siblings at home, on the risk of childhood asthma were estimated using logistic regression. Dose-response effect on childhood asthma risk was assessed for continuous risk factors: number of maternal UTIs during pregnancy, courses of infant antibiotics, and number of older siblings at home. We further assessed and compared the relative importance of these exposures on the asthma risk. In a subgroup of children for whom maternal antibiotic use during pregnancy information was available, the effect of maternal antibiotic use on the risk of childhood asthma was estimated. Results Among 136,098 singleton birth infants, 13.29% developed asthma. In both univariate and adjusted analyses, maternal UTI during pregnancy (odds ratio [OR] 1.2, 95% confidence interval [CI] 1.18, 1.25; adjusted OR [AOR] 1.04, 95%CI 1.02, 1.07 for every additional UTI) and infant antibiotic use (OR 1.21, 95%CI 1.20, 1.22; AOR 1.16, 95%CI 1.15, 1.17 for every additional course) were associated with an increased risk of childhood asthma, while having older siblings at home (OR 0.92, 95%CI 0.91, 0.93; AOR 0.85, 95%CI 0.84, 0.87 for each additional sibling) was associated with a decreased risk of childhood asthma, in a dose-dependent manner. Compared with vaginal

  2. Addition of missing loops and domains to protein models by x-ray solution scattering.

    PubMed Central

    Petoukhov, Maxim V; Eady, Nigel A J; Brown, Katherine A; Svergun, Dmitri I

    2002-01-01

    Inherent flexibility and conformational heterogeneity in proteins can often result in the absence of loops and even entire domains in structures determined by x-ray crystallographic or NMR methods. X-ray solution scattering offers the possibility of obtaining complementary information regarding the structures of these disordered protein regions. Methods are presented for adding missing loops or domains by fixing a known structure and building the unknown regions to fit the experimental scattering data obtained from the entire particle. Simulated annealing was used to minimize a scoring function containing the discrepancy between the experimental and calculated patterns and the relevant penalty terms. In low-resolution models where interface location between known and unknown parts is not available, a gas of dummy residues represents the missing domain. In high-resolution models where the interface is known, loops or domains are represented as interconnected chains (or ensembles of residues with spring forces between the C(alpha) atoms), attached to known position(s) in the available structure. Native-like folds of missing fragments can be obtained by imposing residue-specific constraints. After validation in simulated examples, the methods have been applied to add missing loops or domains to several proteins where partial structures were available. PMID:12496082

  3. Modification of the protein corona-nanoparticle complex by physiological factors.

    PubMed

    Braun, Nicholas J; DeBrosse, Madeleine C; Hussain, Saber M; Comfort, Kristen K

    2016-07-01

    Nanoparticle (NP) effects in a biological system are driven through the formation and structure of the protein corona-NP complex, which is dynamic by nature and dependent upon factors from both the local environment and NP physicochemical parameters. To date, considerable data has been gathered regarding the structure and behavior of the protein corona in blood, plasma, and traditional cell culture medium. However, there exists a knowledge gap pertaining to the protein corona in additional biological fluids and following incubation in a dynamic environment. Using 13nm gold NPs (AuNPs), functionalized with either polyethylene glycol or tannic acid, we demonstrated that both particle characteristics and the associated protein corona were altered when exposed to artificial physiological fluids and under dynamic flow. Furthermore, the magnitude of observed behavioral shifts were dependent upon AuNP surface chemistry. Lastly, we revealed that exposure to interstitial fluid produced protein corona modifications, reshaping of the nano-cellular interface, modified AuNP dosimetry, and induction of previously unseen cytotoxicity. This study highlights the need to elucidate both NP and protein corona behavior in biologically representative environments in an effort to increase accurate interpretation of data and transfer of this knowledge to efficacy, behavior, and safety of nano-based applications. PMID:27127026

  4. Modification of the protein corona-nanoparticle complex by physiological factors.

    PubMed

    Braun, Nicholas J; DeBrosse, Madeleine C; Hussain, Saber M; Comfort, Kristen K

    2016-07-01

    Nanoparticle (NP) effects in a biological system are driven through the formation and structure of the protein corona-NP complex, which is dynamic by nature and dependent upon factors from both the local environment and NP physicochemical parameters. To date, considerable data has been gathered regarding the structure and behavior of the protein corona in blood, plasma, and traditional cell culture medium. However, there exists a knowledge gap pertaining to the protein corona in additional biological fluids and following incubation in a dynamic environment. Using 13nm gold NPs (AuNPs), functionalized with either polyethylene glycol or tannic acid, we demonstrated that both particle characteristics and the associated protein corona were altered when exposed to artificial physiological fluids and under dynamic flow. Furthermore, the magnitude of observed behavioral shifts were dependent upon AuNP surface chemistry. Lastly, we revealed that exposure to interstitial fluid produced protein corona modifications, reshaping of the nano-cellular interface, modified AuNP dosimetry, and induction of previously unseen cytotoxicity. This study highlights the need to elucidate both NP and protein corona behavior in biologically representative environments in an effort to increase accurate interpretation of data and transfer of this knowledge to efficacy, behavior, and safety of nano-based applications.

  5. Profiling lethal factor interacting proteins from human stomach using T7 phage display screening.

    PubMed

    Cardona-Correa, Albin; Rios-Velazquez, Carlos

    2016-05-01

    The anthrax lethal factor (LF) is a zinc dependent metalloproteinase that cleaves the majority of mitogen-activated protein kinase kinases and a member of NOD-like receptor proteins, inducing cell apoptosis. Despite efforts to fully understand the Bacillus anthracis toxin components, the gastrointestinal (GI) anthrax mechanisms have not been fully elucidated. Previous studies demonstrated gastric ulceration, and a substantial bacterial growth rate in Peyer's patches. However, the complete molecular pathways of the disease that results in tissue damage by LF proteolytic activity remains unclear. In the present study, to identify the profile of the proteins potentially involved in GI anthrax, protein‑protein interactions were investigated using human stomach T7 phage display (T7PD) cDNA libraries. T7PD is a high throughput technique that allows the expression of cloned DNA sequences as peptides on the phage surface, enabling the selection and identification of protein ligands. A wild type and mutant LF (E687A) were used to differentiate interaction sites. A total of 124 clones were identified from 194 interacting‑phages, at both the DNA and protein level, by in silico analysis. Databases revealed that the selected candidates were proteins from different families including lipase, peptidase‑A1 and cation transport families, among others. Furthermore, individual T7PD candidates were tested against LF in order to detect their specificity to the target molecule, resulting in 10 LF‑interacting peptides. With a minimum concentration of LF for interaction at 1 µg/ml, the T7PD isolated pepsin A3 pre‑protein (PAP) demonstrated affinity to both types of LF. In addition, PAP was isolated in various lengths for the same protein, exhibiting common regions following PRALINE alignment. These findings will help elucidate and improve the understanding of the molecular pathogenesis of GI anthrax, and aid in the development of potential therapeutic agents. PMID

  6. Insulin-like growth factor factor binding protein-2 is a novel mediator of p53 inhibition of insulin-like growth factor signaling.

    PubMed

    Grimberg, Adda; Coleman, Carrie M; Shi, Zonggao; Burns, Timothy F; MacLachlan, Timothy K; Wang, Wenge; El-Deiry, Wafik S

    2006-10-01

    The p53 tumor suppressor induces cellular growth arrest and apoptosis in response to DNA damage by transcriptionally activating or repressing target genes and also through protein-protein interactions and direct mitochondrial activities. In 1995, insulin-like growth factor binding protein (IGFBP)-3 was identified as one of the genes transcriptionally activated by p53. IGFBP-3 is one of six closely related IGFBP's, with additional IGFBP-related proteins belonging to the IGFBP superfamily. Here we show that IGFBP-2 is also a p53 target. Like IGFBP-3, IGFBP-2 secretion is reduced when p53+/+ lung cancer cells are transfected with human papillomavirus E6, which targets p53 for degradation. IGFBP-2 mRNA is induced by irradiation in vivo in a p53-dependent manner. p53 protein binds IGFBP-2 intronic sequences in an electrophoretic mobility shift assay, and activates transcription in a luciferase assay. Loss of IGFBP-2 inhibits the ability of p53 to inhibit the activation of extracellular signal-regulated kinase (ERK)1 by IGF-I. Thus, p53 effects on the IGF axis are more complex than previously appreciated, and overall transform the axis from IGF-mediated mitogenesis to growth inhibition and apoptosis. This has significant implications for how growth hormone and IGF-I can induce growth without also inducing cancer.

  7. STEROIDOGENIC FACTOR-1 IS A SPHINGOLIPID BINDING PROTEIN

    PubMed Central

    Urs, Aarti N.; Dammer, Eric; Kelly, Samuel; Wang, Elaine; Merrill, Alfred H.; Sewer, Marion B.

    2007-01-01

    Steroidogenic factor (SF1, NR5A1, Ad4BP) is an orphan nuclear receptor that is essential for steroid hormone-biosynthesis and endocrine development. Studies have found that the ability of this receptor to increase target gene expression can be regulated by post-translational modification, subnuclear localization, and protein-protein interactions. Recent crystallographic studies and our mass spectrometric analyses of the endogenous receptor have demonstrated an integral role for ligand-binding in the control of SF1 transactivation activity. Herein, we discuss our findings that sphingosine is an endogenous ligand for SF1. These studies and the structural findings of others have demonstrated that the receptor can bind both sphingolipids and phospholipids. Thus, it is likely that multiple bioactive lipids are ligands for SF1 and that these lipids will differentially act to control SF1 activity in a context-dependent manner. Finally, these findings highlight a central role for bioactive lipids as mediators of trophic-hormone stimulated steroid hormone biosynthesis. PMID:17196738

  8. Solvation free energy of the peptide group: its model dependence and implications for the additive-transfer free-energy model of protein stability.

    PubMed

    Tomar, Dheeraj S; Asthagiri, D; Weber, Valéry

    2013-09-17

    The group-additive decomposition of the unfolding free energy of a protein in an osmolyte solution relative to that in water poses a fundamental paradox: whereas the decomposition describes the experimental results rather well, theory suggests that a group-additive decomposition of free energies is, in general, not valid. In a step toward resolving this paradox, here we study the peptide-group transfer free energy. We calculate the vacuum-to-solvent (solvation) free energies of (Gly)n and cyclic diglycine (cGG) and analyze the data according to experimental protocol. The solvation free energies of (Gly)n are linear in n, suggesting group additivity. However, the slope interpreted as the free energy of a peptide unit differs from that for cGG scaled by a factor of half, emphasizing the context dependence of solvation. However, the water-to-osmolyte transfer free energies of the peptide unit are relatively independent of the peptide model, as observed experimentally. To understand these observations, a way to assess the contribution to the solvation free energy of solvent-mediated correlation between distinct groups is developed. We show that linearity of solvation free energy with n is a consequence of uniformity of the correlation contributions, with apparent group-additive behavior in the water-to-osmolyte transfer arising due to their cancellation. Implications for inferring molecular mechanisms of solvent effects on protein stability on the basis of the group-additive transfer model are suggested.

  9. 21 CFR 1311.115 - Additional requirements for two-factor authentication.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ..., such as a password or response to a challenge question. (2) Something the practitioner is, biometric... modules or one-time-password devices. (c) If one factor is a biometric, the biometric subsystem...

  10. 21 CFR 1311.115 - Additional requirements for two-factor authentication.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ..., such as a password or response to a challenge question. (2) Something the practitioner is, biometric... modules or one-time-password devices. (c) If one factor is a biometric, the biometric subsystem...

  11. Activity of the Human Rhinovirus 3C Protease Studied in Various Buffers, Additives and Detergents Solutions for Recombinant Protein Production.

    PubMed

    Ullah, Raheem; Shah, Majid Ali; Tufail, Soban; Ismat, Fouzia; Imran, Muhammad; Iqbal, Mazhar; Mirza, Osman; Rhaman, Moazur

    2016-01-01

    Proteases are widely used to remove affinity and solubility tags from recombinant proteins to avoid potential interference of these tags with the structure and function of the fusion partner. In recent years, great interest has been seen in use of the human rhinovirus 3C protease owing to its stringent sequence specificity and enhanced activity. Like other proteases, activity of the human rhinovirus 3C protease can be affected in part by the buffer components and additives that are generally employed for purification and stabilization of proteins, hence, necessitate their removal by tedious and time-consuming procedures before proteolysis can occur. To address this issue, we examined the effect of elution buffers used for common affinity based purifications, salt ions, stability/solubility and reducing agents, and detergents on the activity of the human rhinovirus 3C protease using three different fusion proteins at 4°C, a temperature of choice for purification of many proteins. The results show that the human rhinovirus 3C protease performs better at 4°C than the frequently used tobacco etch virus protease and its activity was insensitive to most of the experimental conditions tested. Though number of fusion proteins tested is limited, we expect that these finding will facilitate the use of the human rhinovirus 3C protease in recombinant protein production for pharmaceutical and biotechnological applications. PMID:27093053

  12. Activity of the Human Rhinovirus 3C Protease Studied in Various Buffers, Additives and Detergents Solutions for Recombinant Protein Production.

    PubMed

    Ullah, Raheem; Shah, Majid Ali; Tufail, Soban; Ismat, Fouzia; Imran, Muhammad; Iqbal, Mazhar; Mirza, Osman; Rhaman, Moazur

    2016-01-01

    Proteases are widely used to remove affinity and solubility tags from recombinant proteins to avoid potential interference of these tags with the structure and function of the fusion partner. In recent years, great interest has been seen in use of the human rhinovirus 3C protease owing to its stringent sequence specificity and enhanced activity. Like other proteases, activity of the human rhinovirus 3C protease can be affected in part by the buffer components and additives that are generally employed for purification and stabilization of proteins, hence, necessitate their removal by tedious and time-consuming procedures before proteolysis can occur. To address this issue, we examined the effect of elution buffers used for common affinity based purifications, salt ions, stability/solubility and reducing agents, and detergents on the activity of the human rhinovirus 3C protease using three different fusion proteins at 4°C, a temperature of choice for purification of many proteins. The results show that the human rhinovirus 3C protease performs better at 4°C than the frequently used tobacco etch virus protease and its activity was insensitive to most of the experimental conditions tested. Though number of fusion proteins tested is limited, we expect that these finding will facilitate the use of the human rhinovirus 3C protease in recombinant protein production for pharmaceutical and biotechnological applications.

  13. Activity of the Human Rhinovirus 3C Protease Studied in Various Buffers, Additives and Detergents Solutions for Recombinant Protein Production

    PubMed Central

    Tufail, Soban; Ismat, Fouzia; Imran, Muhammad; Iqbal, Mazhar; Mirza, Osman; Rhaman, Moazur

    2016-01-01

    Proteases are widely used to remove affinity and solubility tags from recombinant proteins to avoid potential interference of these tags with the structure and function of the fusion partner. In recent years, great interest has been seen in use of the human rhinovirus 3C protease owing to its stringent sequence specificity and enhanced activity. Like other proteases, activity of the human rhinovirus 3C protease can be affected in part by the buffer components and additives that are generally employed for purification and stabilization of proteins, hence, necessitate their removal by tedious and time-consuming procedures before proteolysis can occur. To address this issue, we examined the effect of elution buffers used for common affinity based purifications, salt ions, stability/solubility and reducing agents, and detergents on the activity of the human rhinovirus 3C protease using three different fusion proteins at 4°C, a temperature of choice for purification of many proteins. The results show that the human rhinovirus 3C protease performs better at 4°C than the frequently used tobacco etch virus protease and its activity was insensitive to most of the experimental conditions tested. Though number of fusion proteins tested is limited, we expect that these finding will facilitate the use of the human rhinovirus 3C protease in recombinant protein production for pharmaceutical and biotechnological applications. PMID:27093053

  14. Growth factors and extracellular matrix proteins in interactions of cumulus-oocyte complex, spermatozoa and oviduct.

    PubMed

    Einspanier, R; Gabler, C; Bieser, B; Einspanier, A; Berisha, B; Kosmann, M; Wollenhaupt, K; Schams, D

    1999-01-01

    The expression and localization of selected growth factor systems and extracellular matrix (ECM) components that may influence oocyte maturation and fertilization within the mammalian oviduct are reported. Fibroblast growth factor (FGF) and vascular endothelial growth factor (VEGF) systems could be detected by use of RT-PCR, RNase protection assay (RPA) and immunohistochemistry in bovine follicles, bovine cumulus-oocyte complexes (COC) and bovine and marmoset oviducts. Two different subtypes of the FGF receptor (FGFR-1 and -2) were identified in distinct cell types, indicating a functional difference. A complete epidermal growth factor (EGF) system was found in the porcine, but not in the bovine, oviduct. There were additional differences between bovine and primate oviducts: FGF-1/2 and FGFR were increased in the marmoset around ovulation, in contrast to an increase in FGF-1 in the cow. Immunohistochemistry revealed accumulation and storage of FGF and VEGF on the surface of the epithelium, possibly due to their binding property on heparanglycoproteins. Other ECM components, matrix metalloproteinase 1 (MMP-1) and tissue inhibitor of metalloproteinase 1 (TIMP-1), were found to be modulated in the ovarian follicle, COC and oviduct during the cycle. An oviduct-mediated depletion of sperm surface proteins (BSP1-3) was discovered as well as a sperm-induced novel oviductal mRNA related to an anti-oxidant protein family. Associated systems of growth factors and ECM components can be suggested as paracrine or autocrine mediators during fertilization in a species-, cycle- and tissue-dependent manner.

  15. Implementing a Rational and Consistent Nomenclature for Serine/Arginine-Rich Protein Splicing Factors (SR Proteins) in Plants

    PubMed Central

    Barta, Andrea; Kalyna, Maria; Reddy, Anireddy S.N.

    2010-01-01

    Growing interest in alternative splicing in plants and the extensive sequencing of new plant genomes necessitate more precise definition and classification of genes coding for splicing factors. SR proteins are a family of RNA binding proteins, which function as essential factors for constitutive and alternative splicing. We propose a unified nomenclature for plant SR proteins, taking into account the newly revised nomenclature of the mammalian SR proteins and a number of plant-specific properties of the plant proteins. We identify six subfamilies of SR proteins in Arabidopsis thaliana and rice (Oryza sativa), three of which are plant specific. The proposed subdivision of plant SR proteins into different subfamilies will allow grouping of paralogous proteins and simple assignment of newly discovered SR orthologs from other plant species and will promote functional comparisons in diverse plant species. PMID:20884799

  16. Cloning and characterization of additional members of the G protein-coupled receptor family.

    PubMed

    Lee, D K; Lynch, K R; Nguyen, T; Im, D S; Cheng, R; Saldivia, V R; Liu, Y; Liu, I S; Heng, H H; Seeman, P; George, S R; O'Dowd, B F; Marchese, A

    2000-02-29

    A search of the expressed sequence tag (EST) database retrieved a human cDNA sequence which partially encoded a novel G protein-coupled receptor (GPCR) GPR26. A human genomic DNA fragment encoding a partial open reading frame (ORF) and a rat cDNA encoding the full length ORF of GPR26 were obtained by library screening. The rat GPR26 cDNA encoded a protein of 317 amino acids, most similar (albeit distantly related) to the serotonin 5-HT(5A) and gastrin releasing hormone BB2 receptors. GPR26 mRNA expression analysis revealed signals in the striatum, pons, cerebellum and cortex. HEK293 and Rh7777 cells transfected with GPR26 cDNA displayed high basal cAMP levels, slow growth rate of clonal populations and derangements of normal cell shape. We also used a sequence reported only in the patent literature encoding GPR57 (a.k.a. HNHCI32) to PCR amplify a DNA fragment which was used to screen a human genomic library. This resulted in the cloning of a genomic fragment containing a pseudogene, psiGPR57, with a 99.6% nucleotide identity to GPR57. Based on shared sequence identities, the receptor encoded by GPR57 was predicted to belong to a novel subfamily of GPCRs together with GPR58 (a.k.a. phBL5, reported only in the patent literature), putative neurotransmitter receptor (PNR) and a 5-HT(4) pseudogene. Analysis of this subfamily revealed greatest identities (approximately 56%) between the receptors encoded by GPR57 and GPR58, each with shared identities of approximately 40% with PNR. Furthermore, psiGPR57, GPR58, PNR and the 5-HT(4) pseudogene were mapped in a cluster localized to chromosome 6q22-24. PNR and GPR58 were expressed in COS cells, however no specific binding was observed for various serotonin receptor-specific ligands.

  17. Evolutionary Dynamics of Floral Homeotic Transcription Factor Protein-Protein Interactions.

    PubMed

    Bartlett, Madelaine; Thompson, Beth; Brabazon, Holly; Del Gizzi, Robert; Zhang, Thompson; Whipple, Clinton

    2016-06-01

    Protein-protein interactions (PPIs) have widely acknowledged roles in the regulation of development, but few studies have addressed the timing and mechanism of shifting PPIs over evolutionary history. The B-class MADS-box transcription factors, PISTILLATA (PI) and APETALA3 (AP3) are key regulators of floral development. PI-like (PI(L)) and AP3-like (AP3(L)) proteins from a number of plants, including Arabidopsis thaliana (Arabidopsis) and the grass Zea mays (maize), bind DNA as obligate heterodimers. However, a PI(L) protein from the grass relative Joinvillea can bind DNA as a homodimer. To ascertain whether Joinvillea PI(L) homodimerization is an anomaly or indicative of broader trends, we characterized PI(L) dimerization across the Poales and uncovered unexpected evolutionary lability. Both obligate B-class heterodimerization and PI(L) homodimerization have evolved multiple times in the order, by distinct molecular mechanisms. For example, obligate B-class heterodimerization in maize evolved very recently from PI(L) homodimerization. A single amino acid change, fixed during domestication, is sufficient to toggle one maize PI(L) protein between homodimerization and obligate heterodimerization. We detected a signature of positive selection acting on residues preferentially clustered in predicted sites of contact between MADS-box monomers and dimers, and in motifs that mediate MADS PPI specificity in Arabidopsis. Changing one positively selected residue can alter PI(L) dimerization activity. Furthermore, ectopic expression of a Joinvillea PI(L) homodimer in Arabidopsis can homeotically transform sepals into petals. Our results provide a window into the evolutionary remodeling of PPIs, and show that novel interactions have the potential to alter plant form in a context-dependent manner. PMID:26908583

  18. Identification of Epigenetic Factor Proteins Expressed in Human Embryonic Stem Cell-Derived Trophoblasts and in Human Placental Trophoblasts.

    PubMed

    Sarkar, Prasenjit; Mischler, Adam; Randall, Shan M; Collier, Timothy S; Dorman, Karen F; Boggess, Kim A; Muddiman, David C; Rao, Balaji M

    2016-08-01

    Human embryonic stem cells (hESCs) have been used to derive trophoblasts through differentiation in vitro. Intriguingly, mouse ESCs are prevented from differentiation to trophoblasts by certain epigenetic factor proteins such as Dnmt1, thus necessitating the study of epigenetic factor proteins during hESC differentiation to trophoblasts. We used stable isotope labeling by amino acids in cell culture and quantitative proteomics to study changes in the nuclear proteome during hESC differentiation to trophoblasts and identified changes in the expression of 30 epigenetic factor proteins. Importantly, the DNA methyltransferases DNMT1, DNMT3A, and DNMT3B were downregulated. Additionally, we hypothesized that nuclear proteomics of hESC-derived trophoblasts may be used for screening epigenetic factor proteins expressed by primary trophoblasts in human placental tissue. Accordingly, we conducted immunohistochemistry analysis of six epigenetic factor proteins identified from hESC-derived trophoblasts-DNMT1, DNMT3B, BAF155, BAF60A, BAF57, and ING5-in 6-9 week human placentas. Indeed, expression of these proteins was largely, though not fully, consistent with that observed in 6-9 week placental trophoblasts. Our results support the use of hESC-derived trophoblasts as a model for placental trophoblasts, which will enable further investigation of epigenetic factors involved in human trophoblast development. PMID:27378238

  19. Impact of bentonite additions during vinification on protein stability and volatile compounds of Albariño wines.

    PubMed

    Lira, Eugenio; Rodríguez-Bencomo, Juan José; Salazar, Fernando N; Orriols, Ignacio; Fornos, Daniel; López, Francisco

    2015-03-25

    Today, bentonite continues to be one of the most used products to remove proteins in white wines in order to avoid their precipitation in bottles. However, excessive use of bentonite has negative effects on the aroma of final wine, so the optimization of the dose and the time of its application are important for winemakers. This paper analyzes how applying an equal dose of bentonite at different stages (must clarification; beginning, middle, and end of fermentation) affects the macromolecular profile, protein stability, physical-chemical characteristics and aromatic profile of the wine obtained. The results showed the addition during fermentation (especially in the middle and at the end) reduced the total dose required for protein stabilization of Albariño wines and maintained the sensory characteristics of this variety. PMID:25751284

  20. Role of diacylglycerol-regulated protein kinase C isotypes in growth factor activation of the Raf-1 protein kinase.

    PubMed

    Cai, H; Smola, U; Wixler, V; Eisenmann-Tappe, I; Diaz-Meco, M T; Moscat, J; Rapp, U; Cooper, G M

    1997-02-01

    The Raf protein kinases function downstream of Ras guanine nucleotide-binding proteins to transduce intracellular signals from growth factor receptors. Interaction with Ras recruits Raf to the plasma membrane, but the subsequent mechanism of Raf activation has not been established. Previous studies implicated hydrolysis of phosphatidylcholine (PC) in Raf activation; therefore, we investigated the role of the epsilon isotype of protein kinase C (PKC), which is stimulated by PC-derived diacylglycerol, as a Raf activator. A dominant negative mutant of PKC epsilon inhibited both proliferation of NIH 3T3 cells and activation of Raf in COS cells. Conversely, overexpression of active PKC epsilon stimulated Raf kinase activity in COS cells and overcame the inhibitory effects of dominant negative Ras in NIH 3T3 cells. PKC epsilon also stimulated Raf kinase in baculovirus-infected Spodoptera frugiperda Sf9 cells and was able to directly activate Raf in vitro. Consistent with its previously reported activity as a Raf activator in vitro, PKC alpha functioned similarly to PKC epsilon in both NIH 3T3 and COS cell assays. In addition, constitutively active mutants of both PKC alpha and PKC epsilon overcame the inhibitory effects of dominant negative mutants of the other PKC isotype, indicating that these diacylglycerol-regulated PKCs function as redundant activators of Raf-1 in vivo.

  1. Simultaneous separation of acidic and basic proteins using gemini pyrrolidinium surfactants and hexafluoroisopropanol as dynamic coating additives in capillary electrophoresis.

    PubMed

    Tian, Yu; Li, Yunfang; Mei, Jie; Cai, Bo; Dong, Jinfeng; Shi, Zhiguo; Xiao, Yuxiu

    2015-09-18

    The separation of acidic and basic proteins using CE has been limited in part due to the adsorption of proteins onto the capillary wall. In this work, the efficient control of EOF and the simultaneous separation of acidic and basic proteins are achieved by use of C18-4-C18PB as a dynamic coating additive, which is a representative surfactant for 1,1'-(butane-1,s-alkyl)bis(1-alkylpyrrolidinium) bromide (Cn-4-CnPB, n=10, 12, 14, 16 and 18). C18-4-C18PB exhibits a powerful capability in the reversal of EOF, and a low concentration even less than 0.001 mM is sufficient to reverse EOF at the tested pH values (3.0-9.0). Baseline separation of eight proteins with sharp peaks and high efficiencies (54,000-297,000 plates/m) is obtained with 30 mM NaH2PO4 buffer (pH 5.0) containing 4 mM C18-4-C18PB. At the same buffer condition, the Cn-4-CnPB with shorter alkyl chain (n=10, 12, 14, 16) cannot achieve the same effective protein separation as C18-4-C18PB. However, the combined use of small amounts (≤0.5%, v/v) of hexafluoroisopropanol (HFIP) and Cn-4-CnPB (n=10, 12, 14, 16) as additives can completely separate all eight proteins with high efficiencies of 81,000-318,000 plates/m. The RSDs of migration time are less than 0.80% and 5.84% for run-to-run and day-to-day assays (n=5), respectively, and the protein recoveries are larger than 90.15%. To the best of our knowledge, this is the first report on the simultaneous separation of acidic and basic proteins using Cn-4-CnPB surfactants or Cn-4-CnPB surfactants combined with HFIP as dynamic coating additives.

  2. Calcifying nanoparticles (nanobacteria): an additional potential factor for urolithiasis in space flight crews.

    PubMed

    Jones, Jeffrey A; Ciftcioglu, Neva; Schmid, Josef F; Barr, Yael R; Griffith, Donald

    2009-01-01

    Spaceflight-induced microgravity appears to be a risk factor for the development of urinary calculi, resulting in urolithiasis during and after spaceflight. Calcifying nanoparticles, or nanobacteria, multiply more rapidly in simulated microgravity and create external shells of calcium phosphate. The question arises whether calcifying nanoparticles are nidi for calculi and contribute to the development of clinically significant urolithiasis in those who are predisposed to the development of urinary calculi because of intrinsic or extrinsic factors. This case report describes a calculus recovered after flight from an astronaut that, on morphologic and immunochemical analysis (including specific monoclonal antibody staining), demonstrated characteristics of calcifying nanoparticles. PMID:18718644

  3. Effect of antibiotic, Lacto-lase and probiotic addition in chicken feed on protein and fat content of chicken meat

    NASA Astrophysics Data System (ADS)

    Azhar, Noor Amiza; Abdullah, Aminah

    2015-09-01

    This research was conducted to investigate the effect of chicken feed additives (antibiotic, Lacto-lase® and probiotic) on protein and fat content of chicken meat. Chicken fed with control diet (corn-soy based diet) served as a control. The treated diets were added with zinc bacitracin (antibiotic), different amount of Lacto-lase® (a mixture of probiotic and enzyme) and probiotic. Chicken were slaughtered at the age of 43-48 days. Each chicken was divided into thigh, breast, drumstick, drumette and wing. Protein content in chicken meat was determined by using macro-Kjeldahl method meanwhile Soxhlet method was used to analyse fat content. The result of the study showed that the protein content of chicken breast was significantly higher (p≤0.05) while thigh had the lowest protein content (p≤0.05). Antibiotic fed chicken was found to have the highest protein content among the treated chickens but there was no significant different with 2g/kg Lacto-lase® fed chicken (p>0.05). All thighs were significantly higher (p≤0.05) in fat content except for drumette of control chicken while breast contained the lowest fat content compared to other chicken parts studied. The control chicken meat contained significantly higher (p≤0.05) amount of fat compared to the other treated chickens. Chicken fed with 2g/kg Lacto-lase® had the lowest (p≤0.05) fat content. The result of this study indicated that the addition of Lacto-lase® as a replacement of antibiotic in chicken feed will not affect the content of protein and fat of chicken meat.

  4. A role for the perlecan protein core in the activation of the keratinocyte growth factor receptor.

    PubMed Central

    Ghiselli, G; Eichstetter, I; Iozzo, R V

    2001-01-01

    Perlecan, a widespread heparan sulphate (HS) proteoglycan, is directly involved in the storing of angiogenic growth factors, mostly members of the fibroblast growth factor (FGF) gene family. We have previously shown that antisense targeting of the perlecan gene causes a reduced growth and responsiveness to FGF7 [also known as keratinocyte growth factor (KGF)] in human cancer cells, and that the perlecan protein core interacts specifically with FGF7. In the present paper, we have investigated human colon carcinoma cells in which the perlecan gene was disrupted by targeted homologous recombination. After screening over 1000 clones, we obtained two clones heterozygous for the null mutation with no detectable perlecan, indicating that the other allele was non-functioning. The perlecan-deficient cells grew more slowly, did not respond to FGF7 with or without the addition of heparin, and were less tumorigenic than control cells. Paradoxically, the perlecan-deficient cells displayed increased FGF7 surface binding. However, the perlecan protein core was required for functional activation of the KGF receptor and downstream signalling. Because heparin could not substitute for perlecan, the HS chains are not critical for FGF7-mediated signalling in this cell system. These results provide the first genetic evidence that the perlecan protein core is a molecular entity implicated in FGF7 binding and activation of its receptor. PMID:11563979

  5. Modified yeast-two-hybrid system to identify proteins interacting with the growth factor progranulin.

    PubMed

    Tian, Qing-Yun; Zhao, Yun-Peng; Liu, Chuan-ju

    2012-01-17

    Progranulin (PGRN), also known as granulin epithelin precursor (GEP), is a 593-amino-acid autocrine growth factor. PGRN is known to play a critical role in a variety of physiologic and disease processes, including early embryogenesis, wound healing, inflammation, and host defense. PGRN also functions as a neurotrophic factor, and mutations in the PGRN gene resulting in partial loss of the PGRN protein cause frontotemporal dementia. Our recent studies have led to the isolation of PGRN as an important regulator of cartilage development and degradation. Although PGRN, discovered nearly two decades ago, plays crucial roles in multiple physiological and pathological conditions, efforts to exploit the actions of PGRN and understand the mechanisms involved have been significantly hampered by our inability to identify its binding receptor(s). To address this issue, we developed a modified yeast two-hybrid (MY2H) approach based on the most commonly used GAL4 based 2-hybrid system. Compared with the conventional yeast two-hybrid screen, MY2H dramatically shortens the screen process and reduces the number of false positive clones. In addition, this approach is reproducible and reliable, and we have successfully employed this system in isolating the binding proteins of various baits, including ion channel, extracellular matrix protein, and growth factor. In this paper, we describe this MY2H experimental procedure in detail using PGRN as an example that led to the identification of TNFR2 as the first known PGRN-associated receptor.

  6. Probing of some compounds as anti-aggregatory additives in the protein refolding process from Escherichia coli inclusion bodies.

    PubMed

    Zilinskas, Albinas; Sereikaite, Jolanta

    2011-01-01

    Five compounds of different chemical structure were tested for aggregation suppression during the refolding of porcine and mink growth hormones as model proteins from Escherichia coli inclusion bodies by the dilution method. Of all compounds tested in this work, 3-guanidinopropionic acid (GPA) containing a guanidinium group was the most effective additive for aggregation suppression. Anti-aggregatory properties of GPA were compared with the ones of l-arginine.

  7. 21 CFR 1311.115 - Additional requirements for two-factor authentication.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ...) separate from the computer to which the practitioner is gaining access. (b) If one factor is a hard token, it must be separate from the computer to which it is gaining access and must meet at least the criteria of FIPS 140-2 Security Level 1, as incorporated by reference in § 1311.08, for...

  8. The effect of nutritional additives on anti-infective factors in human milk.

    PubMed

    Quan, R; Yang, C; Rubinstein, S; Lewiston, N J; Stevenson, D K; Kerner, J A

    1994-06-01

    It has become a common practice to supplement human milk with a variety of additives to improve the nutritive content of the feeding for the premature infant. Twenty-two freshly frozen human milk samples were measured for lysozyme activity, total IgA, and specific IgA to Escherichia coli serotypes 01, 04, and 06. One mL aliquots were mixed with the following: 1 mL of Similac, Similac Special Care, Enfamil, Enfamil Premature Formula, and sterile water; 33 mL of Poly-Vi-Sol, 33 mg of Moducal, and 38 mg of breast-milk fortifier, and then reanalyzed. Significant decreases (41% to 74%) in lysozyme activity were seen with the addition of all formulas; breast-milk fortifier reduced activity by 19%, while no differences were seen with Moducal, sterile water, or Poly-Vi-Sol. No differences were seen in total IgA content, but some decreases were seen in specific IgA to E. coli serotypes 04 and 06. E. coli growth was determined after 3 1/2 hours of incubation at 37 degrees C after mixing. All cow-milk formulas enhanced E. coli growth; soy formulas and other additives preserved inhibition of bacterial growth. Nutritional additives can impair anti-infective properties of human milk, and such interplay should be considered in the decision on the feeding regimen of premature infants.

  9. 34 CFR 377.22 - What additional factors does the Secretary consider in making grants?

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... (Continued) OFFICE OF SPECIAL EDUCATION AND REHABILITATIVE SERVICES, DEPARTMENT OF EDUCATION DEMONSTRATION PROJECTS TO INCREASE CLIENT CHOICE PROGRAM How Does the Secretary Make an Award? § 377.22 What additional... strategies to increase client choice, in order to ensure that a variety of approaches are demonstrated...

  10. 34 CFR 377.22 - What additional factors does the Secretary consider in making grants?

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... (Continued) OFFICE OF SPECIAL EDUCATION AND REHABILITATIVE SERVICES, DEPARTMENT OF EDUCATION DEMONSTRATION PROJECTS TO INCREASE CLIENT CHOICE PROGRAM How Does the Secretary Make an Award? § 377.22 What additional... strategies to increase client choice, in order to ensure that a variety of approaches are demonstrated...

  11. Additive and Synergistic Bactericidal Activity of Antibodies Directed against Minor Outer Membrane Proteins of Neisseria meningitidis▿

    PubMed Central

    Weynants, Vincent E.; Feron, Christiane M.; Goraj, Karine K.; Bos, Martine P.; Denoël, Philippe A.; Verlant, Vincent G.; Tommassen, Jan; Peak, Ian R. A.; Judd, Ralph C.; Jennings, Michael P.; Poolman, Jan T.

    2007-01-01

    Neisseria meningitidis serogroup B is a major cause of bacterial meningitis in younger populations. The available vaccines are based on outer membrane vesicles obtained from wild-type strains. In children less than 2 years old they confer protection only against strains expressing homologous PorA, a major, variable outer membrane protein (OMP). We genetically modified a strain in order to eliminate PorA and to overproduce one or several minor and conserved OMPs. Using a mouse model mimicking children's PorA-specific bactericidal activity, it was demonstrated that overproduction of more than one minor OMP is required to elicit antibodies able to induce complement-mediated killing of strains expressing heterologous PorA. It is concluded that a critical density of bactericidal antibodies needs to be reached at the surface of meningococci to induce complement-mediated killing. With minor OMPs, this threshold is reached when more than one antigen is targeted, and this allows cross-protection. PMID:17664268

  12. Molecular Basis and Therapeutic Strategies to Rescue Factor IX Variants That Affect Splicing and Protein Function.

    PubMed

    Tajnik, Mojca; Rogalska, Malgorzata Ewa; Bussani, Erica; Barbon, Elena; Balestra, Dario; Pinotti, Mirko; Pagani, Franco

    2016-05-01

    Mutations that result in amino acid changes can affect both pre-mRNA splicing and protein function. Understanding the combined effect is essential for correct diagnosis and for establishing the most appropriate therapeutic strategy at the molecular level. We have identified a series of disease-causing splicing mutations in coagulation factor IX (FIX) exon 5 that are completely recovered by a modified U1snRNP particle, through an SRSF2-dependent enhancement mechanism. We discovered that synonymous mutations and missense substitutions associated to a partial FIX secretion defect represent targets for this therapy as the resulting spliced-corrected proteins maintains normal FIX coagulant specific activity. Thus, splicing and protein alterations contribute to define at the molecular level the disease-causing effect of a number of exonic mutations in coagulation FIX exon 5. In addition, our results have a significant impact in the development of splicing-switching therapies in particular for mutations that affect both splicing and protein function where increasing the amount of a correctly spliced protein can circumvent the basic functional defects. PMID:27227676

  13. Eukaryotic Initiation Factor 6, an evolutionarily conserved regulator of ribosome biogenesis and protein translation

    SciTech Connect

    Guo, Jianjun; Jin, Zhaoqing; Yang, Xiaohan; Li, Jian-Feng; Chen, Jay

    2011-01-01

    We recently identified Receptor for Activated C Kinase 1 (RACK1) as one of the molecular links between abscisic acid (ABA) signaling and its regulation on protein translation. Moreover, we identified Eukaryotic Initiation Factor 6 (eIF6) as an interacting partner of RACK1. Because the interaction between RACK1 and eIF6 in mammalian cells is known to regulate the ribosome assembly step of protein translation initiation, it was hypothesized that the same process of protein translation in Arabidopsis is also regulated by RACK1 and eIF6. In this article, we analyzed the amino acid sequences of eIF6 in different species from different lineages and discovered some intriguing differences in protein phosphorylation sites that may contribute to its action in ribosome assembly and biogenesis. In addition, we discovered that, distinct from non-plant organisms in which eIF6 is encoded by a single gene, all sequenced plant genomes contain two or more copies of eIF6 genes. While one copy of plant eIF6 is expressed ubiquitously and might possess the conserved function in ribosome biogenesis and protein translation, the other copy seems to be only expressed in specific organs and therefore may have gained some new functions. We proposed some important studies that may help us better understand the function of eIF6 in plants.

  14. Molecular Basis and Therapeutic Strategies to Rescue Factor IX Variants That Affect Splicing and Protein Function

    PubMed Central

    Bussani, Erica; Barbon, Elena; Pinotti, Mirko; Pagani, Franco

    2016-01-01

    Mutations that result in amino acid changes can affect both pre-mRNA splicing and protein function. Understanding the combined effect is essential for correct diagnosis and for establishing the most appropriate therapeutic strategy at the molecular level. We have identified a series of disease-causing splicing mutations in coagulation factor IX (FIX) exon 5 that are completely recovered by a modified U1snRNP particle, through an SRSF2-dependent enhancement mechanism. We discovered that synonymous mutations and missense substitutions associated to a partial FIX secretion defect represent targets for this therapy as the resulting spliced-corrected proteins maintains normal FIX coagulant specific activity. Thus, splicing and protein alterations contribute to define at the molecular level the disease-causing effect of a number of exonic mutations in coagulation FIX exon 5. In addition, our results have a significant impact in the development of splicing-switching therapies in particular for mutations that affect both splicing and protein function where increasing the amount of a correctly spliced protein can circumvent the basic functional defects. PMID:27227676

  15. Yeast ortholog of the Drosophila crooked neck protein promotes spliceosome assembly through stable U4/U6.U5 snRNP addition.

    PubMed Central

    Chung, S; McLean, M R; Rymond, B C

    1999-01-01

    Mutants in the Drosophila crooked neck (crn) gene show an embryonic lethal phenotype with severe developmental defects. The unusual crn protein consists of sixteen tandem repeats of the 34 amino acid tetratricopeptide (TPR) protein recognition domain. Crn-like TPR elements are found in several RNA processing proteins, although it is unknown how the TPR repeats or the crn protein contribute to Drosophila development. We have isolated a Saccharomyces cerevisiae gene, CLF1, that encodes a crooked neck-like factor. CLF1 is an essential gene but the lethal phenotype of a clf1::HIS3 chromosomal null mutant can be rescued by plasmid-based expression of CLF1 or the Drosophila crn open reading frame. Clf1p is required in vivo and in vitro for pre-mRNA 5' splice site cleavage. Extracts depleted of Clf1p arrest spliceosome assembly after U2 snRNP addition but prior to productive U4/U6.U5 association. Yeast two-hybrid analyses and in vitro binding studies show that Clf1p interacts specifically and differentially with the U1 snRNP-Prp40p protein and the yeast U2AF65 homolog, Mud2p. Intriguingly, Prp40p and Mud2p also bind the phylogenetically conserved branchpoint binding protein (BBP/SF1). Our results indicate that Clf1p acts as a scaffolding protein in spliceosome assembly and suggest that Clf1p may support the cross-intron bridge during the prespliceosome-to-spliceosome transition. PMID:10445879

  16. Additive transgene expression and genetic introgression in multiple green-fluorescent protein transgenic crop x weed hybrid generations.

    PubMed

    Halfhill, M D; Millwood, R J; Weissinger, A K; Warwick, S I; Stewart, C N

    2003-11-01

    The level of transgene expression in crop x weed hybrids and the degree to which crop-specific genes are integrated into hybrid populations are important factors in assessing the potential ecological and agricultural risks of gene flow associated with genetic engineering. The average transgene zygosity and genetic structure of transgenic hybrid populations change with the progression of generations, and the green fluorescent protein (GFP) transgene is an ideal marker to quantify transgene expression in advancing populations. The homozygous T(1) single-locus insert GFP/ Bacillus thuringiensis (Bt) transgenic canola ( Brassica napus, cv Westar) with two copies of the transgene fluoresced twice as much as hemizygous individuals with only one copy of the transgene. These data indicate that the expression of the GFP gene was additive, and fluorescence could be used to determine zygosity status. Several hybrid generations (BC(1)F(1), BC(2)F(1)) were produced by backcrossing various GFP/Bt transgenic canola ( B. napus, cv Westar) and birdseed rape ( Brassica rapa) hybrid generations onto B. rapa. Intercrossed generations (BC(2)F(2) Bulk) were generated by crossing BC(2)F(1) individuals in the presence of a pollinating insect ( Musca domestica L.). The ploidy of plants in the BC(2)F(2) Bulk hybrid generation was identical to the weedy parental species, B. rapa. AFLP analysis was used to quantify the degree of B. napus introgression into multiple backcross hybrid generations with B. rapa. The F(1) hybrid generations contained 95-97% of the B. napus-specific AFLP markers, and each successive backcross generation demonstrated a reduction of markers resulting in the 15-29% presence in the BC(2)F(2) Bulk population. Average fluorescence of each successive hybrid generation was analyzed, and homozygous canola lines and hybrid populations that contained individuals homozygous for GFP (BC(2)F(2) Bulk) demonstrated significantly higher fluorescence than hemizygous hybrid

  17. Additive transgene expression and genetic introgression in multiple green-fluorescent protein transgenic crop x weed hybrid generations.

    PubMed

    Halfhill, M D; Millwood, R J; Weissinger, A K; Warwick, S I; Stewart, C N

    2003-11-01

    The level of transgene expression in crop x weed hybrids and the degree to which crop-specific genes are integrated into hybrid populations are important factors in assessing the potential ecological and agricultural risks of gene flow associated with genetic engineering. The average transgene zygosity and genetic structure of transgenic hybrid populations change with the progression of generations, and the green fluorescent protein (GFP) transgene is an ideal marker to quantify transgene expression in advancing populations. The homozygous T(1) single-locus insert GFP/ Bacillus thuringiensis (Bt) transgenic canola ( Brassica napus, cv Westar) with two copies of the transgene fluoresced twice as much as hemizygous individuals with only one copy of the transgene. These data indicate that the expression of the GFP gene was additive, and fluorescence could be used to determine zygosity status. Several hybrid generations (BC(1)F(1), BC(2)F(1)) were produced by backcrossing various GFP/Bt transgenic canola ( B. napus, cv Westar) and birdseed rape ( Brassica rapa) hybrid generations onto B. rapa. Intercrossed generations (BC(2)F(2) Bulk) were generated by crossing BC(2)F(1) individuals in the presence of a pollinating insect ( Musca domestica L.). The ploidy of plants in the BC(2)F(2) Bulk hybrid generation was identical to the weedy parental species, B. rapa. AFLP analysis was used to quantify the degree of B. napus introgression into multiple backcross hybrid generations with B. rapa. The F(1) hybrid generations contained 95-97% of the B. napus-specific AFLP markers, and each successive backcross generation demonstrated a reduction of markers resulting in the 15-29% presence in the BC(2)F(2) Bulk population. Average fluorescence of each successive hybrid generation was analyzed, and homozygous canola lines and hybrid populations that contained individuals homozygous for GFP (BC(2)F(2) Bulk) demonstrated significantly higher fluorescence than hemizygous hybrid

  18. Vitellogenin-RNAi and ovariectomy each increase lifespan, increase protein storage, and decrease feeding, but are not additive in grasshoppers.

    PubMed

    Tetlak, Alicia G; Burnett, Jacob B; Hahn, Daniel A; Hatle, John D

    2015-12-01

    Reduced reproduction has been shown to increase lifespan in many animals, yet the mechanisms behind this trade-off are unclear. We addressed this question by combining two distinct, direct means of life-extension via reduced reproduction, to test whether they were additive. In the lubber grasshopper, Romalea microptera, ovariectomized (OVX) individuals had a ~20% increase in lifespan and a doubling of storage relative to controls (Sham operated). Similarly, young female grasshoppers treated with RNAi against vitellogenin (the precursor to egg yolk protein) had increased fat body mass and halted ovarian growth. In this study, we compared VgRNAi to two control groups that do not reduce reproduction, namely buffer injection (Buffer) and injection with RNAi against a hexameric storage protein (Hex90RNAi). Each injection treatment was tested with and without ovariectomy. Hence, we tested feeding, storage, and lifespans in six groups: OVX and Buffer, OVX and Hex90RNAi, OVX and VgRNAi, Sham and Buffer, Sham and Hex90RNAi, and Sham and VgRNAi. Ovariectomized grasshoppers and VgRNAi grasshoppers each had similar reductions in feeding (~40%), increases in protein storage in the hemolymph (150-300%), and extensions in lifespan (13-21%). Ovariectomized grasshoppers had higher vitellogenin protein levels than did VgRNAi grasshoppers. Last but not least, when ovariectomy and VgRNAi were applied together, there was no greater effect on feeding, protein storage, or longevity. Hence, feeding regulation, and protein storage in insects, may be conserved components of life-extension via reduced reproduction. PMID:26298568

  19. Somatomedin-1 binding protein-3: insulin-like growth factor-1 binding protein-3, insulin-like growth factor-1 carrier protein.

    PubMed

    2003-01-01

    Somatomedin-1 binding protein-3 [insulin-like growth factor-1 binding protein-3, SomatoKine] is a recombinant complex of insulin-like growth factor-1 (rhIGF-1) and binding protein-3 (IGFBP-3), which is the major circulating somatomedin (insulin-like growth factor) binding protein; binding protein-3 regulates the delivery of somatomedin-1 to target tissues. Somatomedin-1 binding protein-3 has potential as replacement therapy for somatomedin-1 which may become depleted in indications such as major surgery, organ damage/failure and traumatic injury, resulting in catabolism. It also has potential for the treatment of osteoporosis; diseases associated with protein wasting including chronic renal failure, cachexia and severe trauma; and to attenuate cardiac dysfunction in a variety of disease states, including after severe burn trauma. Combined therapy with somatomedin-1 and somatomedin-1 binding protein-3 would prolong the duration of action of somatomedin-1 and would reduce or eliminate some of the undesirable effects associated with somatomedin-1 monotherapy. Somatomedin-1 is usually linked to binding protein-3 in the normal state of the body, and particular proteases clip them apart in response to stresses and release somatomedin-1 as needed. Therefore, somatomedin-1 binding protein-3 is a self-dosing system and SomatoKine would augment the natural supply of these linked compounds. Somatomedin-1 binding protein-3 was developed by Celtrix using its proprietary recombinant protein production technology. Subsequently, Celtrix was acquired by Insmed Pharmaceuticals on June 1 2000. Insmed and Avecia, UK, have signed an agreement for the manufacturing of SomatoKine and its components, IGF-1 and binding protein-3. CGMP clinical production of SomatoKine and its components will be done in Avecia's Advanced Biologics Centre, Billingham, UK, which manufactures recombinant-based medicines and vaccines with a capacity of up to 1000 litres. In 2003, manufacturing of SomatoKine is

  20. CHARMM additive all-atom force field for carbohydrate derivatives and its utility in polysaccharide and carbohydrate-protein modeling

    PubMed Central

    Guvench, Olgun; Mallajosyula, Sairam S.; Raman, E. Prabhu; Hatcher, Elizabeth; Vanommeslaeghe, Kenno; Foster, Theresa J.; Jamison, Francis W.; MacKerell, Alexander D.

    2011-01-01

    Monosaccharide derivatives such as xylose, fucose, N-acetylglucosamine (GlcNAc), N-acetylgalactosamine (GlaNAc), glucuronic acid, iduronic acid, and N-acetylneuraminic acid (Neu5Ac) are important components of eukaryotic glycans. The present work details development of force-field parameters for these monosaccharides and their covalent connections to proteins via O-linkages to serine or threonine sidechains and via N-linkages to asparagine sidechains. The force field development protocol was designed to explicitly yield parameters that are compatible with the existing CHARMM additive force field for proteins, nucleic acids, lipids, carbohydrates, and small molecules. Therefore, when combined with previously developed parameters for pyranose and furanose monosaccharides, for glycosidic linkages between monosaccharides, and for proteins, the present set of parameters enables the molecular simulation of a wide variety of biologically-important molecules such as complex carbohydrates and glycoproteins. Parametrization included fitting to quantum mechanical (QM) geometries and conformational energies of model compounds, as well as to QM pair interaction energies and distances of model compounds with water. Parameters were validated in the context of crystals of relevant monosaccharides, as well NMR and/or x-ray crystallographic data on larger systems including oligomeric hyaluronan, sialyl Lewis X, O- and N-linked glycopeptides, and a lectin:sucrose complex. As the validated parameters are an extension of the CHARMM all-atom additive biomolecular force field, they further broaden the types of heterogeneous systems accessible with a consistently-developed force-field model. PMID:22125473

  1. Hepatitis C Virus E1 and E2 Proteins Used as Separate Immunogens Induce Neutralizing Antibodies with Additive Properties

    PubMed Central

    Beaumont, Elodie; Roch, Emmanuelle; Chopin, Lucie; Roingeard, Philippe

    2016-01-01

    Various strategies involving the use of hepatitis C virus (HCV) E1 and E2 envelope glycoproteins as immunogens have been developed for prophylactic vaccination against HCV. However, the ideal mode of processing and presenting these immunogens for effective vaccination has yet to be determined. We used our recently described vaccine candidate based on full-length HCV E1 or E2 glycoproteins fused to the heterologous hepatitis B virus S envelope protein to compare the use of the E1 and E2 proteins as separate immunogens with their use as the E1E2 heterodimer, in terms of immunogenetic potential and the capacity to induce neutralizing antibodies. The specific anti-E1 and anti-E2 antibody responses induced in animals immunized with vaccine particles harboring the heterodimer were profoundly impaired with respect to those in animals immunized with particles harboring E1 and E2 separately. Moreover, the anti-E1 and anti-E2 antibodies had additive neutralizing properties that increase the cross-neutralization of heterologous strains of various HCV genotypes, highlighting the importance of including both E1 and E2 in the vaccine for an effective vaccination strategy. Our study has important implications for the optimization of HCV vaccination strategies based on HCV envelope proteins, regardless of the platform used to present these proteins to the immune system. PMID:26966906

  2. Hepatitis C Virus E1 and E2 Proteins Used as Separate Immunogens Induce Neutralizing Antibodies with Additive Properties.

    PubMed

    Beaumont, Elodie; Roch, Emmanuelle; Chopin, Lucie; Roingeard, Philippe

    2016-01-01

    Various strategies involving the use of hepatitis C virus (HCV) E1 and E2 envelope glycoproteins as immunogens have been developed for prophylactic vaccination against HCV. However, the ideal mode of processing and presenting these immunogens for effective vaccination has yet to be determined. We used our recently described vaccine candidate based on full-length HCV E1 or E2 glycoproteins fused to the heterologous hepatitis B virus S envelope protein to compare the use of the E1 and E2 proteins as separate immunogens with their use as the E1E2 heterodimer, in terms of immunogenetic potential and the capacity to induce neutralizing antibodies. The specific anti-E1 and anti-E2 antibody responses induced in animals immunized with vaccine particles harboring the heterodimer were profoundly impaired with respect to those in animals immunized with particles harboring E1 and E2 separately. Moreover, the anti-E1 and anti-E2 antibodies had additive neutralizing properties that increase the cross-neutralization of heterologous strains of various HCV genotypes, highlighting the importance of including both E1 and E2 in the vaccine for an effective vaccination strategy. Our study has important implications for the optimization of HCV vaccination strategies based on HCV envelope proteins, regardless of the platform used to present these proteins to the immune system.

  3. Hepatoma-derived growth factor-related protein-3 is a novel angiogenic factor.

    PubMed

    LeBlanc, Michelle E; Wang, Weiwen; Caberoy, Nora B; Chen, Xiuping; Guo, Feiye; Alvarado, Gabriela; Shen, Chen; Wang, Feng; Wang, Hui; Chen, Rui; Liu, Zhao-Jun; Webster, Keith; Li, Wei

    2015-01-01

    Hepatoma-derived growth factor-related protein-3 (Hdgfrp3 or HRP-3) was recently reported as a neurotrophic factor and is upregulated in hepatocellular carcinoma to promote cancer cell survival. Here we identified HRP-3 as a new endothelial ligand and characterized its in vitro and in vivo functional roles and molecular signaling. We combined open reading frame phage display with multi-round in vivo binding selection to enrich retinal endothelial ligands, which were systematically identified by next generation DNA sequencing. One of the identified endothelial ligands was HRP-3. HRP-3 expression in the retina and brain was characterized by Western blot and immunohistochemistry. Cell proliferation assay showed that HRP-3 stimulated the growth of human umbilical vein endothelial cells (HUVECs). HRP-3 induced tube formation of HUVECs in culture. Wound healing assay indicated that HRP-3 promoted endothelial cell migration. HRP-3 was further confirmed for its in vitro angiogenic activity by spheroid sprouting assay. HRP-3 extrinsically activated the extracellular-signal-regulated kinase ½ (ERK1/2) pathway in endothelial cells. The angiogenic activity of HRP-3 was independently verified by mouse cornea pocket assay. Furthermore, in vivo Matrigel plug assay corroborated HRP-3 activity to promote new blood vessel formation. These results demonstrated that HRP-3 is a novel angiogenic factor.

  4. Impact of antinutritional factors in food proteins on the digestibility of protein and the bioavailability of amino acids and on protein quality.

    PubMed

    Sarwar Gilani, G; Wu Xiao, Chao; Cockell, Kevin A

    2012-08-01

    Dietary antinutritional factors have been reported to adversely affect the digestibility of protein, bioavailability of amino acids and protein quality of foods. Published data on these negative effects of major dietary antinutritional factors are summarized in this manuscript. Digestibility and the quality of mixed diets in developing countries are considerably lower than of those in developed regions. For example, the digestibility of protein in traditional diets from developing countries such as India, Guatemala and Brazil is considerably lower compared to that of protein in typical North American diets (54-78 versus 88-94 %). Poor digestibility of protein in the diets of developing countries, which are based on less refined cereals and grain legumes as major sources of protein, is due to the presence of less digestible protein fractions, high levels of insoluble fibre, and/or high concentrations of antinutritional factors present endogenously or formed during processing. Examples of naturally occurring antinutritional factors include glucosinolates in mustard and canola protein products, trypsin inhibitors and haemagglutinins in legumes, tannins in legumes and cereals, gossypol in cottonseed protein products, and uricogenic nucleobases in yeast protein products. Heat/alkaline treatments of protein products may yield Maillard reaction compounds, oxidized forms of sulphur amino acids, D-amino acids and lysinoalanine (LAL, an unnatural nephrotoxic amino acid derivative). Among common food and feed protein products, soyabeans are the most concentrated source of trypsin inhibitors. The presence of high levels of dietary trypsin inhibitors from soyabeans, kidney beans or other grain legumes have been reported to cause substantial reductions in protein and amino acid digestibility (up to 50 %) and protein quality (up to 100 %) in rats and/or pigs. Similarly, the presence of high levels of tannins in sorghum and other cereals, fababean and other grain legumes can cause

  5. Protein modification during anti-viral heat-treatment bioprocessing of factor VIII concentrates, factor IX concentrates, and model proteins in the presence of sucrose.

    PubMed

    Smales, C Mark; Pepper, Duncan S; James, David C

    2002-01-01

    To ensure the optimal safety of plasma derived and new generation recombinant proteins, heat treatment is customarily applied in the manufacturing of such biopharmaceuticals as a means of viral inactivation. In subjecting proteins to anti-viral heat-treatment it is necessary to use high concentrations of thermostabilizing excipients to prevent protein damage, and it is therefore imperative that the correct balance between bioprocessing conditions, maintenance of protein integrity and virus kill is found. In this study we have utilized model proteins (lysozyme, fetuin, and human serum albumin) and plasma-derived therapeutic proteins (factor VIII and factor IX) to investigate the protein modifications that occur during anti-viral heat treatment. Specifically, we investigated the relationship between bioprocessing conditions and the type and extent of protein modification under a variety of industrially relevant wet and lyophilized heat treatments using sucrose as a thermostabilizing agent. Heat treatment led to the formation of disulfide crosslinks and aggregates in proteins containing free cysteine residues. Terminal oligosaccharide sialic acid residues were hydrolyzed from the glycan moieties of glycoproteins during anti-viral heat treatment. Heat treatment promoted sucrose hydrolysis to yield glucose and fructose, leading, in turn, to the glycation of lysine amino groups in those proteins containing di-lysine motifs. During extended hear treatments, 1,2-dicarbonyl type advanced glycation end-products were also formed. Glycation-type modifications were more prevalent in wet heat-treated protein formulations.

  6. BRCA1 protein level is not affected by peptide growth factors in MCF10A cell line.

    PubMed

    Aprelikova, O; Kuthiala, A; Bessho, M; Ethier, S; Liu, E T

    1996-12-01

    The breast cancer susceptibility gene (BRCA1) has been identified as a putative tumor suppressor on chromosome 17. We raised antibody against Ring-finger domain of BRCA1. The antibody recognizes a specific BRCA1 protein doublet of about 220 kD. The majority of BRCA1 protein is localized to the nuclear fraction of untreated MCF10A cells. Though BRCA1 is thought to be a growth suppressor gene, no change in BRCA1 protein level was found when MCF10A cells were arrested by growth factor deprivation or stimulation of cell proliferation by re-addition of growth factors. Furthermore the subcellular localization of the BRCA1 protein does not change throughout the cell cycle. These results suggest that BRCA1 may not be directly involved in the regulation of the cell cycle of breast cancer cell line.

  7. Kinetoplastid Membrane Protein-11 as a Vaccine Candidate and a Virulence Factor in Leishmania

    PubMed Central

    de Mendonça, Sergio Coutinho Furtado; Cysne-Finkelstein, Léa; Matos, Denise Cristina de Souza

    2015-01-01

    Kinetoplastid membrane protein-11 (KMP-11), a protein present in all kinetoplastid protozoa, is considered a potential candidate for a leishmaniasis vaccine. In Leishmania amazonensis, KMP-11 is expressed in promastigotes and amastigotes. In both stages, the protein was found in association with membrane structures at the cell surface, flagellar pocket, and intracellular vesicles. More importantly, its surface expression is higher in amastigotes than in promastigotes and increases during metacyclogenesis. The increased expression of KMP-11 in metacyclic promastigotes, and especially in amastigotes, indicates a role for this molecule in the parasite relationship with the mammalian host. In this connection, we have shown that addition of KMP-11 exacerbates L. amazonensis infection in peritoneal macrophages from BALB/c mice by increasing interleukin (IL)-10 secretion and arginase activity while reducing nitric oxide production. The doses of KMP-11, the IL-10 levels, and the intracellular amastigote loads were strongly, positively, and significantly correlated. The increase in parasite load induced by KMP-11 was inhibited by anti-KMP-11 or anti-IL-10-neutralizing antibodies, but not by isotype controls. The neutralizing antibodies, but not the isotype controls, were also able to significantly decrease the parasite load in macrophages cultured without the addition of KMP-11, demonstrating that KMP-11-induced exacerbation of the infection is not dependent on the addition of exogenous KMP-11 and that the protein naturally expressed by the parasite is able to promote it. All these data indicate that KMP-11 acts as a virulence factor in L. amazonensis infection. PMID:26528287

  8. Antigenicity of Recombinant Maltose Binding Protein-Mycobacterium avium subsp. paratuberculosis Fusion Proteins with and without Factor Xa Cleaving

    PubMed Central

    Begg, Douglas J.; Purdie, Auriol C.; Bannantine, John P.; Whittington, Richard J.

    2013-01-01

    Mycobacterium avium subsp. paratuberculosis causes Johne's disease (JD) in ruminants. Proteomic studies have shown that M. avium subsp. paratuberculosis expresses certain proteins when exposed to in vitro physiological stress conditions similar to the conditions experienced within a host during natural infection. Such proteins are hypothesized to be expressed in vivo, are recognized by the host immune system, and may be of potential use in the diagnosis of JD. In this study, 50 recombinant maltose binding protein (MBP)-M. avium subsp. paratuberculosis fusion proteins were evaluated using serum samples from sheep infected with M. avium subsp. paratuberculosis, and 29 (58%) were found to be antigenic. Among 50 fusion proteins, 10 were evaluated in MBP fusion and factor Xa-cleaved forms. A total of 31 proteins (62%) were found to be antigenic in either MBP fusion or factor Xa-cleaved forms. Antigenicity after cleavage and removal of the MBP tag was marginally enhanced. PMID:24132604

  9. Aitchbone hanging and ageing period are additive factors influencing pork eating quality.

    PubMed

    Channon, H A; Taverner, M R; D'Souza, D N; Warner, R D

    2014-01-01

    The effects of abattoir, carcase weight (60 or 80 kg HCW), hanging method (Achilles or aitchbone) and ageing period (2 or 7 day post-slaughter) on eating quality attributes of pork were investigated in this 3×2×2×2 factorial study. A total of 144 Large White×Landrace female pigs were slaughtered at one of three abattoirs and sides hung from either the Achilles tendon or the aitchbone. After 24 h chilling, loin (M. longissimus thoracis et lumborum) and topside (M. semimembranosus) muscles were individually vacuum packaged and aged for 2 or 7 days post-slaughter. Consumers (n=852) evaluated eating quality. Neither abattoir nor carcase weight influenced tenderness, flavour or overall liking of pork. Improvements in tenderness, flavour and overall liking were found due to aitchbone hanging (P<0.001) and ageing (P<0.001) for 7 days compared with Achilles-hung carcases and pork aged for 2 days, respectively. This study demonstrated that aitchbone hanging and 7 day ageing can improve eating quality, but these effects were additive as the interaction term was not significant. PMID:24013699

  10. Homologous Transcription Factors DUX4 and DUX4c Associate with Cytoplasmic Proteins during Muscle Differentiation

    PubMed Central

    Ansseau, Eugénie; Matteotti, Christel; Yip, Cassandre; Liu, Jian; Leroy, Baptiste; Hubeau, Céline; Gerbaux, Cécile; Cloet, Samuel; Wauters, Armelle; Zorbo, Sabrina; Meyer, Pierre; Pirson, Isabelle; Laoudj-Chenivesse, Dalila; Wattiez, Ruddy; Harper, Scott Q.; Belayew, Alexandra; Coppée, Frédérique

    2016-01-01

    Hundreds of double homeobox (DUX) genes map within 3.3-kb repeated elements dispersed in the human genome and encode DNA-binding proteins. Among these, we identified DUX4, a potent transcription factor that causes facioscapulohumeral muscular dystrophy (FSHD). In the present study, we performed yeast two-hybrid screens and protein co-purifications with HaloTag-DUX fusions or GST-DUX4 pull-down to identify protein partners of DUX4, DUX4c (which is identical to DUX4 except for the end of the carboxyl terminal domain) and DUX1 (which is limited to the double homeodomain). Unexpectedly, we identified and validated (by co-immunoprecipitation, GST pull-down, co-immunofluorescence and in situ Proximal Ligation Assay) the interaction of DUX4, DUX4c and DUX1 with type III intermediate filament protein desmin in the cytoplasm and at the nuclear periphery. Desmin filaments link adjacent sarcomere at the Z-discs, connect them to sarcolemma proteins and interact with mitochondria. These intermediate filament also contact the nuclear lamina and contribute to positioning of the nuclei. Another Z-disc protein, LMCD1 that contains a LIM domain was also validated as a DUX4 partner. The functionality of DUX4 or DUX4c interactions with cytoplasmic proteins is underscored by the cytoplasmic detection of DUX4/DUX4c upon myoblast fusion. In addition, we identified and validated (by co-immunoprecipitation, co-immunofluorescence and in situ Proximal Ligation Assay) as DUX4/4c partners several RNA-binding proteins such as C1QBP, SRSF9, RBM3, FUS/TLS and SFPQ that are involved in mRNA splicing and translation. FUS and SFPQ are nuclear proteins, however their cytoplasmic translocation was reported in neuronal cells where they associated with ribonucleoparticles (RNPs). Several other validated or identified DUX4/DUX4c partners are also contained in mRNP granules, and the co-localizations with cytoplasmic DAPI-positive spots is in keeping with such an association. Large muscle RNPs were

  11. Homologous Transcription Factors DUX4 and DUX4c Associate with Cytoplasmic Proteins during Muscle Differentiation.

    PubMed

    Ansseau, Eugénie; Eidahl, Jocelyn O; Lancelot, Céline; Tassin, Alexandra; Matteotti, Christel; Yip, Cassandre; Liu, Jian; Leroy, Baptiste; Hubeau, Céline; Gerbaux, Cécile; Cloet, Samuel; Wauters, Armelle; Zorbo, Sabrina; Meyer, Pierre; Pirson, Isabelle; Laoudj-Chenivesse, Dalila; Wattiez, Ruddy; Harper, Scott Q; Belayew, Alexandra; Coppée, Frédérique

    2016-01-01

    Hundreds of double homeobox (DUX) genes map within 3.3-kb repeated elements dispersed in the human genome and encode DNA-binding proteins. Among these, we identified DUX4, a potent transcription factor that causes facioscapulohumeral muscular dystrophy (FSHD). In the present study, we performed yeast two-hybrid screens and protein co-purifications with HaloTag-DUX fusions or GST-DUX4 pull-down to identify protein partners of DUX4, DUX4c (which is identical to DUX4 except for the end of the carboxyl terminal domain) and DUX1 (which is limited to the double homeodomain). Unexpectedly, we identified and validated (by co-immunoprecipitation, GST pull-down, co-immunofluorescence and in situ Proximal Ligation Assay) the interaction of DUX4, DUX4c and DUX1 with type III intermediate filament protein desmin in the cytoplasm and at the nuclear periphery. Desmin filaments link adjacent sarcomere at the Z-discs, connect them to sarcolemma proteins and interact with mitochondria. These intermediate filament also contact the nuclear lamina and contribute to positioning of the nuclei. Another Z-disc protein, LMCD1 that contains a LIM domain was also validated as a DUX4 partner. The functionality of DUX4 or DUX4c interactions with cytoplasmic proteins is underscored by the cytoplasmic detection of DUX4/DUX4c upon myoblast fusion. In addition, we identified and validated (by co-immunoprecipitation, co-immunofluorescence and in situ Proximal Ligation Assay) as DUX4/4c partners several RNA-binding proteins such as C1QBP, SRSF9, RBM3, FUS/TLS and SFPQ that are involved in mRNA splicing and translation. FUS and SFPQ are nuclear proteins, however their cytoplasmic translocation was reported in neuronal cells where they associated with ribonucleoparticles (RNPs). Several other validated or identified DUX4/DUX4c partners are also contained in mRNP granules, and the co-localizations with cytoplasmic DAPI-positive spots is in keeping with such an association. Large muscle RNPs were

  12. Transcription factors, chromatin proteins and the diversification of Hemiptera.

    PubMed

    Vidal, Newton M; Grazziotin, Ana Laura; Iyer, Lakshminarayan M; Aravind, L; Venancio, Thiago M

    2016-02-01

    Availability of complete genomes provides a means to explore the evolution of enormous developmental, morphological, and behavioral diversity among insects. Hemipterans in particular show great diversity of both morphology and life history within a single order. To better understand the role of transcription regulators in the diversification of hemipterans, using sequence profile searches and hidden Markov models we computationally analyzed transcription factors (TFs) and chromatin proteins (CPs) in the recently available Rhodnius prolixus genome along with 13 other insect and 4 non-insect arthropod genomes. We generated a comprehensive collection of TFs and CPs across arthropods including 303 distinct types of domains in TFs and 139 in CPs. This, along with the availability of two hemipteran genomes, R. prolixus and Acyrthosiphon pisum, helped us identify possible determinants for their dramatic morphological and behavioral divergence. We identified five domain families (i.e. Pipsqueak, SAZ/MADF, THAP, FLYWCH and BED finger) as having undergone differential patterns of lineage-specific expansion in hemipterans or within hemipterans relative to other insects. These expansions appear to be at least in part driven by transposons, with the DNA-binding domains of transposases having provided the raw material for emergence of new TFs. Our analysis suggests that while R. prolixus probably retains a state closer to the ancestral hemipteran, A. pisum represents a highly derived state, with the emergence of asexual reproduction potentially favoring genome duplication and transposon expansion. Both hemipterans are predicted to possess active DNA methylation systems. However, in the course of their divergence, aphids seem to have expanded the ancestral hemipteran DNA methylation along with a distinctive linkage to the histone methylation system, as suggested by expansion of SET domain methylases, including those fused to methylated CpG recognition domains. Thus

  13. Epidermal growth factor (EGF)-receptor is phosphorylated at threonine-654 in A431 cells following EGF addition

    SciTech Connect

    Whiteley, B.; Glaser, L.

    1986-05-01

    It has been shown that activation of protein kinase C by tumor-promoting phorbol diesters causes phorphorylation of the EGF-receptor at threonine-654 and is believed to thereby regulate the EGF receptor tyrosine kinase and EGF binding activity. In their present studies, /sup 32/P-labeled A431 cells were treated with and without 10 nM phorbol 12-myristate 13-acetate (PMA), or with 200 ng/ml EGF. Analysis of /sup 32/P-labeled EGF receptor tryptic phosphopeptides by reverse-phase HPLC confirmed the known effects of PMA and revealed that EGF caused phosphorylation at threonine-654 as well as various tyrosine residues. This effect occurred as early as 1 minute after EGF addition and was maximal after 5 minutes. The magnitude of the response appears to be 50% of a 15 minute treatment with 10 nM PMA. Direct measurement of diacylglycerol using an E. coli diacylglycerol kinase confirmed that EGF-stimulated phosphoinositide turnover could cause very rapid activation of protein kinase C. These results imply that protein kinase C is playing a role in negative modulation of EGF-receptor activity following EGF addition to A431 cells.

  14. Intrauterine growth restriction inhibits expression of eukaryotic elongation factor 2 kinase, a regulator of protein translation.

    PubMed

    McKnight, Robert A; Yost, Christian C; Zinkhan, Erin K; Fu, Qi; Callaway, Christopher W; Fung, Camille M

    2016-08-01

    Nutrient deprivation suppresses protein synthesis by blocking peptide elongation. Transcriptional upregulation and activation of eukaryotic elongation factor 2 kinase (eEF2K) blocks peptide elongation by phosphorylating eukaryotic elongation factor 2. Previous studies examining placentas from intrauterine growth restricted (IUGR) newborn infants show decreased eEF2K expression and activity despite chronic nutrient deprivation. However, the effect of IUGR on hepatic eEF2K expression in the fetus is unknown. We, therefore, examined the transcriptional regulation of hepatic eEF2K gene expression in a Sprague-Dawley rat model of IUGR. We found decreased hepatic eEF2K mRNA and protein levels in IUGR offspring at birth compared with control, consistent with previous placental observations. Furthermore, the CpG island within the eEF2K promoter demonstrated increased methylation at a critical USF 1/2 transcription factor binding site. In vitro methylation of this binding site caused near complete loss of eEF2K promoter activity, designating this promoter as methylation sensitive. The eEF2K promotor in IUGR offspring also lost the protective histone covalent modifications associated with unmethylated CGIs. In addition, the +1 nucleosome was displaced 3' and RNA polymerase loading was reduced at the IUGR eEF2K promoter. Our findings provide evidence to explain why IUGR-induced chronic nutrient deprivation does not result in the upregulation of eEF2K gene transcription. PMID:27317589

  15. Glycan structure of Gc Protein-derived Macrophage Activating Factor as revealed by mass spectrometry.

    PubMed

    Borges, Chad R; Rehder, Douglas S

    2016-09-15

    Disagreement exists regarding the O-glycan structure attached to human vitamin D binding protein (DBP). Previously reported evidence indicated that the O-glycan of the Gc1S allele product is the linear core 1 NeuNAc-Gal-GalNAc-Thr trisaccharide. Here, glycan structural evidence is provided from glycan linkage analysis and over 30 serial glycosidase-digestion experiments which were followed by analysis of the intact protein by electrospray ionization mass spectrometry (ESI-MS). Results demonstrate that the O-glycan from the Gc1F protein is the same linear trisaccharide found on the Gc1S protein and that the hexose residue is galactose. In addition, the putative anti-cancer derivative of DBP known as Gc Protein-derived Macrophage Activating Factor (GcMAF, which is formed by the combined action of β-galactosidase and neuraminidase upon DBP) was analyzed intact by ESI-MS, revealing that the activating E. coli β-galactosidase cleaves nothing from the protein-leaving the glycan structure of active GcMAF as a Gal-GalNAc-Thr disaccharide, regardless of the order in which β-galactosidase and neuraminidase are applied. Moreover, glycosidase digestion results show that α-N-Acetylgalactosamindase (nagalase) lacks endoglycosidic function and only cleaves the DBP O-glycan once it has been trimmed down to a GalNAc-Thr monosaccharide-precluding the possibility of this enzyme removing the O-glycan trisaccharide from cancer-patient DBP in vivo.

  16. Glycan structure of Gc Protein-derived Macrophage Activating Factor as revealed by mass spectrometry.

    PubMed

    Borges, Chad R; Rehder, Douglas S

    2016-09-15

    Disagreement exists regarding the O-glycan structure attached to human vitamin D binding protein (DBP). Previously reported evidence indicated that the O-glycan of the Gc1S allele product is the linear core 1 NeuNAc-Gal-GalNAc-Thr trisaccharide. Here, glycan structural evidence is provided from glycan linkage analysis and over 30 serial glycosidase-digestion experiments which were followed by analysis of the intact protein by electrospray ionization mass spectrometry (ESI-MS). Results demonstrate that the O-glycan from the Gc1F protein is the same linear trisaccharide found on the Gc1S protein and that the hexose residue is galactose. In addition, the putative anti-cancer derivative of DBP known as Gc Protein-derived Macrophage Activating Factor (GcMAF, which is formed by the combined action of β-galactosidase and neuraminidase upon DBP) was analyzed intact by ESI-MS, revealing that the activating E. coli β-galactosidase cleaves nothing from the protein-leaving the glycan structure of active GcMAF as a Gal-GalNAc-Thr disaccharide, regardless of the order in which β-galactosidase and neuraminidase are applied. Moreover, glycosidase digestion results show that α-N-Acetylgalactosamindase (nagalase) lacks endoglycosidic function and only cleaves the DBP O-glycan once it has been trimmed down to a GalNAc-Thr monosaccharide-precluding the possibility of this enzyme removing the O-glycan trisaccharide from cancer-patient DBP in vivo. PMID:27503803

  17. Tissue Factor Pathway Inhibitor: Multiple Anticoagulant Activities for a Single Protein.

    PubMed

    Mast, Alan E

    2016-01-01

    Tissue factor (TF) pathway inhibitor (TFPI) is an anticoagulant protein that inhibits early phases of the procoagulant response. Alternatively spliced isoforms of TFPI are differentially expressed by endothelial cells and human platelets and plasma. The TFPIβ isoform localizes to the endothelium surface where it is a potent inhibitor of TF-factor VIIa complexes that initiate blood coagulation. The TFPIα isoform is present in platelets. TFPIα contains a stretch of 9 amino acids nearly identical to those found in the B-domain of factor V that are well conserved in mammals. These amino acids provide exosite binding to activated factor V, which allows for TFPIα to inhibit prothrombinase during the initiation phase of blood coagulation. Endogenous inhibition at this point in the coagulation cascade was only recently recognized and has provided a biochemical rationale to explain the pathophysiological mechanisms underlying several clinical disorders. These include the east Texas bleeding disorder that is caused by production of an altered form of factor V with high affinity for TFPI and a paradoxical procoagulant effect of heparins. In addition, these findings have led to ideas for pharmacological targeting of TFPI that may reduce bleeding in hemophilia patients.

  18. Sequentially Integrated Optimization of the Conditions to Obtain a High-Protein and Low-Antinutritional Factors Protein Isolate from Edible Jatropha curcas Seed Cake

    PubMed Central

    León-López, Liliana; Dávila-Ortiz, Gloria; Jiménez-Martínez, Cristian; Hernández-Sánchez, Humberto

    2013-01-01

    Jatropha curcas seed cake is a protein-rich byproduct of oil extraction which could be used to produce protein isolates. The purpose of this study was the optimization of the protein isolation process from the seed cake of an edible provenance of J. curcas by an alkaline extraction followed by isoelectric precipitation method via a sequentially integrated optimization approach. The influence of four different factors (solubilization pH, extraction temperature, NaCl addition, and precipitation pH) on the protein and antinutritional compounds content of the isolate was evaluated. The estimated optimal conditions were an extraction temperature of 20°C, a precipitation pH of 4, and an amount of NaCl in the extraction solution of 0.6 M for a predicted protein content of 93.3%. Under these conditions, it was possible to obtain experimentally a protein isolate with 93.21% of proteins, 316.5 mg 100 g−1 of total phenolics, 2891.84 mg 100 g−1 of phytates and 168 mg 100 g−1 of saponins. The protein content of the this isolate was higher than the content reported by other authors. PMID:25937971

  19. [PPR proteins--modular factors regulating expression of organellar genomes].

    PubMed

    Zapisek, Bartosz; Piątkowski, Jakub

    2015-01-01

    PPR proteins make up the most numerous family of RNA-binding proteins identified to date. They localize almost exclusively to plastids and mitochondria of eukaryotic organisms. The most striking feature of this family is the expansion of PPR protein-encoding genes in vascular plants, which likely coincided with plants colonizing land. PPR proteins participate in stabilizing, editing, splicing, degradation and processing of policistronic transcripts, as well as translation activation in mitochondria and plastids. Although the number of PPR proteins in non-plant organisms is significantly smaller than in plants, they still play a crucial role in regulating the expression of mtDNA. Disruptions of PPR protein-encoding genes usually result in severe phenotypic consequences. Plant PPR proteins bind RNA in a sequence-specific manner, where a single PPR motif recognizes an individual nucleotide in a given sequence. This opens up possibilities for engineering de novo synthetic protein sequences that would interact with precisely determined organellar sequences, thus enabling modulation of mtDNA and ctDNA expression.

  20. Screening for Host Factors Directly Interacting with RSV Protein: Microfluidics.

    PubMed

    Kipper, Sarit; Avrahami, Dorit; Bajorek, Monika; Gerber, Doron

    2016-01-01

    We present a high-throughput microfluidics platform to identify novel host cell binding partners of respiratory syncytial virus (RSV) matrix (M) protein. The device consists of thousands of reaction chambers controlled by micro-mechanical valves. The microfluidic device is mated to a microarray-printed custom-made gene library. These genes are then transcribed and translated on-chip, resulting in a protein array ready for binding to RSV M protein.Even small viral proteome, such as that of RSV, presents a challenge due to the fact that viral proteins are usually multifunctional and thus their interaction with the host is complex. Protein microarrays technology allows the interrogation of protein-protein interactions, which could possibly overcome obstacles by using conventional high throughput methods. Using microfluidics platform we have identified new host interactors of M involved in various cellular pathways. A number of microfluidics based assays have already provided novel insights into the virus-host interactome, and the results have important implications for future antiviral strategies aimed at targets of viral protein interactions with the host. PMID:27464694

  1. Structural and functional analysis of VQ motif-containing proteins in Arabidopsis as interacting proteins of WRKY transcription factors.

    PubMed

    Cheng, Yuan; Zhou, Yuan; Yang, Yan; Chi, Ying-Jun; Zhou, Jie; Chen, Jian-Ye; Wang, Fei; Fan, Baofang; Shi, Kai; Zhou, Yan-Hong; Yu, Jing-Quan; Chen, Zhixiang

    2012-06-01

    WRKY transcription factors are encoded by a large gene superfamily with a broad range of roles in plants. Recently, several groups have reported that proteins containing a short VQ (FxxxVQxLTG) motif interact with WRKY proteins. We have recently discovered that two VQ proteins from Arabidopsis (Arabidopsis thaliana), SIGMA FACTOR-INTERACTING PROTEIN1 and SIGMA FACTOR-INTERACTING PROTEIN2, act as coactivators of WRKY33 in plant defense by specifically recognizing the C-terminal WRKY domain and stimulating the DNA-binding activity of WRKY33. In this study, we have analyzed the entire family of 34 structurally divergent VQ proteins from Arabidopsis. Yeast (Saccharomyces cerevisiae) two-hybrid assays showed that Arabidopsis VQ proteins interacted specifically with the C-terminal WRKY domains of group I and the sole WRKY domains of group IIc WRKY proteins. Using site-directed mutagenesis, we identified structural features of these two closely related groups of WRKY domains that are critical for interaction with VQ proteins. Quantitative reverse transcription polymerase chain reaction revealed that expression of a majority of Arabidopsis VQ genes was responsive to pathogen infection and salicylic acid treatment. Functional analysis using both knockout mutants and overexpression lines revealed strong phenotypes in growth, development, and susceptibility to pathogen infection. Altered phenotypes were substantially enhanced through cooverexpression of genes encoding interacting VQ and WRKY proteins. These findings indicate that VQ proteins play an important role in plant growth, development, and response to environmental conditions, most likely by acting as cofactors of group I and IIc WRKY transcription factors.

  2. Influence of boron addition to Ti-13Zr-13Nb alloy on MG63 osteoblast cell viability and protein adsorption.

    PubMed

    Majumdar, P; Singh, S B; Dhara, S; Chakraborty, M

    2015-01-01

    Cell proliferation, cell morphology and protein adsorption on near β-type Ti-13Zr-13Nb (TZN) alloy and Ti-13Zr-13Nb-0.5B (TZNB) composite have been investigated and compared to evaluate the effect of boron addition which has been added to the Ti alloy to improve their poor tribological properties by forming in situ TiB precipitates. MG63 cell proliferation on substrates with different chemistry but the same topography was compared. The MTT assay test showed that the cell viability on the TZN alloy was higher than the boron containing TZNB composite after 36 h of incubation and the difference was pronounced after 7 days. However, both the materials showed substantially higher cell attachment than the control (polystyrene). For the same period of incubation in fetal bovine serum (FBS), the amount of protein adsorbed on the surface of boron free TZN samples was higher than that in the case of boron containing TZNB composite. The presence of boron in the TZN alloy influenced protein adsorption and cell response and they are lower in TZNB than in TZN as a result of the associated difference in chemical characteristics.

  3. c-Fos: an AP-1 transcription factor with an additional cytoplasmic, non-genomic lipid synthesis activation capacity.

    PubMed

    Caputto, Beatriz L; Cardozo Gizzi, Andrés M; Gil, Germán A

    2014-09-01

    The mechanisms that co-ordinately activate lipid synthesis when high rates of membrane biogenesis are needed to support cell growth are largely unknown. c-Fos, a well known AP-1 transcription factor, has emerged as a unique protein with the capacity to associate to specific enzymes of the pathway of synthesis of phospholipids at the endoplasmic reticulum and activate their synthesis to accompany genomic decisions of growth. Herein, we discuss this cytoplasmic, non-genomic effect of c-Fos in the context of other mechanisms that have been proposed to regulate lipid synthesis.

  4. Glucocorticoids and protein kinase A coordinately modulate transcription factor recruitment at a glucocorticoid-responsive unit.

    PubMed Central

    Espinás, M L; Roux, J; Pictet, R; Grange, T

    1995-01-01

    The rat tyrosine aminotransferase gene is a model system to study transcriptional regulation by glucocorticoid hormones. We analyzed transcription factor binding to the tyrosine aminotransferase gene glucocorticoid-responsive unit (GRU) at kb -2.5, using in vivo footprinting studies with both dimethyl sulfate and DNase I. At this GRU, glucocorticoid activation triggers a disruption of the nucleosomal structure. We show here that various regulatory pathways affect transcription factor binding to this GRU. The binding differs in two closely related glucocorticoid-responsive hepatoma cell lines. In line H4II, glucocorticoid induction promotes the recruitment of hepatocyte nuclear factor 3 (HNF3), presumably through the nucleosomal disruption. However, the footprint of the glucocorticoid receptor (GR) is not visible, even though a regular but transient interaction of the GR is necessary to maintain HNF3 binding. In contrast, in line FTO2B, HNF3 binds to the GRU in the absence of glucocorticoids and nucleosomal disruption, showing that a "closed" chromatin conformation does not repress the binding of certain transcription factors in a uniform manner. In FTO2B cells, the footprint of the GR is detectable, but this requires the activation of protein kinase A. In addition, protein kinase A stimulation also improves the recruitment of HNF3 independently of glucocorticoids and enhances the glucocorticoid response mediated by this GRU in an HNF3-dependent manner. In conclusion, the differences in the behavior of this regulatory sequence in the two cell lines show that various regulatory pathways are integrated at this GRU through modulation of interrelated events: transcription factor binding to DNA and nucleosomal disruption. PMID:7565684

  5. Effects of antinutritional factors on protein digestibility and amino acid availability in foods.

    PubMed

    Gilani, G Sarwar; Cockell, Kevin A; Sepehr, Estatira

    2005-01-01

    Digestibility of protein in traditional diets from developing countries such as India, Guatemala, and Brazil is considerably lower compared to that of protein in typical North American diets (54-78 versus 88-94%). The presence of less digestible protein fractions, high levels of insoluble fiber, and high concentrations of antinutritional factors in the diets of developing countries, which are based on less refined cereals and grain legumes as major sources of protein, are responsible for poor digestibility of protein. The effects of the presence of some of the important antinutritional factors on protein and amino digestibilities of food and feed products are reviewed in this chapter. Food and feed products may contain a number of antinutritional factors that may adversely affect protein digestibility and amino acid availability. Antinutritional factors may occur naturally, such as glucosinolates in mustard and rapeseed protein products, trypsin inhibitors and hemagglutinins in legumes, tannins in legumes and cereals, phytates in cereals and oilseeds, and gossypol in cottonseed protein products. Antinutritional factors may also be formed during heat/alkaline processing of protein products, yielding Maillard compounds, oxidized forms of sulfur amino acids, D-amino acids, and lysinoalanine (LAL, an unnatural amino acid derivative). The presence of high levels of dietary trypsin inhibitors from soybeans, kidney beans, or other grain legumes can cause substantial reductions in protein and amino acid digestibilities (up to 50%) in rats and pigs. Similarly, the presence of high levels of tannins in cereals, such as sorghum, and grain legumes, such as fababean (Vicia faba L.), can result in significantly reduced protein and amino acid digestibilities (up to 23%) in rats, poultry, and pigs. Studies involving phytase supplementation of production rations for swine or poultry have provided indirect evidence that normally encountered levels of phytates in cereals and legumes

  6. Pig performance increases with the addition of DL-methionine and L-lysine to ensiled cassava leaf protein diets.

    PubMed

    Ly, Nguyen Thi Hoa; Ngoan, Le Duc; Verstegen, Martin Wilhelmus Antonius; Hendriks, Wouter Hendrikus

    2012-01-01

    Two studies were conducted to determine the impact of supplementation of diets containing ensiled cassava leaves as the main protein source with synthetic amino acids, DL-methionine alone or with L-lysine. In study 1, a total of 40 pigs in five units, all cross-breds between Large White and Mong Cai, with an average initial body weight of 20.5 kg were randomly assigned to four treatments consisting of a basal diet containing 45% of dry matter (DM) from ensiled cassava leaves (ECL) and ensiled cassava root supplemented with 0%, 0.05%, 0.1% and 0.15% DL-methionine (as DM). Results showed a significantly improved performance and protein gain by extra methionine. This reduced the feed cost by 2.6%, 7.2% and 7.5%, respectively. In study 2, there were three units and in each unit eight cross-bred (Large White × Mong Cai) pigs with an initial body weight of 20.1 kg were randomly assigned to the four treatments. The four diets were as follows: a basal diet containing 15% ECL (as DM) supplemented with different amounts of amino acids L-lysine and DL-methionine to the control diet. The results showed that diets with 15% of DM as ECL with supplementation of 0.2% lysine +0.1% DL-methionine and 0.1% lysine +0.05% DL-methionine at the 20-50 kg and above 50 kg, respectively, resulted in the best performance, protein gain and lowest costs for cross-bred (Large White × Mong Cai) pigs. Ensiled cassava leaves can be used as a protein supplement for feeding pigs provided the diets contain additional amounts of synthetic lysine and methionine.

  7. Evidence for protein kinase C-dependent and -independent activation of mitogen-activated protein kinase in T cells: potential role of additional diacylglycerol binding proteins.

    PubMed

    Puente, L G; Stone, J C; Ostergaard, H L

    2000-12-15

    Activation of mitogen-activated protein kinases (MAPK) is a critical signal transduction event for CTL activation, but the signaling mechanisms responsible are not fully characterized. Protein kinase C (PKC) is thought to contribute to MAPK activation following TCR stimulation. We have found that dependence on PKC varies with the method used to stimulate the T cells. Extracellular signal-regulated kinase (ERK) activation in CTL stimulated with soluble cross-linked anti-CD3 is completely inhibited by the PKC inhibitor bisindolylmaleimide (BIM). In contrast, only the later time points in the course of ERK activation are sensitive to BIM when CTL are stimulated with immobilized anti-CD3, a condition that stimulates CTL degranulation. Surprisingly, MAPK activation in response to immobilized anti-CD3 is strongly inhibited at all time points by the diacylglycerol (DAG)-binding domain inhibitor calphostin C implicating the contribution of a DAG-dependent but PKC-independent pathway in the activation of ERK in CTL clones. Chronic exposure to phorbol ester down-regulates the expression of DAG-responsive PKC isoforms; however, this treatment of CTL clones does not inhibit anti-CD3-induced activation of MAPK. Phorbol ester-treated cells have reduced expression of several isoforms of PKC but still express the recently described DAG-binding Ras guanylnucleotide-releasing protein. These results indicate that the late phase of MAPK activation in CTL clones in response to immobilized anti-CD3 stimulation requires PKC while the early phase requires a DAG-dependent, BIM-resistant component.

  8. The E-class PPR protein MEF3 of Arabidopsis thaliana can also function in mitochondrial RNA editing with an additional DYW domain.

    PubMed

    Verbitskiy, Daniil; Merwe, Johannes A van der; Zehrmann, Anja; Härtel, Barbara; Takenaka, Mizuki

    2012-02-01

    In plants, RNA editing is observed in mitochondria and plastids, changing selected C nucleotides into Us in both organelles. We here identify the PPR (pentatricopeptide repeat) protein MEF3 (mitochondrial editing factor 3) of the E domain PPR subclass by genetic mapping of a variation between ecotypes Columbia (Col) and Landsberg erecta (Ler) in Arabidopsis thaliana to be required for a specific RNA editing event in mitochondria. The Ler variant of MEF3 differs from Col in two amino acids in repeats 9 and 10, which reduce RNA editing levels at site atp4-89 to about 50% in Ler. In a T-DNA insertion line, editing at this site is completely lost. In Vitis vinifera the gene most similar to MEF3 continues into a DYW extension beyond the common E domain. Complementation assays with various combinations of PPR and E domains from the vine and A. thaliana proteins show that the vine E region can substitute for the A. thaliana E region with or without the DYW domain. These findings suggest that the additional DYW domain does not disturb the MEF3 protein function in mitochondrial RNA editing in A. thaliana.

  9. Template-dependent nucleotide addition in the reverse (3'-5') direction by Thg1-like protein.

    PubMed

    Kimura, Shoko; Suzuki, Tateki; Chen, Meirong; Kato, Koji; Yu, Jian; Nakamura, Akiyoshi; Tanaka, Isao; Yao, Min

    2016-03-01

    Thg1-like protein (TLP) catalyzes the addition of a nucleotide to the 5'-end of truncated transfer RNA (tRNA) species in a Watson-Crick template-dependent manner. The reaction proceeds in two steps: the activation of the 5'-end by adenosine 5'-triphosphate (ATP)/guanosine 5'-triphosphate (GTP), followed by nucleotide addition. Structural analyses of the TLP and its reaction intermediates have revealed the atomic detail of the template-dependent elongation reaction in the 3'-5' direction. The enzyme creates two substrate binding sites for the first- and second-step reactions in the vicinity of one reaction center consisting of two Mg(2+) ions, and the two reactions are executed at the same reaction center in a stepwise fashion. When the incoming nucleotide is bound to the second binding site with Watson-Crick hydrogen bonds, the 3'-OH of the incoming nucleotide and the 5'-triphosphate of the tRNA are moved to the reaction center where the first reaction has occurred. That the 3'-5' elongation enzyme performs this elaborate two-step reaction in one catalytic center suggests that these two reactions have been inseparable throughout the process of protein evolution. Although TLP and Thg1 have similar tetrameric organization, the tRNA binding mode of TLP is different from that of Thg1, a tRNA(His)-specific G-1 addition enzyme. Each tRNA(His) binds to three of the four Thg1 tetramer subunits, whereas in TLP, tRNA only binds to a dimer interface and the elongation reaction is terminated by measuring the accepter stem length through the flexible β-hairpin. Furthermore, mutational analyses show that tRNA(His) is bound to TLP in a similar manner as Thg1, thus indicating that TLP has a dual binding mode.

  10. Template-dependent nucleotide addition in the reverse (3′-5′) direction by Thg1-like protein

    PubMed Central

    Kimura, Shoko; Suzuki, Tateki; Chen, Meirong; Kato, Koji; Yu, Jian; Nakamura, Akiyoshi; Tanaka, Isao; Yao, Min

    2016-01-01

    Thg1-like protein (TLP) catalyzes the addition of a nucleotide to the 5′-end of truncated transfer RNA (tRNA) species in a Watson-Crick template–dependent manner. The reaction proceeds in two steps: the activation of the 5′-end by adenosine 5′-triphosphate (ATP)/guanosine 5′-triphosphate (GTP), followed by nucleotide addition. Structural analyses of the TLP and its reaction intermediates have revealed the atomic detail of the template-dependent elongation reaction in the 3′-5′ direction. The enzyme creates two substrate binding sites for the first- and second-step reactions in the vicinity of one reaction center consisting of two Mg2+ ions, and the two reactions are executed at the same reaction center in a stepwise fashion. When the incoming nucleotide is bound to the second binding site with Watson-Crick hydrogen bonds, the 3′-OH of the incoming nucleotide and the 5′-triphosphate of the tRNA are moved to the reaction center where the first reaction has occurred. That the 3′-5′ elongation enzyme performs this elaborate two-step reaction in one catalytic center suggests that these two reactions have been inseparable throughout the process of protein evolution. Although TLP and Thg1 have similar tetrameric organization, the tRNA binding mode of TLP is different from that of Thg1, a tRNAHis-specific G−1 addition enzyme. Each tRNAHis binds to three of the four Thg1 tetramer subunits, whereas in TLP, tRNA only binds to a dimer interface and the elongation reaction is terminated by measuring the accepter stem length through the flexible β-hairpin. Furthermore, mutational analyses show that tRNAHis is bound to TLP in a similar manner as Thg1, thus indicating that TLP has a dual binding mode. PMID:27051866

  11. Converting nitrogen into protein--beyond 6.25 and Jones' factors.

    PubMed

    Mariotti, François; Tomé, Daniel; Mirand, Philippe Patureau

    2008-02-01

    The protein content in foodstuffs is estimated by multiplying the determined nitrogen content by a nitrogen-to-protein conversion factor. Jones' factors for a series of foodstuffs, including 6.25 as the standard, default conversion factor, have now been used for 75 years. This review provides a brief history of these factors and their underlying paradigm, with an insight into what is meant by "protein." We also review other compelling data on specific conversion factors which may have been overlooked. On the one hand, when 6.25 is used irrespective of the foodstuff, "protein" is simply nitrogen expressed using a different unit and says little about protein (s.s.). On the other hand, conversion factors specific to foodstuffs, such as those provided by Jones, are scientifically flawed. However, the nitrogen:protein ratio does vary according to the foodstuff considered. Therefore, from a scientific point of view, it would be reasonable not to apply current specific factors any longer, but they have continued to be used because scientists fear opening the Pandora's box. But because conversion factors are critical to enabling the simple conversion of determined nitrogen values into protein values and thus accurately evaluating the quantity and the quality of protein in foodstuffs, we propose a set of specific conversion factors for different foodstuffs, together with a default conversion factor (5.6). This would be far more accurate and scientifically sound, and preferable when specifically expressing nitrogen as protein. These factors are of particular importance when "protein" basically means "amino acids," this being the principal nutritional viewpoint.

  12. Additive Promotion of Viral Internal Ribosome Entry Site-Mediated Translation by Far Upstream Element-Binding Protein 1 and an Enterovirus 71-Induced Cleavage Product

    PubMed Central

    Hung, Chuan-Tien; Kung, Yu-An; Li, Mei-Ling; Lee, Kuo-Ming; Liu, Shih-Tung; Shih, Shin-Ru

    2016-01-01

    The 5' untranslated region (5' UTR) of the enterovirus 71 (EV71) RNA genome contains an internal ribosome entry site (IRES) that is indispensable for viral protein translation. Due to the limited coding capacity of their RNA genomes, EV71 and other picornaviruses typically recruit host factors, known as IRES trans-acting factors (ITAFs), to mediate IRES-dependent translation. Here, we show that EV71 viral proteinase 2A is capable of cleaving far upstream element-binding protein 1 (FBP1), a positive ITAF that directly binds to the EV71 5' UTR linker region to promote viral IRES-driven translation. The cleavage occurs at the Gly-371 residue of FBP1 during the EV71 infection process, and this generates a functional cleavage product, FBP11-371. Interestingly, the cleavage product acts to promote viral IRES activity. Footprinting analysis and gel mobility shift assay results showed that FBP11-371 similarly binds to the EV71 5' UTR linker region, but at a different site from full-length FBP1; moreover, FBP1 and FBP11-371 were found to act additively to promote IRES-mediated translation and virus yield. Our findings expand the current understanding of virus-host interactions with regard to viral recruitment and modulation of ITAFs, and provide new insights into translational control during viral infection. PMID:27780225

  13. The Activating Transcription Factor 3 Protein Suppresses the Oncogenic Function of Mutant p53 Proteins*

    PubMed Central

    Wei, Saisai; Wang, Hongbo; Lu, Chunwan; Malmut, Sarah; Zhang, Jianqiao; Ren, Shumei; Yu, Guohua; Wang, Wei; Tang, Dale D.; Yan, Chunhong

    2014-01-01

    Mutant p53 proteins (mutp53) often acquire oncogenic activities, conferring drug resistance and/or promoting cancer cell migration and invasion. Although it has been well established that such a gain of function is mainly achieved through interaction with transcriptional regulators, thereby modulating cancer-associated gene expression, how the mutp53 function is regulated remains elusive. Here we report that activating transcription factor 3 (ATF3) bound common mutp53 (e.g. R175H and R273H) and, subsequently, suppressed their oncogenic activities. ATF3 repressed mutp53-induced NFKB2 expression and sensitized R175H-expressing cancer cells to cisplatin and etoposide treatments. Moreover, ATF3 appeared to suppress R175H- and R273H-mediated cancer cell migration and invasion as a consequence of preventing the transcription factor p63 from inactivation by mutp53. Accordingly, ATF3 promoted the expression of the metastasis suppressor SHARP1 in mutp53-expressing cells. An ATF3 mutant devoid of the mutp53-binding domain failed to disrupt the mutp53-p63 binding and, thus, lost the activity to suppress mutp53-mediated migration, suggesting that ATF3 binds to mutp53 to suppress its oncogenic function. In line with these results, we found that down-regulation of ATF3 expression correlated with lymph node metastasis in TP53-mutated human lung cancer. We conclude that ATF3 can suppress mutp53 oncogenic function, thereby contributing to tumor suppression in TP53-mutated cancer. PMID:24554706

  14. Patagonfibrase modifies protein expression of tissue factor and protein disulfide isomerase in rat skin.

    PubMed

    Peichoto, María Elisa; Santoro, Marcelo Larami

    2016-09-01

    Patagonfibrase is a hemorrhagic metalloproteinase isolated from the venom of the South American rear-fanged snake Philodryas patagoniensis, and is an important contributor to local lesions inflicted by this species. The tissue factor (TF)-factor VIIa complex, besides triggering the coagulation cascade, has been demonstrated to be involved in inflammatory events. Our aim was to determine whether patagonfibrase affects the expression of TF and protein disulfide isomerase (PDI), an enzyme that controls TF biological activity, at the site of patagonfibrase injection, and thus if they may play a role in hemostatic and inflammatory events induced by snake venoms. Patagonfibrase (60 μg/kg) was administered s.c. to rats, and after 3 h blood was collected to evaluate hemostasis parameters, and skin fragments close to the site of injection were taken to assess TF and PDI expression. Patagonfibrase did not alter blood cell counts, plasma fibrinogen levels, or levels of TF activity in plasma. However, by semiquantitative Western blotting, patagonfibrase increased TF expression by 2-fold, and decreased PDI expression by 3-fold in skin samples. In agreement, by immunohistochemical analyses, prominent TF expression was observed in the subcutaneous tissue. Thus, patagonfibrase affects the local expression of TF and PDI without inducing any systemic hemostatic disturbance, although that they may be involved in the local inflammatory events induced by hemorrhagic metalloproteinases. Once antivenom therapy is not totally effective to treat the local injury induced by snake venoms, modulation of the activity and expression of TF and/or PDI might become a strategy for treating snake envenomation. PMID:27390042

  15. Methicillin resistance in Staphylococcus epidermidis. Relationship between the additional penicillin-binding protein and an attachment transpeptidase.

    PubMed

    Gaisford, W C; Reynolds, P E

    1989-10-20

    The penicillin-binding proteins (PBP) of a methicillin-resistant strain of Staphylococcus epidermidis, 100,604 p+m+ and a non-isogenic sensitive strain, p-m- were characterised. The presence of a novel PBP, produced by the methicillin-resistant strain of S. epidermidis, with an Mr identical to that of PBP2' in Staphylococcus aureus 13,136 p-m+, was revealed by sodium dodecyl sulphate/polyacrylamide gel electrophoresis and subsequent fluorography of solubilised membrane proteins isolated from cells labelled with [3H]benzylpenicillin. This novel PBP was only detected in cells which had been grown at 30 degrees C, in media containing beta-lactam antibiotic and 5% NaCl. The sensitivity of an attachment transpeptidation reaction measured under non-growing conditions in the sensitive and resistant strains indicated that the novel PBP catalysed this reaction. The similarity of radiolabelled peptides resulting from partial proteolytic digestion of the novel PBP in S. epidermidis 100,604 p+m+ and from PBP2' in S. aureus 13,136 p+m+ lends support to the theory that the additional DNA encoding PBP2' in S. aureus and the same protein in S. epidermidis has been passed to both species from an unknown source. Studies of the development and loss of resistance of attachment transpeptidase activity, and the appearance and disappearance of the novel protein when cultures of the resistant strain were transferred from conditions allowing the expression of resistance to those not allowing such expression and vice-versa, indicated that there was a strong correlation between the presence of PBP2' and the degree of resistance of the attachment transpeptidation reaction and that the production of this protein was affected by temperature at a regulatory or genetic level. Studies on the induction and loss of beta-lactamase activity and of the novel PBP when the resistant strain was grown in the presence or absence of beta-lactam antibiotics at either 40 degrees C or 30 degrees C suggests that

  16. Methicillin resistance in Staphylococcus epidermidis. Relationship between the additional penicillin-binding protein and an attachment transpeptidase.

    PubMed

    Gaisford, W C; Reynolds, P E

    1989-10-20

    The penicillin-binding proteins (PBP) of a methicillin-resistant strain of Staphylococcus epidermidis, 100,604 p+m+ and a non-isogenic sensitive strain, p-m- were characterised. The presence of a novel PBP, produced by the methicillin-resistant strain of S. epidermidis, with an Mr identical to that of PBP2' in Staphylococcus aureus 13,136 p-m+, was revealed by sodium dodecyl sulphate/polyacrylamide gel electrophoresis and subsequent fluorography of solubilised membrane proteins isolated from cells labelled with [3H]benzylpenicillin. This novel PBP was only detected in cells which had been grown at 30 degrees C, in media containing beta-lactam antibiotic and 5% NaCl. The sensitivity of an attachment transpeptidation reaction measured under non-growing conditions in the sensitive and resistant strains indicated that the novel PBP catalysed this reaction. The similarity of radiolabelled peptides resulting from partial proteolytic digestion of the novel PBP in S. epidermidis 100,604 p+m+ and from PBP2' in S. aureus 13,136 p+m+ lends support to the theory that the additional DNA encoding PBP2' in S. aureus and the same protein in S. epidermidis has been passed to both species from an unknown source. Studies of the development and loss of resistance of attachment transpeptidase activity, and the appearance and disappearance of the novel protein when cultures of the resistant strain were transferred from conditions allowing the expression of resistance to those not allowing such expression and vice-versa, indicated that there was a strong correlation between the presence of PBP2' and the degree of resistance of the attachment transpeptidation reaction and that the production of this protein was affected by temperature at a regulatory or genetic level. Studies on the induction and loss of beta-lactamase activity and of the novel PBP when the resistant strain was grown in the presence or absence of beta-lactam antibiotics at either 40 degrees C or 30 degrees C suggests that

  17. Regulation of myostatin in vivo by growth and differentiation factor-associated serum protein-1: a novel protein with protease inhibitor and follistatin domains.

    PubMed

    Hill, Jennifer J; Qiu, Yongchang; Hewick, Rodney M; Wolfman, Neil M

    2003-06-01

    Myostatin, a member of the TGFbeta superfamily, is a potent and specific negative regulator of skeletal muscle mass. In serum, myostatin circulates as part of a latent complex containing myostatin propeptide and/or follistatin-related gene (FLRG). Here, we report the identification of an additional protein associated with endogenous myostatin in normal mouse and human serum, discovered by affinity purification and mass spectrometry. This protein, which we have named growth and differentiation factor-associated serum protein-1 (GASP-1), contains multiple domains associated with protease-inhibitory proteins, including a whey acidic protein domain, a Kazal domain, two Kunitz domains, and a netrin domain. GASP-1 also contains a domain homologous to the 10-cysteine repeat found in follistatin, a protein that binds and inhibits activin, another member of the TGFbeta superfamily. We have cloned mouse GASP-1 and shown that it inhibits the biological activity of mature myostatin, but not activin, in a luciferase reporter gene assay. Surprisingly, recombinant GASP-1 binds directly not only to mature myostatin, but also to the myostatin propeptide. Thus, GASP-1 represents a novel class of inhibitory TGFbeta binding proteins.

  18. Small G proteins in peroxisome biogenesis: the potential involvement of ADP-ribosylation factor 6

    PubMed Central

    2009-01-01

    Background Peroxisomes execute diverse and vital functions in virtually every eukaryote. New peroxisomes form by budding from pre-existing organelles or de novo by vesiculation of the ER. It has been suggested that ADP-ribosylation factors and COPI coatomer complexes are involved in these processes. Results Here we show that all viable Saccharomyces cerevisiae strains deficient in one of the small GTPases which have an important role in the regulation of vesicular transport contain functional peroxisomes, and that the number of these organelles in oleate-grown cells is significantly upregulated in the arf1 and arf3 null strains compared to the wild-type strain. In addition, we provide evidence that a portion of endogenous Arf6, the mammalian orthologue of yeast Arf3, is associated with the cytoplasmic face of rat liver peroxisomes. Despite this, ablation of Arf6 did neither influence the regulation of peroxisome abundance nor affect the localization of peroxisomal proteins in cultured fetal hepatocytes. However, co-overexpression of wild-type, GTP hydrolysis-defective or (dominant-negative) GTP binding-defective forms of Arf1 and Arf6 caused mislocalization of newly-synthesized peroxisomal proteins and resulted in an alteration of peroxisome morphology. Conclusion These observations suggest that Arf6 is a key player in mammalian peroxisome biogenesis. In addition, they also lend strong support to and extend the concept that specific Arf isoform pairs may act in tandem to regulate exclusive trafficking pathways. PMID:19686593

  19. Stretching human mesenchymal stromal cells on stiffness-customized collagen type I generates a smooth muscle marker profile without growth factor addition

    PubMed Central

    Rothdiener, Miriam; Hegemann, Miriam; Uynuk-Ool, Tatiana; Walters, Brandan; Papugy, Piruntha; Nguyen, Phong; Claus, Valentin; Seeger, Tanja; Stoeckle, Ulrich; Boehme, Karen A.; Aicher, Wilhelm K.; Stegemann, Jan P.; Hart, Melanie L.; Kurz, Bodo; Klein, Gerd; Rolauffs, Bernd

    2016-01-01

    Using matrix elasticity and cyclic stretch have been investigated for inducing mesenchymal stromal cell (MSC) differentiation towards the smooth muscle cell (SMC) lineage but not in combination. We hypothesized that combining lineage-specific stiffness with cyclic stretch would result in a significantly increased expression of SMC markers, compared to non-stretched controls. First, we generated dense collagen type I sheets by mechanically compressing collagen hydrogels. Atomic force microscopy revealed a nanoscale stiffness range known to support myogenic differentiation. Further characterization revealed viscoelasticity and stable biomechanical properties under cyclic stretch with >99% viable adherent human MSC. MSCs on collagen sheets demonstrated a significantly increased mRNA but not protein expression of SMC markers, compared to on culture flasks. However, cyclic stretch of MSCs on collagen sheets significantly increased both mRNA and protein expression of α-smooth muscle actin, transgelin, and calponin versus plastic and non-stretched sheets. Thus, lineage-specific stiffness and cyclic stretch can be applied together for inducing MSC differentiation towards SMCs without the addition of recombinant growth factors or other soluble factors. This represents a novel stimulation method for modulating the phenotype of MSCs towards SMCs that could easily be incorporated into currently available methodologies to obtain a more targeted control of MSC phenotype. PMID:27775041

  20. Hepatocyte growth factor induction of macrophage chemoattractant protein-1 and osteophyte-inducing factors in osteoarthritis.

    PubMed

    Dankbar, Berno; Neugebauer, Katja; Wunrau, Christina; Tibesku, Carsten O; Skwara, Adrian; Pap, Thomas; Fuchs-Winkelmann, Susanne

    2007-05-01

    In osteoarthritis (OA), hepatocyte growth factor (HGF) is supposed to play a role in cartilage repair. Because the development of osteophytes is a major characteristic of OA and thought to be part of an attempted repair process, the purpose of this study was to determine whether HGF may be involved in osteophyte formation. HGF levels in synovial fluids from 41 patients assessed by enzyme immunosorbant assay were correlated with disease severity and osteophyte formation, evaluated by anteroposterior weight-bearing radiographs. Detection of HGF, c-Met, and CD68 in cartilage and synovial tissues was assessed by immunohistochemistry. Effects of HGF on the secretion of TGF-beta1 and BMP-2 by chondrocytes, fibroblast-like synovial cells (FLS), and macrophages as well as HGF-induced secretion of MCP-1 by FLS and chondrocytes were determined by ELISA. HGF was detected in all synovial fluids and concentrations correlated highly with disease severity and osteophyte formation (p < 0.001). Immunohistochemistry revealed weak synovial staining for HGF, whereas increasing numbers of HGF expressing chondrocytes were detected depending on disease severity. In addition, an increased number of macrophages in synovial specimens was observed, which was likewise severity dependent. In a series of subsequent in vitro studies, HGF remarkable induced MCP-1 secretion by FLS in a dose-dependent manner. No effect on TGF-beta1 and BMP-2 secretion by FLS and chondrocytes was evident upon HGF stimulation, whereas secretion of these growth factors by PMA-differentiated THP-1 cells was significantly increased by HGF. The results indicate that HGF may facilitate osteophyte development by promoting MCP-1-mediated entry of monocytes/macrophages into the OA-affected joint and/or by stimulating macrophage-derived growth factors.

  1. A survey of PPR proteins identifies DYW domains like those of land plant RNA editing factors in diverse eukaryotes.

    PubMed

    Schallenberg-Rüdinger, Mareike; Lenz, Henning; Polsakiewicz, Monika; Gott, Jonatha M; Knoop, Volker

    2013-01-01

    The pentatricopeptide repeat modules of PPR proteins are key to their sequence-specific binding to RNAs. Gene families encoding PPR proteins are greatly expanded in land plants where hundreds of them participate in RNA maturation, mainly in mitochondria and chloroplasts. Many plant PPR proteins contain additional carboxyterminal domains and have been identified as essential factors for specific events of C-to-U RNA editing, which is abundant in the two endosymbiotic plant organelles. Among those carboxyterminal domain additions to plant PPR proteins, the so-called DYW domain is particularly interesting given its similarity to cytidine deaminases. The frequency of organelle C-to-U RNA editing and the diversity of DYW-type PPR proteins correlate well in plants and both were recently identified outside of land plants, in the protist Naegleria gruberi. Here we present a systematic survey of PPR protein genes and report on the identification of additional DYW-type PPR proteins in the protists Acanthamoeba castellanii, Malawimonas jakobiformis, and Physarum polycephalum. Moreover, DYW domains were also found in basal branches of multi-cellular lineages outside of land plants, including the alga Nitella flexilis and the rotifers Adineta ricciae and Philodina roseola. Intriguingly, the well-characterized and curious patterns of mitochondrial RNA editing in the slime mold Physarum also include examples of C-to-U changes. Finally, we identify candidate sites for mitochondrial RNA editing in Malawimonas, further supporting a link between DYW-type PPR proteins and C-to-U editing, which may have remained hitherto unnoticed in additional eukaryote lineages.

  2. A survey of PPR proteins identifies DYW domains like those of land plant RNA editing factors in diverse eukaryotes.

    PubMed

    Schallenberg-Rüdinger, Mareike; Lenz, Henning; Polsakiewicz, Monika; Gott, Jonatha M; Knoop, Volker

    2013-01-01

    The pentatricopeptide repeat modules of PPR proteins are key to their sequence-specific binding to RNAs. Gene families encoding PPR proteins are greatly expanded in land plants where hundreds of them participate in RNA maturation, mainly in mitochondria and chloroplasts. Many plant PPR proteins contain additional carboxyterminal domains and have been identified as essential factors for specific events of C-to-U RNA editing, which is abundant in the two endosymbiotic plant organelles. Among those carboxyterminal domain additions to plant PPR proteins, the so-called DYW domain is particularly interesting given its similarity to cytidine deaminases. The frequency of organelle C-to-U RNA editing and the diversity of DYW-type PPR proteins correlate well in plants and both were recently identified outside of land plants, in the protist Naegleria gruberi. Here we present a systematic survey of PPR protein genes and report on the identification of additional DYW-type PPR proteins in the protists Acanthamoeba castellanii, Malawimonas jakobiformis, and Physarum polycephalum. Moreover, DYW domains were also found in basal branches of multi-cellular lineages outside of land plants, including the alga Nitella flexilis and the rotifers Adineta ricciae and Philodina roseola. Intriguingly, the well-characterized and curious patterns of mitochondrial RNA editing in the slime mold Physarum also include examples of C-to-U changes. Finally, we identify candidate sites for mitochondrial RNA editing in Malawimonas, further supporting a link between DYW-type PPR proteins and C-to-U editing, which may have remained hitherto unnoticed in additional eukaryote lineages. PMID:23899506

  3. Proteomic Interaction Patterns between Human Cyclins, the Cyclin-Dependent Kinase Ortholog pUL97 and Additional Cytomegalovirus Proteins.

    PubMed

    Steingruber, Mirjam; Kraut, Alexandra; Socher, Eileen; Sticht, Heinrich; Reichel, Anna; Stamminger, Thomas; Amin, Bushra; Couté, Yohann; Hutterer, Corina; Marschall, Manfred

    2016-01-01

    The human cytomegalovirus (HCMV)-encoded cyclin-dependent kinase (CDK) ortholog pUL97 associates with human cyclin B1 and other types of cyclins. Here, the question was addressed whether cyclin interaction of pUL97 and additional viral proteins is detectable by mass spectrometry-based approaches. Proteomic data were validated by coimmunoprecipitation (CoIP), Western blot, in vitro kinase and bioinformatic analyses. Our findings suggest that: (i) pUL97 shows differential affinities to human cyclins; (ii) pUL97 inhibitor maribavir (MBV) disrupts the interaction with cyclin B1, but not with other cyclin types; (iii) cyclin H is identified as a new high-affinity interactor of pUL97 in HCMV-infected cells; (iv) even more viral phosphoproteins, including all known substrates of pUL97, are detectable in the cyclin-associated complexes; and (v) a first functional validation of pUL97-cyclin B1 interaction, analyzed by in vitro kinase assay, points to a cyclin-mediated modulation of pUL97 substrate preference. In addition, our bioinformatic analyses suggest individual, cyclin-specific binding interfaces for pUL97-cyclin interaction, which could explain the different strengths of interactions and the selective inhibitory effect of MBV on pUL97-cyclin B1 interaction. Combined, the detection of cyclin-associated proteins in HCMV-infected cells suggests a complex pattern of substrate phosphorylation and a role of cyclins in the fine-modulation of pUL97 activities.

  4. Proteomic Interaction Patterns between Human Cyclins, the Cyclin-Dependent Kinase Ortholog pUL97 and Additional Cytomegalovirus Proteins

    PubMed Central

    Steingruber, Mirjam; Kraut, Alexandra; Socher, Eileen; Sticht, Heinrich; Reichel, Anna; Stamminger, Thomas; Amin, Bushra; Couté, Yohann; Hutterer, Corina; Marschall, Manfred

    2016-01-01

    The human cytomegalovirus (HCMV)-encoded cyclin-dependent kinase (CDK) ortholog pUL97 associates with human cyclin B1 and other types of cyclins. Here, the question was addressed whether cyclin interaction of pUL97 and additional viral proteins is detectable by mass spectrometry-based approaches. Proteomic data were validated by coimmunoprecipitation (CoIP), Western blot, in vitro kinase and bioinformatic analyses. Our findings suggest that: (i) pUL97 shows differential affinities to human cyclins; (ii) pUL97 inhibitor maribavir (MBV) disrupts the interaction with cyclin B1, but not with other cyclin types; (iii) cyclin H is identified as a new high-affinity interactor of pUL97 in HCMV-infected cells; (iv) even more viral phosphoproteins, including all known substrates of pUL97, are detectable in the cyclin-associated complexes; and (v) a first functional validation of pUL97-cyclin B1 interaction, analyzed by in vitro kinase assay, points to a cyclin-mediated modulation of pUL97 substrate preference. In addition, our bioinformatic analyses suggest individual, cyclin-specific binding interfaces for pUL97-cyclin interaction, which could explain the different strengths of interactions and the selective inhibitory effect of MBV on pUL97-cyclin B1 interaction. Combined, the detection of cyclin-associated proteins in HCMV-infected cells suggests a complex pattern of substrate phosphorylation and a role of cyclins in the fine-modulation of pUL97 activities. PMID:27548200

  5. Proteomic Interaction Patterns between Human Cyclins, the Cyclin-Dependent Kinase Ortholog pUL97 and Additional Cytomegalovirus Proteins.

    PubMed

    Steingruber, Mirjam; Kraut, Alexandra; Socher, Eileen; Sticht, Heinrich; Reichel, Anna; Stamminger, Thomas; Amin, Bushra; Couté, Yohann; Hutterer, Corina; Marschall, Manfred

    2016-01-01

    The human cytomegalovirus (HCMV)-encoded cyclin-dependent kinase (CDK) ortholog pUL97 associates with human cyclin B1 and other types of cyclins. Here, the question was addressed whether cyclin interaction of pUL97 and additional viral proteins is detectable by mass spectrometry-based approaches. Proteomic data were validated by coimmunoprecipitation (CoIP), Western blot, in vitro kinase and bioinformatic analyses. Our findings suggest that: (i) pUL97 shows differential affinities to human cyclins; (ii) pUL97 inhibitor maribavir (MBV) disrupts the interaction with cyclin B1, but not with other cyclin types; (iii) cyclin H is identified as a new high-affinity interactor of pUL97 in HCMV-infected cells; (iv) even more viral phosphoproteins, including all known substrates of pUL97, are detectable in the cyclin-associated complexes; and (v) a first functional validation of pUL97-cyclin B1 interaction, analyzed by in vitro kinase assay, points to a cyclin-mediated modulation of pUL97 substrate preference. In addition, our bioinformatic analyses suggest individual, cyclin-specific binding interfaces for pUL97-cyclin interaction, which could explain the different strengths of interactions and the selective inhibitory effect of MBV on pUL97-cyclin B1 interaction. Combined, the detection of cyclin-associated proteins in HCMV-infected cells suggests a complex pattern of substrate phosphorylation and a role of cyclins in the fine-modulation of pUL97 activities. PMID:27548200

  6. Identifying food proteins with allergenic potential: evolution of approaches to safety assessment and research to provide additional tools.

    PubMed

    Ladics, Gregory S; Selgrade, MaryJane K

    2009-08-01

    A safety assessment process exists for genetically engineered crops that includes the evaluation of the expressed protein for allergenic potential. The objectives of this evaluation are twofold: (1) to protect allergic consumers from exposure to known allergenic or cross-reactive proteins, and (2) protect the general population from risks associated with the introduction of genes encoding proteins that are likely to become food allergens. The first systematic approach to address these concerns was formulated by Metcalfe et al. [Metcalfe, D.D., Astwood, J.D., Townsend, R., Sampson, H.A., Taylor, S.L., and Fuchs, R.L. 1996. Assessment of the allergenic potential of foods from genetically engineered crop plants. Crit. Rev. Food Sci. Nutr. 36(5), 165-186.] and subsequently Food and Agriculture Organization of the United Nations/World Health Organization (FAO/WHO) [FAO/WHO, 2001. Evaluation of allergenicity of genetically modified foods. Report of a Joint FAO/WHO Expert Consultation on Allergenicity of Foods Derived from Biotechnology. January 22-25, 2001. Rome, Italy]. More recently, Codex [Codex Alimentarius Commission, 2003. Alinorm 03/34: Joint FAO/WHO Food Standard Programme, Codex Alimentarius Commission, Twenty-Fifth Session, Rome, Italy, 30 June-5 July, 2003. Appendix III, Guideline for the conduct of food safety assessment of foods derived from recombinant-DNA plants, and Appendix IV, Annex on the assessment of possible allergenicity. pp. 47-60], noting that no single factor is recognized as an identifier for protein allergenicity, suggested a weight of evidence approach be conducted that takes into account a variety of factors and approaches for an overall assessment of allergenic potential. These various recommendations are based on what is known about allergens, including the history of exposure and safety of the gene(s) source; amino acid sequence identity to human allergens; stability to pepsin digestion in vitro; protein abundance in the crop and

  7. Endosomal cholesterol trafficking: protein factors at a glance.

    PubMed

    Du, Ximing; Yang, Hongyuan

    2013-01-01

    The delivery of low-density lipoprotein-derived cholesterol (LDL-C) from endosomal compartments to the plasma membrane and the endoplasmic reticulum (ER) is an important yet poorly understood cellular process. Niemann-Pick C1 (NPC1), a multi-pass integral membrane protein on the limiting membranes of late endosomes (LE)/lysosomes (Ly), is known to insert lumenal LDL-C to the limiting membrane of LE/Ly. Recent progress has identified novel cytoplasmic proteins that regulate the exit of LDL-C from LE/Ly, such as ORP5, a member of the oxysterol-binding protein-related protein (ORPs) family, and Hrs/VPS27, a well-established regulator of the endosomal sorting complex required for transport pathway. Whereas ORP5/ORPs may serve as cytosolic cholesterol carriers and deliver cholesterol in a non-vesicular manner, how Hrs/VPS27 regulate endosomal cholesterol sorting remains enigmatic. We discuss the functional relationship between NPC1, Hrs, and ORP5, and formulate possible schemes on how LDL-C may be moved from endosomal compartments to other cellular organelles. PMID:23165745

  8. Maize endosperm-specific transcription factors O2 and PBF network the regulation of protein and starch synthesis

    PubMed Central

    Zhang, Zhiyong; Zheng, Xixi; Yang, Jun; Messing, Joachim; Wu, Yongrui

    2016-01-01

    The maize endosperm-specific transcription factors opaque2 (O2) and prolamine-box binding factor (PBF) regulate storage protein zein genes. We show that they also control starch synthesis. The starch content in the PbfRNAi and o2 mutants was reduced by ∼5% and 11%, respectively, compared with normal genotypes. In the double-mutant PbfRNAi;o2, starch was decreased by 25%. Transcriptome analysis reveals that >1,000 genes were affected in each of the two mutants and in the double mutant; these genes were mainly enriched in sugar and protein metabolism. Pyruvate orthophosphate dikinase 1 and 2 (PPDKs) and starch synthase III (SSIII) are critical components in the starch biosynthetic enzyme complex. The expression of PPDK1, PPDK2, and SSIII and their protein levels are further reduced in the double mutants as compared with the single mutants. When the promoters of these genes were analyzed, we found a prolamine box and an O2 box that can be additively transactivated by PBF and O2. Starch synthase IIa (SSIIa, encoding another starch synthase for amylopectin) and starch branching enzyme 1 (SBEI, encoding one of the two main starch branching enzymes) are not directly regulated by PBF and O2, but their protein levels are significantly decreased in the o2 mutant and are further decreased in the double mutant, indicating that o2 and PbfRNAi may affect the levels of some other transcription factor(s) or mRNA regulatory factor(s) that in turn would affect the transcript and protein levels of SSIIa and SBEI. These findings show that three important traits—nutritional quality, calories, and yield—are linked through the same transcription factors. PMID:27621432

  9. Maize endosperm-specific transcription factors O2 and PBF network the regulation of protein and starch synthesis.

    PubMed

    Zhang, Zhiyong; Zheng, Xixi; Yang, Jun; Messing, Joachim; Wu, Yongrui

    2016-09-27

    The maize endosperm-specific transcription factors opaque2 (O2) and prolamine-box binding factor (PBF) regulate storage protein zein genes. We show that they also control starch synthesis. The starch content in the PbfRNAi and o2 mutants was reduced by ∼5% and 11%, respectively, compared with normal genotypes. In the double-mutant PbfRNAi;o2, starch was decreased by 25%. Transcriptome analysis reveals that >1,000 genes were affected in each of the two mutants and in the double mutant; these genes were mainly enriched in sugar and protein metabolism. Pyruvate orthophosphate dikinase 1 and 2 (PPDKs) and starch synthase III (SSIII) are critical components in the starch biosynthetic enzyme complex. The expression of PPDK1, PPDK2, and SSIII and their protein levels are further reduced in the double mutants as compared with the single mutants. When the promoters of these genes were analyzed, we found a prolamine box and an O2 box that can be additively transactivated by PBF and O2. Starch synthase IIa (SSIIa, encoding another starch synthase for amylopectin) and starch branching enzyme 1 (SBEI, encoding one of the two main starch branching enzymes) are not directly regulated by PBF and O2, but their protein levels are significantly decreased in the o2 mutant and are further decreased in the double mutant, indicating that o2 and PbfRNAi may affect the levels of some other transcription factor(s) or mRNA regulatory factor(s) that in turn would affect the transcript and protein levels of SSIIa and SBEI These findings show that three important traits-nutritional quality, calories, and yield-are linked through the same transcription factors. PMID:27621432

  10. Perspectives into factors limiting in vivo digestion of legume proteins: antinutritional compounds or storage proteins?

    PubMed

    Carbonaro, M; Grant, G; Cappelloni, M; Pusztai, A

    2000-03-01

    The in vivo protein digestibility of raw and cooked common bean (Phaseolus vulgaris L.) and faba bean (Vicia faba L.) and of protein fractions extracted from them was determined with growing rats. Overnight-fasted rats were intubated with a protein suspension or fed the same amount of protein added to a basal diet. The rats were killed 1 h later, the contents of stomach and small intestine were washed out, and their protein contents were measured. The in vivo digestibility of proteins of raw common bean flour was 72.4% and not significantly improved after cooking. In contrast, the digestibility of faba bean proteins was decreased from 86.5 to 60.6% by the thermal treatment. Globulins from either species had similar digestibilities (approximately 70%). Proteins in the soluble fraction of cooked beans were more digestible than those in the insoluble fraction, which contained the bulk of the proteins. Hemagglutination assay and trypsin inhibitor determination indicated that after the thermal treatment only very low, nonharmful, levels of both lectin and inhibitor remained. Faba bean contained more polyphenols than common bean samples, with most of the polyphenols being bound to globulins. However, protein-bound polyphenols were markedly decreased after cooking. SDS-PAGE characterization of the gastrointestinal digesta of globulins and amino acid analysis of undigested proteins of whole cooked common bean and faba bean suggested that it is mainly the structural properties of the storage proteins and not their binding of polyphenols, which determines the extent of protein aggregation on autoclaving and may therefore be responsible for their low digestibility. PMID:10725143

  11. Requirement of nucleotide exchange factor for Ypt1 GTPase mediated protein transport.

    PubMed

    Jones, S; Litt, R J; Richardson, C J; Segev, N

    1995-09-01

    Small GTPases of the rab family are involved in the regulation of vesicular transport. It is believed that cycling between the GTP- and GDP-bound forms, and accessory factors regulating this cycling are crucial for rab function. However, an essential role for rab nucleotide exchange factors has not yet been demonstrated. In this report we show the requirement of nucleotide exchange factor activity for Ypt1 GTPase mediated protein transport. The Ypt1 protein, a member of the rab family, plays a role in targeting vesicles to the acceptor compartment and is essential for the first two steps of the yeast secretory pathway. We use two YPT1 dominant mutations that contain alterations in a highly conserved GTP-binding domain, N121I and D124N. YPT1-D124N is a novel mutation that encodes a protein with nucleotide specificity modified from guanine to xanthine. This provides a tool for the study of an individual rab GTPase in crude extracts: a xanthosine triphosphate (XTP)-dependent conditional dominant mutation. Both mutations confer growth inhibition and a block in protein secretion when expressed in vivo. The purified mutant proteins do not bind either GDP or GTP. Moreover, they completely inhibit the ability of the exchange factor to stimulate nucleotide exchange for wild type Ypt1 protein, and are potent inhibitors of ER to Golgi transport in vitro at the vesicle targeting step. The inhibitory effects of the Ypt1-D124N mutant protein on both nucleotide exchange activity and protein transport in vitro can be relieved by XTP, indicating that it is the nucleotide-free form of the mutant protein that is inhibitory. These results suggest that the dominant mutant proteins inhibit protein transport by sequestering the exchange factor from the wild type Ypt1 protein, and that this factor has an essential role in vesicular transport.

  12. Insulin-like growth factor binding protein-5 in osteogenesis: Facilitator or Inhibitor?

    PubMed Central

    Mukherjee, Aditi; Rotwein, Peter

    2007-01-01

    The insulin-like growth factors (IGFs) play a central role in controlling somatic growth in mammals and exert anabolic effects on most tissues, including bone. IGF action is mediated by the IGF-I receptor and additionally is regulated by six high-affinity IGF binding proteins (IGFBP-1 through IGFBP-6), of which IGFBP-4 and IGFBP-5 are most abundant in bone. The focus of this brief review is on the role of IGFBP-5 in bone biology. IGFBP-5 has been implicated as a pro-osteogenic factor in several studies but conversely has been shown to act as an inhibitor of bone formation, primarily by interfering with IGF actions on osteoblasts. These potentially contradictory effects of IGFBP-5 in bone are further complicated by observations indicating that IGFBP-5 additionally may function in an IGF-independent way, and may have been accentuated by differences in both experimental design and methodology among published studies. Suggestions are made for a more systematic approach to help discern the true roles of IGFBP-5 in bone physiology. PMID:17317255

  13. Human chorionic gonadotropin promotes expression of protein absorption factors in the intestine of goldfish (Carassius auratus).

    PubMed

    Zhou, Y; Hao, G; Zhong, H; Wu, Q; Lu, S Q; Zhao, Q; Liu, Z

    2015-07-27

    Protein use is crucial for the ovulation and spawning of fish. Currently, limited information is available regarding the expression of protein absorption factors during the breeding seasons of teleosts and thus how various proteins involved in this process is not well-understood. The expression of CDX2, CREB, gluatamate dehydrogenase, LAT2, aminopeptidase N, PepT1, and SP1 were significantly elevated from the non-breeding season to the breeding season in female goldfish, and all proteins except PepT1 and SP1 were elevated in male goldfish. Injection of human chorionic gonadotropin upregulated the expression of all proteins except for aminopeptidase N in female goldfish and SP1 in male goldfish, suggesting a luteinizing hormone-inductive effect on protein absorption factors. Protein use in the intestine is increased during the breeding seasons as a result of increased luteinizing hormone.

  14. Liquid-Liquid Phase Separation in a Dual Variable Domain Immunoglobulin Protein Solution: Effect of Formulation Factors and Protein-Protein Interactions.

    PubMed

    Raut, Ashlesha S; Kalonia, Devendra S

    2015-09-01

    Dual variable domain immunoglobulin proteins (DVD-Ig proteins) are large molecules (MW ∼ 200 kDa) with increased asymmetry because of their extended Y-like shape, which results in increased formulation challenges. Liquid-liquid phase separation (LLPS) of protein solutions into protein-rich and protein-poor phases reduces solution stability at intermediate concentrations and lower temperatures, and is a serious concern in formulation development as therapeutic proteins are generally stored at refrigerated conditions. In the current work, LLPS was studied for a DVD-Ig protein molecule as a function of solution conditions by measuring solution opalescence. LLPS of the protein was confirmed by equilibrium studies and by visually observing under microscope. The protein does not undergo any structural change after phase separation. Protein-protein interactions were measured by light scattering (kD) and Tcloud (temperature that marks the onset of phase separation). There is a good agreement between kD measured in dilute solution with Tcloud measured in the critical concentration range. Results indicate that the increased complexity of the molecule (with respect to size, shape, and charge distribution on the molecule) increases contribution of specific and nonspecific interactions in solution, which are affected by formulation factors, resulting in LLPS for DVD-Ig protein.

  15. Factor VIIa binding to endothelial cell protein C receptor protects vascular barrier integrity in vivo

    PubMed Central

    SUNDARAM, J.; KESHAVA, S.; GOPALAKRISHNAN, R.; ESMON, C. T.; PENDURTHI, U. R.; RAO, L . V. M.

    2014-01-01

    Summary Background Recent studies have shown that factor VIIa binds to endothelial cell protein C receptor (EPCR), a cellular receptor for protein C and activated protein C. At present, the physiologic significance of FVIIa interaction with EPCR in vivo remains unclear. Objective: To investigate whether exogenously administered FVIIa, by binding to EPCR, induces a barrier protective effect in vivo. Methods Lipopolysaccharide (LPS)-induced vascular leakage in the lung and kidney, and vascular endothelial growth factor (VEGF)-induced vascular leakage in the skin, were used to evaluate the FVIIa-induced barrier protective effect. Wild-type, EPCR-deficient, EPCR-overexpressing and hemophilia A mice were used in the studies. Results Administration of FVIIa reduced LPS-induced vascular leakage in the lung and kidney; the FVIIa-induced barrier protective effect was attenuated in EPCR-deficient mice. The extent of VEGF-induced vascular leakage in the skin was highly dependent on EPCR expression levels. Therapeutic concentrations of FVIIa attenuated VEGF-induced vascular leakage in control mice but not in EPCR-deficient mice. Blockade of FVIIa binding to EPCR with a blocking mAb completely attenuated the FVIIa-induced barrier protective effect. Similarly, administration of protease-activated receptor 1 antagonist blocked the FVIIa-induced barrier protective effect. Hemophilic mice showed increased vascular permeability, and administration of therapeutic concentrations of FVIIa improved barrier integrity in these mice. Conclusions This is the first study to demonstrate that FVIIa binding to EPCR leads to a barrier protective effect in vivo. This finding may have clinical relevance, as it indicates additional advantages of using FVIIa in treating hemophilic patients. PMID:24977291

  16. The La protein counteracts cisplatin-induced cell death by stimulating protein synthesis of anti-apoptotic factor Bcl2

    PubMed Central

    Heise, Tilman; Kota, Venkatesh; Brock, Alexander; Morris, Amanda B.; Rodriguez, Reycel M.; Zierk, Avery W.; Howe, Philip H.; Sommer, Gunhild

    2016-01-01

    Up-regulation of anti-apoptotic factors is a critical mechanism of cancer cell resistance and often counteracts the success of chemotherapeutic treatment. Herein, we identified the cancer-associated RNA-binding protein La as novel factor contributing to cisplatin resistance. Our data demonstrate that depletion of the RNA-binding protein La in head and neck squamous cell carcinoma cells (HNSCC) increases the sensitivity toward cisplatin-induced cell death paralleled by reduced expression of the anti-apoptotic factor Bcl2. Furthermore, it is shown that transient expression of Bcl2 in La-depleted cells protects against cisplatin-induced cell death. By dissecting the underlying mechanism we report herein, that the La protein is required for Bcl2 protein synthesis in cisplatin-treated cells. The RNA chaperone La binds in close proximity to the authentic translation start site and unwinds a secondary structure embedding the authentic AUG. Altogether, our data support a novel model, whereby cancer-associated La protein contributes to cisplatin resistance by stimulating the translation of anti-apoptotic factor Bcl2 in HNSCC cells. PMID:27105491

  17. Interaction between Major Nitrogen Regulatory Protein NIT2 and Pathway-Specific Regulatory Factor NIT4 Is Required for Their Synergistic Activation of Gene Expression in Neurospora crassa

    PubMed Central

    Feng, Bo; Marzluf, George A.

    1998-01-01

    In Neurospora crassa, the major nitrogen regulatory protein, NIT2, a member of the GATA family of transcription factors, controls positively the expression of numerous genes which specify nitrogen catabolic enzymes. Expression of the highly regulated structural gene nit-3, which encodes nitrate reductase, is dependent upon a synergistic interaction of NIT2 with a pathway-specific control protein, NIT4, a member of the GAL4 family of fungal regulatory factors. The NIT2 and NIT4 proteins both bind at specific recognition elements in the nit-3 promoter, but, in addition, we show that a direct protein-protein interaction between NIT2 and NIT4 is essential for optimal expression of the nit-3 structural gene. Neurospora possesses at least five different GATA factors which control different areas of cellular function, but which have a similar DNA binding specificity. Significantly, only NIT2, of the several Neurospora GATA factors examined, interacts with NIT4. We propose that protein-protein interactions of the individual GATA factors with additional pathway-specific regulatory factors determine each of their specific regulatory functions. PMID:9632783

  18. Potential regulation of GnRH gene by a steroidogenic factor-1-like protein.

    PubMed

    Corley, D R; Li, X; Lei, Z M; Rao, C V

    2000-08-01

    Steroidogenic factor-1 (SF-1) is a member of an orphan nuclear hormone receptor superfamily. It plays a critical role in the development and function of the hypothalamic-pituitary-gonadal and adrenal axis. However, whether SF-1 can regulate transcription of gonadotrophin-releasing hormone (GnRH) gene is not known. To examine this possibility, we first over-expressed SF-1 and found that it not only decreased steady state GnRH messenger ribonucleic acid (mRNA) levels but also reduced its promoter activity in GT1-7 neurons. The inhibitory effect of SF-1 was lost when the 5'-flanking region of GnRH gene containing two distal (-1479 to -1474 bp and -1059 to -1054 bp) hexamers was deleted. Gel mobility shift assays showed that GT1-7 cell nuclear extracts contained a protein that formed a specific complex with synthetic oligonucleotides containing the two distal hexamers or a consensus SF-1 binding sequence. The migration of this complex was, however, slower than the complex formed with MA-10 cell nuclear extracts which were shown to contain a 53 kDa SF-1 protein. The addition of anti-SF-1 antibody supershifted the complex formed with MA-10, but not with GT1-7 cell nuclear extracts. The same antibody, however, detected a 60 kDa protein and immunostained nuclei of GT1-7 neurons. These results are consistent with GT1-7 neurons containing an SF-1-like protein that can bind to the distal hexamer sequences in the 5'-flanking region of rat GnRH gene to inhibit its transcription.

  19. Models for the activation pathway of epidermal growth factor receptor protein-tyrosine kinase

    SciTech Connect

    Campion, S.R.; Niyogi, S.K. )

    1991-03-15

    Activation of the epidermal growth factor (EGF) receptor's intrinsic protein-tyrosine kinase activity, which occurs upon formation of the receptor-ligand complex, is the critical regulatory event affecting the subsequent EGF-dependent cellular responses leading to DNA synthesis and cell proliferation. The molecular mechanism by which EGF-dependent activation of receptor kinase activity takes place is not clearly understood. In this study, the growth factor-dependent activation of the EGF receptor tyrosine kinase was examined in vitro using detergent-solubilized, partially purified GEF receptors from A5431 human epidermoid carcinoma cells. Evaluation of the cooperativity observed in the EGF-dependent activation of soluble receptor tyrosine kinase would suggest a mechanism requiring the binding of the EGF peptide to both ligand binding sites on a receptor dimer to induce full receptor kinase activity. Equations describing potential cooperative kinase activation pathways have been examined. The theoretical system which best simulates the allosteric regulation observed in the experimental kinase activation data is that describing multiple essential activation. In addition, studies using mutant analogs of the EGF peptide ligand appear to confirm the requirement for an essential conformational change in the receptor-ligand complex to activate the receptor kinase activity. Several mutant growth factor analogues are able to occupy the ligand binding sites on the receptor without inducing the fully active receptor conformation.

  20. Binding Mode Analysis of Zerumbone to Key Signal Proteins in the Tumor Necrosis Factor Pathway

    PubMed Central

    Fatima, Ayesha; Abdul, Ahmad Bustamam Hj.; Abdullah, Rasedee; Karjiban, Roghayeh Abedi; Lee, Vannajan Sanghiran

    2015-01-01

    Breast cancer is the second most common cancer among women worldwide. Several signaling pathways have been implicated as causative and progression agents. The tumor necrosis factor (TNF) α protein plays a dual role in promoting and inhibiting cancer depending largely on the pathway initiated by the binding of the protein to its receptor. Zerumbone, an active constituent of Zingiber zerumbet, Smith, is known to act on the tumor necrosis factor pathway upregulating tumour necrosis factor related apoptosis inducing ligand (TRAIL) death receptors and inducing apoptosis in cancer cells. Zerumbone is a sesquiterpene that is able to penetrate into the hydrophobic pockets of proteins to exert its inhibiting activity with several proteins. We found a good binding with the tumor necrosis factor, kinase κB (IKKβ) and the Nuclear factor κB (NF-κB) component proteins along the TNF pathway. Our results suggest that zerumbone can exert its apoptotic activities by inhibiting the cytoplasmic proteins. It inhibits the IKKβ kinase that activates the NF-κB and also binds to the NF-κB complex in the TNF pathway. Blocking both proteins can lead to inhibition of cell proliferating proteins to be downregulated and possibly ultimate induction of apoptosis. PMID:25629232

  1. Low edge safety factor operation and passive disruption avoidance in current carrying plasmas by the addition of stellarator rotational transform

    NASA Astrophysics Data System (ADS)

    Pandya, M. D.; ArchMiller, M. C.; Cianciosa, M. R.; Ennis, D. A.; Hanson, J. D.; Hartwell, G. J.; Hebert, J. D.; Herfindal, J. L.; Knowlton, S. F.; Ma, X.; Massidda, S.; Maurer, D. A.; Roberds, N. A.; Traverso, P. J.

    2015-11-01

    Low edge safety factor operation at a value less than two ( q (a )=1 /ι̷tot(a )<2 ) is routine on the Compact Toroidal Hybrid device with the addition of sufficient external rotational transform. Presently, the operational space of this current carrying stellarator extends down to q (a )=1.2 without significant n = 1 kink mode activity after the initial plasma current rise phase of the discharge. The disruption dynamics of these low edge safety factor plasmas depend upon the fraction of helical field rotational transform from external stellarator coils to that generated by the plasma current. We observe that with approximately 10% of the total rotational transform supplied by the stellarator coils, low edge q disruptions are passively suppressed and avoided even though q(a) < 2. When the plasma does disrupt, the instability precursors measured and implicated as the cause are internal tearing modes with poloidal, m, and toroidal, n, helical mode numbers of m /n =3 /2 and 4/3 observed on external magnetic sensors and m /n =1 /1 activity observed on core soft x-ray emissivity measurements. Even though the edge safety factor passes through and becomes much less than q(a) < 2, external n = 1 kink mode activity does not appear to play a significant role in the disruption phenomenology observed.

  2. Structural basis for the requirement of additional factors for MLL1 SET domain activity and recognition of epigenetic marks.

    PubMed

    Southall, Stacey M; Wong, Poon-Sheng; Odho, Zain; Roe, S Mark; Wilson, Jon R

    2009-01-30

    The mixed-lineage leukemia protein MLL1 is a transcriptional regulator with an essential role in early development and hematopoiesis. The biological function of MLL1 is mediated by the histone H3K4 methyltransferase activity of the carboxyl-terminal SET domain. We have determined the crystal structure of the MLL1 SET domain in complex with cofactor product AdoHcy and a histone H3 peptide. This structure indicates that, in order to form a well-ordered active site, a highly variable but essential component of the SET domain must be repositioned. To test this idea, we compared the effect of the addition of MLL complex members on methyltransferase activity and show that both RbBP5 and Ash2L but not Wdr5 stimulate activity. Additionally, we have determined the effect of posttranslational modifications on histone H3 residues downstream and upstream from the target lysine and provide a structural explanation for why H3T3 phosphorylation and H3K9 acetylation regulate activity. PMID:19187761

  3. Yin Yang 1: a multifaceted protein beyond a transcription factor.

    PubMed

    Deng, Zhiyong; Cao, Paul; Wan, Mei Mei; Sui, Guangchao

    2010-01-01

    As a transcription factor, Yin Yang 1 (YY1) regulates the transcription of a dazzling list of genes and the number of its targets still mounts. Recent studies revealed that YY1 possesses functions independent of its DNA binding activity and its regulatory role in tumorigenesis has started to emerge.

  4. Structure of the Get3 targeting factor in complex with its membrane protein cargo

    PubMed Central

    Mateja, Agnieszka; Paduch, Marcin; Chang, Hsin-Yang; Szydlowska, Anna; Kossiakoff, Anthony A.; Hegde, Ramanujan S.; Keenan, Robert J.

    2015-01-01

    Tail-anchored (TA) proteins are a physiologically important class of membrane proteins targeted to the endoplasmic reticulum by the conserved GET pathway. During transit, their hydrophobic transmembrane domains (TMDs) are chaperoned by the cytosolic targeting factor Get3, but the molecular nature of the functional Get3-TA protein targeting complex remains unknown. We reconstituted the physiologic assembly pathway for a functional targeting complex and showed that it comprises a TA protein bound to a Get3 homodimer. Crystal structures of Get3 bound to different TA proteins showed an α-helical TMD occupying a hydrophobic groove that spans the Get3 homodimer. Our data elucidate the mechanism of TA protein recognition and shielding by Get3, and suggest general principles of hydrophobic domain chaperoning by cellular targeting factors. PMID:25745174

  5. Sequence Analysis of Hypothetical Proteins from Helicobacter pylori 26695 to Identify Potential Virulence Factors

    PubMed Central

    Naqvi, Ahmad Abu Turab; Anjum, Farah; Khan, Faez Iqbal; Islam, Asimul; Ahmad, Faizan

    2016-01-01

    Helicobacter pylori is a Gram-negative bacteria that is responsible for gastritis in human. Its spiral flagellated body helps in locomotion and colonization in the host environment. It is capable of living in the highly acidic environment of the stomach with the help of acid adaptive genes. The genome of H. pylori 26695 strain contains 1,555 coding genes that encode 1,445 proteins. Out of these, 340 proteins are characterized as hypothetical proteins (HP). This study involves extensive analysis of the HPs using an established pipeline which comprises various bioinformatics tools and databases to find out probable functions of the HPs and identification of virulence factors. After extensive analysis of all the 340 HPs, we found that 104 HPs are showing characteristic similarities with the proteins with known functions. Thus, on the basis of such similarities, we assigned probable functions to 104 HPs with high confidence and precision. All the predicted HPs contain representative members of diverse functional classes of proteins such as enzymes, transporters, binding proteins, regulatory proteins, proteins involved in cellular processes and other proteins with miscellaneous functions. Therefore, we classified 104 HPs into aforementioned functional groups. During the virulence factors analysis of the HPs, we found 11 HPs are showing significant virulence. The identification of virulence proteins with the help their predicted functions may pave the way for drug target estimation and development of effective drug to counter the activity of that protein. PMID:27729842

  6. Functional and structural properties of a novel protein and virulence factor (Protein sHIP) in Streptococcus pyogenes.

    PubMed

    Wisniewska, Magdalena; Happonen, Lotta; Kahn, Fredrik; Varjosalo, Markku; Malmström, Lars; Rosenberger, George; Karlsson, Christofer; Cazzamali, Giuseppe; Pozdnyakova, Irina; Frick, Inga-Maria; Björck, Lars; Streicher, Werner; Malmström, Johan; Wikström, Mats

    2014-06-27

    Streptococcus pyogenes is a significant bacterial pathogen in the human population. The importance of virulence factors for the survival and colonization of S. pyogenes is well established, and many of these factors are exposed to the extracellular environment, enabling bacterial interactions with the host. In the present study, we quantitatively analyzed and compared S. pyogenes proteins in the growth medium of a strain that is virulent to mice with a non-virulent strain. Particularly, one of these proteins was present at significantly higher levels in stationary growth medium from the virulent strain. We determined the three-dimensional structure of the protein that showed a unique tetrameric organization composed of four helix-loop-helix motifs. Affinity pull-down mass spectrometry analysis in human plasma demonstrated that the protein interacts with histidine-rich glycoprotein (HRG), and the name sHIP (streptococcal histidine-rich glycoprotein-interacting protein) is therefore proposed. HRG has antibacterial activity, and when challenged by HRG, sHIP was found to rescue S. pyogenes bacteria. This and the finding that patients with invasive S. pyogenes infection respond with antibody production against sHIP suggest a role for the protein in S. pyogenes pathogenesis.

  7. Functional and Structural Properties of a Novel Protein and Virulence Factor (Protein sHIP) in Streptococcus pyogenes *

    PubMed Central

    Wisniewska, Magdalena; Happonen, Lotta; Kahn, Fredrik; Varjosalo, Markku; Malmström, Lars; Rosenberger, George; Karlsson, Christofer; Cazzamali, Giuseppe; Pozdnyakova, Irina; Frick, Inga-Maria; Björck, Lars; Streicher, Werner; Malmström, Johan; Wikström, Mats

    2014-01-01

    Streptococcus pyogenes is a significant bacterial pathogen in the human population. The importance of virulence factors for the survival and colonization of S. pyogenes is well established, and many of these factors are exposed to the extracellular environment, enabling bacterial interactions with the host. In the present study, we quantitatively analyzed and compared S. pyogenes proteins in the growth medium of a strain that is virulent to mice with a non-virulent strain. Particularly, one of these proteins was present at significantly higher levels in stationary growth medium from the virulent strain. We determined the three-dimensional structure of the protein that showed a unique tetrameric organization composed of four helix-loop-helix motifs. Affinity pull-down mass spectrometry analysis in human plasma demonstrated that the protein interacts with histidine-rich glycoprotein (HRG), and the name sHIP (streptococcal histidine-rich glycoprotein-interacting protein) is therefore proposed. HRG has antibacterial activity, and when challenged by HRG, sHIP was found to rescue S. pyogenes bacteria. This and the finding that patients with invasive S. pyogenes infection respond with antibody production against sHIP suggest a role for the protein in S. pyogenes pathogenesis. PMID:24825900

  8. New member of the trefoil factor family of proteins is an alpha-macroglobulin protease inhibitor.

    PubMed

    Thøgersen, Ida B; Hammes, Stephen R; Rubenstein, David S; Pizzo, Salvatore V; Valnickova, Zuzana; Enghild, Jan J

    2002-07-29

    The amino acid sequence of the monomeric alpha-macroglobulin (alphaM) from the American bullfrog, Rana catesbiana, was determined. The mature protein consisted of 1469 amino acid residues and shared sequence identity with other members of the alphaM family of protein. The central portion of the frog monomeric alphaM contained Cys residues positioned analogously to the Cys residues in human alpha(2)-macroglobulin (alpha(2)M), known to be involved in disulfide bridges. Additionally, the frog monomeric alphaM contained six Cys residues in a approximately 60 residue COOH-terminal extension not present in previously characterized alphaMs. The spacing of the Cys residues and the overall sequence identity of this COOH-terminal extension were consistent with a trefoil motif. This is the first time a member of the trefoil factor family has been identified in the circulatory system. The "bait region" was located between Arg(675)-Lys(685) and contained mainly basic amino acid residues. The COOH-terminal receptor-binding domain was not exposed prior to proteolysis of this highly susceptible region. The proximity of the receptor-binding and trefoil domains implied that the trefoil domain is similarly concealed before bait region cleavage. PMID:12147353

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

    PubMed

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

    2016-04-01

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

  10. Early decrease in dietary protein:energy ratio by fat addition and ontogenetic changes in muscle growth mechanisms of rainbow trout: short- and long-term effects.

    PubMed

    Alami-Durante, Hélène; Cluzeaud, Marianne; Duval, Carine; Maunas, Patrick; Girod-David, Virginia; Médale, Françoise

    2014-09-14

    As the understanding of the nutritional regulation of muscle growth mechanisms in fish is fragmentary, the present study aimed to (1) characterise ontogenetic changes in muscle growth-related genes in parallel to changes in muscle cellularity; (2) determine whether an early decrease in dietary protein:energy ratio by fat addition affects the muscle growth mechanisms of rainbow trout (Oncorhynchus mykiss) alevins; and (3) determine whether this early feeding of a high-fat (HF) diet to alevins had a long-term effect on muscle growth processes in juveniles fed a commercial diet. Developmental regulation of hyperplasia and hypertrophy was evidenced at the molecular (expression of myogenic regulatory factors, proliferating cell nuclear antigen and myosin heavy chains (MHC)) and cellular (number and diameter of white muscle fibres) levels. An early decrease in dietary protein:energy ratio by fat addition stimulated the body growth of alevins but led to a fatty phenotype, with accumulation of lipids in the anterior part, and less caudal muscle when compared at similar body weights, due to a decrease in both the white muscle hyperplasia and maximum hypertrophy of white muscle fibres. These HF diet-induced cellular changes were preceded by a very rapid down-regulation of the expression of fast-MHC. The present study also demonstrated that early dietary composition had a long-term effect on the subsequent muscle growth processes of juveniles fed a commercial diet for 3 months. When compared at similar body weights, initially HF diet-fed juveniles indeed had a lower mean diameter of white muscle fibres, a smaller number of large white muscle fibres, and lower expression levels of MyoD1 and myogenin. These findings demonstrated the strong effect of early feed composition on the muscle growth mechanisms of trout alevins and juveniles.

  11. Heat-induced Accumulation of Chloroplast Protein Synthesis Elongation Factor, EF-TU, in Winter Wheat

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Chloroplast protein synthesis elongation factor, EF-Tu, has been implicated in heat tolerance in maize (Zea mays L.). Chloroplast EF-Tu is highly conserved, and it is possible that this protein may be of importance to heat tolerance in other species including wheat (Triticum aestivum L.). In this ...

  12. Networks of Host Factors that Interact with NS1 Protein of Influenza A Virus

    PubMed Central

    Thulasi Raman, Sathya N.; Zhou, Yan

    2016-01-01

    Pigs are an important host of influenza A viruses due to their ability to generate reassortant viruses with pandemic potential. NS1 protein of influenza A viruses is a key virulence factor and a major antagonist of innate immune responses. It is also involved in enhancing viral mRNA translation and regulation of virus replication. Being a protein with pleiotropic functions, NS1 has a variety of cellular interaction partners. Hence, studies on swine influenza viruses (SIV) and identification of swine influenza NS1-interacting host proteins is of great interest. Here, we constructed a recombinant SIV carrying a Strep-tag in the NS1 protein and infected primary swine respiratory epithelial cells (SRECs) with this virus. The Strep-tag sequence in the NS1 protein enabled us to purify intact, the NS1 protein and its interacting protein complex specifically. We identified cellular proteins present in the purified complex by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and generated a dataset of these proteins. 445 proteins were identified by LC-MS/MS and among them 192 proteins were selected by setting up a threshold based on MS parameters. The selected proteins were analyzed by bioinformatics and were categorized as belonging to different functional groups including translation, RNA processing, cytoskeleton, innate immunity, and apoptosis. Protein interaction networks were derived using these data and the NS1 interactions with some of the specific host factors were verified by immunoprecipitation. The novel proteins and the networks revealed in our study will be the potential candidates for targeted study of the molecular interaction of NS1 with host proteins, which will provide insights into the identification of new therapeutic targets to control influenza infection and disease pathogenesis. PMID:27199973

  13. Quantitative Proteomics Identifies Serum Response Factor Binding Protein 1 as a Host Factor for Hepatitis C Virus Entry.

    PubMed

    Gerold, Gisa; Meissner, Felix; Bruening, Janina; Welsch, Kathrin; Perin, Paula M; Baumert, Thomas F; Vondran, Florian W; Kaderali, Lars; Marcotrigiano, Joseph; Khan, Abdul G; Mann, Matthias; Rice, Charles M; Pietschmann, Thomas

    2015-08-01

    Hepatitis C virus (HCV) enters human hepatocytes through a multistep mechanism involving, among other host proteins, the virus receptor CD81. How CD81 governs HCV entry is poorly characterized, and CD81 protein interactions after virus binding remain elusive. We have developed a quantitative proteomics protocol to identify HCV-triggered CD81 interactions and found 26 dynamic binding partners. At least six of these proteins promote HCV infection, as indicated by RNAi. We further characterized serum response factor binding protein 1 (SRFBP1), which is recruited to CD81 during HCV uptake and supports HCV infection in hepatoma cells and primary human hepatocytes. SRFBP1 facilitates host cell penetration by all seven HCV genotypes, but not of vesicular stomatitis virus and human coronavirus. Thus, SRFBP1 is an HCV-specific, pan-genotypic host entry factor. These results demonstrate the use of quantitative proteomics to elucidate pathogen entry and underscore the importance of host protein-protein interactions during HCV invasion. PMID:26212323

  14. Inhibition of protein kinase C catalytic activity by additional regions within the human protein kinase Calpha-regulatory domain lying outside of the pseudosubstrate sequence.

    PubMed Central

    Kirwan, Angie F; Bibby, Ashley C; Mvilongo, Thierry; Riedel, Heimo; Burke, Thomas; Millis, Sherri Z; Parissenti, Amadeo M

    2003-01-01

    The N-terminal pseudosubstrate site within the protein kinase Calpha (PKCalpha)-regulatory domain has long been regarded as the major determinant for autoinhibition of catalytic domain activity. Previously, we observed that the PKC-inhibitory capacity of the human PKCalpha-regulatory domain was only reduced partially on removal of the pseudosubstrate sequence [Parissenti, Kirwan, Kim, Colantonio and Schimmer (1998) J. Biol. Chem. 273, 8940-8945]. This finding suggested that one or more additional region(s) contributes to the inhibition of catalytic domain activity. To assess this hypothesis, we first examined the PKC-inhibitory capacity of a smaller fragment of the PKCalpha-regulatory domain consisting of the C1a, C1b and V2 regions [GST-Ralpha(39-177): this protein contained the full regulatory domain of human PKCalpha fused to glutathione S-transferase (GST), but lacked amino acids 1-38 (including the pseudosubstrate sequence) and amino acids 178-270 (including the C2 region)]. GST-Ralpha(39-177) significantly inhibited PKC in a phorbol-independent manner and could not bind the peptide substrate used in our assays. These results suggested that a region within C1/V2 directly inhibits catalytic domain activity. Providing further in vivo support for this hypothesis, we found that expression of N-terminally truncated pseudosubstrate-less bovine PKCalpha holoenzymes in yeast was capable of inhibiting cell growth in a phorbol-dependent manner. This suggested that additional autoinhibitory force(s) remained within the truncated holoenzymes that could be relieved by phorbol ester. Using tandem PCR-mediated mutagenesis, we observed that mutation of amino acids 33-86 within GST-Ralpha(39-177) dramatically reduced its PKC-inhibitory capacity when protamine was used as substrate. Mutagenesis of a broad range of sequences within C2 (amino acids 159-242) also significantly reduced PKC-inhibitory capacity. Taken together, these observations support strongly the existence of

  15. Divergent Protein Motifs Direct Elongation Factor P-Mediated Translational Regulation in Salmonella enterica and Escherichia coli

    PubMed Central

    Hersch, Steven J.; Wang, Mengchi; Zou, S. Betty; Moon, Kyung-Mee; Foster, Leonard J.; Ibba, Michael; Navarre, William Wiley

    2013-01-01

    ABSTRACT Elongation factor P (EF-P) is a universally conserved bacterial translation factor homologous to eukaryotic/archaeal initiation factor 5A. In Salmonella, deletion of the efp gene results in pleiotropic phenotypes, including increased susceptibility to numerous cellular stressors. Only a limited number of proteins are affected by the loss of EF-P, and it has recently been determined that EF-P plays a critical role in rescuing ribosomes stalled at PPP and PPG peptide sequences. Here we present an unbiased in vivo investigation of the specific targets of EF-P by employing stable isotope labeling of amino acids in cell culture (SILAC) to compare the proteomes of wild-type and efp mutant Salmonella. We found that metabolic and motility genes are prominent among the subset of proteins with decreased production in the Δefp mutant. Furthermore, particular tripeptide motifs are statistically overrepresented among the proteins downregulated in efp mutant strains. These include both PPP and PPG but also additional motifs, such as APP and YIRYIR, which were confirmed to induce EF-P dependence by a translational fusion assay. Notably, we found that many proteins containing polyproline motifs are not misregulated in an EF-P-deficient background, suggesting that the factors that govern EF-P-mediated regulation are complex. Finally, we analyzed the specific region of the PoxB protein that is modulated by EF-P and found that mutation of any residue within a specific GSCGPG sequence eliminates the requirement for EF-P. This work expands the known repertoire of EF-P target motifs and implicates factors beyond polyproline motifs that are required for EF-P-mediated regulation. PMID:23611909

  16. Deep proteomics of mouse skeletal muscle enables quantitation of protein isoforms, metabolic pathways, and transcription factors.

    PubMed

    Deshmukh, Atul S; Murgia, Marta; Nagaraj, Nagarjuna; Treebak, Jonas T; Cox, Jürgen; Mann, Matthias

    2015-04-01

    Skeletal muscle constitutes 40% of individual body mass and plays vital roles in locomotion and whole-body metabolism. Proteomics of skeletal muscle is challenging because of highly abundant contractile proteins that interfere with detection of regulatory proteins. Using a state-of-the art MS workflow and a strategy to map identifications from the C2C12 cell line model to tissues, we identified a total of 10,218 proteins, including skeletal muscle specific transcription factors like myod1 and myogenin and circadian clock proteins. We obtain absolute abundances for proteins expressed in a muscle cell line and skeletal muscle, which should serve as a valuable resource. Quantitation of protein isoforms of glucose uptake signaling pathways and in glucose and lipid metabolic pathways provides a detailed metabolic map of the cell line compared with tissue. This revealed unexpectedly complex regulation of AMP-activated protein kinase and insulin signaling in muscle tissue at the level of enzyme isoforms.

  17. Deep Proteomics of Mouse Skeletal Muscle Enables Quantitation of Protein Isoforms, Metabolic Pathways, and Transcription Factors*

    PubMed Central

    Deshmukh, Atul S.; Murgia, Marta; Nagaraj, Nagarjuna; Treebak, Jonas T.; Cox, Jürgen; Mann, Matthias

    2015-01-01

    Skeletal muscle constitutes 40% of individual body mass and plays vital roles in locomotion and whole-body metabolism. Proteomics of skeletal muscle is challenging because of highly abundant contractile proteins that interfere with detection of regulatory proteins. Using a state-of-the art MS workflow and a strategy to map identifications from the C2C12 cell line model to tissues, we identified a total of 10,218 proteins, including skeletal muscle specific transcription factors like myod1 and myogenin and circadian clock proteins. We obtain absolute abundances for proteins expressed in a muscle cell line and skeletal muscle, which should serve as a valuable resource. Quantitation of protein isoforms of glucose uptake signaling pathways and in glucose and lipid metabolic pathways provides a detailed metabolic map of the cell line compared with tissue. This revealed unexpectedly complex regulation of AMP-activated protein kinase and insulin signaling in muscle tissue at the level of enzyme isoforms. PMID:25616865

  18. The transcription factor X-box binding protein-1 in neurodegenerative diseases

    PubMed Central

    2014-01-01

    Endoplasmic reticulum (ER) is the cellular compartment where secreted and integral membrane proteins are folded and matured. The accumulation of unfolded or misfolded proteins triggers a stress that is physiologically controlled by an adaptative protective response called Unfolded Protein Response (UPR). UPR is primordial to induce a quality control response and to restore ER homeostasis. When this adaptative response is defective, protein aggregates overwhelm cells and affect, among other mechanisms, synaptic function, signaling transduction and cell survival. Such dysfunction likely contributes to several neurodegenerative diseases that are indeed characterized by exacerbated protein aggregation, protein folding impairment, increased ER stress and UPR activation. This review briefly documents various aspects of the biology of the transcription factor XBP-1 (X-box Binding Protein-1) and summarizes recent findings concerning its putative contribution to the altered UPR response observed in various neurodegenerative disorders including Parkinson’s and Alzheimer’s diseases. PMID:25216759

  19. Uncoupling Protein 2 Polymorphisms as Risk Factors for Neural Tube Defects

    PubMed Central

    Mitchell, Adam; Pangilinan, Faith; VanderMeer, Julie; Molloy, Anne M.; Troendle, James; Conley, Mary; Kirke, Peadar N.; Scott, John M.; Brody, Lawrence C.; Mills, James L.

    2008-01-01

    BACKGROUND: Both environmental and genetic factors are involved in the etiology of neural tube defects (NTDs). Inadequate folate intake and obesity are important environmental risk factors. Several folate-related genetic variants have been identified as risk factors; however, little is known about how genetic variants relate to the increased risk seen in obese women. Uncoupling Protein 2 (UCP2) is an attractive candidate to screen for NTD risk because of its possible role in obesity as well as energy metabolism, type-2 diabetes, and the regulation of reactive oxygen species. Interestingly, a previous study found that a common UCP2 compound homozygous genotype was associated with a threefold increase in NTD risk. METHODS: We evaluated three polymorphisms, −866G>A, A55V, and the 3′UTR 45bp insertion/deletion, as risk factors for NTDs in Irish NTD cases (N=169), their mothers (N=163), their fathers (N=167) and normal control subjects (N=332). RESULTS: Allele and genotype frequencies were not significantly different when comparing NTD mothers, NTD fathers, or affected children to controls. Additionally, the previously reported risk genotype (combined homozygosity of 55VV and 3′UTR 45bp deletion/deletion) was not present at a higher frequency in any NTD group when compared to controls. CONCLUSIONS: In our Irish study population, UCP2 polymorphisms do not influence NTD risk. Moreover, the prevalence of this allele in other populations was similar to the Irish prevalence but far lower than reported in the previous NTD study, suggesting that this previous finding of an association with NTDs might have been due to an unrepresentative study sample. PMID:19137581

  20. Hepatitis C virus nonstructural protein-5A activates sterol regulatory element-binding protein-1c through transcription factor Sp1

    SciTech Connect

    Xiang, Zhonghua; Qiao, Ling; Zhou, Yan; Babiuk, Lorne A.; Liu, Qiang

    2010-11-19

    Research highlights: {yields} A chimeric subgenomic HCV replicon expresses HCV-3a NS5A in an HCV-1b backbone. {yields} HCV-3a NS5A increases mature SREBP-1c protein level. {yields} HCV-3a NS5A activates SREBP-1c transcription. {yields} Domain II of HCV-3a NS5A is more effective in SREBP-1c promoter activation. {yields} Transcription factor Sp1 is required for SREBP-1c activation by HCV-3a NS5A. -- Abstract: Steatosis is an important clinical manifestation of hepatitis C virus (HCV) infection. The molecular mechanisms of HCV-associated steatosis are not well understood. Sterol regulatory element-binding protein-1c (SREBP-1c) is a key transcription factor which activates the transcription of lipogenic genes. Here we showed that the nuclear, mature SREBP-1c level increases in the nucleus of replicon cells expressing HCV-3a nonstructural protein-5A (NS5A). We further showed that HCV-3a NS5A up-regulates SREBP-1c transcription. Additional analysis showed that transcriptional factor Sp1 is involved in SREBP-1c activation by HCV-3a NS5A because inhibition of Sp1 activity by mithramycin A or a dominant-negative Sp1 construct abrogated SREBP-1c promoter activation by HCV-3a NS5A. In addition, chromatin immunoprecipitation (ChIP) assay demonstrated enhanced binding of Sp1 on the SREBP-1c promoter in HCV-3a NS5A replicon cells. These results showed that HCV-3a NS5A activates SREBP-1c transcription through Sp1. Taken together, our results suggest that HCV-3a NS5A is a contributing factor for steatosis caused by HCV-3a infection.

  1. Local dynamics of proteins and DNA evaluated from crystallographic B factors.

    PubMed

    Schneider, Bohdan; Gelly, Jean Christophe; de Brevern, Alexandre G; Černý, Jiří

    2014-09-01

    The dynamics of protein and nucleic acid structures is as important as their average static picture. The local molecular dynamics concealed in diffraction images is expressed as so-called B factors. To find out how the crystal-derived B factors represent the dynamic behaviour of atoms and residues of proteins and DNA in their complexes, the distributions of scaled B factors from a carefully curated data set of over 700 protein-DNA crystal structures were analyzed [Schneider et al. (2014), Nucleic Acids Res. 42, 3381-3394]. Amino acids and nucleotides were categorized based on their molecular neighbourhood as solvent-accessible, solvent-inaccessible (i.e. forming the protein core) or lying at protein-protein or protein-DNA interfaces; the backbone and side-chain atoms were analyzed separately. The B factors of two types of crystal-ordered water molecules were also analyzed. The analysis confirmed several expected features of protein and DNA dynamics, but also revealed surprising facts. Solvent-accessible amino acids have B factors that are larger than those of residues at the biomolecular interfaces, and core-forming amino acids are the most restricted in their movement. A unique feature of the latter group is that their side-chain and backbone atoms are restricted in their movement to the same extent; in all other amino-acid groups the side chains are more floppy than the backbone. The low values of the B factors of water molecules bridging proteins with DNA and the very large fluctuations of DNA phosphates are surprising. The features discriminating different types of residues are less pronounced in structures with lower crystallographic resolution. Some of the observed trends are likely to be the consequence of improper refinement protocols that may need to be rectified.

  2. Biochemical characterization of a factor X activator protein purified from Walterinnesia aegyptia venom.

    PubMed

    Khan, Sami U; Al-Saleh, Saad S

    2015-10-01

    Factor X of blood coagulation cascade can be activated by both intrinsic and extrinsic activating complex, trypsin and some kind of snake venom. A factor X activator protein is reported in Elapidae snake venom. The aim of this study was to evaluate biochemical properties of factor X activator protein because of its prospective application in biochemical research and therapeutics. Crude venom was fractionated on a HPLC system Gold 126/1667 using a combination of Protein PAK 125 and Protein PAK 60 Columns. Molecular weight was determined using SDS-PAGE. Walterinnesia aegyptia venom was fractionated into several protein peaks, but procoagulant and factor X activation activity coexisted into peak no.6. It appeared as single band on native PAGE and molecular weight was 60,000 ± 3. Purified up to 37-fold over crude venom. It shortened recalcification time, effect was dose-dependent and strictly Ca(2++)-dependent. Factor X activator seems to be able to activate factor X specifically because it showed no activation activity on human prothrombin, plasminogen, or protein C. It did not hydrolyze factor Xa substrate S-2222, thrombin substrate S-2238, plasmin substrate S-2251 or S-2302 and kalikrein substrate S-2266. It did not hydrolyze synthetic ester benzoyl arginine ethyl ester. Procoagulant activity was completely inhibited by irreversible serine protease inhibitors phenylmethylsulphonyl fluoride and N-p-tosylphenylalanine chloromethyl ketone. This study illustrates that factor X activator from W. aegyptia is though different in many aspects from factor X activators of Viperidae and Crotalidae venoms, but shows several properties identical to factor X activators from Elapidae venoms. PMID:26407136

  3. Insulin-like growth factor-II and insulin-like growth factor-binding proteins in bovine cystic ovarian disease.

    PubMed

    Rey, F; Rodríguez, F M; Salvetti, N R; Palomar, M M; Barbeito, C G; Alfaro, N S; Ortega, H H

    2010-01-01

    Cystic ovarian disease (COD) is one of the most common reproductive disorders of cattle and is considered to have multifactorial aetiology. An accepted hypothesis involves neuroendocrinological dysfunction of the hypothalamic-pituitary-gonadal axis; however, the role of growth factors in COD has not been extensively investigated. The present study examines the potential role of members of the insulin-like growth factor (IGF) family in COD. Expression of genes encoding IGF-II and insulin-like growth factor-binding proteins (IGFBPs) was examined and the distribution of IGF-II within the follicular wall was assessed immunohistochemically. Finally, the concentration of IGF-II protein was determined in follicular fluid. There was increased IGF-II mRNA in the wall of cystic follicles, mainly associated with granulosa cells. Additionally, there was significantly more IGF-II protein in granulosa and theca cells in cystic follicles, but no change in the concentration of IGF-II in follicular fluid. Total IGFBPs, assessed by western blotting, were similar in different structures. However, by discriminating each IGFBP a decrease was detected in IGFBP-2 expression in cystic follicles that may be related to the observed higher expression of IGF-II. In summary, the present study provides evidence to suggest that COD in cattle is associated with modifications in the IGF-II system.

  4. THE CANNABINOID WIN 55,212-2 DECREASES SPECIFICITY PROTEIN (Sp) TRANSCRIPTION FACTORS AND THE ONCOGENIC CAP PROTEIN eIF4E IN COLON CANCER CELLS

    PubMed Central

    Sreevalsan, Sandeep; Safe, Stephen

    2013-01-01

    2,3-Dihydro-5-methyl-3-([morpholinyl]methyl)pyrollo(1,2,3-de)-1,4-benzoxazinyl]-[1-naphthaleny]methanone [WIN 55,212-2 (WIN)] is a synthetic cannabinoid that inhibits RKO, HT-29 and SW480 cell growth, induced apoptosis, and downregulated expression of survivin, cyclin D1, epidermal growth factor receptor (EGFR), vascular endothelial growth factor (VEGF) and its receptor (VEGFR1). WIN also decreased expression of specificity protein (Sp) transcription factors Sp1, Sp3 and Sp4, and this is consistent with the observed downregulation of the aforementioned Sp-regulated genes. In addition, we also observed by RNA interference (RNAi) that the oncogenic cap protein eIF4E was an Sp-regulated gene also downregulated by WIN in colon cancer cells. WIN-mediated repression of Sp proteins was not affected by CB receptor antagonists or by knockdown of the receptor but was attenuated by the phosphatase inhibitor sodium orthovanadate or by knockdown of protein phosphatase 2A (PP2A). WIN-mediated repression of Sp1, Sp3 and Sp4 was due to PP2A-dependent downregulation of microRNA-27a (miR-27a) and induction of miR-27a-regulated ZBTB10 which has previously been characterized as an “Sp repressor”. The results demonstrate that the anticancer activity of WIN is due, in part, to PP2A-dependent disruption of miR-27a:ZBTB10 and ZBTB10-mediated repression of Sp transcription factors and Sp-regulated genes including eIF4E. PMID:24030632

  5. Gelation of protein recovered from whole Antarctic krill (Euphausia superba) by isoelectric solubilization/precipitation as affected by functional additives.

    PubMed

    Chen, Yi-Chen; Jaczynski, Jacek

    2007-03-01

    This study demonstrated that the novel isoelectric solubilization/precipitation can be applied to recover functional muscle protein in a continuous mode from whole Antarctic krill. Protein recovered from whole krill had a much lower ash content than whole krill, suggesting good removal of inedible impurities (shell, appendages, etc.). Lipids were retained to a higher degree with krill protein solubilized at acidic rather than basic pH. The viscoelastic modulus (G') showed that recovered krill protein failed to form heat-induced gel unless beef plasma protein (BPP) was added. Therefore, protease inhibitors are suggested for development of krill-derived products. Even with BPP, the G' decreased between 45 and 55 degrees C. However, krill protein solubilized at acidic pH had a higher decrease of the G' than the protein solubilized at basic pH, likely due to krill endogenous cathepsin L. Krill protein-based gels developed from protein solubilized at basic pH, especially pH 12.0, had better texture (torsion and Kramer tests and texture profile analysis) than acidic counterparts, possibly due to higher proteolysis and denaturation at acidic pH. Gels made from protein solubilized at acidic pH were brighter and whiter likely due to a higher lipid content.

  6. alpha-Factor-mediatd modification of a 32P-labeled protein by MATa cells of Saccharomyces cerevisiae.

    PubMed

    Finkelstein, D B; McAlister, L

    1981-03-10

    Addition of the polypeptide mating pheromone alpha-factor to haploid MATa cells of Saccharomyces cerevisiae results in the modification of a 32P-labeled protein (P17) with an apparent Mr of 17,000 to a form having an apparent Mr of 17,500 (P17). 32P associated with both P17 and P17 exhibits an unusually rapid rate of turnover. The conversion of P17 to P17 precedes the appearance of morphologically abnormal cells and, in contrast to other responses elicited by this pheromone, this change in apparent molecular weight does not require protein synthesis. Upon removal of alpha-factor, the P17/P17 ratio returns to pretreatment levels. PMID:7007388

  7. GATA Factor-G-Protein-Coupled Receptor Circuit Suppresses Hematopoiesis

    PubMed Central

    Gao, Xin; Wu, Tongyu; Johnson, Kirby D.; Lahvic, Jamie L.; Ranheim, Erik A.; Zon, Leonard I.; Bresnick, Emery H.

    2016-01-01

    Summary Hematopoietic stem cells (HSCs) originate from hemogenic endothelium within the aorta-gonad-mesonephros (AGM) region of the mammalian embryo. The relationship between genetic circuits controlling stem cell genesis and multi-potency is not understood. A Gata2 cis element (+9.5) enhances Gata2 expression in the AGM and induces the endothelial to HSC transition. We demonstrated that GATA-2 rescued hematopoiesis in +9.5−/− AGMs. As G-protein-coupled receptors (GPCRs) are the most common targets for FDA-approved drugs, we analyzed the GPCR gene ensemble to identify GATA-2-regulated GPCRs. Of the 20 GATA-2-activated GPCR genes, four were GATA-1-activated, and only Gpr65 expression resembled Gata2. Contrasting with the paradigm in which GATA-2-activated genes promote hematopoietic stem and progenitor cell genesis/function, our mouse and zebrafish studies indicated that GPR65 suppressed hematopoiesis. GPR65 established repressive chromatin at the +9.5 site, restricted occupancy by the activator Scl/TAL1, and repressed Gata2 transcription. Thus, a Gata2 cis element creates a GATA-2-GPCR circuit that limits positive regulators that promote hematopoiesis. PMID:26905203

  8. PAP/HIP protein is an obesogenic factor.

    PubMed

    Secq, Veronique; Mallmann, Cecilia; Gironella, Meritxell; Lopez, Belen; Closa, Daniel; Garcia, Stéphane; Christa, Laurence; Montalto, Giuseppe; Dusetti, Nelson; Iovanna, Juan L

    2014-02-01

    In this article we report the obesogenic role of the acute phase protein PAP/HIP. We found that the transgenic TgPAP/HIP mice develop spontaneous obesity under standard nutritional conditions, with high levels of glucose, leptin, and LDL and low levels of triglycerides and HDL in blood. Accordingly, PAP/HIP-deficient mice are skinny under standard nutritional conditions. We also found that expression of PAP/HIP is induced in intestinal epithelial cells in response to gavage with olive oil and this induction is AG490 sensitive. We demonstrated that incubation of 3T3-L1 preadipocytes with a low concentration as 1 ng/ml of recombinant PAP/HIP results in accelerated BrdU incorporation in vitro. PAP/HIP-dependent adipocytes growth is sensitive to the MEK inhibitor U0126. Finally, patients with severe obesity present higher blood levels of PAP/HIP than non-obese control individuals. Altogether our data suggest that PAP/HIP could be a mediator of fat tissue development, released by the intestine and induced by the presence of food into the gut.

  9. Protein-energy malnutrition: a risk factor for various ailments.

    PubMed

    Batool, Rizwana; Butt, Masood Sadiq; Sultan, Muhammad Tauseef; Saeed, Farhan; Naz, Rabia

    2015-01-01

    The wheel of industrialization that spun throughout the last century resulted in urbanization coupled with modifications in lifestyles and dietary habits. However, the communities living in developing economies are facing many problems related to their diet and health. Amongst, the prevalence of nutritional problems especially protein-energy malnutrition (PEM) and micronutrients deficiencies are the rising issues. Moreover, the immunity or susceptibility to infect-parasitic diseases is also directly linked with the nutritional status of the host. Likewise, disease-related malnutrition that includes an inflammatory component is commonly observed in clinical practice thus affecting the quality of life. The PEM is treatable but early detection is a key for its appropriate management. However, controlling the menace of PEM requires an aggressive partnership between the physician and the dietitian. This review mainly attempts to describe the pathophysiology, prevalence and consequences of PEM and aims to highlight the importance of this clinical syndrome and the recent growth in our understanding of the processes behind its development. Some management strategies/remedies to overcome PEM are also the limelight of the article. In the nutshell, early recognition, prompt management, and robust follow up are critical for best outcomes in preventing and treating PEM.

  10. 25 CFR 39.1101 - Addition of pre-kindergarten as a weight factor to the Indian School Equalization Formula in...

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 25 Indians 1 2012-04-01 2011-04-01 true Addition of pre-kindergarten as a weight factor to the Indian School Equalization Formula in fiscal year 1982. 39.1101 Section 39.1101 Indians BUREAU OF INDIAN... Programs § 39.1101 Addition of pre-kindergarten as a weight factor to the Indian School...

  11. 25 CFR 39.1101 - Addition of pre-kindergarten as a weight factor to the Indian School Equalization Formula in...

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 25 Indians 1 2011-04-01 2011-04-01 false Addition of pre-kindergarten as a weight factor to the Indian School Equalization Formula in fiscal year 1982. 39.1101 Section 39.1101 Indians BUREAU OF INDIAN... Programs § 39.1101 Addition of pre-kindergarten as a weight factor to the Indian School...

  12. 25 CFR 39.1101 - Addition of pre-kindergarten as a weight factor to the Indian School Equalization Formula in...

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 25 Indians 1 2014-04-01 2014-04-01 false Addition of pre-kindergarten as a weight factor to the Indian School Equalization Formula in fiscal year 1982. 39.1101 Section 39.1101 Indians BUREAU OF INDIAN... Programs § 39.1101 Addition of pre-kindergarten as a weight factor to the Indian School...

  13. 25 CFR 39.1101 - Addition of pre-kindergarten as a weight factor to the Indian School Equalization Formula in...

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 25 Indians 1 2010-04-01 2010-04-01 false Addition of pre-kindergarten as a weight factor to the Indian School Equalization Formula in fiscal year 1982. 39.1101 Section 39.1101 Indians BUREAU OF INDIAN... Programs § 39.1101 Addition of pre-kindergarten as a weight factor to the Indian School...

  14. 25 CFR 39.1101 - Addition of pre-kindergarten as a weight factor to the Indian School Equalization Formula in...

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 25 Indians 1 2013-04-01 2013-04-01 false Addition of pre-kindergarten as a weight factor to the Indian School Equalization Formula in fiscal year 1982. 39.1101 Section 39.1101 Indians BUREAU OF INDIAN... Programs § 39.1101 Addition of pre-kindergarten as a weight factor to the Indian School...

  15. Identification of Tetrahymena 14-nm filament-associated protein as elongation factor 1 alpha.

    PubMed

    Kurasawa, Y; Numata, O; Katoh, M; Hirano, H; Chiba, J; Watanabe, Y

    1992-11-01

    Tetrahymena 14-nm filament-forming protein has dual functions as a citrate synthase in mitochondria and as a cytoskeletal protein involved in oral morphogenesis and in pronuclear behavior during conjugation. By immunoblotting using monoclonal and polyclonal antibodies following two-dimensional gel electrophoresis, we demonstrated that the 14-nm filament protein fraction contained two 49-kDa proteins whose isoelectric points were 8.0 and 9.0; a monoclonal antibody (MAb) 26B4 and a polyclonal antibody 49KI reacted only to a pI 8.0 protein, while two other MAbs, 11B6 and 11B8, reacted only to a pI 9.0 protein. From the N-terminal amino acid sequences, the pI 8.0 protein was identified as the previously reported 14-nm filament-forming protein/citrate synthase, but the pI 9.0 protein N-terminal sequence had no similarity with that of the pI 8.0 protein. The pI 9.0 protein is considered to be a 14-nm filament-associated protein since the pI 9.0 protein copurifies with the pI 8.0 protein during two cycles of an assembly and disassembly purification protocol. Cloning and sequencing the pI 9.0 protein gene from a Tetrahymena pyriformis cDNA library, we identified the pI 9.0 protein as elongation factor 1 alpha (EF-1 alpha) based on it sharing 73-76% sequence identity with EF-1 alpha from several species. PMID:1385189

  16. FRS2 proteins recruit intracellular signaling pathways by binding to diverse targets on fibroblast growth factor and nerve growth factor receptors.

    PubMed

    Ong, S H; Guy, G R; Hadari, Y R; Laks, S; Gotoh, N; Schlessinger, J; Lax, I

    2000-02-01

    The docking protein FRS2 was implicated in the transmission of extracellular signals from the fibroblast growth factor (FGF) or nerve growth factor (NGF) receptors to the Ras/mitogen-activated protein kinase signaling cascade. The two members of the FRS2 family, FRS2alpha and FRS2beta, are structurally very similar. Each is composed of an N-terminal myristylation signal, a phosphotyrosine-binding (PTB) domain, and a C-terminal tail containing multiple binding sites for the SH2 domains of the adapter protein Grb2 and the protein tyrosine phosphatase Shp2. Here we show that the PTB domains of both the alpha and beta isoforms of FRS2 bind directly to the FGF or NGF receptors. The PTB domains of the FRS2 proteins bind to a highly conserved sequence in the juxtamembrane region of FGFR1. While FGFR1 interacts with FRS2 constitutively, independent of ligand stimulation and tyrosine phosphorylation, NGF receptor (TrkA) binding to FRS2 is strongly dependent on receptor activation. Complex formation with TrkA is dependent on phosphorylation of Y490, a canonical PTB domain binding site that also functions as a binding site for Shc (NPXpY). Using deletion and alanine scanning mutagenesis as well as peptide competition assays, we demonstrate that the PTB domains of the FRS2 proteins specifically recognize two different primary structures in two different receptors in a phosphorylation-dependent or -independent manner. In addition, NGF-induced tyrosine phosphorylation of FRS2alpha is diminished in cells that overexpress a kinase-inactive mutant of FGFR1. This experiment suggests that FGFR1 may regulate signaling via NGF receptors by sequestering a common key element which both receptors utilize for transmitting their signals. The multiple interactions mediated by FRS2 appear to play an important role in target selection and in defining the specificity of several families of receptor tyrosine kinases. PMID:10629055

  17. Amblyomma americanum tick saliva insulin-like growth factor binding protein-related protein 1 binds insulin but not insulin-like growth factors.

    PubMed

    Radulović, Ž M; Porter, L M; Kim, T K; Bakshi, M; Mulenga, A

    2015-10-01

    Silencing Amblyomma americanum insulin-like growth factor binding protein-related protein 1 (AamIGFBP-rP1) mRNA prevented ticks from feeding to repletion. In this study, we used recombinant (r)AamIGFBP-rP1 in a series of assays to obtain further insight into the role(s) of this protein in tick feeding regulation. Our results suggest that AamIGFBP-1 is an antigenic protein that is apparently exclusively expressed in salivary glands. We found that both males and females secrete AamIGFBP-rP1 into the host during feeding and confirmed that female ticks secrete this protein from within 24-48 h after attachment. Our data suggest that native AamIGFBP-rP1 is a functional insulin binding protein in that both yeast- and insect cell-expressed rAamIGFBP-rP1 bound insulin, but not insulin-like growth factors. When subjected to anti-blood clotting and platelet aggregation assays, rAamIGFBP-rP1 did not have any effect. Unlike human IGFBP-rP1, which is controlled by trypsinization, rAamIGFBP-rP1 is resistant to digestion, suggesting that the tick protein may not be under mammalian host control at the tick feeding site. The majority of tick-borne pathogens are transmitted 48 h after the tick has attached. Thus, the demonstrated antigenicity and secretion into the host within 24-48 h of the tick starting to feed makes AamIGFBP-rP1 an attractive target for antitick vaccine development.

  18. Multimodular biosensors reveal a novel platform for activation of G proteins by growth factor receptors

    PubMed Central

    Midde, Krishna K.; Aznar, Nicolas; Laederich, Melanie B.; Ma, Gary S.; Kunkel, Maya T.; Newton, Alexandra C.; Ghosh, Pradipta

    2015-01-01

    Environmental cues are transmitted to the interior of the cell via a complex network of signaling hubs. Receptor tyrosine kinases (RTKs) and trimeric G proteins are two such major signaling hubs in eukaryotes. Conventionally, canonical signal transduction via trimeric G proteins is thought to be triggered exclusively by G protein-coupled receptors. Here we used molecular engineering to develop modular fluorescent biosensors that exploit the remarkable specificity of bimolecular recognition, i.e., of both G proteins and RTKs, and reveal the workings of a novel platform for activation of G proteins by RTKs in single living cells. Comprised of the unique modular makeup of guanidine exchange factor Gα-interacting vesicle-associated protein (GIV)/girdin, a guanidine exchange factor that links G proteins to a variety of RTKs, these biosensors provide direct evidence that RTK–GIV–Gαi ternary complexes are formed in living cells and that Gαi is transactivated within minutes after growth factor stimulation at the plasma membrane. Thus, GIV-derived biosensors provide a versatile strategy for visualizing, monitoring, and manipulating the dynamic association of Gαi with RTKs for noncanonical transactivation of G proteins in cells and illuminate a fundamental signaling event regulated by GIV during diverse cellular processes and pathophysiologic states. PMID:25713130

  19. Multimodular biosensors reveal a novel platform for activation of G proteins by growth factor receptors.

    PubMed

    Midde, Krishna K; Aznar, Nicolas; Laederich, Melanie B; Ma, Gary S; Kunkel, Maya T; Newton, Alexandra C; Ghosh, Pradipta

    2015-03-01

    Environmental cues are transmitted to the interior of the cell via a complex network of signaling hubs. Receptor tyrosine kinases (RTKs) and trimeric G proteins are two such major signaling hubs in eukaryotes. Conventionally, canonical signal transduction via trimeric G proteins is thought to be triggered exclusively by G protein-coupled receptors. Here we used molecular engineering to develop modular fluorescent biosensors that exploit the remarkable specificity of bimolecular recognition, i.e., of both G proteins and RTKs, and reveal the workings of a novel platform for activation of G proteins by RTKs in single living cells. Comprised of the unique modular makeup of guanidine exchange factor Gα-interacting vesicle-associated protein (GIV)/girdin, a guanidine exchange factor that links G proteins to a variety of RTKs, these biosensors provide direct evidence that RTK-GIV-Gαi ternary complexes are formed in living cells and that Gαi is transactivated within minutes after growth factor stimulation at the plasma membrane. Thus, GIV-derived biosensors provide a versatile strategy for visualizing, monitoring, and manipulating the dynamic association of Gαi with RTKs for noncanonical transactivation of G proteins in cells and illuminate a fundamental signaling event regulated by GIV during diverse cellular processes and pathophysiologic states.

  20. Renal protein synthesis in diabetes mellitus: effects of insulin and insulin-like growth factor I

    SciTech Connect

    Barac-Nieto, M.; Lui, S.M.; Spitzer, A. )

    1991-06-01

    Is increased synthesis of proteins responsible for the hypertrophy of kidney cells in diabetes mellitus Does the lack of insulin, and/or the effect of insulin-like growth factor I (IGFI) on renal tubule protein synthesis play a role in diabetic renal hypertrophy To answer these questions, we determined the rates of 3H-valine incorporation into tubule proteins and the valine-tRNA specific activity, in the presence or absence of insulin and/or IGFI, in proximal tubule suspension isolated from kidneys of streptozotocin diabetic and control rats. The rate of protein synthesis increased, while the stimulatory effects of insulin and IGFI on tubule protein synthesis were reduced, early (96 hours) after induction of experimental diabetes. Thus, hypertrophy of the kidneys in experimental diabetes mellitus is associated with increases in protein synthesis, rather than with decreases in protein degradation. Factor(s) other than the lack of insulin, or the effects of IGFI, must be responsible for the high rate of protein synthesis present in the hypertrophying tubules of diabetic rats.

  1. CHEMOSENSITIZATION BY A NON-APOPTOGENIC HEAT SHOCK PROTEIN 70-BINDING APOPTOSIS INDUCING FACTOR MUTANT

    EPA Science Inventory

    Chemosensitization by a non-apoptogenic heat shock protein 70-binding apoptosis inducing factor mutant

    Abstract
    HSP70 inhibits apoptosis by neutralizing the caspase activator Apaf-1 and by interacting with apoptosis inducing factor (AIF), a mitochondrial flavoprotein wh...

  2. Effects of Ethanol Addition on the Efficiency of Subcritical Water Extraction of Proteins and Amino Acids from Porcine Placenta

    PubMed Central

    2015-01-01

    In a previous study, hydrolysates of porcine placenta were obtained and the extraction efficiency for proteins and amino acids was compared between sub- and super-critical water extraction systems; optimum efficiency was found to be achieved using subcritical water (170℃, 10 bar). In this study, the effects of adding ethanol to the subcritical water system were investigated. The lowest-molecular-weight extraction product detected weighed 434 Da, and the efficiency of extraction for low-molecular-weight products was increased when either the concentration of ethanol was decreased, or the extraction time was lengthened from 10 min to 30 min. The highest concentration of free amino acids (approximately 8 mM) was observed following 30 min extraction using pure distilled water. The concentration of free amino acids was significantly lower when ethanol was added or a shorter extraction time was used (p<0.05). Color change of the solution following extraction was measured. There were no significant differences in color between lysates produced with different extraction times when using distilled water (p>0.05); however, using different extraction times produced significant differences in color when using 20% or 50% ethanol solution for subcritical extraction (p<0.05). The range of pH for the hydrolysate solutions was 6.4-7.5. In conclusion, the investigated extraction system was successful in the extraction of ≤ 500 Da hydrolysates from porcine placenta, but addition of ethanol did not yield higher production of low-molecular-weight hydrolysates than that achieved by DW alone. PMID:26761837

  3. Effects of Ethanol Addition on the Efficiency of Subcritical Water Extraction of Proteins and Amino Acids from Porcine Placenta.

    PubMed

    Park, Sung Hee; Kim, Jae-Hyeong; Min, Sang-Gi; Jo, Yeon-Ji; Chun, Ji-Yeon

    2015-01-01

    In a previous study, hydrolysates of porcine placenta were obtained and the extraction efficiency for proteins and amino acids was compared between sub- and super-critical water extraction systems; optimum efficiency was found to be achieved using subcritical water (170℃, 10 bar). In this study, the effects of adding ethanol to the subcritical water system were investigated. The lowest-molecular-weight extraction product detected weighed 434 Da, and the efficiency of extraction for low-molecular-weight products was increased when either the concentration of ethanol was decreased, or the extraction time was lengthened from 10 min to 30 min. The highest concentration of free amino acids (approximately 8 mM) was observed following 30 min extraction using pure distilled water. The concentration of free amino acids was significantly lower when ethanol was added or a shorter extraction time was used (p<0.05). Color change of the solution following extraction was measured. There were no significant differences in color between lysates produced with different extraction times when using distilled water (p>0.05); however, using different extraction times produced significant differences in color when using 20% or 50% ethanol solution for subcritical extraction (p<0.05). The range of pH for the hydrolysate solutions was 6.4-7.5. In conclusion, the investigated extraction system was successful in the extraction of ≤ 500 Da hydrolysates from porcine placenta, but addition of ethanol did not yield higher production of low-molecular-weight hydrolysates than that achieved by DW alone. PMID:26761837

  4. Essential ribosome assembly factor Fap7 regulates a hierarchy of RNA–protein interactions during small ribosomal subunit biogenesis

    PubMed Central

    Hellmich, Ute A.; Weis, Benjamin L.; Lioutikov, Anatoli; Wurm, Jan Philip; Kaiser, Marco; Christ, Nina A.; Hantke, Katharina; Kötter, Peter; Entian, Karl-Dieter; Schleiff, Enrico; Wöhnert, Jens

    2013-01-01

    Factor activating Pos9 (Fap7) is an essential ribosome biogenesis factor important for the assembly of the small ribosomal subunit with an uncommon dual ATPase and adenylate kinase activity. Depletion of Fap7 or mutations in its ATPase motifs lead to defects in small ribosomal subunit rRNA maturation, the absence of ribosomal protein Rps14 from the assembled subunit, and retention of the nascent small subunit in a quality control complex with the large ribosomal subunit. The molecular basis for the role of Fap7 in ribosome biogenesis is, however, not yet understood. Here we show that Fap7 regulates multiple interactions between the precursor rRNA, ribosomal proteins, and ribosome assembly factors in a hierarchical manner. Fap7 binds to Rps14 with a very high affinity. Fap7 binding blocks both rRNA-binding elements of Rps14, suggesting that Fap7 inhibits premature interactions of Rps14 with RNA. The Fap7/Rps14 interaction is modulated by nucleotide binding to Fap7. Rps14 strongly activates the ATPase activity but not the adenylate kinase activity of Fap7, identifying Rps14 as an example of a ribosomal protein functioning as an ATPase-activating factor. In addition, Fap7 inhibits the RNA cleavage activity of Nob1, the endonuclease responsible for the final maturation step of the small subunit rRNA, in a nucleotide independent manner. Thus, Fap7 may regulate small subunit biogenesis at multiple stages. PMID:24003121

  5. Polysomes of Trypanosoma brucei: Association with Initiation Factors and RNA-Binding Proteins.

    PubMed

    Klein, Cornelia; Terrao, Monica; Inchaustegui Gil, Diana; Clayton, Christine

    2015-01-01

    We report here the results of experiments designed to identify RNA-binding proteins that might be associated with Trypanosoma brucei polysomes. After some preliminary mass spectrometry of polysomal fractions, we investigated the distributions of selected tagged proteins using sucrose gradients and immunofluorescence. As expected, the polysomal fractions contained nearly all annotated ribosomal proteins, the translation-associated protein folding complex, and many translation factors, but also many other abundant proteins. Results suggested that cap-binding proteins EIF4E3 and EIF4E4 were associated with both free and membrane-bound polysomes. The EIF4E binding partners EIF4G4 and EIF4G3 were present but the other EIF4E and EIF4G paralogues were not detected. The dominant EIF4E in the polysomal fraction is EIF4E4 and very few polysomal mRNAs are associated with EIF4G. Thirteen potential mRNA-binding proteins were detected in the polysomes, including the known polysome-associated protein RBP42. The locations of two of the other proteins were tested after epitope tagging: RBP29 was in the nucleus and ZC3H29 was in the cytoplasm. Quantitative analyses showed that specific association of an RNA-binding protein with the polysome fraction in sucrose gradients will not be detected if the protein is in more than 25-fold molar excess over its target binding sites.

  6. Polysomes of Trypanosoma brucei: Association with Initiation Factors and RNA-Binding Proteins

    PubMed Central

    Klein, Cornelia; Terrao, Monica; Inchaustegui Gil, Diana; Clayton, Christine

    2015-01-01

    We report here the results of experiments designed to identify RNA-binding proteins that might be associated with Trypanosoma brucei polysomes. After some preliminary mass spectrometry of polysomal fractions, we investigated the distributions of selected tagged proteins using sucrose gradients and immunofluorescence. As expected, the polysomal fractions contained nearly all annotated ribosomal proteins, the translation-associated protein folding complex, and many translation factors, but also many other abundant proteins. Results suggested that cap-binding proteins EIF4E3 and EIF4E4 were associated with both free and membrane-bound polysomes. The EIF4E binding partners EIF4G4 and EIF4G3 were present but the other EIF4E and EIF4G paralogues were not detected. The dominant EIF4E in the polysomal fraction is EIF4E4 and very few polysomal mRNAs are associated with EIF4G. Thirteen potential mRNA-binding proteins were detected in the polysomes, including the known polysome-associated protein RBP42. The locations of two of the other proteins were tested after epitope tagging: RBP29 was in the nucleus and ZC3H29 was in the cytoplasm. Quantitative analyses showed that specific association of an RNA-binding protein with the polysome fraction in sucrose gradients will not be detected if the protein is in more than 25-fold molar excess over its target binding sites. PMID:26287607

  7. Intracellular protein delivery activity of peptides derived from insulin-like growth factor binding proteins 3 and 5

    SciTech Connect

    Goda, Natsuko; Tenno, Takeshi; Inomata, Kosuke; Shirakawa, Masahiro; Tanaka, Toshiki; Hiroaki, Hidekazu

    2008-08-01

    Insulin-like growth factor binding proteins (IGFBPs) have various IGF-independent cellular activities, including receptor-independent cellular uptake followed by transcriptional regulation, although mechanisms of cellular entry remain unclear. Herein, we focused on their receptor-independent cellular entry mechanism in terms of protein transduction domain (PTD) activity, which is an emerging technique useful for clinical applications. The peptides of 18 amino acid residues derived from IGFBP-3 and IGFBP-5, which involve heparin-binding regions, mediated cellular delivery of an exogenous protein into NIH3T3 and HeLa cells. Relative protein delivery activities of IGFBP-3/5-derived peptides were approximately 20-150% compared to that of the HIV-Tat peptide, a potent PTD. Heparin inhibited the uptake of the fusion proteins with IGFBP-3 and IGFBP-5, indicating that the delivery pathway is heparin-dependent endocytosis, similar to that of HIV-Tat. The delivery of GST fused to HIV-Tat was competed by either IGFBP-3 or IGFBP-5-derived synthetic peptides. Therefore, the entry pathways of the three PTDs are shared. Our data has shown a new approach for designing protein delivery systems using IGFBP-3/5 derived peptides based on the molecular mechanisms of IGF-independent activities of IGFBPs.

  8. Effect of annatto addition and bleaching treatments on ultrafiltration flux during production of 80% whey protein concentrate and 80% serum protein concentrate.

    PubMed

    Adams, Michael C; Zulewska, Justyna; Barbano, David M

    2013-04-01

    The goals of this study were to determine if adding annatto color to milk or applying a bleaching process to whey or microfiltration (MF) permeate influenced ultrafiltration (UF) flux, diafiltration (DF) flux, or membrane fouling during production of 80% whey protein concentrate (WPC80) or 80% serum protein concentrate (SPC80). Separated Cheddar cheese whey (18 vats using 900 kg of whole milk each) and MF permeate of skim milk (18 processing runs using 800 kg of skim milk each) were produced to make WPC80 and SPC80, respectively. The 6 treatments, replicated 3 times each, that constituted the 18 processing runs within either whey or MF permeate UF were as follows: (1) no annatto; (2) no annatto+benzoyl peroxide (BPO); (3) no annatto+hydrogen peroxide (H2O2); (4) annatto; (5) annatto+BPO; and (6) annatto+H2O2. Approximately 700 kg of whey or 530 kg of MF permeate from each treatment were heated to 50°C and processed in 2 stages (UF and DF) with the UF system in batch recirculation mode using a polyethersulfone spiral-wound UF membrane with a molecular weight cutoff of 10,000 Da. Addition of annatto color had no effect on UF or DF flux. The processes of bleaching whey or MF permeate with or without added color improved flux during processing. Bleaching with H2O2 usually produced higher flux than bleaching with BPO. Bleaching with BPO increased WPC80 flux to a greater extent than it did SPC80 flux. Though no differences in mean flux were observed for a common bleaching treatment between the WPC80 and SPC80 production processes during the UF stage, mean flux during WPC80 DF was higher than mean flux during SPC80 DF for each bleaching treatment. Water flux values before and after processing were used to calculate a fouling coefficient that demonstrated differences in fouling which were consistent with flux differences among treatments. In both processes, bleaching with H2O2 led to the largest reduction in fouling. No effect of annatto on fouling was observed. The

  9. Presence of cytoplasmic factors functional in peroxisomal protein import implicates organelle-associated defects in several human peroxisomal disorders.

    PubMed Central

    Wendland, M; Subramani, S

    1993-01-01

    Cells from patients with peroxisome-deficient disorders contain membrane ghosts devoid of most matrix contents instead of normal peroxisomes indicating that the underlying molecular defects impair the import of matrix proteins into these peroxisome ghosts. Genetic heterogeneity for the molecular defects was inferred from the assignment of patients with peroxisome-deficient disorders into nine complementation groups. The aim of our studies was to analyze cell lines from six different complementation groups in a systematic manner for the presence of peroxisome ghosts, the ability to import Ser-Lys-Leu-containing proteins into peroxisome ghosts and for the presence of cytosolic factors required for peroxisomal protein import. We show that each of the cell lines analyzed contains peroxisome ghosts, but is unable to import matrix proteins as judged by a peroxisomal import assay using permeabilized cells. The addition of wild type cytosol did not restore the capacity to import matrix proteins but cytosol prepared from these cell lines was functional in stimulation of peroxisomal protein import in a heterologous system. These results implicate organelle-associated molecular defects in each of the six cell lines analyzed. Images PMID:7693762

  10. Trefoil factors: Gastrointestinal-specific proteins associated with gastric cancer.

    PubMed

    Xiao, Ping; Ling, Hui; Lan, Gang; Liu, Jiao; Hu, Haobin; Yang, Ruirui

    2015-10-23

    Trefoil factor family (TFF), composed of TFF1, TFF2, and TFF3, is a cluster of secreted peptides characterized by trefoil domain (s) and C-terminal dimerization domain. TFF1, a gastric tumor suppressor, is a single trefoil peptide originally detected in breast cancer cell lines but expressed mainly in the stomach; TFF2, a candidate of gastric cancer suppressor with two trefoil domains, is abundant in the stomach and duodenal Brunner's glands; and TFF3 is another single trefoil peptide expressed throughout the intestine which can promote the development of gastric carcinoma. According to multiple studies, TFFs play a regulatory function in the mammals' digestive system, namely in mucosal protection and epithelial cell reconstruction, tumor suppression or promotion, signal transduction and the regulation of proliferation and apoptosis. Action mechanisms of TFFs remain unresolved, but the recent demonstration of a GKN (gastrokine) 2-TFF1 heterodimer implicates structural and functional interplay with gastrokines. This review aims to encapsulate the structural and biological characteristics of TFF.

  11. NSs Virulence Factor of Rift Valley Fever Virus Engages the F-Box Proteins FBXW11 and β-TRCP1 To Degrade the Antiviral Protein Kinase PKR

    PubMed Central

    Kainulainen, Markus; Lau, Simone; Samuel, Charles E.; Hornung, Veit

    2016-01-01

    ABSTRACT Rift Valley fever virus (RVFV, family Bunyaviridae, genus Phlebovirus) is a relevant pathogen of both humans and livestock in Africa. The nonstructural protein NSs is a major virulence factor known to suppress the type I interferon (IFN) response by inhibiting host cell transcription and by proteasomal degradation of a major antiviral IFN effector, the translation-inhibiting protein kinase PKR. Here, we identified components of the modular SCF (Skp1, Cul1, F-box protein)-type E3 ubiquitin ligases as mediators of PKR destruction by NSs. Small interfering RNAs (siRNAs) against the conserved SCF subunit Skp1 protected PKR from NSs-mediated degradation. Consequently, RVFV replication was severely reduced in Skp1-depleted cells when PKR was present. SCF complexes have a variable F-box protein subunit that determines substrate specificity for ubiquitination. We performed an siRNA screen for all (about 70) human F-box proteins and found FBXW11 to be involved in PKR degradation. The partial stabilization of PKR by FBXW11 depletion upregulated PKR autophosphorylation and phosphorylation of the PKR substrate eIF2α and caused a shutoff of host cell protein synthesis in RVFV-infected cells. To maximally protect PKR from the action of NSs, knockdown of structurally and functionally related FBXW1 (also known as β-TRCP1), in addition to FBXW11 deletion, was necessary. Consequently, NSs was found to interact with both FBXW11 and β-TRCP1. Thus, NSs eliminates the antiviral kinase PKR by recruitment of SCF-type E3 ubiquitin ligases containing FBXW11 and β-TRCP1 as substrate recognition subunits. This antagonism of PKR by NSs is essential for efficient RVFV replication in mammalian cells. IMPORTANCE Rift Valley fever virus is a pathogen of humans and animals that has the potential to spread from Africa and the Arabian Peninsula to other regions. A major virulence mechanism is the proteasomal degradation of the antiviral kinase PKR by the viral protein NSs. Here, we

  12. Adhesion properties of Lactobacillus rhamnosus mucus-binding factor to mucin and extracellular matrix proteins.

    PubMed

    Nishiyama, Keita; Nakamata, Koichi; Ueno, Shintaro; Terao, Akari; Aryantini, Ni Putu Desy; Sujaya, I Nengah; Fukuda, Kenji; Urashima, Tadasu; Yamamoto, Yuji; Mukai, Takao

    2015-01-01

    We previously described potential probiotic Lactobacillus rhamnosus strains, isolated from fermented mare milk produced in Sumbawa Island, Indonesia, which showed high adhesion to porcine colonic mucin (PCM) and extracellular matrix (ECM) proteins. Recently, mucus-binding factor (MBF) was found in the GG strain of L. rhamnosus as a mucin-binding protein. In this study, we assessed the ability of recombinant MBF protein from the FSMM22 strain, one of the isolates of L. rhamnosus from fermented Sumbawa mare milk, to adhere to PCM and ECM proteins by overlay dot blot and Biacore assays. MBF bound to PCM, laminin, collagen IV, and fibronectin with submicromolar dissociation constants. Adhesion of the FSMM22 mbf mutant strain to PCM and ECM proteins was significantly less than that of the wild-type strain. Collectively, these results suggested that MBF contribute to L. rhamnosus host colonization via mucin and ECM protein binding.

  13. Gβγ Inhibits Exocytosis via Interaction with Critical Residues on Soluble N-Ethylmaleimide-Sensitive Factor Attachment Protein-25

    PubMed Central

    Wells, Christopher A.; Zurawski, Zack; Betke, Katherine M.; Yim, Yun Young; Hyde, Karren; Rodriguez, Shelagh; Alford, Simon

    2012-01-01

    Spatial and temporal regulation of neurotransmitter release is a complex process accomplished by the exocytotic machinery working in tandem with numerous regulatory proteins. G-protein βγ dimers regulate the core process of exocytosis by interacting with the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins soluble N-ethylmaleimide-sensitive factor attachment protein-25 (SNAP-25), syntaxin 1A, and synaptobrevin. Gβγ binding to ternary SNAREs overlaps with calcium-dependent binding of synaptotagmin, inhibiting synaptotagmin-1 binding and fusion of the synaptic vesicle. To further explore the binding sites of Gβγ on SNAP-25, peptides based on the sequence of SNAP-25 were screened for Gβγ binding. Peptides that bound Gβγ were subjected to alanine scanning mutagenesis to determine their relevance to the Gβγ-SNAP-25 interaction. Peptides from this screen were tested in protein-protein interaction assays for their ability to modulate the interaction of Gβγ with SNAP-25. A peptide from the C terminus, residues 193 to 206, significantly inhibited the interaction. In addition, Ala mutants of SNAP-25 residues from the C terminus of SNAP-25, as well as from the amino-terminal region decreased binding to Gβ1γ1. When SNAP-25 with eight residues mutated to alanine was assembled with syntaxin 1A, there was significantly reduced affinity of this mutated t-SNARE for Gβγ, but it still interacted with synaptotagmin-1 in a Ca2+-dependent manner and reconstituted evoked exocytosis in botulinum neurotoxin E-treated neurons. However, the mutant SNAP-25 could no longer support 5-hydroxytryptamine-mediated inhibition of exocytosis. PMID:22962332

  14. Additional cytosine inside mitochondrial C-tract D-loop as a progression risk factor in oral precancer cases

    PubMed Central

    Pandey, Rahul; Mehrotra, Divya; Mahdi, Abbas Ali; Sarin, Rajiv; Kowtal, Pradnya

    2014-01-01

    Introduction Alterations inside Polycytosine tract (C-tract) of mitochondrial DNA (mtDNA) have been described in many different tumor types. The Poly-Cytosine region is located within the mtDNA D-loop region which acts as point of mitochondrial replication origin. A suggested pathogenesis is that it interferes with the replication process of mtDNA which in turn affects the mitochondrial functioning and generates disease. Methodology 100 premalignant cases (50 leukoplakia & 50 oral submucous fibrosis) were selected and the mitochondrial DNA were isolated from the lesion tissues and from the blood samples. Polycytosine tract in mtDNA was sequenced by direct capillary sequencing. Results 40 (25 leukoplakia & 15 oral submucous fibrosis) patients harbored lesions that displayed one additional cytosine after nucleotide thymidine (7CT6C) at nt position 316 in C-tract of mtDNA which were absent in corresponding mtDNA derived from blood samples. Conclusion Our results show an additional cytosine in the mtDNA at polycytosine site in oral precancer cases. It is postulated that any increase/decrease in the number of cytosine residues in the Poly-Cytosine region may affect the rate of mtDNA replication by impairing the binding of polymerase and other transacting factors. By promoting mitochondrial genomic instability, it may have a central role in the dysregulation of mtDNA functioning, for example alterations in energy metabolism that may promote tumor development. We, therefore, report and propose that this alteration may represent the early development of oral cancer. Further studies with large number of samples are needed in to confirm the role of such mutation in carcinogenesis. PMID:25737911

  15. Bone morphogenetic protein 4: a ventralizing factor in early Xenopus development.

    PubMed

    Dale, L; Howes, G; Price, B M; Smith, J C

    1992-06-01

    The mesoderm of amphibian embryos such as Xenopus laevis arises through an inductive interaction in which cells of the vegetal hemisphere of the embryo act on overlying equatorial and animal pole cells. Three classes of 'mesoderm-inducing factor' (MIF) that might be responsible for this interaction in vivo have been discovered. These are members of the transforming growth factor type beta (TGF-beta), fibroblast growth factor (FGF) and Wnt families. Among the most potent MIFs are the activins, members of the TGF-beta family, but RNA for activin A and B is not detectable in the Xenopus embryo until neurula and late blastula stages, respectively, and this is probably too late for the molecules to act as natural inducers. In this paper, we use the polymerase chain reaction to clone additional members of the TGF-beta family that might possess mesoderm-inducing activity. We show that transcripts encoding Xenopus bone morphogenetic protein 4 (XBMP-4) are detectable in the unfertilized egg, and that injection of XBMP-4 RNA into the animal hemisphere of Xenopus eggs causes animal caps isolated from the resulting blastulae to express mesoderm-specific markers. Surprisingly, however, XBMP-4 preferentially induces ventral mesoderm, whereas the closely related activin induces axial tissues. Furthermore, the action of XBMP-4 is 'dominant' over that of activin. In this respect, XBMP-4 differs from basic FGF, another ventral inducer, where simultaneous treatment with FGF and activin results in activin-like responses. The dominance of XBMP-4 over activin may account for the ability of injected XBMP-4 RNA to 'ventralize' whole Xenopus embryos. It is interesting, however, that blastopore formation in such embryos can occur perfectly normally. This contrasts with embryos ventralized by UV-irradiation and suggests that XBMP-4-induced ventralization occurs after the onset of gastrulation. PMID:1425340

  16. Mapping transcription factor interactome networks using HaloTag protein arrays.

    PubMed

    Yazaki, Junshi; Galli, Mary; Kim, Alice Y; Nito, Kazumasa; Aleman, Fernando; Chang, Katherine N; Carvunis, Anne-Ruxandra; Quan, Rosa; Nguyen, Hien; Song, Liang; Alvarez, José M; Huang, Shao-Shan Carol; Chen, Huaming; Ramachandran, Niroshan; Altmann, Stefan; Gutiérrez, Rodrigo A; Hill, David E; Schroeder, Julian I; Chory, Joanne; LaBaer, Joshua; Vidal, Marc; Braun, Pascal; Ecker, Joseph R

    2016-07-19

    Protein microarrays enable investigation of diverse biochemical properties for thousands of proteins in a single experiment, an unparalleled capacity. Using a high-density system called HaloTag nucleic acid programmable protein array (HaloTag-NAPPA), we created high-density protein arrays comprising 12,000 Arabidopsis ORFs. We used these arrays to query protein-protein interactions for a set of 38 transcription factors and transcriptional regulators (TFs) that function in diverse plant hormone regulatory pathways. The resulting transcription factor interactome network, TF-NAPPA, contains thousands of novel interactions. Validation in a benchmarked in vitro pull-down assay revealed that a random subset of TF-NAPPA validated at the same rate of 64% as a positive reference set of literature-curated interactions. Moreover, using a bimolecular fluorescence complementation (BiFC) assay, we confirmed in planta several interactions of biological interest and determined the interaction localizations for seven pairs. The application of HaloTag-NAPPA technology to plant hormone signaling pathways allowed the identification of many novel transcription factor-protein interactions and led to the development of a proteome-wide plant hormone TF interactome network.

  17. Mapping transcription factor interactome networks using HaloTag protein arrays.

    PubMed

    Yazaki, Junshi; Galli, Mary; Kim, Alice Y; Nito, Kazumasa; Aleman, Fernando; Chang, Katherine N; Carvunis, Anne-Ruxandra; Quan, Rosa; Nguyen, Hien; Song, Liang; Alvarez, José M; Huang, Shao-Shan Carol; Chen, Huaming; Ramachandran, Niroshan; Altmann, Stefan; Gutiérrez, Rodrigo A; Hill, David E; Schroeder, Julian I; Chory, Joanne; LaBaer, Joshua; Vidal, Marc; Braun, Pascal; Ecker, Joseph R

    2016-07-19

    Protein microarrays enable investigation of diverse biochemical properties for thousands of proteins in a single experiment, an unparalleled capacity. Using a high-density system called HaloTag nucleic acid programmable protein array (HaloTag-NAPPA), we created high-density protein arrays comprising 12,000 Arabidopsis ORFs. We used these arrays to query protein-protein interactions for a set of 38 transcription factors and transcriptional regulators (TFs) that function in diverse plant hormone regulatory pathways. The resulting transcription factor interactome network, TF-NAPPA, contains thousands of novel interactions. Validation in a benchmarked in vitro pull-down assay revealed that a random subset of TF-NAPPA validated at the same rate of 64% as a positive reference set of literature-curated interactions. Moreover, using a bimolecular fluorescence complementation (BiFC) assay, we confirmed in planta several interactions of biological interest and determined the interaction localizations for seven pairs. The application of HaloTag-NAPPA technology to plant hormone signaling pathways allowed the identification of many novel transcription factor-protein interactions and led to the development of a proteome-wide plant hormone TF interactome network. PMID:27357687

  18. A fragment of anthrax lethal factor delivers proteins to the cytosol without requiring protective antigen

    PubMed Central

    Kushner, Nicholas; Zhang, Dong; Touzjian, Neal; Essex, Max; Lieberman, Judy; Lu, Yichen

    2003-01-01

    Anthrax protective antigen (PA) is a 735-aa polypeptide that facilitates the exit of anthrax lethal factor (LF) from the endosome to the cytosol where the toxin acts. We recently found, however, that a fusion protein of the detoxified N-terminal domain of lethal factor (LFn) with a foreign peptide could induce CD8 T cell immune responses in the absence of PA. Because CD8 T cells recognize peptides derived from proteins degraded in the cytosol, this result suggests that lethal factor may be capable of entering the cytosol independently of PA. To investigate this further, the intracellular trafficking of an LFn-enhanced green fluorescent protein fusion protein (LFn-GFP) in the presence or absence of PA was examined by using confocal microscopy. LFn-GFP is able to enter the cytosol without PA. Moreover, it efficiently colocalizes with the proteosome 20s subunit, which degrades proteins into peptides for presentation to CD8 T cells by the MHC class I pathway. We further demonstrate that in the presence of an immune adjuvant LFn fusion protein without PA is able to effectively elicit anti-HIV cytotoxic T lymphocyte in inbred mice. These results indicate that LFn may be used without PA in a protein vaccine as a carrier to deliver antigens into the cytosol for efficient induction of T lymphocyte responses. Furthermore, these results enable us to propose a modified molecular mechanism of anthrax lethal toxin. PMID:12740437

  19. [Mechanisms underlying physiological functions of food factors via non-specific interactions with biological proteins].

    PubMed

    Murakami, Akira

    2015-01-01

      We previously reported that zerumbone, a sesquiterpene found in Zingiber zerumbet SMITH, showed notable cancer preventive effects in various organs of experimental rodents. This agent up-regulated nuclear factor-E2-related factor (Nrf2)-dependent expressions of anti-oxidative and xenobiotics-metabolizing enzymes, leading to an increased self-defense capacity. On the other hand, zerumbone markedly suppressed the expression of cyclooxygenase-2, an inducible pro-inflammatory enzyme, by disrupting mRNA stabilizing processes. Binding experiments using a biotin derivative of zerumbone demonstrated that Keap1, an Nrf2 repressive protein, is one of its major binding proteins that promotes their dissociation for inducing Nrf2 transactivation. We then generated a specific antibody against zerumbone-modified proteins and found that zerumbone modified numerous cellular proteins in a non-specific manner, with global distribution of the modified proteins seen not only in cytoplasm but also the nucleus. Based on those observations, zerumbone was speculated to cause proteo-stress, a notion supported by previous findings that it increased the C-terminus of Hsc70 interacting protein-dependent protein ubiquitination and also promoted aggresome formation. Interestingly, zerumbone counteracted proteo-stress and heat stress via up-regulation of the protein quality control systems (PQCs), e.g., heat shock proteins (HSPs), ubiquitin-proteasome, and autophagy. Meanwhile, several phytochemicals, including ursolic acid and curcumin, were identified as marked HSP70 inducers, whereas most nutrients tested were scarcely active. Recent studies have revealed that PQCs play important roles in the prevention of many lifestyle related diseases, such as cancer, thus non-specific binding of phytochemicals to cellular proteins may be a novel and unique mechanism underlying their physiological activities.

  20. Properties of proteins in cancer procoagulant preparations that are detected by anti-tissue factor antibodies.

    PubMed

    Raasi, Shari; Mielicki, Wojciech P; Gordon, Stuart G; Korte, Wolfgang

    2004-08-15

    Cancer procoagulant (CP) and tissue factor (TF; only in complex with Factor VIIa (FVIIa)) can activate FX to FXa. Controversy still exists whether or not CP is an entity different from TF, or whether CP activity is due to contamination of CP preparations with TF/FVIIa complex. We therefore looked for proteins in CP preparations that were detected by anti-TF antibodies and then sequenced these proteins. One- and two-dimensional gels of CP and TF were used to identify proteins immunoreactive to monoclonal anti-CP and anti-TF antibodies (Mabs). Those proteins in the CP preparation recognized by anti-TF antibodies were sequenced. Angiotensinogen precursor, alpha-1-antitrypsin precursor, and vitamin D-binding protein were identified along with one so far unidentified sequence; however, no TF-sequences were identified. Also, no proteins with the correct molecular weight for TF were identified using anti-TF antibodies. It seems possible that CP preparations contain proteins that have some epitopes similar to the epitopes recognized in TF by anti-TF Mab. However, these proteins do neither have the molecular weight nor the amino acid sequence of TF.

  1. Heterologous expression of Translocated promoter region protein, Tpr, identified as a transcription factor from Rattus norvegicus.

    PubMed

    Agarwal, Shivani; Yadav, Sunita Kumari; Dixit, Aparna

    2011-05-01

    Our earlier studies have demonstrated that the 35 kDa isoform of Translocated promoter region protein (Tpr) of Rattus norvegicus was able to augment c-jun transcription efficiently. Identification of direct targets that may in part downregulate c-jun transcription might prove to be an ideal target to curtail the proliferation of normal cells under pathophysiological conditions. In order to evaluate its potential as a pharmaceutical target, the protein must be produced and purified in sufficiently high yields. In the present study, we report the high level expression of Tpr protein of R. norvegicus employing heterologous host, Escherichia coli, to permit its structural characterization in great detail. We here demonstrate that the Tpr protein was expressed in soluble form and approximately 90 mg/L of the purified protein at the shake flask level could be achieved to near homogeneity using single step-metal chelate affinity chromatography. The amino acid sequence of the protein was confirmed by mass spectroscopic analysis. The highly unstable and disordered Tpr protein was imparted structural and functional stability by the addition of glycerol and it has been shown that the natively unfolded Tpr protein retains DNA binding ability under these conditions only. Thus, the present study emphasizes the significance of an efficient prokaryotic system, which results in a high level soluble expression of a DNA binding protein of eukaryotic origin. Thus, the present strategy employed for purification of the R. norvegicus Tpr protein bypasses the need for the tedious expression strategies associated with the eukaryotic expression systems.

  2. Apoptosis-inducing factor (AIF) inhibits protein synthesis by interacting with the eukaryotic translation initiation factor 3 subunit p44 (eIF3g).

    PubMed

    Kim, Jong-Tae; Kim, Kwang Dong; Song, Eun Young; Lee, Hee Gu; Kim, Jae Wha; Kim, Jung Woo; Chae, Suhn-Kee; Kim, Eunhee; Lee, Myeong-Sok; Yang, Young; Lim, Jong-Seok

    2006-11-27

    Apoptosis-inducing factor (AIF) is a ubiquitous FAD-binding flavoprotein comprised of 613 amino acids and plays an important role in caspase-independent apoptosis. During apoptotic induction, AIF is translocated from the mitochondrial intermembrane space to the nucleus, where it interacts with DNA and activates a nuclear endonuclease. By performing a yeast two-hybrid screen with mature AIF, we have isolated the eukaryotic translation initiation factor 3 subunit p44 (eIF3g). Our deletion mutant analysis revealed that the eIF3g N-terminus interacts with the C-terminal region of AIF. The direct interaction between AIF and eIF3g was confirmed in a GST pull-down assay and also verified by the results of co-immunoprecipitation and confocal microscopy studies. Using an in vitro TNT coupled transcription-translation system, we found that mature AIF could inhibit newly-translated protein synthesis and this inhibition was significantly blocked by eIF3g competitively. These results were also confirmed in cells. In addition, mature AIF overexpression specifically resulted in the activation of caspase-7, thereby amplifying the inhibition of protein synthesis including eIF3g cleavage. Our data suggest that eIF3g is one of the cytosolic targets that interacts with mature AIF, and provide insight into the AIF's cellular functions of the inhibition of protein synthesis during apoptosis.

  3. A common site within factor H SCR 7 responsible for binding heparin, C-reactive protein and streptococcal M protein.

    PubMed

    Giannakis, Eleni; Jokiranta, T Sakari; Male, Dean A; Ranganathan, Shoba; Ormsby, Rebecca J; Fischetti, Vince A; Mold, Carolyn; Gordon, David L

    2003-04-01

    The complement inhibitor factor H (fH) interacts via its seventh short consensus repeat (SCR) domain with multiple ligands including heparin, streptococcal M protein and C-reactive protein (CRP). The aim of this study was to localize the residues in SCR 7 required for these interactions. We initially built a homology model of fH SCR 6-7 using the averaged NMR structures of fH SCR 15-16 and vaccinia control protein SCR 3-4 as templates. Electrostatic potentials of the model's surface demonstrated a co-localization of three clusters of positively charged residues on SCR 7, labeled site A (R369 and K370), site B (R386 and K387) and site C (K392). These residues, localized to the linker region preceding SCR 7 and to the end of a "hypervariable loop" in SCR 7, were systematically replaced with uncharged alanine residues in an fH construct containing SCR 1-7. The resulting proteins were expressed in the methylotrophic yeast, Pichia pastoris. By ELISA analysis we demonstrated: first, that substituting site A inhibited heparin and CRP binding; secondly, that substituting site B inhibited binding to heparin, CRP and M protein; and thirdly, that substituting site C clearly inhibited only heparin binding.

  4. Effects of buffer additives and thermal processing methods on the solubility of shrimp (Penaeus monodon) proteins and the immunoreactivity of its major allergen.

    PubMed

    Lasekan, Adeseye O; Nayak, Balunkeswar

    2016-06-01

    This study examines the potential of two buffer additives (Tween 20 and DTT) to improve the solubility of proteins from shrimp subjected to different heat treatments and the allergenicity of tropomyosin in the extracts. The concentration of soluble proteins extracted by all the buffers from processed shrimp was significantly reduced compared with untreated samples. The concentration of total soluble proteins from heat treated shrimp increased significantly when phosphate buffer containing both surfactant and reducing agent was used as the extraction buffer. However, the concentrations of heat-stable proteins in the buffers were mostly similar. The electrophoretic profile of extracted proteins showed that tropomyosin is very stable under the different heat treatment methods used in this study except for high pressure steaming where the intensity of tropomyosin band was reduced. Competitive inhibition ELISA showed that high pressure steaming reduced the allergenicity of tropomyosin compared with other heat treatments methods.

  5. Dimerization of complement factor H-related proteins modulates complement activation in vivo.

    PubMed

    Goicoechea de Jorge, Elena; Caesar, Joseph J E; Malik, Talat H; Patel, Mitali; Colledge, Matthew; Johnson, Steven; Hakobyan, Svetlana; Morgan, B Paul; Harris, Claire L; Pickering, Matthew C; Lea, Susan M

    2013-03-19

    The complement system is a key component regulation influences susceptibility to age-related macular degeneration, meningitis, and kidney disease. Variation includes genomic rearrangements within the complement factor H-related (CFHR) locus. Elucidating the mechanism underlying these associations has been hindered by the lack of understanding of the biological role of CFHR proteins. Here we present unique structural data demonstrating that three of the CFHR proteins contain a shared dimerization motif and that this hitherto unrecognized structural property enables formation of both homodimers and heterodimers. Dimerization confers avidity for tissue-bound complement fragments and enables these proteins to efficiently compete with the physiological complement inhibitor, complement factor H (CFH), for ligand binding. Our data demonstrate that these CFHR proteins function as competitive antagonists of CFH to modulate complement activation in vivo and explain why variation in the CFHRs predisposes to disease.

  6. Eukaryotic translation initiation factor 4GI is a cellular target for NS1 protein, a translational activator of influenza virus.

    PubMed

    Aragón, T; de la Luna, S; Novoa, I; Carrasco, L; Ortín, J; Nieto, A

    2000-09-01

    Influenza virus NS1 protein is an RNA-binding protein whose expression alters several posttranscriptional regulatory processes, like polyadenylation, splicing, and nucleocytoplasmic transport of cellular mRNAs. In addition, NS1 protein enhances the translational rate of viral, but not cellular, mRNAs. To characterize this effect, we looked for targets of NS1 influenza virus protein among cellular translation factors. We found that NS1 coimmunoprecipitates with eukaryotic initiation factor 4GI (eIF4GI), the large subunit of the cap-binding complex eIF4F, either in influenza virus-infected cells or in cells transfected with NS1 cDNA. Affinity chromatography studies using a purified His-NS1 protein-containing matrix showed that the fusion protein pulls down endogenous eIF4GI from COS-1 cells and labeled eIF4GI translated in vitro, but not the eIF4E subunit of the eIF4F factor. Similar in vitro binding experiments with eIF4GI deletion mutants indicated that the NS1-binding domain of eIF4GI is located between residues 157 and 550, in a region where no other component of the translational machinery is known to interact. Moreover, using overlay assays and pull-down experiments, we showed that NS1 and eIF4GI proteins interact directly, in an RNA-independent manner. Mapping of the eIF4GI-binding domain in the NS1 protein indicated that the first 113 N-terminal amino acids of the protein, but not the first 81, are sufficient to bind eIF4GI. The first of these mutants has been previously shown to act as a translational enhancer, while the second is defective in this activity. Collectively, these and previously published data suggest a model where NS1 recruits eIF4GI specifically to the 5' untranslated region (5' UTR) of the viral mRNA, allowing for the preferential translation of the influenza virus messengers.

  7. Fibroblast growth factor, but not activin, is a potent activator of mitogen-activated protein kinase in Xenopus explants.

    PubMed Central

    Graves, L M; Northrop, J L; Potts, B C; Krebs, E G; Kimelman, D

    1994-01-01

    Isolated explants from the animal hemisphere of Xenopus embryos were incubated with Xenopus basic fibroblast growth factor (XbFGF) or human activin A. XbFGF incubation resulted in the rapid activation of mitogen-activated protein kinase (MAPK) and ribosomal S6 protein kinase (pp90rsk) in a dose-dependent manner with the highest levels of activation occurring at 50 ng/ml. Maximal activation occurred within 6-10 min after the addition of growth factor, and the activity of both kinases declined to unstimulated levels after 30 min. Activin was unable to activate either MAPK or pp90rsk in the Xenopus explants to a substantial level, although it induced dorsal mesoderm better than XbFGF under the same experimental conditions. The regulatory protein Xwnt-8 did not activate MAPK, nor did it enhance the activation of MAPK by XbFGF. XbFGF was able to activate MAPK through at least the midgastrula stage, suggesting that this family of growth factors may have a role in gastrula-stage events. Images PMID:7510404

  8. Overlapping binding sites of the frataxin homologue assembly factor and the heat shock protein 70 transfer factor on the Isu iron-sulfur cluster scaffold protein.

    PubMed

    Manicki, Mateusz; Majewska, Julia; Ciesielski, Szymon; Schilke, Brenda; Blenska, Anna; Kominek, Jacek; Marszalek, Jaroslaw; Craig, Elizabeth A; Dutkiewicz, Rafal

    2014-10-31

    In mitochondria FeS clusters, prosthetic groups critical for the activity of many proteins, are first assembled on Isu, a 14-kDa scaffold protein, and then transferred to recipient apoproteins. The assembly process involves interaction of Isu with both Nfs1, the cysteine desulfurase serving as a sulfur donor, and the yeast frataxin homolog (Yfh1) serving as a regulator of desulfurase activity and/or iron donor. Here, based on the results of biochemical experiments with purified wild-type and variant proteins, we report that interaction of Yfh1 with both Nfs1 and Isu are required for formation of a stable tripartite assembly complex. Disruption of either Yfh1-Isu or Nfs1-Isu interactions destabilizes the complex. Cluster transfer to recipient apoprotein is known to require the interaction of Isu with the J-protein/Hsp70 molecular chaperone pair, Jac1 and Ssq1. Here we show that the Yfh1 interaction with Isu involves the PVK sequence motif, which is also the site key for the interaction of Isu with Hsp70 Ssq1. Coupled with our previous observation that Nfs1 and Jac1 binding to Isu is mutually exclusive due to partially overlapping binding sites, we propose that such mutual exclusivity of cluster assembly factor (Nfs1/Yfh1) and cluster transfer factor (Jac1/Ssq1) binding to Isu has functional consequences for the transition from the assembly process to the transfer process, and thus regulation of the biogenesis of FeS cluster proteins.

  9. Interacting factors and cellular localization of SR protein-specific kinase Dsk1

    SciTech Connect

    Tang, Zhaohua; Luca, Maria; Taggart-Murphy, Laura; Portillio, Jessica; Chang, Cathey; Guven, Ayse; Lin, Ren-Jang; Murray, Johanne; Carr, Antony

    2012-10-01

    Schizosaccharomyces pombe Dsk1 is an SR protein-specific kinase (SRPK), whose homologs have been identified in every eukaryotic organism examined. Although discovered as a mitotic regulator with protein kinase activity toward SR splicing factors, it remains largely unknown about what and how Dsk1 contributes to cell cycle and pre-mRNA splicing. In this study, we investigated the Dsk1 function by determining interacting factors and cellular localization of the kinase. Consistent with its reported functions, we found that pre-mRNA processing and cell cycle factors are prominent among the proteins co-purified with Dsk1. The identification of these factors led us to find Rsd1 as a novel Dsk1 substrate, as well as the involvement of Dsk1 in cellular distribution of poly(A){sup +} RNA. In agreement with its role in nuclear events, we also found that Dsk1 is mainly localized in the nucleus during G{sub 2} phase and at mitosis. Furthermore, we revealed the oscillation of Dsk1 protein in a cell cycle-dependent manner. This paper marks the first comprehensive analysis of in vivo Dsk1-associated proteins in fission yeast. Our results reflect the conserved role of SRPK family in eukaryotic organisms, and provide information about how Dsk1 functions in pre-mRNA processing and cell-division cycle.

  10. Binding of Streptococcus mutans SR protein to human monocytes: production of tumor necrosis factor, interleukin 1, and interleukin 6.

    PubMed

    Soell, M; Holveck, F; Schöller, M; Wachsmann, R D; Klein, J P

    1994-05-01

    To examine the possible implication of protein SR, an I/II-related antigen from Streptococcus mutans OMZ 175 (serotype f), in inflammatory reactions, we tested the immunomodulatory effects of protein SR on human monocytes. Using biotinylated protein, we provide evidence that protein SR binds to human monocytes in dose-, time-, and calcium-dependent manners through specific interactions. These results were confirmed by competition experiments using either soluble human monocyte extract or anti-SR immunoglobulin G. Binding occurred through lectin-like interactions between SR and carbohydrate portions of monocyte membrane glycoproteins, since binding could be inhibited by several sugars, especially fucose and N-acetylneuraminic acid (NANA), which were confirmed by ligand blotting to be the primer ligands recognized by SR on human monocyte extracts. The ability of protein SR to stimulate the production of cytokines by human circulating monocytes was then examined. The release of tumor necrosis factor alpha (TNF-alpha), interleukin 1 beta, and interleukin 6 is time and dose dependent and not affected by the addition of polymyxin B. Activation of monocytes resulted from specific binding of SR to NANA and fucose present on cell surface glycoproteins since TNF-alpha release could be inhibited by sialidase and pronase treatment of monocytes and by NANA and fucose. These results confirm that sialic acid and fucose present on cell surface macromolecules and especially glycoproteins are needed for the binding of SR to monocytes and for the release of TNF-alpha. PMID:8168943

  11. Binding of Streptococcus mutans SR protein to human monocytes: production of tumor necrosis factor, interleukin 1, and interleukin 6.

    PubMed Central

    Soell, M; Holveck, F; Schöller, M; Wachsmann, R D; Klein, J P

    1994-01-01

    To examine the possible implication of protein SR, an I/II-related antigen from Streptococcus mutans OMZ 175 (serotype f), in inflammatory reactions, we tested the immunomodulatory effects of protein SR on human monocytes. Using biotinylated protein, we provide evidence that protein SR binds to human monocytes in dose-, time-, and calcium-dependent manners through specific interactions. These results were confirmed by competition experiments using either soluble human monocyte extract or anti-SR immunoglobulin G. Binding occurred through lectin-like interactions between SR and carbohydrate portions of monocyte membrane glycoproteins, since binding could be inhibited by several sugars, especially fucose and N-acetylneuraminic acid (NANA), which were confirmed by ligand blotting to be the primer ligands recognized by SR on human monocyte extracts. The ability of protein SR to stimulate the production of cytokines by human circulating monocytes was then examined. The release of tumor necrosis factor alpha (TNF-alpha), interleukin 1 beta, and interleukin 6 is time and dose dependent and not affected by the addition of polymyxin B. Activation of monocytes resulted from specific binding of SR to NANA and fucose present on cell surface glycoproteins since TNF-alpha release could be inhibited by sialidase and pronase treatment of monocytes and by NANA and fucose. These results confirm that sialic acid and fucose present on cell surface macromolecules and especially glycoproteins are needed for the binding of SR to monocytes and for the release of TNF-alpha. Images PMID:8168943

  12. Isolation and molecular genetic characterization of the Bacillus subtilis gene (infB) encoding protein synthesis initiation factor 2.

    PubMed Central

    Shazand, K; Tucker, J; Chiang, R; Stansmore, K; Sperling-Petersen, H U; Grunberg-Manago, M; Rabinowitz, J C; Leighton, T

    1990-01-01

    Western blot (immunoblot) analysis of Bacillus subtilis cell extracts detected two proteins that cross-reacted with monospecific polyclonal antibody raised against Escherichia coli initiation factor 2 alpha (IF2 alpha). Subsequent Southern blot analysis of B. subtilis genomic DNA identified a 1.3-kilobase (kb) HindIII fragment which cross-hybridized with both E. coli and Bacillus stearothermophilus IF2 gene probes. This DNA was cloned from a size-selected B. subtilis plasmid library. The cloned HindIII fragment, which was shown by DNA sequence analysis to encode the N-terminal half of the B. subtilis IF2 protein and 0.2 kb of upstream flanking sequence, was utilized as a homologous probe to clone an overlapping 2.76-kb ClaI chromosomal fragment containing the entire IF2 structural gene. The HindIII fragment was also used as a probe to obtain overlapping clones from a lambda gt11 library which contained additional upstream and downstream flanking sequences. Sequence comparisons between the B. subtilis IF2 gene and the other bacterial homologs from E. coli, B. stearothermophilus, and Streptococcus faecium displayed extensive nucleic acid and protein sequence homologies. The B. subtilis infB gene encodes two proteins, IF2 alpha (78.6 kilodaltons) and IF2 beta (68.2 kilodaltons); both were expressed in B. subtilis and E. coli. These two proteins cross-reacted with antiserum to E. coli IF2 alpha and were able to complement in vivo an E. coli infB gene disruption. Four-factor recombination analysis positioned the infB gene at 145 degrees on the B. subtilis chromosome, between the polC and spcB loci. This location is distinct from those of the other major ribosomal protein and rRNA gene clusters of B. subtilis. Images PMID:2110148

  13. Activation of p38 Mitogen-Activated Protein Kinase Promotes Epidermal Growth Factor Receptor Internalization

    PubMed Central

    Vergarajauregui, Silvia; Miguel, Anitza San; Puertollano, Rosa

    2006-01-01

    Endocytic trafficking plays an important role in the regulation of the epidermal growth factor receptor (EGFR). To address if cellular kinases regulate EGFR internalization, we used anisomycin, a potent activator of kinase cascades in mammalian cells, especially the stress-activated mitogen-activated protein (MAP) kinase subtypes. Here, we report that activation of p38 MAP kinase by anisomycin is sufficient to induce internalization of EGFR. Anisomycin and EGF employ different mechanisms to promote EGFR endocytosis as anisomycin-induced internalization does not require tyrosine kinase activity or ubiquitination of the receptor. In addition, anisomycin treatment did not result in delivery and degradation of EGFR at lysosomes. Incubation with a specific inhibitor of p38, or depletion of endogenous p38 by small interfering RNAs, abolished anisomycin-induced internalization of EGFR while having no effect on transferrin endocytosis, indicating that the effect of p38 activation on EGFR endocytosis is specific. Interestingly, inhibition of p38 activation also abolished endocytosis of EGFR induced by UV radiation. Our results reveal a novel role for p38 in the regulation of EGFR endocytosis and suggest that stimulation of EGFR internalization by p38 might represent a general mechanism to prevent generation of proliferative or anti-apoptotic signals under stress conditions. PMID:16683917

  14. Effects of Factor XIII Deficiency on Thromboelastography. Thromboelastography with Calcium and Streptokinase Addition is more Sensitive than Solubility Tests

    PubMed Central

    Martinuzzo, M.; Barrera, L.; Altuna, D.; Baña, F. Tisi; Bieti, J.; Amigo, Q.; D’Adamo, M.; López, M.S.; Oyhamburu, J.; Otaso, J.C.

    2016-01-01

    Background Homozygous or double heterozygous factor XIII (FXIII) deficiency is characterized by soft tissue hematomas, intracranial and delayed spontaneous bleeding. Alterations of thromboelastography (TEG) parameters in these patients have been reported. The aim of the study was to show results of TEG, TEG Lysis (Lys 60) induced by subthreshold concentrations of streptokinase (SK), and to compare them to the clot solubility studies results in samples of a 1-year-old girl with homozygous or double heterozygous FXIII deficiency. Case A year one girl with a history of bleeding from the umbilical cord. During her first year of life, several hematomas appeared in soft upper limb tissue after punctures for vaccination and a gluteal hematoma. One additional sample of a heterozygous patient and three samples of acquired FXIII deficiency were also evaluated. Materials and Methods Clotting tests, von Willebrand factor (vWF) antigen and activity, plasma FXIII-A subunit (pFXIII-A) were measured by an immunoturbidimetric assay in a photo-optical coagulometer. Solubility tests were performed with Ca2+-5 M urea and thrombin-2% acetic acid. Basal and post-FXIII concentrate infusion samples were studied. TEG was performed with CaCl2 or CaCl2 + SK (3.2 U/mL) in a Thromboelastograph. Results Prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time, fibrinogen, factor VIIIc, vWF, and platelet aggregation were normal. Antigenic pFXIII-A subunit was < 2%. TEG, evaluated at diagnosis and post FXIII concentrate infusion (pFXIII-A= 37%), presented a normal reaction time (R), 8 min, prolonged k (14 and 11min respectively), a low Maximum-Amplitude (MA) ( 39 and 52 mm respectively), and Clot Lysis (Lys60) slightly increased (23 and 30% respectively). In the sample at diagnosis, clot solubility was abnormal, 50 and 45 min with Ca-Urea and thrombin-acetic acid, respectively, but normal (>16 hours) 1-day post-FXIII infusion. Analysis of FXIII deficient and normal

  15. Effects of Factor XIII Deficiency on Thromboelastography. Thromboelastography with Calcium and Streptokinase Addition is more Sensitive than Solubility Tests

    PubMed Central

    Martinuzzo, M.; Barrera, L.; Altuna, D.; Baña, F. Tisi; Bieti, J.; Amigo, Q.; D’Adamo, M.; López, M.S.; Oyhamburu, J.; Otaso, J.C.

    2016-01-01

    Background Homozygous or double heterozygous factor XIII (FXIII) deficiency is characterized by soft tissue hematomas, intracranial and delayed spontaneous bleeding. Alterations of thromboelastography (TEG) parameters in these patients have been reported. The aim of the study was to show results of TEG, TEG Lysis (Lys 60) induced by subthreshold concentrations of streptokinase (SK), and to compare them to the clot solubility studies results in samples of a 1-year-old girl with homozygous or double heterozygous FXIII deficiency. Case A year one girl with a history of bleeding from the umbilical cord. During her first year of life, several hematomas appeared in soft upper limb tissue after punctures for vaccination and a gluteal hematoma. One additional sample of a heterozygous patient and three samples of acquired FXIII deficiency were also evaluated. Materials and Methods Clotting tests, von Willebrand factor (vWF) antigen and activity, plasma FXIII-A subunit (pFXIII-A) were measured by an immunoturbidimetric assay in a photo-optical coagulometer. Solubility tests were performed with Ca2+-5 M urea and thrombin-2% acetic acid. Basal and post-FXIII concentrate infusion samples were studied. TEG was performed with CaCl2 or CaCl2 + SK (3.2 U/mL) in a Thromboelastograph. Results Prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time, fibrinogen, factor VIIIc, vWF, and platelet aggregation were normal. Antigenic pFXIII-A subunit was < 2%. TEG, evaluated at diagnosis and post FXIII concentrate infusion (pFXIII-A= 37%), presented a normal reaction time (R), 8 min, prolonged k (14 and 11min respectively), a low Maximum-Amplitude (MA) ( 39 and 52 mm respectively), and Clot Lysis (Lys60) slightly increased (23 and 30% respectively). In the sample at diagnosis, clot solubility was abnormal, 50 and 45 min with Ca-Urea and thrombin-acetic acid, respectively, but normal (>16 hours) 1-day post-FXIII infusion. Analysis of FXIII deficient and normal

  16. Growth Factor Tethering to Protein Nanoparticles via Coiled-Coil Formation for Targeted Drug Delivery.

    PubMed

    Assal, Yasmine; Mizuguchi, Yoshinori; Mie, Masayasu; Kobatake, Eiry

    2015-08-19

    Protein-based nanoparticles are attractive carriers for drug delivery because they are biodegradable and can be genetically designed. Moreover, modification of protein-based nanoparticles with cell-specific ligands allows for active targeting abilities. Previously, we developed protein nanoparticles comprising genetically engineered elastin-like polypeptides (ELPs) with fused polyaspartic acid tails (ELP-D). Epidermal growth factor (EGF) was displayed on the surface of the ELP-D nanoparticles via genetic design to allow for active cell-targeting abilities. Herein, we focused on the coiled-coil structural motif as a means for noncovalent tethering of growth factor to ELP-D. Specifically, two peptides known to form a heterodimer via a coiled-coil structural motif were fused to ELP-D and single-chain vascular endothelial growth factor (scVEGF121), to facilitate noncovalent tethering upon formation of the heterodimer coiled-coil structure. Drug-loaded growth factor-tethered ELP-Ds were found to be effective against cancer cells by provoking cell apoptosis. These results demonstrate that tethering growth factor to protein nanoparticles through coiled-coil formation yields a promising biomaterial candidate for targeted drug delivery.

  17. The F-BAR Protein PACSIN2 Regulates Epidermal Growth Factor Receptor Internalization

    PubMed Central

    de Kreuk, Bart-Jan; Anthony, Eloise C.; Geerts, Dirk; Hordijk, Peter L.

    2012-01-01

    Signaling via growth factor receptors, including the epidermal growth factor (EGF) receptor, is key to various cellular processes, such as proliferation, cell survival, and cell migration. In a variety of human diseases such as cancer, aberrant expression and activation of growth factor receptors can lead to disturbed signaling. Intracellular trafficking is crucial for proper signaling of growth factor receptors. As a result, the level of cell surface expression of growth factor receptors is an important determinant for the outcome of downstream signaling. BAR domain-containing proteins represent an important family of proteins that regulate membrane dynamics. In this study, we identify a novel role for the F-BAR protein PACSIN2 in the regulation of EGF receptor signaling. We show that internalized EGF as well as the (activated) EGF receptor translocated to PACSIN2-positive endosomes. Furthermore, loss of PACSIN2 increased plasma membrane expression of the EGF receptor in resting cells and increased EGF-induced phosphorylation of the EGF receptor. As a consequence, EGF-induced activation of Erk and Akt as well as cell proliferation were enhanced in PACSIN2-depleted cells. In conclusion, this study identifies a novel role for the F-BAR-domain protein PACSIN2 in regulating EGF receptor surface levels and EGF-induced downstream signaling. PMID:23129763

  18. Synergies of phosphatidylserine and protein disulfide isomerase in tissue factor activation

    PubMed Central

    Langer, Florian; Ruf, Wolfram

    2014-01-01

    Summary Tissue factor (TF), the cellular receptor and cofactor for factor VII/VIIa, initiates haemostasis and thrombosis. Initial tissue distribution studies suggested that TF was sequestered from the circulation and only present at perivascular sites. However, there is now clear evidence that TF also exists as a blood-borne form with critical contributions not only to arterial thrombosis following plaque rupture and to venous thrombosis following endothelial perturbation, but also to various other clotting abnormalities associated with trauma, infection, or cancer. Because thrombin generation, fibrin deposition, and platelet aggregation in the contexts of haemostasis, thrombosis, and pathogen defence frequently occur without TF de novo synthesis, considerable efforts are still directed to understanding the molecular events underlying the conversion of predominantly non-coagulant or cryptic TF on the surface of haematopoietic cells to a highly procoagulant molecule following cellular injury or stimulation. This article will review some of the still controversial mechanisms implicated in cellular TF activation or decryption with particular focus on the coordinated effects of outer leaflet phosphatidylserine exposure and thiol-disulfide exchange pathways involving protein disulfide isomerase (PDI). In this regard, our recent findings of ATP-triggered stimulation of the purinergic P2X7 receptor on myeloid and smooth muscle cells resulting in potent TF activation and shedding of procoagulant microparticles as well as of rapid monocyte TF decryption following antithymocyte globulin-dependent membrane complement fixation have delineated specific PDI-dependent pathways of cellular TF activation and thus illustrated additional and novel links in the coupling of inflammation and coagulation. PMID:24452853

  19. Association of atypical protein kinase C isotypes with the docker protein FRS2 in fibroblast growth factor signaling.

    PubMed

    Lim, Y P; Low, B C; Lim, J; Wong, E S; Guy, G R

    1999-07-01

    FRS2 is a docker protein that recruits signaling proteins to the plasma membrane in fibroblast growth factor signal transduction. We report here that FRS2 was associated with PKC lambda when Swiss 3T3 cells were stimulated with basic fibroblast growth factor. PKC zeta, the other member of the atypical PKC subfamily, could also bind FRS2. The association between FRS2 and PKC lambda is likely to be direct as shown by yeast two-hybrid analysis. The C-terminal fragments of FRS2 (amino acid residues 300-508) and SNT2 (amino acids 281-492), an isoform bearing 50% identity to FRS2, interacted with PKC lambda at a region (amino acids 240-562) that encompasses the catalytic domain. In vitro kinase assays revealed neither FRS2 nor SNT2 was a substrate of PKC lambda or zeta. Mutation of the alanine residue (Ala-120) to glutamate in the pseudo-substrate region of PKC lambda results in a constitutively active kinase that exhibited more than 2-fold greater binding to FRS2 in vitro than its "closed" wild-type counterpart. Tyrosine phosphorylation of FRS2 did not affect its binding to the constitutively active PKC lambda mutant, suggesting that the activation of PKC lambda is necessary and sufficient for its association with FRS2. It is likely that FRS2 serves as an anchoring protein for targeting activated atypical PKCs to the cell plasma membrane in signaling pathways.

  20. Hydrolyzed casein and whey protein meals comparably stimulate net whole-body protein synthesis in COPD patients with nutritional depletion without an additional effect of leucine co-ingestion

    PubMed Central

    Jonker, Renate; Deutz, Nicolaas EP; Erbland, Marcia L; Anderson, Paula J; Engelen, Mariëlle PKJ

    2013-01-01

    Background & Aims Muscle wasting commonly occurs in COPD, negatively affecting outcome. The aim was to examine the net whole-body protein synthesis response to two milk protein meals with comparable absorption rates (hydrolyzed casein (hCAS) vs. hydrolyzed whey (hWHEY)) and the effects of co-ingesting leucine. Methods Twelve COPD patients (GOLD stage II-IV) with nutritional depletion, were studied following intake of a 15g hCAS or hWHEY protein meal with or without leucine-co-ingestion, according to a double-blind randomized cross-over design. The isotopic tracers L-[ring-2H5]-Phenylalanine, L-[ring-2H2]-Tyrosine, L-[2H3]-3-Methylhistidine (given via continuous intravenous infusion), and L-[15N]-Phenylalanine (added to the protein meals) were used to measure endogenous whole-body protein breakdown (WbPB), whole-body protein synthesis (WbPS), net protein synthesis (NetPS), splanchnic extraction and myofibrillar protein breakdown (MPB). Analyses were done in arterialized-venous plasma by LC/MS/MS. Results WbPS was greater after intake of the hCAS protein meal (P<0.05) whereas the hWHEY protein meal reduced WbPB more (P<0.01). NetPS was stimulated comparably, with a protein conversion rate greater than 70%. Addition of leucine did not modify the insulin, WbPB, WbPS or MPB response. Conclusions Hydrolyzed casein and whey protein meals comparably and efficiently stimulate whole-body protein anabolism in COPD patients with nutritional depletion without an additional effect of leucine co-ingestion. PMID:23886411

  1. The TITAN5 gene of Arabidopsis encodes a protein related to the ADP ribosylation factor family of GTP binding proteins.

    PubMed

    McElver, J; Patton, D; Rumbaugh, M; Liu, C; Yang, L J; Meinke, D

    2000-08-01

    The titan (ttn) mutants of Arabidopsis exhibit dramatic alterations in mitosis and cell cycle control during seed development. Endosperm development in these mutants is characterized by the formation of giant polyploid nuclei with enlarged nucleoli. Embryo development is accompanied by significant cell enlargement in some mutants (ttn1 and ttn5) but not others (ttn2 and ttn3). We describe here the molecular cloning of TTN5 using a T-DNA-tagged allele. A second allele with a similar phenotype contains a nonsense mutation in the same coding region. The predicted protein is related to ADP ribosylation factors (ARFs), members of the RAS family of small GTP binding proteins that regulate various cellular functions in eukaryotes. TTN5 is most closely related in sequence to the ARL2 class of ARF-like proteins isolated from humans, rats, and mice. Although the cellular functions of ARL proteins remain unclear, the ttn5 phenotype is consistent with the known roles of ARFs in the regulation of intracellular vesicle transport.

  2. Effect of addition of thermally modified cowpea protein on sensory acceptability and textural properties of wheat bread and sponge cake.

    PubMed

    Campbell, Lydia; Euston, Stephen R; Ahmed, Mohamed A

    2016-03-01

    This paper investigates the sensory acceptability and textural properties of leavened wheat bread and sponge cake fortified with cow protein isolates that had been denatured and glycated by thermal treatment. Defatted cowpea flour was prepared from cow pea beans and the protein isolate was prepared (CPI) and thermally denatured (DCPI). To prepare glycated cowpea protein isolate (GCPI) the cowpea flour slurry was heat treated before isolation of the protein. CPI was more susceptible to thermal denaturation than GCPI as determined by turbidity and sulphydryl groups resulting in greater loss of solubility. This is attributed to the higher glycation degree and higher carbohydrate content of GCPI as demonstrated by glycoprotein staining of SDS PAGE gels. Water absorption of bread dough was significantly enhanced by DCPI and to a larger extent GCPI compared to the control, resulting in softer texture. CPI resulted in significantly increased crumb hardness in baked bread than the control whereas DCPI or GCPI resulted in significantly softer crumb. Bread fortified with 4% DCPI or GCPI was similar to control as regards sensory and textural properties whereas 4% CPI was significantly different, limiting its inclusion level to 2%. There was a trend for higher sensory acceptability scores for GCPI containing bread compared DCPI. Whole egg was replaced by 20% by GCPI (3.5%) in sponge cake without affecting the sensory acceptability, whereas CPI and DCPI supplemented cakes were significantly different than the control. PMID:26471676

  3. Conversion of canola meal into a high-protein feed additive via solid-state fungal incubation process

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The study goal was to determine the optimal fungal culture to reduce glucosinolates (GLS), fiber, and residual sugars while increasing the protein content and nutritional value of canola meal. Solid-state incubation conditions were used to enhance filamentous growth of the fungi. Flask trials were p...

  4. Effect of addition of thermally modified cowpea protein on sensory acceptability and textural properties of wheat bread and sponge cake.

    PubMed

    Campbell, Lydia; Euston, Stephen R; Ahmed, Mohamed A

    2016-03-01

    This paper investigates the sensory acceptability and textural properties of leavened wheat bread and sponge cake fortified with cow protein isolates that had been denatured and glycated by thermal treatment. Defatted cowpea flour was prepared from cow pea beans and the protein isolate was prepared (CPI) and thermally denatured (DCPI). To prepare glycated cowpea protein isolate (GCPI) the cowpea flour slurry was heat treated before isolation of the protein. CPI was more susceptible to thermal denaturation than GCPI as determined by turbidity and sulphydryl groups resulting in greater loss of solubility. This is attributed to the higher glycation degree and higher carbohydrate content of GCPI as demonstrated by glycoprotein staining of SDS PAGE gels. Water absorption of bread dough was significantly enhanced by DCPI and to a larger extent GCPI compared to the control, resulting in softer texture. CPI resulted in significantly increased crumb hardness in baked bread than the control whereas DCPI or GCPI resulted in significantly softer crumb. Bread fortified with 4% DCPI or GCPI was similar to control as regards sensory and textural properties whereas 4% CPI was significantly different, limiting its inclusion level to 2%. There was a trend for higher sensory acceptability scores for GCPI containing bread compared DCPI. Whole egg was replaced by 20% by GCPI (3.5%) in sponge cake without affecting the sensory acceptability, whereas CPI and DCPI supplemented cakes were significantly different than the control.

  5. Local dynamics of proteins and DNA evaluated from crystallographic B factors

    SciTech Connect

    Schneider, Bohdan; Gelly, Jean-Christophe; Brevern, Alexandre G. de; Černý, Jiří

    2014-09-01

    Distributions of scaled B factors from 704 protein–DNA complexes reflect primarily the neighbourhood of amino-acid and nucleotide residues: their flexibility grows from the protein core to protein–protein and protein–DNA interfaces, to solvent-exposed residues. Some of the findings clearly observed at higher resolution structures can no longer be observed for structures at low resolution indicating problems in refinement protocols. The dynamics of protein and nucleic acid structures is as important as their average static picture. The local molecular dynamics concealed in diffraction images is expressed as so-called B factors. To find out how the crystal-derived B factors represent the dynamic behaviour of atoms and residues of proteins and DNA in their complexes, the distributions of scaled B factors from a carefully curated data set of over 700 protein–DNA crystal structures were analyzed [Schneider et al. (2014 ▶), Nucleic Acids Res.42, 3381–3394]. Amino acids and nucleotides were categorized based on their molecular neighbourhood as solvent-accessible, solvent-inaccessible (i.e. forming the protein core) or lying at protein–protein or protein–DNA interfaces; the backbone and side-chain atoms were analyzed separately. The B factors of two types of crystal-ordered water molecules were also analyzed. The analysis confirmed several expected features of protein and DNA dynamics, but also revealed surprising facts. Solvent-accessible amino acids have B factors that are larger than those of residues at the biomolecular interfaces, and core-forming amino acids are the most restricted in their movement. A unique feature of the latter group is that their side-chain and backbone atoms are restricted in their movement to the same extent; in all other amino-acid groups the side chains are more floppy than the backbone. The low values of the B factors of water molecules bridging proteins with DNA and the very large fluctuations of DNA phosphates are

  6. Relationship between Sonic hedgehog protein, brain-derived neurotrophic factor and oxidative stress in autism spectrum disorders.

    PubMed

    Al-Ayadhi, Laila Y

    2012-02-01

    The etiology of autism spectrum disorders (ASD) is not well known but oxidative stress has been suggested to play a pathological role. We report here that the serum levels of Sonic hedgehog (SHH) protein and brain-derived neurotrophic factor (BDNF) might be linked to oxidative stress in ASD. By using the whole blood or polymorphonuclear leukocytes, we demonstrated that autistic children produced a significantly higher level of oxygen free radicals (OFR). In addition, we found significantly higher levels of serum SHH protein in children with mild as well as severe form of autism. We also found that the serum level of BDNF was significantly reduced in autistic children with mild form of the disorder but not with severe form of the disorder. Our findings are the first to report a correlation between SHH, BDNF and OFR in autistic children, suggesting a pathological role of oxidative stress and SHH in autism spectrum disorders. PMID:21984201

  7. Class I and II Small Heat Shock Proteins Together with HSP101 Protect Protein Translation Factors during Heat Stress1[OPEN

    PubMed Central

    Basha, Eman; Fowler, Mary E.; Kim, Minsoo; Bordowitz, Juliana; Katiyar-Agarwal, Surekha

    2016-01-01

    The ubiquitous small heat shock proteins (sHSPs) are well documented to act in vitro as molecular chaperones to prevent the irreversible aggregation of heat-sensitive proteins. However, the in vivo activities of sHSPs remain unclear. To investigate the two most abundant classes of plant cytosolic sHSPs (class I [CI] and class II [CII]), RNA interference (RNAi) and overexpression lines were created in Arabidopsis (Arabidopsis thaliana) and shown to have reduced and enhanced tolerance, respectively, to extreme heat stress. Affinity purification of CI and CII sHSPs from heat-stressed seedlings recovered eukaryotic translation elongation factor (eEF) 1B (α-, β-, and γ-subunits) and eukaryotic translation initiation factor 4A (three isoforms), although the association with CI sHSPs was stronger and additional proteins involved in translation were recovered with CI sHSPs. eEF1B subunits became partially insoluble during heat stress and, in the CI and CII RNAi lines, showed reduced recovery to the soluble cell fraction after heat stress, which was also dependent on HSP101. Furthermore, after heat stress, CI sHSPs showed increased retention in the insoluble fraction in the CII RNAi line and vice versa. Immunolocalization revealed that both CI and CII sHSPs were present in cytosolic foci, some of which colocalized with HSP101 and with eEF1Bγ and eEF1Bβ. Thus, CI and CII sHSPs have both unique and overlapping functions and act either directly or indirectly to protect specific translation factors in cytosolic stress granules. PMID:27474115

  8. Pollen specific expression of maize genes encoding actin depolymerizing factor-like proteins.

    PubMed Central

    Lopez, I; Anthony, R G; Maciver, S K; Jiang, C J; Khan, S; Weeds, A G; Hussey, P J

    1996-01-01

    In pollen development, a dramatic reorganization of the actin cytoskeleton takes place during the passage of the pollen grain into dormancy and on activation of pollen tube growth. A role for actin-binding proteins is implicated and we report here the identification of a small gene family in maize that encodes actin depolymerizing factor (ADF)-like proteins. The ADF group of proteins are believed to control actin polymerization and depolymerization in response to both intracellular and extracellular signals. Two of the maize genes ZmABP1 and ZmABP2 are expressed specifically in pollen and germinating pollen suggesting that the protein products may be involved in pollen actin reorganization. A third gene, ZmABP3, encodes a protein only 56% and 58% identical to ZmABP1 and ZmABP2, respectively, and its expression is suppressed in pollen and germinated pollen. The fundamental biochemical characteristics of the ZmABP proteins has been elucidated using bacterially expressed ZmABP3 protein. This has the ability to bind monomeric actin (G-actin) and filamentous actin (F-actin). Moreover, it decreases the viscosity of polymerized actin solutions consistent with an ability to depolymerize filaments. These biochemical characteristics, taken together with the sequence comparisons, support the inclusion of the ZmABP proteins in the ADF group. Images Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:8693008

  9. Visna virus Tat protein: a potent transcription factor with both activator and suppressor domains.

    PubMed Central

    Carruth, L M; Hardwick, J M; Morse, B A; Clements, J E

    1994-01-01

    Visna virus is a pathogenic lentivirus of sheep tat is distantly related to the primate lentiviruses, including human immunodeficiency virus type 1. The visna virus genome encodes a small regulatory protein, Tat, which is necessary for efficient viral replication and enhanced viral transcription. To investigate the mechanism of action of the visna Tat protein and to localize the protein domain(s) responsible for transcriptional activation, chimeric proteins containing visna virus Tat sequences fused to the DNA binding domain of the yeast transactivation factor GAL4 (residues 1 to 147) were made. The GAL4-Tat fusion proteins were transfected into cells and tested for the ability to activate the adenovirus E1b promoter via upstream GAL4 DNA binding sites. Full-length GAL4-Tat fusion proteins were weak transactivators in this system, giving only a two- to fourfold increase in transcription in several cell types, including HeLa and sheep choroid plexus cells. In contrast, fusion of the N-terminal region of the Tat protein to GAL4 revealed a potent activation domain. Amino acids 13 to 38 appeared to be the most critical for activation. No other region of the protein showed any activation in the GAL4 system. This N-terminal region of the visna virus Tat protein has a large number of acidic and hydrophobic residues, suggesting that Tat has an acidic activation domain common to many transcriptional transactivators. Mutations in hydrophobic and bulky aromatic residues dramatically reduced the activity of the chimeric protein. Competition experiments suggest that mechanism of the visna virus Tat activation domain may closely resemble that of the herpesvirus activator VP16 and human immunodeficiency virus Tat, a related lentivirus activator, since both significantly reduce the level of visna virus Tat activation. Finally, a domain between residues 39 and 53 was identified in the Tat protein that, in the GAL4 system, negatively regulates activation by Tat. Images PMID:8083955

  10. Bone morphogenetic protein-4 strongly potentiates growth factor-induced proliferation of mammary epithelial cells

    SciTech Connect

    Montesano, Roberto Sarkoezi, Rita; Schramek, Herbert

    2008-09-12

    Bone morphogenetic proteins (BMPs) are multifunctional cytokines that elicit pleiotropic effects on biological processes such as cell proliferation, cell differentiation and tissue morphogenesis. With respect to cell proliferation, BMPs can exert either mitogenic or anti-mitogenic activities, depending on the target cells and their context. Here, we report that in low-density cultures of immortalized mammary epithelial cells, BMP-4 did not stimulate cell proliferation by itself. However, when added in combination with suboptimal concentrations of fibroblast growth factor (FGF)-2, FGF-7, FGF-10, epidermal growth factor (EGF) or hepatocyte growth factor (HGF), BMP-4 potently enhanced growth factor-induced cell proliferation. These results reveal a hitherto unsuspected interplay between BMP-4 and growth factors in the regulation of mammary epithelial cell proliferation. We suggest that the ability of BMP-4 to potentiate the mitogenic activity of multiple growth factors may contribute to mammary gland ductal morphogenesis as well as to breast cancer progression.

  11. Protein interactions of MADS box transcription factors involved in flowering in Lolium perenne.

    PubMed

    Ciannamea, Stefano; Kaufmann, Kerstin; Frau, Marta; Tonaco, Isabella A Nougalli; Petersen, Klaus; Nielsen, Klaus K; Angenent, Gerco C; Immink, Richard G H

    2006-01-01

    Regulation of flowering time is best understood in the dicot model species Arabidopsis thaliana. Molecular analyses revealed that genes belonging to the MADS box transcription factor family play pivotal regulatory roles in both the vernalization- and photoperiod-regulated flowering pathways. Here the analysis of three APETALA1 (AP1)-like MADS box proteins (LpMADS1-3) and a SHORT VEGETATIVE PHASE (SVP)-like MADS box protein (LpMADS10) from the monocot perennial grass species Lolium perenne is reported. Features of these MADS box proteins were studied by yeast two-hybrid assays. Protein-protein interactions among the Lolium proteins and with members of the Arabidopsis MADS box family have been studied. The expression pattern for LpMADS1 and the protein properties suggest that not the Arabidopsis AP1 gene, but the SUPPRESSOR OF CONSTANS1 (SOC1) gene, is the functional equivalent of LpMADS1. To obtain insight into the molecular mechanism underlying the regulation of LpMADS1 gene expression in vernalization-sensitive and -insensitive Lolium accessions, the upstream sequences of this gene from a winter and spring growth habit variety were compared with respect to MADS box protein binding. In both promoter elements, a putative MADS box transcription factor-binding site (CArG-box) is present; however, the putative spring promoter has a short deletion adjacent to this DNA motif. Experiments using yeast one-hybrid and gel retardation assays demonstrated that the promoter element is bound by an LpMADS1-LpMADS10 higher order protein complex and, furthermore, that this complex binds efficiently to the promoter element from the winter variety only. This strongly supports the model that LpMADS1 together with LpMADS10 controls the vernalization-dependent regulation of the LpMADS1 gene, which is part of the vernalization-induced flowering process in Lolium. PMID:17005923

  12. Multiplex immunoassays for quantification of cytokines, growth factors, and other proteins in stem cell communication.

    PubMed

    Valekova, Ivona; Skalnikova, Helena Kupcova; Jarkovska, Karla; Motlik, Jan; Kovarova, Hana

    2015-01-01

    Immunoassays represent valuable and broadly used techniques for detection and quantification of proteins. Thanks to their high sensitivity, such techniques are powerful for analyzing growth factors, trophic factors, angiogenic factors, hormones, cytokines, chemokines, soluble receptors, and other proteins which play key roles in intercellular communication and operate as potent regulators of stem cell survival, proliferation, differentiation, or cell death. Multiplex immunological assays, in contrast to ELISA, offer simultaneous quantification of tens of proteins across multiple samples, and have been developed to save time, costs, and sample volumes. Among them, planar antibody microarrays and xMAP(®) bead-based assays have become particularly popular for characterization of proteins secreted by stem cells, as they are relatively easy, highly accurate, multiplex to a high degree and a broad spectrum of analytes can be measured. Here, we describe protocols for multiplex quantification of secreted proteins using Quantibody(®) microarrays (RayBiotech) and xMAP(®) assays (Luminex and its partners). PMID:25063502

  13. Elongation Factor Tu and Heat Shock Protein 70 Are Membrane-Associated Proteins from Mycoplasma ovipneumoniae Capable of Inducing Strong Immune Response in Mice.

    PubMed

    Jiang, Fei; He, Jinyan; Navarro-Alvarez, Nalu; Xu, Jian; Li, Xia; Li, Peng; Wu, Wenxue

    2016-01-01

    Chronic non-progressive pneumonia, a disease that has become a worldwide epidemic has caused considerable loss to sheep industry. Mycoplasma ovipneumoniae (M. ovipneumoniae) is the causative agent of interstitial pneumonia in sheep, goat and bighorn. We here have identified by immunogold and immunoblotting that elongation factor Tu (EF-Tu) and heat shock protein 70 (HSP 70) are membrane-associated proteins on M. ovipneumonaiea. We have evaluated the humoral and cellular immune responses in vivo by immunizing BALB/c mice with both purified recombinant proteins rEF-Tu and rHSP70. The sera of both rEF-Tu and rHSP70 treated BALB/c mice demonstrated increased levels of IgG, IFN-γ, TNF-α, IL-12(p70), IL-4, IL-5 and IL-6. In addition, ELISPOT assay showed significant increase in IFN-γ+ secreting lymphocytes in the rHSP70 group when compared to other groups. Collectively our study reveals that rHSP70 induces a significantly better cellular immune response in mice, and may act as a Th1 cytokine-like adjuvant in immune response induction. Finally, growth inhibition test (GIT) of M. ovipneumoniae strain Y98 showed that sera from rHSP70 or rEF-Tu-immunized mice inhibited in vitro growth of M. ovipneumoniae. Our data strongly suggest that EF-Tu and HSP70 of M. ovipneumoniae are membrane-associated proteins capable of inducing antibody production, and cytokine secretion. Therefore, these two proteins may be potential candidates for vaccine development against M. ovipneumoniae infection in sheep.

  14. Elongation Factor Tu and Heat Shock Protein 70 Are Membrane-Associated Proteins from Mycoplasma ovipneumoniae Capable of Inducing Strong Immune Response in Mice

    PubMed Central

    Jiang, Fei; He, Jinyan; Navarro-Alvarez, Nalu; Xu, Jian; Li, Xia; Li, Peng; Wu, Wenxue

    2016-01-01

    Chronic non-progressive pneumonia, a disease that has become a worldwide epidemic has caused considerable loss to sheep industry. Mycoplasma ovipneumoniae (M. ovipneumoniae) is the causative agent of interstitial pneumonia in sheep, goat and bighorn. We here have identified by immunogold and immunoblotting that elongation factor Tu (EF-Tu) and heat shock protein 70 (HSP 70) are membrane-associated proteins on M. ovipneumonaiea. We have evaluated the humoral and cellular immune responses in vivo by immunizing BALB/c mice with both purified recombinant proteins rEF-Tu and rHSP70. The sera of both rEF-Tu and rHSP70 treated BALB/c mice demonstrated increased levels of IgG, IFN-γ, TNF-α, IL-12(p70), IL-4, IL-5 and IL-6. In addition, ELISPOT assay showed significant increase in IFN-γ+ secreting lymphocytes in the rHSP70 group when compared to other groups. Collectively our study reveals that rHSP70 induces a significantly better cellular immune response in mice, and may act as a Th1 cytokine-like adjuvant in immune response induction. Finally, growth inhibition test (GIT) of M. ovipneumoniae strain Y98 showed that sera from rHSP70 or rEF-Tu-immunized mice inhibited in vitro growth of M. ovipneumoniae. Our data strongly suggest that EF-Tu and HSP70 of M. ovipneumoniae are membrane-associated proteins capable of inducing antibody production, and cytokine secretion. Therefore, these two proteins may be potential candidates for vaccine development against M. ovipneumoniae infection in sheep. PMID:27537186

  15. Elongation Factor Tu and Heat Shock Protein 70 Are Membrane-Associated Proteins from Mycoplasma ovipneumoniae Capable of Inducing Strong Immune Response in Mice.

    PubMed

    Jiang, Fei; He, Jinyan; Navarro-Alvarez, Nalu; Xu, Jian; Li, Xia; Li, Peng; Wu, Wenxue

    2016-01-01

    Chronic non-progressive pneumonia, a disease that has become a worldwide epidemic has caused considerable loss to sheep industry. Mycoplasma ovipneumoniae (M. ovipneumoniae) is the causative agent of interstitial pneumonia in sheep, goat and bighorn. We here have identified by immunogold and immunoblotting that elongation factor Tu (EF-Tu) and heat shock protein 70 (HSP 70) are membrane-associated proteins on M. ovipneumonaiea. We have evaluated the humoral and cellular immune responses in vivo by immunizing BALB/c mice with both purified recombinant proteins rEF-Tu and rHSP70. The sera of both rEF-Tu and rHSP70 treated BALB/c mice demonstrated increased levels of IgG, IFN-γ, TNF-α, IL-12(p70), IL-4, IL-5 and IL-6. In addition, ELISPOT assay showed significant increase in IFN-γ+ secreting lymphocytes in the rHSP70 group when compared to other groups. Collectively our study reveals that rHSP70 induces a significantly better cellular immune response in mice, and may act as a Th1 cytokine-like adjuvant in immune response induction. Finally, growth inhibition test (GIT) of M. ovipneumoniae strain Y98 showed that sera from rHSP70 or rEF-Tu-immunized mice inhibited in vitro growth of M. ovipneumoniae. Our data strongly suggest that EF-Tu and HSP70 of M. ovipneumoniae are membrane-associated proteins capable of inducing antibody production, and cytokine secretion. Therefore, these two proteins may be potential candidates for vaccine development against M. ovipneumoniae infection in sheep. PMID:27537186

  16. Characterization of group A streptococci (Streptococcus pyogenes): correlation of M-protein and emm-gene type with T-protein agglutination pattern and serum opacity factor.

    PubMed

    Johnson, Dwight R; Kaplan, Edward L; VanGheem, Amy; Facklam, Richard R; Beall, Bernard

    2006-02-01

    Strain characterization of group A streptococci (GAS) has traditionally been based on serological identification of M protein. Additional tests to determine T-protein serotype and production of streptococcal serum opacity factor (SOF) provide important information both to aid in and to supplement M-protein serotyping. Advances in DNA-sequencing technology in the late twentieth century resulted in the development of a method for determining the M type of GAS from the sequence of the gene encoding M protein, the emm gene. Although emm-sequence typing has largely replaced M typing in many laboratories, information provided by T typing and SOF determination continues to provide valuable supplementary information for strain characterization. A comprehensive summary of the correlation of T pattern and SOF production with M type was last published in 1993, several years before emm typing became widely available. Since then, the ease of M-type identification afforded by emm typing has resulted in an increase in the number of confirmed M/emm types of more than 50 %. However, comprehensive information about T-protein serotype and the correlation of SOF production with these new M/emm types is not widely available. This report presents a comprehensive summary of this information, not only for newly described types, but also updated information for previously described types. This information was extracted from combined records from streptococcal reference laboratories at the University of Minnesota and at the Centers for Disease Control and Prevention in Atlanta. Data from more than 40,000 strains (representing uncomplicated GAS infections, systemic invasive infections and strains associated with non-suppurative sequelae, collected from the US and diverse locations worldwide) were analysed.

  17. Mapping transcription factor interactome networks using HaloTag protein arrays

    PubMed Central

    Yazaki, Junshi; Galli, Mary; Kim, Alice Y.; Nito, Kazumasa; Aleman, Fernando; Chang, Katherine N.; Quan, Rosa; Nguyen, Hien; Song, Liang; Alvarez, José M.; Huang, Shao-shan Carol; Chen, Huaming; Ramachandran, Niroshan; Altmann, Stefan; Gutiérrez, Rodrigo A.; Schroeder, Julian I.; Chory, Joanne; LaBaer, Joshua; Vidal, Marc; Braun, Pascal; Ecker, Joseph R.

    2016-01-01

    Protein microarrays enable investigation of diverse biochemical properties for thousands of proteins in a single experiment, an unparalleled capacity. Using a high-density system called HaloTag nucleic acid programmable protein array (HaloTag-NAPPA), we created high-density protein arrays comprising 12,000 Arabidopsis ORFs. We used these arrays to query protein–protein interactions for a set of 38 transcription factors and transcriptional regulators (TFs) that function in diverse plant hormone regulatory pathways. The resulting transcription factor interactome network, TF-NAPPA, contains thousands of novel interactions. Validation in a benchmarked in vitro pull-down assay revealed that a random subset of TF-NAPPA validated at the same rate of 64% as a positive reference set of literature-curated interactions. Moreover, using a bimolecular fluorescence complementation (BiFC) assay, we confirmed in planta several interactions of biological interest and determined the interaction localizations for seven pairs. The application of HaloTag-NAPPA technology to plant hormone signaling pathways allowed the identification of many novel transcription factor–protein interactions and led to the development of a proteome-wide plant hormone TF interactome network. PMID:27357687

  18. Interaction of Shiga toxin 2 with complement regulators of the factor H protein family.

    PubMed

    Poolpol, Kulwara; Orth-Höller, Dorothea; Speth, Cornelia; Zipfel, Peter F; Skerka, Christine; de Córdoba, Santiago Rodriguez; Brockmeyer, Jens; Bielaszewska, Martina; Würzner, Reinhard

    2014-03-01

    Shiga toxin 2 (Stx2) is believed to be a major virulence factor of enterohemorrhagic Escherichia coli (EHEC) contributing to hemolytic uremic syndrome (HUS). The complement system has recently been found to be involved in the pathogenesis of EHEC-associated HUS. Stx2 was shown to activate complement via the alternative pathway, to bind factor H (FH) at short consensus repeats (SCRs) 6-8 and 18-20 and to delay and reduce FH cofactor activity on the cell surface. We now show that complement factor H-related protein 1 (FHR-1) and factor H-like protein 1 (FHL-1), proteins of the FH protein family that show amino acid sequence and regulatory function similarities with FH, also bind to Stx2. The FHR-1 binding site for Stx2 was located at SCRs 3-5 and the binding capacity of FHR-1*A allotype was higher than that of FHR-1*B. FHR-1 and FHL-1 competed with FH for Stx2 binding, and in the case of FHR-1 this competition resulted in a reduction of FH cofactor activity. FHL-1 retained its cofactor activity in the fluid phase when bound to Stx2. In conclusion, multiple interactions of key complement inhibitors FH, FHR-1 and FHL-1 with Stx2 corroborate our hypothesis of a direct role of complement in EHEC-associated HUS.

  19. The Fimbrial Protein is a Virulence Factor and Potential Vaccine Antigen of Avibacterium paragallinarum.

    PubMed

    Liu, C-C; Ou, S-C; Tan, D-H; Hsieh, M-K; Shien, J-H; Chang, P-C

    2016-09-01

    Fimbriae are recognized as virulence factors and potential vaccine antigens of several pathogenic bacteria, but the function of the fimbriae from Avibacterium paragallinarum is not well known. In this study, a gene encoding the fimbrial protein FlfA was identified in A. paragallinarum . Sequencing analysis of the putative promoter region of flfA suggests that flfA expression in A. paragallinarum might be controlled by phase variation. The flfA gene from A. paragallinarum was expressed as a recombinant protein (r-FlfA) in Escherichia coli . Immunization with r-FlfA conferred chickens protection against challenge infection with A. paragallinarum . Virulence assays showed that the flfA-deficient mutants of A. paragallinarum were less virulent than their parental wild-type strains. These results indicated that the fimbrial protein FlfA is a virulence factor and potential vaccine antigen from A. paragallinarum . PMID:27610725

  20. Regulation of insulin-like growth factor-binding protein messenger ribonucleic acid levels in sheep thyroid cells.

    PubMed

    Bachrach, L K; Eggo, M C; Burrow, G N; Liu, F; Tram, T; Powell, D R

    1991-04-01

    The insulin-like growth factors (IGFs) exist primarily bound to cell surface receptors or complexed to specific binding proteins (IGFBPs). The IGFBPs modulate the bioavailability of the IGFs and may enhance or inhibit IGF actions. Several distinct forms of IGFBPs have been described on the basis of size, immunological determinants, and distribution in biological fluids; the IGFBPs may differ as well in their biological function. Sheep thyroid cells produce IGFBPs under hormonal regulation. Cells grown in basal medium or with six-hormone (6H) medium supplements (transferrin, glycyl-histidyl-lysine, hydrocortisone, somatostatin, insulin, and TSH) release nonglycosylated BPs that migrate at 24, 27, 29, and 32 kDa on Western ligand blot. Cells cultured with the thyroid mitogens epidermal growth factor and phorbol ester release additional glycosylated IGFBPs of 40-44 kDa. Immunoprecipitation experiments indicate that 29- and 32-kDa IGFBPs are antigenically related to IGFBP-2, and the 40- to 44-kDa proteins are related to IGFBP-3. Using specific cDNA probes IGFBP-1, -2, and -3, we examined the regulation of IGFBP mRNA levels in sheep thyroid cultures. The rat IGFBP-2 cDNA probe hybridized to an approximately 1.6-kilobase mRNA species in cells under all culture conditions. However, IGFBP-3 mRNA was detectable only in epidermal growth factor- or phorbol ester-treated cells and appeared within 4 h, preceding the release of IGFBP-3 protein into the medium. The 6H additives, which stimulate differentiated function in thyroid cells, inhibited the mRNA levels of both IGFBP-2 and IGFBP-3. IGFBP-1 mRNA was not detectable. The distinct regulation of these IGFBPs suggest that they may play different biological roles in modulating thyroid physiology. PMID:1706262

  1. Mexican American First-Generation Students' Perceptions of Siblings and Additional Factors Influencing Their College Choice Process

    ERIC Educational Resources Information Center

    Elias McAllister, Dora

    2012-01-01

    The purpose of this study was to understand the factors influencing the college choice process of Mexican American first-generation students who had an older sibling with college experience. While a considerable amount of research exists on factors influencing the college choice process of first-generation college students, and a few studies…

  2. Fat and starch as additive risk factors for milk fat depression in dairy diets containing corn dried distillers grains with solubles.

    PubMed

    Ramirez Ramirez, H A; Castillo Lopez, E; Harvatine, K J; Kononoff, P J

    2015-03-01

    Two experiments were conducted to evaluate the additive effects of starch and fat as risk factors associated with milk fat depression in dairy diets containing corn dried distillers grains with solubles. In experiment 1, 4 multiparous ruminally cannulated Holstein cows, averaging 114±14 d in milk and 662±52 kg of body weight, were randomly assigned to 4 treatments in a 4×4 Latin square to determine the effect of these risk factors on rumen fermentation and milk fatty acid profile. In each 21-d period, cows were assigned to 1 of 4 dietary treatments: a control diet (CON; ether extract 5.2%, starch 19%); CON with added oil (OL; ether extract 6.4%, starch 18%); CON with added starch (STR; ether extract 5.5%, starch 22%); and CON with added oil and starch (COMBO; ether extract 6.5%, starch 23%). After completion of experiment 1, milk production response was evaluated in a second experiment with a similar approach to diet formulation. Twenty Holstein cows, 12 primiparous and 8 multiparous, averaging 117±17 d in milk and 641±82 kg, were used in replicated 4×4 Latin squares with 21-d periods. Results from experiment 1 showed that ruminal pH was not affected by treatment averaging 5.87±0.08. Molar proportion of propionate in rumen fluid was greatest on the COMBO diet, followed by OL and STR, and lowest for CON. The concentration of trans-10,cis-12 conjugated linoleic acid in milk fat increased with the COMBO diet. Adding oil, starch, or a combination of both resulted in lower concentration and yield of fatty acids<16 carbons. Compared with the control, OL and STR resulted in 13% lower concentration, whereas the COMBO diet resulted in a 27% reduction; similarly yield was reduced by 24% with the OL and STR treatments and 54% with the COMBO diet. In experiment 2, milk yield, milk protein percentage, and milk protein yield were similar across treatments, averaging 26.6±1.01 kg/d, 3.2±0.05%, and 0.84±0.03 kg/d, respectively. Fat-corrected milk was greatest for CON, 26

  3. Wheat (Triticum aestivum) seedlings secrete proteases from the roots and, after protein addition, grow well on medium without inorganic nitrogen.

    PubMed

    Adamczyk, B; Godlewski, M; Zimny, J; Zimny, A

    2008-11-01

    This paper reports on the role of proteases secreted by roots in nitrogen capture by plants. The study was conducted on aseptically cultivated wheat seedlings (Triticum aestivum cv. Tacher) obtained from embryos isolated from grains. Seedlings were cultivated for 21 days on deionised water, Murashige Skoog medium (MS), MS without inorganic nitrogen (IN), and MS without IN, in which IN was replaced by casein (0.01%, 0.1% or 1%). Comparison of seedlings grown on these media showed that casein entirely compensated for the lack of inorganic nitrogen in the medium. Shoots and roots of seedlings cultivated on MS medium with this protein had higher fresh weight than those cultivated on MS medium without casein. The increase in fresh weight of seedlings was correlated with casein concentration and proteolytic activity in the medium. In conclusion, wheat that uses proteases secreted by the roots can directly utilise proteins in the medium as a source of nitrogen without prior digestion by microbial proteases and without protein mineralisation. These results suggest the important role of organic nitrogen fertilisers in increasing wheat yield. PMID:18950429

  4. Impaired immunogenicity of a meningococcal factor H-binding protein vaccine engineered to eliminate factor h binding.

    PubMed

    Beernink, Peter T; Shaughnessy, Jutamas; Ram, Sanjay; Granoff, Dan M

    2010-07-01

    Meningococcal factor H-binding protein (fHbp) is a promising antigen that is part of two vaccines in clinical development. The protein specifically binds human complement factor H (fH), which downregulates complement activation on the bacterial surface and enables the organism to evade host defenses. In humans, the vaccine antigen forms a complex with fH, which may affect anti-fHbp antibody repertoire and decrease serum bactericidal activity by covering important fHbp epitopes. In a recent study, fHbp residues in contact with fH were identified from a crystal structure. Two fHbp glutamate residues that mediated ion-pair interactions with fH were replaced with alanine, and the resulting E218A/E239A mutant no longer bound the fH fragment. In the present study, we generated the E218A/E239A mutant recombinant protein and confirmed the lack of fH binding. By enzyme-linked immunosorbent assay (ELISA), the mutant fHbp showed similar respective concentration-dependent inhibition of binding of four bactericidal anti-fHbp monoclonal antibodies (MAbs) to fHbp, compared with inhibition by the soluble wild-type protein. In two mouse strains, the mutant fHbp elicited up to 4-fold-lower IgG anti-fHbp antibody titers and up to 20-fold-lower serum bactericidal titers than those elicited by the wild-type fHbp vaccine. Thus, although introduction of the two alanine substitutions to eliminate fH binding did not appear to destabilize the molecule globally, the mutations resulted in decreased immunogenicity in mouse models in which neither the mutant nor the wild-type control vaccine bound fH. These results cast doubt on the vaccine potential in humans of this mutant fHbp.

  5. Regulation of Myogenesis by Fibroblast Growth Factors Requires Beta-Gamma Subunits of Pertussis Toxin-Sensitive G Proteins

    PubMed Central

    Fedorov, Yuri V.; Jones, Nathan C.; Olwin, Bradley B.

    1998-01-01

    Terminal differentiation of skeletal muscle cells in culture is inhibited by a number of different growth factors whose subsequent intracellular signaling events are poorly understood. In this study, we have investigated the role of heterotrimeric G proteins in mediating fibroblast growth factor (FGF)-dependent signals that regulate myogenic differentiation. Pertussis toxin, which ADP-ribosylates and inactivates susceptible G proteins, promotes terminal differentiation in the presence of FGF-2, suggesting that Gα or Gβγ subunits or both are involved in transducing the FGF-dependent signal(s) that inhibits myogenesis. We found that Gβγ subunits are likely to be involved since the expression of the C terminus of β-adrenergic receptor kinase 1, a Gβγ subunit-sequestering agent, promotes differentiation in the presence of FGF-2, and expression of the free Gβγ dimer can replace FGF-2, rescuing cells from pertussis toxin-induced differentiation. Addition of pertussis toxin also blocked FGF-2-mediated activation of mitogen-activated protein kinases (MAPKs). Ectopic expression of dominant active mutants in the Ras/MAPK pathway rescued cells from pertussis toxin-induced terminal differentiation, suggesting that the Gβγ subunits act upstream of the Ras/MAPK pathway. It is unlikely that the pertussis toxin-sensitive pathway is activated by other, as yet unidentified FGF receptors since PDGF (platelet-derived growth factor)-stimulated MM14 cells expressing a chimeric receptor containing the FGF receptor-1 intracellular domain and the PDGF receptor extracellular domain were sensitive to pertussis toxin. Our data suggest that FGF-mediated signals involved in repression of myogenic differentiation are transduced by a pertussis toxin-sensitive G-protein-coupled mechanism. This signaling pathway requires the action of Gβγ subunits and activation of MAPKs to repress skeletal muscle differentiation. PMID:9742095

  6. Mammalian splicing factor SF1 interacts with SURP domains of U2 snRNP-associated proteins.

    PubMed

    Crisci, Angela; Raleff, Flore; Bagdiul, Ivona; Raabe, Monika; Urlaub, Henning; Rain, Jean-Christophe; Krämer, Angela

    2015-12-01

    Splicing factor 1 (SF1) recognizes the branch point sequence (BPS) at the 3' splice site during the formation of early complex E, thereby pre-bulging the BPS adenosine, thought to facilitate subsequent base-pairing of the U2 snRNA with the BPS. The 65-kDa subunit of U2 snRNP auxiliary factor (U2AF65) interacts with SF1 and was shown to recruit the U2 snRNP to the spliceosome. Co-immunoprecipitation experiments of SF1-interacting proteins from HeLa cell extracts shown here are consistent with the presence of SF1 in early splicing complexes. Surprisingly almost all U2 snRNP proteins were found associated with SF1. Yeast two-hybrid screens identified two SURP domain-containing U2 snRNP proteins as partners of SF1. A short, evolutionarily conserved region of SF1 interacts with the SURP domains, stressing their role in protein-protein interactions. A reduction of A complex formation in SF1-depleted extracts could be rescued with recombinant SF1 containing the SURP-interaction domain, but only partial rescue was observed with SF1 lacking this sequence. Thus, SF1 can initially recruit the U2 snRNP to the spliceosome during E complex formation, whereas U2AF65 may stabilize the association of the U2 snRNP with the spliceosome at later times. In addition, these findings may have implications for alternative splicing decisions. PMID:26420826

  7. Mammalian splicing factor SF1 interacts with SURP domains of U2 snRNP-associated proteins.

    PubMed

    Crisci, Angela; Raleff, Flore; Bagdiul, Ivona; Raabe, Monika; Urlaub, Henning; Rain, Jean-Christophe; Krämer, Angela

    2015-12-01

    Splicing factor 1 (SF1) recognizes the branch point sequence (BPS) at the 3' splice site during the formation of early complex E, thereby pre-bulging the BPS adenosine, thought to facilitate subsequent base-pairing of the U2 snRNA with the BPS. The 65-kDa subunit of U2 snRNP auxiliary factor (U2AF65) interacts with SF1 and was shown to recruit the U2 snRNP to the spliceosome. Co-immunoprecipitation experiments of SF1-interacting proteins from HeLa cell extracts shown here are consistent with the presence of SF1 in early splicing complexes. Surprisingly almost all U2 snRNP proteins were found associated with SF1. Yeast two-hybrid screens identified two SURP domain-containing U2 snRNP proteins as partners of SF1. A short, evolutionarily conserved region of SF1 interacts with the SURP domains, stressing their role in protein-protein interactions. A reduction of A complex formation in SF1-depleted extracts could be rescued with recombinant SF1 containing the SURP-interaction domain, but only partial rescue was observed with SF1 lacking this sequence. Thus, SF1 can initially recruit the U2 snRNP to the spliceosome during E complex formation, whereas U2AF65 may stabilize the association of the U2 snRNP with the spliceosome at later times. In addition, these findings may have implications for alternative splicing decisions.

  8. Calcitriol inhibits tumor necrosis factor alpha and macrophage inflammatory protein-2 during lipopolysaccharide-induced acute lung injury in mice.

    PubMed

    Tan, Zhu-Xia; Chen, Yuan-Hua; Xu, Shen; Qin, Hou-Ying; Wang, Hua; Zhang, Cheng; Xu, De-Xiang; Zhao, Hui

    2016-08-01

    Acute lung injury is a common complication of sepsis in intensive care unit patients with an extremely high mortality. The present study investigated the effects of calcitriol, the active form of vitamin D, on tumor necrosis factor alpha (TNF-α) and macrophage inflammatory protein-2 (MIP-2) in sepsis-induced acute lung injury. Mice were intraperitoneally (i.p.) injected with lipopolysaccharide (LPS, 1.0mg/kg) to establish the animal model of sepsis-induced acute lung injury. Some mice were i.p. injected with calcitriol (1.0μg/kg) before LPS injection. An obvious infiltration of inflammatory cells in the lungs was observed beginning at 1h after LPS injection. Correspondingly, TNF-α and MIP-2 in sera and lung homogenates were markedly elevated in LPS-treated mice. Interestingly, calcitriol obviously alleviated LPS-induced infiltration of inflammatory cells in the lungs. Moreover, calcitriol markedly attenuated LPS-induced elevation of TNF-α and MIP-2 in sera and lung homogenates. Further analysis showed that calcitriol repressed LPS-induced p38 mitogen-activated protein kinase (MAPK) and protein kinase B (Akt) phosphorylation. In addition, calcitriol blocked LPS-induced nuclear translocation of nuclear factor kappa B (NF-κB) p65 and p50 subunit in the lungs. Taken together, these results suggest that calcitriol inhibits inflammatory cytokines production in LPS-induced acute lung injury.

  9. Regulation of the germinal center gene program by interferon (IFN) regulatory factor 8/IFN consensus sequence-binding protein.

    PubMed

    Lee, Chang Hoon; Melchers, Mark; Wang, Hongsheng; Torrey, Ted A; Slota, Rebecca; Qi, Chen-Feng; Kim, Ji Young; Lugar, Patricia; Kong, Hee Jeong; Farrington, Lila; van der Zouwen, Boris; Zhou, Jeff X; Lougaris, Vassilios; Lipsky, Peter E; Grammer, Amrie C; Morse, Herbert C

    2006-01-23

    Interferon (IFN) consensus sequence-binding protein/IFN regulatory factor 8 (IRF8) is a transcription factor that regulates the differentiation and function of macrophages, granulocytes, and dendritic cells through activation or repression of target genes. Although IRF8 is also expressed in lymphocytes, its roles in B cell and T cell maturation or function are ill defined, and few transcriptional targets are known. Gene expression profiling of human tonsillar B cells and mouse B cell lymphomas showed that IRF8 transcripts were expressed at highest levels in centroblasts, either from secondary lymphoid tissue or transformed cells. In addition, staining for IRF8 was most intense in tonsillar germinal center (GC) dark-zone centroblasts. To discover B cell genes regulated by IRF8, we transfected purified primary tonsillar B cells with enhanced green fluorescent protein-tagged IRF8, generated small interfering RNA knockdowns of IRF8 expression in a mouse B cell lymphoma cell line, and examined the effects of a null mutation of IRF8 on B cells. Each approach identified activation-induced cytidine deaminase (AICDA) and BCL6 as targets of transcriptional activation. Chromatin immunoprecipitation studies demonstrated in vivo occupancy of 5' sequences of both genes by IRF8 protein. These results suggest previously unappreciated roles for IRF8 in the transcriptional regulation of B cell GC reactions that include direct regulation of AICDA and BCL6.

  10. Calcitriol inhibits tumor necrosis factor alpha and macrophage inflammatory protein-2 during lipopolysaccharide-induced acute lung injury in mice.

    PubMed

    Tan, Zhu-Xia; Chen, Yuan-Hua; Xu, Shen; Qin, Hou-Ying; Wang, Hua; Zhang, Cheng; Xu, De-Xiang; Zhao, Hui

    2016-08-01

    Acute lung injury is a common complication of sepsis in intensive care unit patients with an extremely high mortality. The present study investigated the effects of calcitriol, the active form of vitamin D, on tumor necrosis factor alpha (TNF-α) and macrophage inflammatory protein-2 (MIP-2) in sepsis-induced acute lung injury. Mice were intraperitoneally (i.p.) injected with lipopolysaccharide (LPS, 1.0mg/kg) to establish the animal model of sepsis-induced acute lung injury. Some mice were i.p. injected with calcitriol (1.0μg/kg) before LPS injection. An obvious infiltration of inflammatory cells in the lungs was observed beginning at 1h after LPS injection. Correspondingly, TNF-α and MIP-2 in sera and lung homogenates were markedly elevated in LPS-treated mice. Interestingly, calcitriol obviously alleviated LPS-induced infiltration of inflammatory cells in the lungs. Moreover, calcitriol markedly attenuated LPS-induced elevation of TNF-α and MIP-2 in sera and lung homogenates. Further analysis showed that calcitriol repressed LPS-induced p38 mitogen-activated protein kinase (MAPK) and protein kinase B (Akt) phosphorylation. In addition, calcitriol blocked LPS-induced nuclear translocation of nuclear factor kappa B (NF-κB) p65 and p50 subunit in the lungs. Taken together, these results suggest that calcitriol inhibits inflammatory cytokines production in LPS-induced acute lung injury. PMID:27216047

  11. Chromatin assembly factor I and Hir proteins contribute to building functional kinetochores in S. cerevisiae

    PubMed Central

    Sharp, Judith A.; Franco, Alexa A.; Osley, Mary Ann; Kaufman, Paul D.

    2002-01-01

    Budding yeast centromeres are comprised of ∼125-bp DNA sequences that direct formation of the kinetochore, a specialized chromatin structure that mediates spindle attachment to chromosomes. We report here a novel role for the histone deposition complex chromatin assembly factor I (CAF-I) in building centromeric chromatin. The contribution of CAF-I to kinetochore function overlaps that of the Hir proteins, which have also been implicated in nucleosome formation and heterochromatic gene silencing. cacΔ hirΔ double mutant cells lacking both CAF-I and Hir proteins are delayed in anaphase entry in a spindle assembly checkpoint-dependent manner. Further, cacΔ and hirΔ deletions together cause increased rates of chromosome missegregation, genetic synergies with mutations in kinetochore protein genes, and alterations in centromeric chromatin structure. Finally, CAF-I subunits and Hir1 are enriched at centromeres, indicating that these proteins make a direct contribution to centromeric chromatin structures. PMID:11782447

  12. Structural Basis for Protein anti-Aggregation Activity of the Trigger Factor Chaperone*

    PubMed Central

    Saio, Tomohide; Guan, Xiao; Rossi, Paolo; Economou, Anastassios; Kalodimos, Charalampos G.

    2014-01-01

    Molecular chaperones prevent aggregation and misfolding of proteins but scarcity of structural data has impeded an understanding of the recognition and anti-aggregation mechanisms. Here we report the solution structure, dynamics and energetics of three Trigger Factor (TF) chaperone molecules in complex with alkaline phosphatase (PhoA) captured in the unfolded state. Our data show that TF uses multiple sites to bind to several regions of the PhoA substrate protein primarily through hydrophobic contacts. NMR relaxation experiments show that TF interacts with PhoA in a highly dynamic fashion but as the number and length of the PhoA regions engaged by TF increases, a more stable complex gradually emerges. Multivalent binding keeps the substrate protein in an extended, unfolded conformation. The results show how molecular chaperones recognize unfolded polypeptides and how by acting as unfoldases and holdases prevent the aggregation and premature (mis)folding of unfolded proteins. PMID:24812405

  13. Implication of tubby proteins as transcription factors by structure-based functional analysis.

    PubMed

    Boggon, T J; Shan, W S; Santagata, S; Myers, S C; Shapiro, L

    1999-12-10

    Tubby-like proteins (TULPs) are found in a broad range of multicellular organisms. In mammals, genetic mutation of tubby or other TULPs can result in one or more of three disease phenotypes: obesity (from which the name "tubby" is derived), retinal degeneration, and hearing loss. These disease phenotypes indicate a vital role for tubby proteins; however, no biochemical function has yet been ascribed to any member of this protein family. A structure-directed approach was employed to investigate the biological function of these proteins. The crystal structure of the core domain from mouse tubby was determined at a resolution of 1.9 angstroms. From primarily structural clues, experiments were devised, the results of which suggest that TULPs are a unique family of bipartite transcription factors. PMID:10591637

  14. Improving protein delivery of fibroblast growth factor-2 from bacterial inclusion bodies used as cell culture substrates.

    PubMed

    Seras-Franzoso, Joaquin; Peebo, Karl; García-Fruitós, Elena; Vázquez, Esther; Rinas, Ursula; Villaverde, Antonio

    2014-03-01

    Bacterial inclusion bodies (IBs) have recently been used to generate biocompatible cell culture interfaces, with diverse effects on cultured cells such as cell adhesion enhancement, stimulation of cell growth or induction of mesenchymal stem cell differentiation. Additionally, novel applications of IBs as sustained protein delivery systems with potential applications in regenerative medicine have been successfully explored. In this scenario, with IBs gaining significance in the biomedical field, the fine tuning of this functional biomaterial is crucial. In this work, the effect of temperature on fibroblast growth factor-2 (FGF-2) IB production and performance has been evaluated. FGF-2 was overexpressed in Escherichia coli at 25 and 37 °C, producing IBs with differences in size, particle structure and biological activity. Cell culture topographies made with FGF-2 IBs biofabricated at 25 °C showed higher levels of biological activity as well as a looser supramolecular structure, enabling a higher protein release from the particles. In addition, the controlled use of FGF-2 protein particles enabled the generation of functional topographies with multiple biological activities being effective on diverse cell types.

  15. Localization of complement factor H gene expression and protein distribution in the mouse outer retina

    PubMed Central

    Smit-McBride, Zeljka; Oltjen, Sharon L.; Radu, Roxana A.; Estep, Jason; Nguyen, Anthony T.; Gong, Qizhi

    2015-01-01

    Purpose To determine the localization of complement factor H (Cfh) mRNA and its protein in the mouse outer retina. Methods Quantitative real-time PCR (qPCR) was used to determine the expression of Cfh and Cfh-related (Cfhr) transcripts in the RPE/choroid. In situ hybridization (ISH) was performed using the novel RNAscope 2.0 FFPE assay to localize the expression of Cfh mRNA in the mouse outer retina. Immunohistochemistry (IHC) was used to localize Cfh protein expression, and western blots were used to characterize CFH antibodies used for IHC. Results Cfh and Cfhr2 transcripts were detected in the mouse RPE/choroid using qPCR, while Cfhr1, Cfhr3, and Cfhrc (Gm4788) were not detected. ISH showed abundant Cfh mRNA in the RPE of all mouse strains (C57BL/6, BALB/c, 129/Sv) tested, with the exception of the Cfh−/− eye. Surprisingly, the Cfh protein was detected by immunohistochemistry in photoreceptors rather than in RPE cells. The specificity of the CFH antibodies was tested by western blotting. Our CFH antibodies recognized purified mouse Cfh protein, serum Cfh protein in wild-type C57BL/6, BALB/c, and 129/Sv, and showed an absence of the Cfh protein in the serum of Cfh−/− mice. Greatly reduced Cfh protein immunohistological signals in the Cfh−/− eyes also supported the specificity of the Cfh protein distribution results. Conclusions Only Cfh and Cfhr2 genes are expressed in the mouse outer retina. Only Cfh mRNA was detected in the RPE, but no protein. We hypothesize that the steady-state concentration of Cfh protein is low in the cells due to secretion, and therefore is below the detection level for IHC. PMID:25684976

  16. Structure/function analysis of human factor XII using recombinant deletion mutants. Evidence for an additional region involved in the binding to negatively charged surfaces.

    PubMed

    Citarella, F; Ravon, D M; Pascucci, B; Felici, A; Fantoni, A; Hack, C E

    1996-05-15

    The binding site of human factor XII (FXII) for negatively charged surfaces has been proposed to be localized in the N-terminal region of factor XII. We have generated two recombinant factor XII proteins that lack this region: one protein consisting of the second growth-factor-like domain, the kringle domain, the proline-rich region and the catalytic domain of FXII (rFXII-U-like), and another consisting of only 16 amino acids of the proline-rich region of the heavy-chain region and the catalytic domain (rFXII-1pc). Each recombinant truncated protein, as well as recombinant full-length FXII (rFXII), were produced in HepG2 cells and purified by immunoaffinity chromatography. The capability of these recombinant proteins to bind to negatively charged surfaces and to initiate contact activation was studied. Radiolabeled rFXII-U-like and, to a lesser extent, rFXII-lpc bound to glass in a concentration-dependent manner, yet with lower efficiency than rFXII. The binding of the recombinant proteins was inhibited by a 100-fold molar excess of non-labeled native factor XII. On native polyacrylamide gel electrophoresis, both truncated proteins appeared to bind also to dextran sulfate, a soluble negatively charged compound. Glass-bound rFXII-U-like was able to activate prekallikrein in FXII-deficient plasma (assessed by measuring the generation of kallikrein-C1-inhibitor complexes), but less efficiently than rFXII, rFXII-U-like and rFXII-lpc exhibited coagulant activity, but this activity was significantly lower than that of rFXII. These data confirm that the N-terminal part of the heavy-chain region of factor XII contains a binding site for negatively charged activating surfaces, and indicate that other sequences, possibly located on the second epidermal-growth-factor-like domain and/or the kringle domain, contribute to the binding of factor XII to these surfaces.

  17. Reduction of Factor VIII Inhibitor Titers During Immune Tolerance Induction With Recombinant Factor VIII-Fc Fusion Protein.

    PubMed

    Groomes, Charles L; Gianferante, David M; Crouch, Gary D; Parekh, Dina S; Scott, David W; Lieuw, Kenneth

    2016-05-01

    The development of inhibitors toward factor VIII (FVIII) is a common and serious complication of hemophilia A (HA) therapy. Patients with hemophilia who develop inhibitors often undergo time- and resource-intensive immune tolerance induction (ITI) protocols. We report a 15-month-old male with severe HA and a high-titer inhibitor that occurred while receiving prophylactic treatment with recombinant FVIII (rFVIII), in whom significant inhibitor titer reduction was achieved with thrice weekly infusions of a new, prolonged half-life rFVIII-Fc fusion protein product (trade name Eloctate). Further studies are warranted to explore the potential of Eloctate in ITI protocols. PMID:26739399

  18. Different Protein Kinase C Isoforms Determine Growth Factor Specificity in Neuronal Cells

    PubMed Central

    Corbit, Kevin C.; Soh, Jae-Won; Yoshida, Keiko; Eves, Eva M.; Weinstein, I. Bernard; Rosner, Marsha Rich

    2000-01-01

    Although mitogenic and differentiating factors often activate a number of common signaling pathways, the mechanisms leading to their distinct cellular outcomes have not been elucidated. In a previous report, we demonstrated that mitogen-activated protein (MAP) kinase (ERK) activation by the neurogenic agents fibroblast growth factor (FGF) and nerve growth factor is dependent on protein kinase Cδ (PKCδ), whereas MAP kinase activation in response to the mitogen epidermal growth factor (EGF) is independent of PKCδ in rat hippocampal (H19-7) and pheochromocytoma (PC12) cells. We now show that EGF activates MAP kinase through a PKCζ-dependent pathway involving phosphatidylinositol 3-kinase and PDK1 in H19-7 cells. PKCζ, like PKCδ, acts upstream of MEK, and PKCζ can potentiate Raf-1 activation by EGF. Inhibition of PKCζ also blocks EGF-induced DNA synthesis as monitored by bromodeoxyuridine incorporation in H19-7 cells. Finally, in embryonic rat brain hippocampal cell cultures, inhibitors of PKCζ or PKCδ suppress MAP kinase activation by EGF or FGF, respectively, indicating that these factors activate distinct signaling pathways in primary as well as immortalized neural cells. Taken together, these results implicate different PKC isoforms as determinants of growth factor signaling specificity within the same cell. Furthermore, these data provide a mechanism whereby different growth factors can differentially activate a common signaling intermediate and thereby generate biological diversity. PMID:10891480

  19. An integrated model of transcription factor diffusion shows the importance of intersegmental transfer and quaternary protein structure for target site finding.

    PubMed

    Schmidt, Hugo G; Sewitz, Sven; Andrews, Steven S; Lipkow, Karen

    2014-01-01

    We present a computational model of transcription factor motion that explains both the observed rapid target finding of transcription factors, and how this motion influences protein and genome structure. Using the Smoldyn software, we modelled transcription factor motion arising from a combination of unrestricted 3D diffusion in the nucleoplasm, sliding along the DNA filament, and transferring directly between filament sections by intersegmental transfer. This presents a fine-grain picture of the way in which transcription factors find their targets two orders of magnitude faster than 3D diffusion alone allows. Eukaryotic genomes contain sections of nucleosome free regions (NFRs) around the promoters; our model shows that the presence and size of these NFRs can be explained as their acting as antennas on which transcription factors slide to reach their targets. Additionally, our model shows that intersegmental transfer may have shaped the quaternary structure of transcription factors: sequence specific DNA binding proteins are unusually enriched in dimers and tetramers, perhaps because these allow intersegmental transfer, which accelerates target site finding. Finally, our model shows that a 'hopping' motion can emerge from 3D diffusion on small scales. This explains the apparently long sliding lengths that have been observed for some DNA binding proteins observed in vitro. Together, these results suggest that transcription factor diffusion dynamics help drive the evolution of protein and genome structure.

  20. An Integrated Model of Transcription Factor Diffusion Shows the Importance of Intersegmental Transfer and Quaternary Protein Structure for Target Site Finding

    PubMed Central

    Schmidt, Hugo G.; Sewitz, Sven; Andrews, Steven S.; Lipkow, Karen

    2014-01-01

    We present a computational model of transcription factor motion that explains both the observed rapid target finding of transcription factors, and how this motion influences protein and genome structure. Using the Smoldyn software, we modelled transcription factor motion arising from a combination of unrestricted 3D diffusion in the nucleoplasm, sliding along the DNA filament, and transferring directly between filament sections by intersegmental transfer. This presents a fine-grain picture of the way in which transcription factors find their targets two orders of magnitude faster than 3D diffusion alone allows. Eukaryotic genomes contain sections of nucleosome free regions (NFRs) around the promoters; our model shows that the presence and size of these NFRs can be explained as their acting as antennas on which transcription factors slide to reach their targets. Additionally, our model shows that intersegmental transfer may have shaped the quaternary structure of transcription factors: sequence specific DNA binding proteins are unusually enriched in dimers and tetramers, perhaps because these allow intersegmental transfer, which accelerates target site finding. Finally, our model shows that a ‘hopping’ motion can emerge from 3D diffusion on small scales. This explains the apparently long sliding lengths that have been observed for some DNA binding proteins observed in vitro. Together, these results suggest that transcription factor diffusion dynamics help drive the evolution of protein and genome structure. PMID:25333780

  1. Characterization of Differential Protein Tethering at the Plasma Membrane in Response to Epidermal Growth Factor Signaling

    PubMed Central

    Looyenga, Brendan D.; MacKeigan, Jeffrey P.

    2013-01-01

    Physical tethering of membrane proteins to the cortical actin cytoskeleton provides functional organization to the plasma membrane and contributes to diverse cellular processes including cell signaling, vesicular trafficking, endocytosis, and migration. For these processes to occur, membrane protein tethering must be dynamically regulated in response to environmental cues. In this study, we describe a novel biochemical scheme for isolating the complement of plasma membrane proteins that are physically tethered to the actin cytoskeleton. We utilized this method in combination with tandem liquid chromatography/mass spectrometry (LC–MS/MS) to demonstrate that cytoskeletal tethering of membrane proteins is acutely regulated by epidermal growth factor (EGF) in normal human kidney (HK2) cells. Our results indicate that several proteins known to be involved in EGF signaling, as well as other proteins not traditionally associated with this pathway, are tethered to the cytoskeleton in dynamic fashion. Further analysis of one hit from our proteomic survey, the receptor phosphotyrosine phosphatase PTPRS, revealed a correlation between cytoskeletal tethering and endosomal trafficking in response to EGF. This finding parallels previous indications that PTPRS is involved in the desensitization of EGFR and provides a potential mechanism to coordinate localization of these two membrane proteins in the same compartment upon EGFR activation. PMID:22559174

  2. Effects of doxepin on brain-derived neurotrophic factor, tumor necrosis factor alpha, mitogen-activated protein kinase 14, and AKT1 genes expression in rat hippocampus

    PubMed Central

    Eidelkhani, Nastaran; Radahmadi, Maryam; Kazemi, Mohammad; Rafiee, Laleh; Alaei, Hojjatallah; Reisi, Parham

    2015-01-01

    Background: It has been suggested that doxepin in addition to enhancement of noradrenaline and serotonin levels may have neuroprotective effects. Therefore, this study investigated the effect of doxepin on gene expression of brain-derived neurotrophic factor (BDNF), tumor necrosis factor alpha (TNF-α), mitogen-activated protein kinase 14 (MAPK14), and serine-threonine protein kinase AKT1 in rat hippocampus. Materials and Methods: Male rats were divided randomly into three groups: Control, doxepin 1 mg/kg, and doxepin 5 mg/kg. Rats received an i.p injection of doxepin for 21 days. Then the hippocampi were dissected for the measurement of the expression of BDNF, TNF-α, MAPK14, and AKT1 genes. Results: Our results showed no significant effects of doxepin on gene expression of BDNF, TNF-α, MAPK14, and AKT1 genes in the hippocampus. Conclusions: These results did not show significant effects of doxepin on the genes that affect the neuronal survival in intact animals. However, more studies need to be done, especially in models associated with neuronal damage. PMID:26601091

  3. Modification of water absorption capacity of a plastic based on bean protein using gamma irradiated starches as additives

    NASA Astrophysics Data System (ADS)

    Köber, E.; Gonzalez, M. E.; Gavioli, N.; Salmoral, E. M.

    2007-01-01

    Some properties of a bean protein-starch plastic were modified by irradiation of the starch. Two kinds of starch from bean and cassava were irradiated with doses until 50 kGy before their inclusion in the composite. Water absorption of the resultant product was reduced by 36% and 60% in materials containing bean and cassava starch, respectively. A large decline in the elongation is observed till 10 kGy in both materials, while tensile strength diminished by 11% in the cassava composite.

  4. Fibroblast growth factor 3, a protein with a dual subcellular fate, is interacting with human ribosomal protein S2

    SciTech Connect

    Antoine, Marianne; Reimers, Kerstin; Wirz, Werner; Gressner, Axel M.; Mueller, Robert; Kiefer, Paul . E-mail: pkiefer@ukaachen.de

    2005-12-16

    The secreted isoform of fibroblast growth factor 3 (FGF3) induces a mitogenic cell response, while the nuclear form inhibits cell proliferation. Recently, we identified a nucleolar FGF3-binding protein which is implicated in processing of pre-rRNA as a possible target of nuclear FGF3 signalling. Here, we report a second candidate protein identified by a yeast two-hybrid screen for nuclear FGF3 action, ribosomal protein S2, rpS2. Recombinant rpS2 binds to in vitro translated FGF3 and to nuclear FGF3 extracted from transfected COS-1 cells. Characterization of the FGF3 binding domain of rpS2 showed that both the Arg-Gly-rich N-terminal region and a short carboxyl-terminal sequence of rpS2 are necessary for FGF3 binding. Mapping the S2 binding domains of FGF3 revealed that these domains are important for both NoBP and rpS2 interaction. Transient co-expression of rpS2 and nuclear FGF3 resulted in a reduced nucleolar localization of the FGF. These findings suggest that the nuclear form of FGF3 inhibits cell proliferation by interfering with ribosomal biogenesis.

  5. Osteoblast-specific factor 2: cloning of a putative bone adhesion protein with homology with the insect protein fasciclin I.

    PubMed Central

    Takeshita, S; Kikuno, R; Tezuka, K; Amann, E

    1993-01-01

    A cDNA library prepared from the mouse osteoblastic cell line MC3T3-E1 was screened for the presence of specifically expressed genes by employing a combined subtraction hybridization/differential screening approach. A cDNA was identified and sequenced which encodes a protein designated osteoblast-specific factor 2 (OSF-2) comprising 811 amino acids. OSF-2 has a typical signal sequence, followed by a cysteine-rich domain, a fourfold repeated domain and a C-terminal domain. The protein lacks a typical transmembrane region. The fourfold repeated domain of OSF-2 shows homology with the insect protein fasciclin I. RNA analyses revealed that OSF-2 is expressed in bone and to a lesser extent in lung, but not in other tissues. Mouse OSF-2 cDNA was subsequently used as a probe to clone the human counterpart. Mouse and human OSF-2 show a high amino acid sequence conservation except for the signal sequence and two regions in the C-terminal domain in which 'in-frame' insertions or deletions are observed, implying alternative splicing events. On the basis of the amino acid sequence homology with fasciclin I, we suggest that OSF-2 functions as a homophilic adhesion molecule in bone formation. Images Figure 3 Figure 4 Figure 5 Figure 6 PMID:8363580

  6. Active G protein-coupled receptors (GPCR), matrix metalloproteinases 2/9 (MMP2/9), heparin-binding epidermal growth factor (hbEGF), epidermal growth factor receptor (EGFR), erbB2, and insulin-like growth factor 1 receptor (IGF-1R) are necessary for trenbolone acetate-induced alterations in protein turnover rate of fused bovine satellite cell cultures.

    PubMed

    Thornton, K J; Kamanga-Sollo, E; White, M E; Dayton, W R

    2016-06-01

    Trenbolone acetate (TBA), a testosterone analog, increases protein synthesis and decreases protein degradation in fused bovine satellite cell (BSC) cultures. However, the mechanism through which TBA alters these processes remains unknown. Recent studies indicate that androgens improve rate and extent of muscle growth through a nongenomic mechanism involving G protein-coupled receptors (GPCR), matrix metalloproteinases (MMP), heparin-binding epidermal growth factor (hbEGF), the epidermal growth factor receptor (EGFR), erbB2, and the insulin-like growth factor-1 receptor (IGF-1R). We hypothesized that TBA activates GPCR, resulting in activation of MMP2/9 that releases hbEGF, which activates the EGFR and/or erbB2. To determine whether the proposed nongenomic pathway is involved in TBA-mediated alterations in protein turnover, fused BSC cultures were treated with TBA in the presence or absence of inhibitors for GPCR, MMP2/9, hbEGF, EGFR, erbB2, or IGF-1R, and resultant protein synthesis and degradation rates were analyzed. Assays were replicated at least 9 times for each inhibitor experiment utilizing BSC cultures obtained from at least 3 different steers that had no previous exposure to steroid compounds. As expected, fused BSC cultures treated with 10 n TBA exhibited increased ( < 0.05) protein synthesis rates and decreased ( < 0.05) protein degradation rates when compared to control cultures. Treatment of fused BSC cultures with 10 n TBA in the presence of inhibitors for GPCR, MMP2/9, hbEGF, EGFR, erbB2, or IGF-1R suppressed ( < 0.05) TBA-mediated increases in protein synthesis rate. Alternatively, inhibition of GPCR, MMP2/9, hbEGF, EGFR, erbB2, or IGF-1R in the presence of 10 n TBA each had no ( > 0.05) effect on TBA-mediated decreases in protein degradation. However, inhibition of both EGFR and erbB2 in the presence of 10 n TBA resulted in decreased ( < 0.05) ability of TBA to decrease protein degradation rate. Additionally, fused BSC cultures treated with 10 n

  7. Active G protein-coupled receptors (GPCR), matrix metalloproteinases 2/9 (MMP2/9), heparin-binding epidermal growth factor (hbEGF), epidermal growth factor receptor (EGFR), erbB2, and insulin-like growth factor 1 receptor (IGF-1R) are necessary for trenbolone acetate-induced alterations in protein turnover rate of fused bovine satellite cell cultures.

    PubMed

    Thornton, K J; Kamanga-Sollo, E; White, M E; Dayton, W R

    2016-06-01

    Trenbolone acetate (TBA), a testosterone analog, increases protein synthesis and decreases protein degradation in fused bovine satellite cell (BSC) cultures. However, the mechanism through which TBA alters these processes remains unknown. Recent studies indicate that androgens improve rate and extent of muscle growth through a nongenomic mechanism involving G protein-coupled receptors (GPCR), matrix metalloproteinases (MMP), heparin-binding epidermal growth factor (hbEGF), the epidermal growth factor receptor (EGFR), erbB2, and the insulin-like growth factor-1 receptor (IGF-1R). We hypothesized that TBA activates GPCR, resulting in activation of MMP2/9 that releases hbEGF, which activates the EGFR and/or erbB2. To determine whether the proposed nongenomic pathway is involved in TBA-mediated alterations in protein turnover, fused BSC cultures were treated with TBA in the presence or absence of inhibitors for GPCR, MMP2/9, hbEGF, EGFR, erbB2, or IGF-1R, and resultant protein synthesis and degradation rates were analyzed. Assays were replicated at least 9 times for each inhibitor experiment utilizing BSC cultures obtained from at least 3 different steers that had no previous exposure to steroid compounds. As expected, fused BSC cultures treated with 10 n TBA exhibited increased ( < 0.05) protein synthesis rates and decreased ( < 0.05) protein degradation rates when compared to control cultures. Treatment of fused BSC cultures with 10 n TBA in the presence of inhibitors for GPCR, MMP2/9, hbEGF, EGFR, erbB2, or IGF-1R suppressed ( < 0.05) TBA-mediated increases in protein synthesis rate. Alternatively, inhibition of GPCR, MMP2/9, hbEGF, EGFR, erbB2, or IGF-1R in the presence of 10 n TBA each had no ( > 0.05) effect on TBA-mediated decreases in protein degradation. However, inhibition of both EGFR and erbB2 in the presence of 10 n TBA resulted in decreased ( < 0.05) ability of TBA to decrease protein degradation rate. Additionally, fused BSC cultures treated with 10 n

  8. N,N,N',N'-tetramethyl-1,3-butanediamine as effective running electrolyte additive for efficient electrophoretic separation of basic proteins in bare fused-silica capillaries.

    PubMed

    Corradini, D; Cannarsa, G

    1995-04-01

    The effect of N,N,N',N'-tetramethyl-1,3-butanediamine (TMBD) in the running electrolyte on the electroosmotic flow and the migration behavior of four standard basic proteins in bare fused-silica capillaries was examined at pH 4.0, 5.5, and 6.5. Depending on the electrolyte pH and additive concentration the electroosmotic flow was either cathodic or anodic. A similar Langmuirian-type dependence of the electroosmotic flow on the concentration of TMBD in the running electrolyte was found at the three experimented pH values, which may be indicative of the specific adsorption of the additive in the immobilized region of the electric double layer at the interface between the capillary wall and the electrolyte solution. Electrophoretic separations of the four standard basic proteins performed at the three above pH values, showed well-resolved, efficient and symmetric peaks, demonstrating the utility of this additive for protein electrophoresis in bare fused-silica capillaries. The variations in separation efficiency, peak capacity, resolution and reproducibility of migration times as a function of the additive concentration at pH 6.5 were also examined.

  9. MrpL36p, a highly diverged L31 ribosomal protein homolog with additional functional domains in Saccharomyces cerevisiae mitochondria.

    PubMed Central

    Williams, Elizabeth H; Perez-Martinez, Xochitl; Fox, Thomas D

    2004-01-01

    Translation in mitochondria utilizes a large complement of ribosomal proteins. Many mitochondrial ribosomal components are clearly homologous to eubacterial ribosomal proteins, but others appear unique to the mitochondrial system. A handful of mitochondrial ribosomal proteins appear to be eubacterial in origin but to have evolved additional functional domains. MrpL36p is an essential mitochondrial ribosomal large-subunit component in Saccharomyces cerevisiae. Increased dosage of MRPL36 also has been shown to suppress certain types of translation defects encoded within the mitochondrial COX2 mRNA. A central domain of MrpL36p that is similar to eubacterial ribosomal large-subunit protein L31 is sufficient for general mitochondrial translation but not suppression, and proteins bearing this domain sediment with the ribosomal large subunit in sucrose gradients. In contrast, proteins lacking the L31 domain, but retaining a novel N-terminal sequence and a C-terminal sequence with weak similarity to the Escherichia coli signal recognition particle component Ffh, are sufficient for dosage suppression and do not sediment with the large subunit of the ribosome. Interestingly, the activity of MrpL36p as a dosage suppressor exhibits gene and allele specificity. We propose that MrpL36p represents a highly diverged L31 homolog with derived domains functioning in mRNA selection in yeast mitochondria. PMID:15166137

  10. Epidermal growth factor, but not insulin, stimulates tyrosine phosphorylation of an endogenous protein of Mr 95,000 in triton extracts of human placental syncytiotrophoblast membranes.

    PubMed Central

    Tavaré, J M; Diggle, T A; Denton, R M

    1987-01-01

    1. Triton extracts of syncytiotrophoblast membranes were incubated with [gamma-32P]ATP, MgCl2 and MnCl2. Addition of epidermal growth factor (EGF) resulted in increased phosphorylation not only of the EGF receptor and a Mr-35,000 protein as previously described, but also a protein of Mr 95,000 on both tyrosine and serine residues. In addition, a small increase in the phosphorylation of a protein of Mr 105,000 was observed. Spermine had a similar effect on the phosphorylation of the Mr-95,000 protein, without affecting the phosphorylation of the other proteins. In the absence of MnCl2, the effect of spermine on the phosphorylation of Mr-95,000 protein was still evident, whereas that of EGF was greatly diminished. 2. The Mr-95,000 protein bound poorly to wheat-germ-lectin-Sepharose and was not precipitated by antisera specific for insulin and EGF receptors. The protein continued to exhibit serine and tyrosine phosphorylation on addition of [gamma-32P]ATP, MgCl2 and MnCl2 to a glycoprotein-depleted fraction prepared by chromatography on wheat-germ-lectin-Sepharose. The extent of phosphorylation was no longer increased by spermine or EGF, but was inhibited by heparin. 3. It is suggested that the Mr-95,000 protein not only is a possible direct substrate for the EGF-receptor (but not the insulin receptor) tyrosine kinase but is a substrate for other endogenous kinases, including a protein tyrosine kinase which is probably not a glycoprotein, and a protein serine kinase with properties similar to those of casein kinase II. Images Fig. 1. Fig. 2. Fig. 3. PMID:3328613

  11. Yeast GAL11 protein is a distinctive type transcription factor that enhances basal transcription in vitro.

    PubMed Central

    Sakurai, H; Hiraoka, Y; Fukasawa, T

    1993-01-01

    The yeast auxiliary transcription factor GAL11, a candidate for the coactivator, was partially purified from yeast cells, and its function was characterized in a cell-free transcription system. The partially purified GAL11 protein stimulated basal transcription from the CYC1 core promoter by a factor of 4-5 at the step of preinitiation complex formation. GAL11 protein also enhanced transcription activated by general regulatory factor 1, GAL4-AH, or GAL4-VP16 to the same extent as the basal transcription. Therefore, the apparent potentiation of the activators by GAL11 was attributable to the stimulation of basal transcription. The wild-type GAL11 protein (but not a mutant-type protein) produced in bacteria stimulated transcription as effectively as GAL11 from yeast. These results suggest that GAL11 functions as a positive cofactor of basal and activator-induced transcription in a cell-free transcription system. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:8378310

  12. Complement factor H–related hybrid protein deregulates complement in dense deposit disease

    PubMed Central

    Chen, Qian; Wiesener, Michael; Eberhardt, Hannes U.; Hartmann, Andrea; Uzonyi, Barbara; Kirschfink, Michael; Amann, Kerstin; Buettner, Maike; Goodship, Tim; Hugo, Christian; Skerka, Christine; Zipfel, Peter F.

    2013-01-01

    The renal disorder C3 glomerulopathy with dense deposit disease (C3G-DDD) pattern results from complement dysfunction and primarily affects children and young adults. There is no effective treatment, and patients often progress to end-stage renal failure. A small fraction of C3G-DDD cases linked to factor H or C3 gene mutations as well as autoantibodies have been reported. Here, we examined an index family with 2 patients with C3G-DDD and identified a chromosomal deletion in the complement factor H–related (CFHR) gene cluster. This deletion resulted in expression of a hybrid CFHR2-CFHR5 plasma protein. The recombinant hybrid protein stabilized the C3 convertase and reduced factor H–mediated convertase decay. One patient was refractory to plasma replacement and exchange therapy, as evidenced by the hybrid protein quickly returning to pretreatment plasma levels. Subsequently, complement inhibitors were tested on serum from the patient for their ability to block activity of CFHR2-CFHR5. Soluble CR1 restored defective C3 convertase regulation; however, neither eculizumab nor tagged compstatin had any effect. Our findings provide insight into the importance of CFHR proteins for C3 convertase regulation and identify a genetic variation in the CFHR gene cluster that promotes C3G-DDD. Monitoring copy number and sequence variations in the CFHR gene cluster in C3G-DDD and kidney patients with C3G-DDD variations will help guide treatment strategies. PMID:24334459

  13. Xeroderma Pigmentosum Group A Protein Loads as a Separate Factor onto DNA Lesions

    PubMed Central

    Rademakers, Suzanne; Volker, Marcel; Hoogstraten, Deborah; Nigg, Alex L.; Moné, Martijn J.; van Zeeland, Albert A.; Hoeijmakers, Jan H. J.; Houtsmuller, Adriaan B.; Vermeulen, Wim

    2003-01-01

    Nucleotide excision repair (NER) is the main DNA repair pathway in mammals for removal of UV-induced lesions. NER involves the concerted action of more than 25 polypeptides in a coordinated fashion. The xeroderma pigmentosum group A protein (XPA) has been suggested to function as a central organizer and damage verifier in NER. How XPA reaches DNA lesions and how the protein is distributed in time and space in living cells are unknown. Here we studied XPA in vivo by using a cell line stably expressing physiological levels of functional XPA fused to green fluorescent protein and by applying quantitative fluorescence microscopy. The majority of XPA moves rapidly through the nucleoplasm with a diffusion rate different from those of other NER factors tested, arguing against a preassembled XPA-containing NER complex. DNA damage induced a transient (∼5-min) immobilization of maximally 30% of XPA. Immobilization depends on XPC, indicating that XPA is not the initial lesion recognition protein in vivo. Moreover, loading of replication protein A on NER lesions was not dependent on XPA. Thus, XPA participates in NER by incorporation of free diffusing molecules in XPC-dependent NER-DNA complexes. This study supports a model for a rapid consecutive assembly of free NER factors, and a relatively slow simultaneous disassembly, after repair. PMID:12897146

  14. Factors influencing subcellular localization of the human papillomavirus L2 minor structural protein

    SciTech Connect

    Kieback, Elisa; Mueller, Martin . E-mail: Martin.Mueller@dkfz.de

    2006-02-05

    Two structural proteins form the capsids of papillomaviruses. The major structural protein L1 is the structural determinant of the capsids and is present in 360 copies arranged in 72 pentamers. The minor structural protein L2 is estimated to be present in twelve copies per capsid. Possible roles for L2 in interaction with cell surface receptors and in virion uptake have been suggested. As previously reported, L2 localizes in subnuclear domains identified as nuclear domain 10 (ND10). As it was demonstrated that L2 is able to recruit viral and cellular proteins to ND10, a possible role for L2 as a mediator in viral assembly has been proposed. In this study, we determined factors influencing the localization of L2 at ND10. Under conditions of moderate L2 expression level and in the absence of heterologous viral components, we observed that, in contrast to previous reports, L2 is mainly distributed homogeneously throughout the nucleus. L2, however, is recruited to ND10 at a higher expression level or in the presence of viral components derived from vaccinia virus or from Semliki Forest virus. We observed that translocation of L2 to ND10 is not a concentration-dependent accumulation but rather seems to be triggered by yet unidentified cellular factors. In contrast to HPV 11 and 16 L2, the HPV 18 L2 protein seems to require L1 for efficient nuclear accumulation.

  15. Neural regeneration protein is a novel chemoattractive and neuronal survival-promoting factor

    SciTech Connect

    Gorba, Thorsten; Bradoo, Privahini; Antonic, Ana; Marvin, Keith; Liu, Dong-Xu; Lobie, Peter E.; Reymann, Klaus G.; Gluckman, Peter D.; Sieg, Frank . E-mail: fsieg@neurenpharma.com

    2006-10-01

    Neurogenesis and neuronal migration are the prerequisites for the development of the central nervous system. We have identified a novel rodent gene encoding for a neural regeneration protein (NRP) with an activity spectrum similar to the chemokine stromal-derived factor (SDF)-1, but with much greater potency. The Nrp gene is encoded as a forward frameshift to the hypothetical alkylated DNA repair protein AlkB. The predicted protein sequence of NRP contains domains with homology to survival-promoting peptide (SPP) and the trefoil protein TFF-1. The Nrp gene is first expressed in neural stem cells and expression continues in glial lineages. Recombinant NRP and NRP-derived peptides possess biological activities including induction of neural migration and proliferation, promotion of neuronal survival, enhancement of neurite outgrowth and promotion of neuronal differentiation from neural stem cells. NRP exerts its effect on neuronal survival by phosphorylation of the ERK1/2 and Akt kinases, whereas NRP stimulation of neural migration depends solely on p44/42 MAP kinase activity. Taken together, the expression profile of Nrp, the existence in its predicted protein structure of domains with similarities to known neuroprotective and migration-inducing factors and the high potency of NRP-derived synthetic peptides acting in femtomolar concentrations suggest it to be a novel gene of relevance in cellular and developmental neurobiology.

  16. Nuclear translocation of doublecortin-like protein kinase and phosphorylation of a transcription factor JDP2

    SciTech Connect

    Nagamine, Tadashi; Nomada, Shohgo; Onouchi, Takashi; Kameshita, Isamu; Sueyoshi, Noriyuki

    2014-03-28

    Highlights: • Doublecortin-like protein kinase (DCLK) is a microtubule-associated protein kinase. • In living cells, DCLK was cleaved into two functional fragments. • zDCLK(kinase) was translocated into the nucleus by osmotic stresses. • Jun dimerization protein 2 (JDP2) was identified as zDCLK(kinase)-binding protein. • JDP2 was efficiently phosphorylated by zDCLK(kinase) only when histone was present. - Abstract: Doublecortin-like protein kinase (DCLK) is a microtubule-associated protein kinase predominantly expressed in brain. In a previous paper, we reported that zebrafish DCLK2 (zDCLK) was cleaved into two functional fragments; the N-terminal zDCLK(DC + SP) with microtubule-binding activity and the C-terminal zDCLK(kinase) with a Ser/Thr protein kinase activity. In this study, we demonstrated that zDCLK(kinase) was widely distributed in the cytoplasm and translocated into the nucleus when the cells were treated under hyperosmotic conditions with NaCl or mannitol. By two-hybrid screening using the C-terminal domain of DCLK, Jun dimerization protein 2 (JDP2), a nuclear transcription factor, was identified as zDCLK(kinase)-binding protein. Furthermore, JDP2 served as an efficient substrate for zDCLK(kinase) only when histone was present. These results suggest that the kinase fragment of DCLK is translocated into the nucleus upon hyperosmotic stresses and that the kinase efficiently phosphorylates JDP2, a possible target in the nucleus, with the aid of histones.

  17. Binding affinity prediction for protein-ligand complexes based on β contacts and B factor.

    PubMed

    Liu, Qian; Kwoh, Chee Keong; Li, Jinyan

    2013-11-25

    Accurate determination of protein-ligand binding affinity is a fundamental problem in biochemistry useful for many applications including drug design and protein-ligand docking. A number of scoring functions have been proposed for the prediction of protein-ligand binding affinity. However, accurate prediction is still a challenging problem because poor performance is often seen in the evaluation under the leave-one-cluster-out cross-validation (LCOCV). We introduce a new scoring function named B2BScore to improve the prediction performance. B2BScore integrates two physicochemical properties for protein-ligand binding affinity prediction. One is the property of β contacts. A β contact between two atoms requires no other atoms to interrupt the atomic contact and assumes that the two atoms should have enough direct contact area. The other is the property of B factor to capture the atomic mobility in the dynamic protein-ligand binding process. Tested on the PDBBind2009 data set, B2BScore shows superior prediction performance to existing methods on independent test data as well as under the LCOCV evaluation framework. In particular, B2BScore achieves a significant LCOCV improvement across 26 protein clusters-a big increase of the averaged Pearson's correlation coefficients from 0.418 to 0.518 and a significant decrease of standard deviation of the coefficients from 0.352 to 0.196. We also identified several important and intuitive contact descriptors of protein-ligand binding through the random forest learning in B2BScore. Some of these descriptors are closely related to contacts between carbon atoms without covalent-bond oxygen/nitrogen, preferred contacts of metal ions, interfacial backbone atoms from proteins, or π rings. Some others are negative descriptors relating to those contacts with nitrogen atoms without covalent-bond hydrogens or nonpreferred contacts of metal ions. These descriptors can be directly used to guide protein-ligand docking.

  18. Enhanced Production of Insulin-like Growth Factor I Protein in Escherichia coli by Optimization of Five Key Factors

    PubMed Central

    Ranjbari, Javad; Babaeipour, Valiollah; Vahidi, Hossein; Moghimi, Hamidreza; Mofid, Mohammad Reza; Namvaran, Mohammad Mehdi; Jafari, Sevda

    2015-01-01

    Human insulin-like growth factor I (hIGF-I) is a kind of growth factor with clinical significance in medicine. Up to now, E. coli expression system has been widely used as a host to produce rhIGF-1 with high yields. Batch cultures as non-continuous fermentations were carried out to overproduce rhIGF-I in E. coli. The major objective of this study is over- production of recombinant human insulin-like growth factor I (rhIGF-I) through a developed process by recruiting effective factors in order to achieve the most recombinant protein. In this study we investigated the effect of culture medium, induction temperature and amount of inducer on cell growth and IGF-1 production. Taguchi design of experiments (DOE) method was used as the statistical method. Analysis of experimental data showed that maximum production of rhIGF-I was occurred in 32y culture medium at 32 °C and 0.05 Mm IPTG. Under this condition, 0.694 g/L of rhIGF-I was produced as the inclusion bodies. Following optimization of these three factors, we have also optimized the amount of glucose and induction time in 5 liter top bench bioreactor. Full factorial design of experiment method was used for these two factors as the statistical method. 10 g/L and OD600=5 were selected as the optimum point of Glucose amount and induction time, respectively. Finally, we reached to a concentration of 1.26 g/L rhIGF-1 at optimum condition. PMID:26330880

  19. Ets proteins: new factors that regulate immunoglobulin heavy-chain gene expression.

    PubMed

    Rivera, R R; Stuiver, M H; Steenbergen, R; Murre, C

    1993-11-01

    We used a DNA-protein interaction screening method to isolate a cDNA, Erg-3, whose product binds to a site, designated pi, present in the immunoglobulin (Ig) heavy-chain gene enhancer. Erg-3 is an alternatively spliced product of the erg gene and contains an Ets DNA-binding domain. Fli-1 and PU.1, related Ets proteins, also bind to the same site. In addition, PU.1 binds to a second site, designated microB, in the Ig heavy-chain enhancer. We demonstrate that the pi binding site is crucial for Ig heavy-chain gene enhancer function. In addition, we show that Erg-3 and Fli.1, but not PU.1, can activate a reporter construct containing a multimer of protein-binding sites, synergistically with helix-loop-helix protein E12. We discuss how combinatorial interactions between members of the helix-loop-helix and Ets families may account for the tissue specificity of these proteins.

  20. Dissection of CR1, factor H, membrane cofactor protein, and factor B binding and functional sites in the third complement component.

    PubMed

    Lambris, J D; Lao, Z; Oglesby, T J; Atkinson, J P; Hack, C E; Becherer, J D

    1996-06-15

    Previous studies have suggested that the residues 727-768 of human (Hu) C3 contain the binding sites for CR1, factor H, and factor B. Here, we have (1) characterized further some of the C3 structural requirements for its binding to CR1, H, and B, (2) investigated the functions associated with these C3-ligand interactions, and (3) studied the relationship of MCP-binding sites in C3 with those for CR1, H, and B. Hu C3 molecules in which residues 727-768 were deleted (designated C3delta727-768) or substituted with the corresponding segment of cobra venom factor, Xenopus, or trout C3 (chimeric C3s) were expressed in the baculovirus system and analyzed for their reactivity with C3-binding proteins. In contrast to wild-type iC3 which, in the presence of CR1, is cleaved by factor I to iC3b-a and C3c-a and C3dg, all chimeric C3s were cleaved only to iC3b-a. In addition, the cleavage of deleted (C3delta727-768) iC3 to iC3b-a by factor I in the presence of CR1 was significantly reduced, whereas it remained unaltered in the presence of MCP. Cleavage of iC3 to iC3b-a by factor I and H was similar in all expressed C3s except C3delta727-768, whose cleavage was significantly reduced. All of the expressed molecules except C3delta727-768 were capable of forming the fluid-phase alternative pathway C3 convertase, and all reacted with properdin. These results suggest that during cleavage of iC3 by factor I and CR1, or H, CR1 and H bind to at least two sites on C3 and that the MCP binding site(s) on C3b are different from those for CR1. They also indicate that some or all of the C3 residues that are directly involved in, or contribute to, the structure of one of the CR1 and H binding sites are located within residues 727-768. These studies also demonstrate that, although this segment of C3 may be involved in C3-factor B interaction, other residues in addition to 736EE (previously implicated in B binding) must also contribute significantly to this interaction.

  1. Human Lineage-Specific Transcriptional Regulation through GA-Binding Protein Transcription Factor Alpha (GABPa)

    PubMed Central

    Perdomo-Sabogal, Alvaro; Nowick, Katja; Piccini, Ilaria; Sudbrak, Ralf; Lehrach, Hans; Yaspo, Marie-Laure; Warnatz, Hans-Jörg; Querfurth, Robert

    2016-01-01

    A substantial fraction of phenotypic differences between closely related species are likely caused by differences in gene regulation. While this has already been postulated over 30 years ago, only few examples of evolutionary changes in gene regulation have been verified. Here, we identified and investigated binding sites of the transcription factor GA-binding protein alpha (GABPa) aiming to discover cis-regulatory adaptations on the human lineage. By performing chromatin immunoprecipitation-sequencing experiments in a human cell line, we found 11,619 putative GABPa binding sites. Through sequence comparisons of the human GABPa binding regions with orthologous sequences from 34 mammals, we identified substitutions that have resulted in 224 putative human-specific GABPa binding sites. To experimentally assess the transcriptional impact of those substitutions, we selected four promoters for promoter-reporter gene assays using human and African green monkey cells. We compared the activities of wild-type promoters to mutated forms, where we have introduced one or more substitutions to mimic the ancestral state devoid of the GABPa consensus binding sequence. Similarly, we introduced the human-specific substitutions into chimpanzee and macaque promoter backgrounds. Our results demonstrate that the identified substitutions are functional, both in human and nonhuman promoters. In addition, we performed GABPa knock-down experiments and found 1,215 genes as strong candidates for primary targets. Further analyses of our data sets link GABPa to cognitive disorders, diabetes, KRAB zinc finger (KRAB-ZNF), and human-specific genes. Thus, we propose that differences in GABPa binding sites played important roles in the evolution of human-specific phenotypes. PMID:26814189

  2. An Additional Potential Factor for Kidney Stone Formation during Space Flights: Calcifying Nanoparticles (Nanobacteria): A Case Report

    NASA Technical Reports Server (NTRS)

    Jones, Jeffrey A.; Ciftcioglu, Neva; Schmid, Joseph; Griffith, Donald

    2007-01-01

    Spaceflight-induced microgravity appears to be a risk factor for the development of urinary calculi due to skeletal calcium liberation and other undefined factors, resulting in stone disease in crewmembers during and after spaceflight. Calcifying nanoparticles, or nanobacteria, reproduce at a more rapid rate in simulated microgravity conditions and create external shells of calcium phosphate in the form of apatite. The questions arises whether calcifying nanoparticles are niduses for calculi and contribute to the development of clinical stone disease in humans, who possess environmental factors predisposing to the development of urinary calculi and potentially impaired immunological defenses during spaceflight. A case of a urinary calculus passed from an astronaut post-flight with morphological characteristics of calcifying nanoparticles and staining positive for a calcifying nanoparticle unique antigen, is presented.

  3. Activation of G Proteins by Guanine Nucleotide Exchange Factors Relies on GTPase Activity.

    PubMed

    Stanley, Rob J; Thomas, Geraint M H

    2016-01-01

    G proteins are an important family of signalling molecules controlled by guanine nucleotide exchange and GTPase activity in what is commonly called an 'activation/inactivation cycle'. The molecular mechanism by which guanine nucleotide exchange factors (GEFs) catalyse the activation of monomeric G proteins is well-established, however the complete reversibility of this mechanism is often overlooked. Here, we use a theoretical approach to prove that GEFs are unable to positively control G protein systems at steady-state in the absence of GTPase activity. Instead, positive regulation of G proteins must be seen as a product of the competition between guanine nucleotide exchange and GTPase activity--emphasising a central role for GTPase activity beyond merely signal termination. We conclude that a more accurate description of the regulation of G proteins via these processes is as a 'balance/imbalance' mechanism. This result has implications for the understanding of intracellular signalling processes, and for experimental strategies that rely on modulating G protein systems. PMID:26986850

  4. Immunotherapy for Prostate Cancer with Gc Protein-Derived Macrophage-Activating Factor, GcMAF.

    PubMed

    Yamamoto, Nobuto; Suyama, Hirofumi; Yamamoto, Nobuyuki

    2008-07-01

    Serum Gc protein (known as vitamin D(3)-binding protein) is the precursor for the principal macrophage-activating factor (MAF). The MAF precursor activity of serum Gc protein of prostate cancer patients was lost or reduced because Gc protein was deglycosylated by serum alpha-N-acetylgalactosaminidase (Nagalase) secreted from cancerous cells. Therefore, macrophages of prostate cancer patients having deglycosylated Gc protein cannot be activated, leading to immunosuppression. Stepwise treatment of purified Gc protein with immobilized beta-galactosidase and sialidase generated the most potent MAF (termed GcMAF) ever discovered, which produces no adverse effect in humans. Macrophages activated by GcMAF develop a considerable variation of receptors that recognize the abnormality in malignant cell surface and are highly tumoricidal. Sixteen nonanemic prostate cancer patients received weekly administration of 100 ng of GcMAF. As the MAF precursor activity increased, their serum Nagalase activity decreased. Because serum Nagalase activity is proportional to tumor burden, the entire time course analysis for GcMAF therapy was monitored by measuring the serum Nagalase activity. After 14 to 25 weekly administrations of GcMAF (100 ng/week), all 16 patients had very low serum Nagalase levels equivalent to those of healthy control values, indicating that these patients are tumor-free. No recurrence occurred for 7 years. PMID:18633461

  5. Mitochondrial Bol1 and Bol3 function as assembly factors for specific iron-sulfur proteins

    PubMed Central

    Uzarska, Marta A; Nasta, Veronica; Weiler, Benjamin D; Spantgar, Farah; Ciofi-Baffoni, Simone; Saviello, Maria Rosaria; Gonnelli, Leonardo; Mühlenhoff, Ulrich; Banci, Lucia; Lill, Roland

    2016-01-01

    Assembly of mitochondrial iron-sulfur (Fe/S) proteins is a key process of cells, and defects cause many rare diseases. In the first phase of this pathway, ten Fe/S cluster (ISC) assembly components synthesize and insert [2Fe-2S] clusters. The second phase is dedicated to the assembly of [4Fe-4S] proteins, yet this part is poorly understood. Here, we characterize the BOLA family proteins Bol1 and Bol3 as specific mitochondrial ISC assembly factors that facilitate [4Fe-4S] cluster insertion into a subset of mitochondrial proteins such as lipoate synthase and succinate dehydrogenase. Bol1-Bol3 perform largely overlapping functions, yet cannot replace the ISC protein Nfu1 that also participates in this phase of Fe/S protein biogenesis. Bol1 and Bol3 form dimeric complexes with both monothiol glutaredoxin Grx5 and Nfu1. Complex formation differentially influences the stability of the Grx5-Bol-shared Fe/S clusters. Our findings provide the biochemical basis for explaining the pathological phenotypes of patients with mutations in BOLA3. DOI: http://dx.doi.org/10.7554/eLife.16673.001 PMID:27532772

  6. Mitochondrial Bol1 and Bol3 function as assembly factors for specific iron-sulfur proteins.

    PubMed

    Uzarska, Marta A; Nasta, Veronica; Weiler, Benjamin D; Spantgar, Farah; Ciofi-Baffoni, Simone; Saviello, Maria Rosaria; Gonnelli, Leonardo; Mühlenhoff, Ulrich; Banci, Lucia; Lill, Roland

    2016-08-17

    Assembly of mitochondrial iron-sulfur (Fe/S) proteins is a key process of cells, and defects cause many rare diseases. In the first phase of this pathway, ten Fe/S cluster (ISC) assembly components synthesize and insert [2Fe-2S] clusters. The second phase is dedicated to the assembly of [4Fe-4S] proteins, yet this part is poorly understood. Here, we characterize the BOLA family proteins Bol1 and Bol3 as specific mitochondrial ISC assembly factors that facilitate [4Fe-4S] cluster insertion into a subset of mitochondrial proteins such as lipoate synthase and succinate dehydrogenase. Bol1-Bol3 perform largely overlapping functions, yet cannot replace the ISC protein Nfu1 that also participates in this phase of Fe/S protein biogenesis. Bol1 and Bol3 form dimeric complexes with both monothiol glutaredoxin Grx5 and Nfu1. Complex formation differentially influences the stability of the Grx5-Bol-shared Fe/S clusters. Our findings provide the biochemical basis for explaining the pathological phenotypes of patients with mutations in BOLA3.

  7. Involvement of an SHP-2-Rho small G protein pathway in hepatocyte growth factor/scatter factor-induced cell scattering.

    PubMed

    Kodama, A; Matozaki, T; Fukuhara, A; Kikyo, M; Ichihashi, M; Takai, Y

    2000-08-01

    Hepatocyte growth factor/scatter factor (HGF/SF) induces cell scattering through the tyrosine kinase-type HGF/SF receptor c-Met. We have previously shown that Rho small G protein (Rho) is involved in the HGF/SF-induced scattering of Madin-Darby canine kidney (MDCK) cells by regulating at least the assembly and disassembly of stress fibers and focal adhesions, but it remains unknown how c-Met regulates Rho activity. We have found here a novel signaling pathway of c-Met consisting of SHP-2-Rho that regulates the assembly and disassembly of stress fibers and focal adhesions in MDCK cells. SHP-2 is a protein-tyrosine phosphatase that contains src homology-2 domains. Expression of a dominant negative mutant of SHP-2 (SHP-2-C/S) markedly increased the formation of stress fibers and focal adhesions in MDCK cells and inhibited their scattering. C3, a Clostridium botulinum ADP-ribosyltransferase, and Y-27632, a specific inhibitor for ROCK, reversed the stimulatory effect of SHP-2-C/S on stress fiber formation and the inhibitory effect on cell scattering. Vav2 is a GDP/GTP exchange protein for Rho. Expression of a dominant negative mutant of Vav2 blocked the stimulatory effect of SHP-2-C/S on stress fiber formation. Conversely, expression of mutants of Vav2 that increased stress fiber formation inhibited HGF/SF-induced cell scattering. These results indicate that SHP-2 physiologically modulates the activity of Rho to form stress fibers and focal adhesions and thereby regulates HGF/SF-induced cell scattering. In addition, Vav2 may be involved in the SHP-2-Rho pathway.

  8. The sequence-specific transcription factor c-Jun targets Cockayne syndrome protein B to regulate transcription and chromatin structure.

    PubMed

    Lake, Robert J; Boetefuer, Erica L; Tsai, Pei-Fang; Jeong, Jieun; Choi, Inchan; Won, Kyoung-Jae; Fan, Hua-Ying

    2014-04-01

    Cockayne syndrome is an inherited premature aging disease associated with numerous developmental and neurological defects, and mutations in the gene encoding the CSB protein account for the majority of Cockayne syndrome cases. Accumulating evidence suggests that CSB functions in transcription regulation, in addition to its roles in DNA repair, and those defects in this transcriptional activity might contribute to the clinical features of Cockayne syndrome. Transcription profiling studies have so far uncovered CSB-dependent effects on gene expression; however, the direct targets of CSB's transcriptional activity remain largely unknown. In this paper, we report the first comprehensive analysis of CSB genomic occupancy during replicative cell growth. We found that CSB occupancy sites display a high correlation to regions with epigenetic features of promoters and enhancers. Furthermore, we found that CSB occupancy is enriched at sites containing the TPA-response element. Consistent with this binding site preference, we show that CSB and the transcription factor c-Jun can be found in the same protein-DNA complex, suggesting that c-Jun can target CSB to specific genomic regions. In support of this notion, we observed decreased CSB occupancy of TPA-response elements when c-Jun levels were diminished. By modulating CSB abundance, we found that CSB can influence the expression of nearby genes and impact nucleosome positioning in the vicinity of its binding site. These results indicate that CSB can be targeted to specific genomic loci by sequence-specific transcription factors to regulate transcription and local chromatin structure. Additionally, comparison of CSB occupancy sites with the MSigDB Pathways database suggests that CSB might function in peroxisome proliferation, EGF receptor transactivation, G protein signaling and NF-κB activation, shedding new light on the possible causes and mechanisms of Cockayne syndrome.

  9. Tumor necrosis factor α-induced protein-3 protects zinc transporter 8 against proinflammatory cytokine-induced downregulation

    PubMed Central

    Cheng, Liqing; Zhang, Dongmei; Chen, Bing

    2016-01-01

    Zinc transporter 8 (ZnT8) is exclusively expressed in the pancreatic islet and is essential for insulin crystallization, hexamerization and secretion. Tumor necrosis factor α-induced protein-3 (TNFAIP3) is a zinc finger protein that serves a major role in the negative feedback regulation of NF-κB signaling in response to multiple stimuli, and is a central regulator of immunopathology. Although the role of TNFAIP3 in diabetes has been extensively studied, its effect on ZnT8 has not been fully elucidated. The present study aimed to verify whether proinflammatory cytokines, tumor necrosis factor α (TNF-α) and interleukin-1β (IL-1β), are able to affect ZnT8 expression in islet cells. In addition, the study aimed to determine the effect of TNFAIP3 overexpression on cytokine-altered ZnT8 activity, considering its effect on NF-κB signaling. Cell-based studies using NIT-1 cells overexpressing TNFAIP3 were used to assess the effect of cytokines on ZnT8 and NF-κB activation, as well as the effect of TNFAIP3 on ZnT8 expression. Western blot analysis and immunofluorescence staining were employed to determine the protein expression and NF-κB activation, respectively. The results indicated that cytokine stimulation led to TNFAIP3 upregulation, ZnT8 downregulation and NF-κB activation. Furthermore, TNFAIP3 overexpression protected ZnT8 from cytokine-induced downregulation. In conclusion, the current results suggest that inflammation or TNFAIP3 dysfunction may be involved in the pathogenesis of diabetes via ZnT8 expression, besides from islet cell apoptosis. In addition, restricting inflammation and enhancing TNFAIP3 expression may exert a positive effect in diabetes prevention, treatment and pancreatic cell transplantation. PMID:27588072

  10. Employing Lead Thiocyanate Additive to Reduce the Hysteresis and Boost the Fill Factor of Planar Perovskite Solar Cells.

    PubMed

    Ke, Weijun; Xiao, Chuanxiao; Wang, Changlei; Saparov, Bayrammurad; Duan, Hsin-Sheng; Zhao, Dewei; Xiao, Zewen; Schulz, Philip; Harvey, Steven P; Liao, Weiqiang; Meng, Weiwei; Yu, Yue; Cimaroli, Alexander J; Jiang, Chun-Sheng; Zhu, Kai; Al-Jassim, Mowafak; Fang, Guojia; Mitzi, David B; Yan, Yanfa

    2016-07-01

    Lead thiocyanate in the perovskite precursor can increase the grain size of a perovskite thin film and reduce the conductivity of the grain boundaries, leading to perovskite solar cells with reduced hysteresis and enhanced fill factor. A planar perovskite solar cell with grain boundary and interface passivation achieves a steady-state efficiency of 18.42%.

  11. Identification and validation of genetic variants that influence transcription factor and cell signaling protein levels.

    PubMed

    Hause, Ronald J; Stark, Amy L; Antao, Nirav N; Gorsic, Lidija K; Chung, Sophie H; Brown, Christopher D; Wong, Shan S; Gill, Daniel F; Myers, Jamie L; To, Lida Anita; White, Kevin P; Dolan, M Eileen; Jones, Richard Baker

    2014-08-01

    Many genetic variants associated with human disease have been found to be associated with alterations in mRNA expression. Although it is commonly assumed that mRNA expression changes will lead to consequent changes in protein levels, methodological challenges have limited our ability to test the degree to which this assumption holds true. Here, we further developed the micro-western array approach and globally examined relationships between human genetic variation and cellular protein levels. We collected more than 250,000 protein level measurements comprising 441 transcription factor and signaling protein isoforms across 68 Yoruba (YRI) HapMap lymphoblastoid cell lines (LCLs) and identified 12 cis and 160 trans protein level QTLs (pQTLs) at a false discovery rate (FDR) of 20%. Whereas up to two thirds of cis mRNA expression QTLs (eQTLs) were also pQTLs, many pQTLs were not associated with mRNA expression. Notably, we replicated and functionally validated a trans pQTL relationship between the KARS lysyl-tRNA synthetase locus and levels of the DIDO1 protein. This study demonstrates proof of concept in applying an antibody-based microarray approach to iteratively measure the levels of human proteins and relate these levels to human genome variation and other genomic data sets. Our results suggest that protein-based mechanisms might functionally buffer genetic alterations that influence mRNA expression levels and that pQTLs might contribute phenotypic diversity to a human population independently of influences on mRNA expression.

  12. Comparative analysis of Klebsiella pneumoniae genomes identifies a phospholipase D family protein as a novel virulence factor

    PubMed Central

    2014-01-01

    Background Klebsiella pneumoniae strains are pathogenic to animals and humans, in which they are both a frequent cause of nosocomial infections and a re-emerging cause of severe community-acquired infections. K. pneumoniae isolates of the capsular serotype K2 are among the most virulent. In order to identify novel putative virulence factors that may account for the severity of K2 infections, the genome sequence of the K2 reference strain Kp52.145 was determined and compared to two K1 and K2 strains of low virulence and to the reference strains MGH 78578 and NTUH-K2044. Results In addition to diverse functions related to host colonization and virulence encoded in genomic regions common to the four strains, four genomic islands specific for Kp52.145 were identified. These regions encoded genes for the synthesis of colibactin toxin, a putative cytotoxin outer membrane protein, secretion systems, nucleases and eukaryotic-like proteins. In addition, an insertion within a type VI secretion system locus included sel1 domain containing proteins and a phospholipase D family protein (PLD1). The pld1 mutant was avirulent in a pneumonia model in mouse. The pld1 mRNA was expressed in vivo and the pld1 gene was associated with K. pneumoniae isolates from severe infections. Analysis of lipid composition of a defective E. coli strain complemented with pld1 suggests an involvement of PLD1 in cardiolipin metabolism. Conclusions Determination of the complete genome of the K2 reference strain identified several genomic islands comprising putative elements of pathogenicity. The role of PLD1 in pathogenesis was demonstrated for the first time and suggests that lipid metabolism is a novel virulence mechanism of K. pneumoniae. PMID:24885329

  13. Epidermal growth factor (EGF) promotes phosphorylation at threonine-654 of the EGF receptor: possible role of protein kinase C in homologous regulation of the EGF receptor

    SciTech Connect

    Whiteley, B.; Glaser, L.

    1986-10-01

    Treatment of cells with tumor-promoting phorbol diesters, which causes activation of protein kinase C, leads to phosphorylation of the epidermal growth factor (EGF) receptor at threonine-654. Addition of phorbol diesters to intact cells causes inhibition of the EGF-induced tyrosine-protein kinase activity of the EGF receptor and it has been suggested that this effect of phorbol diesters is mediated by the phosphorylation of the receptor by protein kinase C. The authors measured the activity of protein kinase C in A431 cells by determining the incorporation of (/sup 32/P)phosphate into peptides containing threonine-654 obtained by trypsin digestion of EGF receptors. After 3 h of exposure to serum-free medium, A431 cells had no detectable protein kinase C activity. Addition of EGF to these cells resulted in (/sup 32/P) incorporation into threonine-654 as well as into tyrosine residues. This indicates that EGF promotes the activation of protein kinase C in A431 cells. The phophorylation of threonine-654 induced by EGF was maximal after only 5 min of EGF addition and the (/sup 32/P) incorporation into threonine-654 reached 50% of the (/sup 32/P) in a tyrosine-containing peptide. This indicates that a significant percentage of the total EGF receptors are phosphorylated by protein kinase C. A variety of external stimuli activate Na/sup +//H/sup +/ exchange, including EGF, phorbol diesters, and hypertonicity. To ascertain whether activation of protein kinase C is an intracellular common effector of all of these systems, the authors measured the activity of protein kinase C after exposure of A431 cells to hyperosmotic conditions and observed no effect on phosphorylation of threonine-654, therefore, activation of Na/sup +//H/sup +/ exchange by hypertonic medium is independent of protein kinase C activity.

  14. Metallothionein gene expression is regulated by serum factors and activators of protein kinase C.

    PubMed Central

    Imbra, R J; Karin, M

    1987-01-01

    The exact physiological role of metallothionein (MT) is not clear. It has been suggested that these low-molecular-weight, highly inducible, heavy-metal-binding proteins serve in the regulation of intracellular Zn metabolism. Among the Zn-requiring systems are several enzymes involved in DNA replication and repair. Therefore, during periods of active DNA synthesis there is likely to be an increased demand for Zn, which could be met by elevated MT synthesis. For that reason, we examined whether stimulation of cellular proliferation leads to increased expression of MT. We report here that treatment of cultured mammalian cells with serum growth factors and activators of protein kinase C, all of which are known to have growth stimulatory activity, led to induction of MT mRNA. One of the required steps in the signal transduction pathways triggered by these agents, ending in MT induction, appears to be the activation of protein kinase C. Images PMID:3600629

  15. Physical activity in individuals with haemophilia and experience with recombinant factor VIII Fc fusion protein and recombinant factor IX Fc fusion protein for the treatment of active patients: a literature review and case reports

    PubMed Central

    Wang, Michael; Álvarez-Román, María Teresa; Chowdary, Pratima; Quon, Doris V.; Schafer, Kim

    2016-01-01

    The World Federation of Hemophilia and the National Hemophilia Foundation encourage people with haemophilia (PWH) to participate in routine physical activity. The benefits of physical activity for PWH include improvements in joint, bone, and muscle health. Accordingly, a number of studies suggest that levels of physical activity among PWH are similar to those of their healthy peers, especially among individuals who began prophylaxis at an early age (≤3 years). Importantly, several studies found either no increased risk or only a transient increase in risk of bleeding with more intensive physical activity compared with less intensive physical activity. Data on optimal prophylaxis regimens for PWH who participate in physical/sporting activities; however, remain sparse. Long-acting recombinant factor VIII Fc fusion protein (rFVIIIFc) and recombinant factor IX Fc fusion protein (rFIXFc) demonstrated efficacy for the prevention and treatment of bleeding episodes in Phase 3 clinical trials of participants with haemophilia A and B, respectively, with most individuals able to maintain or increase their physical activities. This manuscript reviews the current literature that describes physical activity in PWH. Additionally, case studies are presented to provide supplemental information to clinicians illustrating the use of rFVIIIFc and rFIXFc in physically active patients with haemophilia A and B, respectively. These case reports demonstrate that it is possible for patients to be physically active and maintain good control of their haemophilia with extended interval prophylactic dosing using rFVIIIFc or rFIXFc. PMID:27116081

  16. Competition between antagonistic complement factors for a single protein on N. meningitidis rules disease susceptibility

    PubMed Central

    Caesar, Joseph JE; Lavender, Hayley; Ward, Philip N; Exley, Rachel M; Eaton, Jack; Chittock, Emily; Malik, Talat H; Goiecoechea De Jorge, Elena; Pickering, Matthew C; Tang, Christoph M; Lea, Susan M

    2014-01-01

    Genome-wide association studies have found variation within the complement factor H gene family links to host susceptibility to meningococcal disease caused by infection with Neisseria meningitidis (Davila et al., 2010). Mechanistic insights have been challenging since variation within this locus is complex and biological roles of the factor H-related proteins, unlike factor H, are incompletely understood. N. meningitidis subverts immune responses by hijacking a host-immune regulator, complement factor H (CFH), to the bacterial surface (Schneider et al., 2006; Madico et al., 2007; Schneider et al., 2009). We demonstrate that complement factor-H related 3 (CFHR3) promotes immune activation by acting as an antagonist of CFH. Conserved sequences between CFH and CFHR3 mean that the bacterium cannot sufficiently distinguish between these two serum proteins to allow it to hijack the regulator alone. The level of protection from complement attack achieved by circulating N. meningitidis therefore depends on the relative levels of CFH and CFHR3 in serum. These data may explain the association between genetic variation in both CFH and CFHR3 and susceptibility to meningococcal disease. DOI: http://dx.doi.org/10.7554/eLife.04008.001 PMID:25534642

  17. 14-3-3 Protein Masks the DNA Binding Interface of Forkhead Transcription Factor FOXO4*

    PubMed Central

    Silhan, Jan; Vacha, Petr; Strnadova, Pavla; Vecer, Jaroslav; Herman, Petr; Sulc, Miroslav; Teisinger, Jan; Obsilova, Veronika; Obsil, Tomas

    2009-01-01

    The role of 14-3-3 proteins in the regulation of FOXO forkhead transcription factors is at least 2-fold. First, the 14-3-3 binding inhibits the interaction between the FOXO and the target DNA. Second, the 14-3-3 proteins prevent nuclear reimport of FOXO factors by masking their nuclear localization signal. The exact mechanisms of these processes are still unclear, mainly due to the lack of structural data. In this work, we used fluorescence spectroscopy to investigate the mechanism of the 14-3-3 protein-dependent inhibition of FOXO4 DNA-binding properties. Time-resolved fluorescence measurements revealed that the 14-3-3 binding affects fluorescence properties of 5-(((acetylamino)ethyl)amino) naphthalene-1-sulfonic acid moiety attached at four sites within the forkhead domain of FOXO4 that represent important parts of the DNA binding interface. Observed changes in 5-(((acetylamino)ethyl)amino) naphthalene-1-sulfonic acid fluorescence strongly suggest physical contacts between the 14-3-3 protein and labeled parts of the FOXO4 DNA binding interface. The 14-3-3 protein binding, however, does not cause any dramatic conformational change of FOXO4 as documented by the results of tryptophan fluorescence experiments. To build a realistic model of the FOXO4·14-3-3 complex, we measured six distances between 14-3-3 and FOXO4 using Förster resonance energy transfer time-resolved fluorescence experiments. The model of the complex suggests that the forkhead domain of FOXO4 is docked within the central channel of the 14-3-3 protein dimer, consistent with our hypothesis that 14-3-3 masks the DNA binding interface of FOXO4. PMID:19416966

  18. Collapsin Response Mediator Protein-2 (CRMP2) is a Plausible Etiological Factor and Potential Therapeutic Target in Alzheimer’s Disease: Comparison and Contrast with Microtubule-Associated Protein Tau

    PubMed Central

    Hensley, Kenneth; Kursula, Petri

    2016-01-01

    Alzheimer’s disease (AD) has long been viewed as a pathology that must be caused either by aberrant amyloid-β protein precursor (AβPP) processing, dysfunctional tau protein processing, or a combination of these two factors. This is a reasonable assumption because amyloid-β peptide (Aβ) accumulation and tau hyperphosphorylation are the defining histological features in AD, and because AβPP and tau mutations can cause AD in humans or AD-like features in animal models. Nonetheless, other protein players are emerging that one can argue are significant etiological players in subsets of AD and potentially novel, druggable targets. In particular, the microtubule-associated protein CRMP2 (collapsin response mediator protein-2) bears striking analogies to tau and is similarly relevant to AD. Like tau, CRMP2 dynamically regulates microtubule stability; it is acted upon by the same kinases; collects similarly in neurofibrillary tangles (NFTs); and when sequestered in NFTs, complexes with critical synapse-stabilizing factors. Additionally, CRMP2 is becoming recognized as an important adaptor protein involved in vesicle trafficking, amyloidogenesis and autophagy, in ways that tau is not. This review systematically compares the biology of CRMP2 to that of tau in the context of AD and explores the hypothesis that CRMP2 is an etiologically significant protein in AD and participates in pathways that can be rationally engaged for therapeutic benefit. PMID:27079722

  19. Structure-factor analysis of femtosecond microdiffraction patterns from protein nanocrystals

    NASA Astrophysics Data System (ADS)

    Kirian, R. A.; White, T. A.; Holton, J. M.; Chapman, H. N.; Fromme, P.; Barty, A.; Lomb, L.; Aquila, A.

    2011-03-01

    A complete set of structure factors has been extracted from hundreds of thousands of femtosecond X-ray diffraction patterns from randomly oriented Photosystem I membrane protein nanocrystals, using the Monte Carlo method of intensity integration. The data, collected at the Linac Coherent Light Source, are compared with conventional single-crystal data collected at a synchrotron source, and the quality of each data set was found to be similar.

  20. Factors affecting the microbial and chemical composition of silage. III. Effect of urea additions on maize silage.

    PubMed

    Mahmoud, S A; Abd-el-Hafez, A; Zaki, M M; Saleh, E A

    1978-01-01

    The effect of urea additions on the microbiological and chemical properties of silage, produced from young maize plants (Darawa stage), was studied. Urea treatments, i.e., 0.25%, 0.50%, 0.75%, and 1.00%, stimulated higher densities of the desired microorganisms than the control, while undesired organisms showed lower counts (proteolytic and saccharolytic anaerobes). Addition of 0.25 to 0.50% or urea resulted in the production of high quality silage with pleasant small and high nutritive value, as confirmed by the various microbiological and chemical analyses conducted. Higher levels (0.75 and 1.00%) of urea decreased the quality of the product. PMID:29417

  1. Pairwise additivity of energy components in protein-ligand binding: the HIV II protease-Indinavir case.

    PubMed

    Ucisik, Melek N; Dashti, Danial S; Faver, John C; Merz, Kenneth M

    2011-08-28

    An energy expansion (binding energy decomposition into n-body interaction terms for n ≥ 2) to express the receptor-ligand binding energy for the fragmented HIV II protease-Indinavir system is described to address the role of cooperativity in ligand binding. The outcome of this energy expansion is compared to the total receptor-ligand binding energy at the Hartree-Fock, density functional theory, and semiempirical levels of theory. We find that the sum of the pairwise interaction energies approximates the total binding energy to ∼82% for HF and to >95% for both the M06-L density functional and PM6-DH2 semiempirical method. The contribution of the three-body interactions amounts to 18.7%, 3.8%, and 1.4% for HF, M06-L, and PM6-DH2, respectively. We find that the expansion can be safely truncated after n=3. That is, the contribution of the interactions involving more than three parties to the total binding energy of Indinavir to the HIV II protease receptor is negligible. Overall, we find that the two-body terms represent a good approximation to the total binding energy of the system, which points to pairwise additivity in the present case. This basic principle of pairwise additivity is utilized in fragment-based drug design approaches and our results support its continued use. The present results can also aid in the validation of non-bonded terms contained within common force fields and in the correction of systematic errors in physics-based score functions. PMID:21895219

  2. Pairwise additivity of energy components in protein-ligand binding: The HIV II protease-Indinavir case

    PubMed Central

    Ucisik, Melek N.; Dashti, Danial S.; Faver, John C.; Merz, Kenneth M.

    2011-01-01

    An energy expansion (binding energy decomposition into n-body interaction terms for n ≥ 2) to express the receptor-ligand binding energy for the fragmented HIV II protease-Indinavir system is described to address the role of cooperativity in ligand binding. The outcome of this energy expansion is compared to the total receptor-ligand binding energy at the Hartree-Fock, density functional theory, and semiempirical levels of theory. We find that the sum of the pairwise interaction energies approximates the total binding energy to ∼82% for HF and to >95% for both the M06-L density functional and PM6-DH2 semiempirical method. The contribution of the three-body interactions amounts to 18.7%, 3.8%, and 1.4% for HF, M06-L, and PM6-DH2, respectively. We find that the expansion can be safely truncated after n = 3. That is, the contribution of the interactions involving more than three parties to the total binding energy of Indinavir to the HIV II protease receptor is negligible. Overall, we find that the two-body terms represent a good approximation to the total binding energy of the system, which points to pairwise additivity in the present case. This basic principle of pairwise additivity is utilized in fragment-based drug design approaches and our results support its continued use. The present results can also aid in the validation of non-bonded terms contained within common force fields and in the correction of systematic errors in physics-based score functions. PMID:21895219

  3. Evolutionarily Conserved Binding of Translationally Controlled Tumor Protein to Eukaryotic Elongation Factor 1B*

    PubMed Central

    Wu, Huiwen; Gong, Weibin; Yao, Xingzhe; Wang, Jinfeng; Perrett, Sarah; Feng, Yingang

    2015-01-01

    Translationally controlled tumor protein (TCTP) is an abundant protein that is highly conserved in eukaryotes. However, its primary function is still not clear. Human TCTP interacts with the metazoan-specific eukaryotic elongation factor 1Bδ (eEF1Bδ) and inhibits its guanine nucleotide exchange factor (GEF) activity, but the structural mechanism remains unknown. The interaction between TCTP and eEF1Bδ was investigated by NMR titration, structure determination, paramagnetic relaxation enhancement, site-directed mutagenesis, isothermal titration calorimetry, and HADDOCK docking. We first demonstrated that the catalytic GEF domain of eEF1Bδ is not responsible for binding to TCTP but rather a previously unnoticed central acidic region (CAR) domain in eEF1Bδ. The mutagenesis data and the structural model of the TCTP-eEF1Bδ CAR domain complex revealed the key binding residues. These residues are highly conserved in eukaryotic TCTPs and in eEF1B GEFs, including the eukaryotically conserved eEF1Bα, implying the interaction may be conserved in all eukaryotes. Interactions were confirmed between TCTP and the eEF1Bα CAR domain for human, fission yeast, and unicellular photosynthetic microalgal proteins, suggesting that involvement in protein translation through the conserved interaction with eEF1B represents a primary function of TCTP. PMID:25635048

  4. Protein kinase A represses skeletal myogenesis by targeting myocyte enhancer factor 2D.

    PubMed

    Du, Min; Perry, Robert L S; Nowacki, Nathaniel B; Gordon, Joseph W; Salma, Jahan; Zhao, Jianzhong; Aziz, Arif; Chan, Joseph; Siu, K W Michael; McDermott, John C

    2008-05-01

    Activation of protein kinase A (PKA) by elevation of the intracellular cyclic AMP (cAMP) level inhibits skeletal myogenesis. Previously, an indirect modulation of the myogenic regulatory factors (MRFs) was implicated as the mechanism. Because myocyte enhancer factor 2 (MEF2) proteins are key regulators of myogenesis and obligatory partners for the MRFs, here we assessed whether these proteins could be involved in PKA-mediated myogenic repression. Initially, in silico analysis revealed several consensus PKA phosphoacceptor sites on MEF2, and subsequent analysis by in vitro kinase assays indicated that PKA directly and efficiently phosphorylates MEF2D. Using mass spectrometric determination of phosphorylated residues, we document that MEF2D serine 121 and serine 190 are targeted by PKA. Transcriptional reporter gene assays to assess MEF2D function revealed that PKA potently represses the transactivation properties of MEF2D. Furthermore, engineered mutation of MEF2D PKA phosphoacceptor sites (serines 121 and 190 to alanine) rendered a PKA-resistant MEF2D protein, which efficiently rescues myogenesis from PKA-mediated repression. Concomitantly, increased intracellular cAMP-mediated PKA activation also resulted in an enhanced nuclear accumulation of histone deacetylase 4 (HDAC4) and a subsequent increase in the MEF2D-HDAC4 repressor complex. Collectively, these data identify MEF2D as a primary target of PKA signaling in myoblasts that leads to inhibition of the skeletal muscle differentiation program.

  5. Respiratory syncytial virus M2-1 protein induces the activation of nuclear factor kappa B

    SciTech Connect

    Reimers, Kerstin . E-mail: reimers.kerstin@mh-hannover.de; Buchholz, Katja; Werchau, Hermann

    2005-01-20

    Respiratory syncytial virus (RSV) induces the production of a number of cytokines and chemokines by activation of nuclear factor kappa B (NF-{kappa}B). The activation of NF-{kappa}B has been shown to depend on viral replication in the infected cells. In this study, we demonstrate that expression of RSV M2-1 protein, a transcriptional processivity and anti-termination factor, is sufficient to activate NF-{kappa}B in A549 cells. Electromobility shift assays show increased NF-{kappa}B complexes in the nuclei of M2-1-expressing cells. M2-1 protein is found in nuclei of M2-1-expressing cells and in RSV-infected cells. Co-immunoprecipitations of nuclear extracts of M2-1-expressing cells and of RSV-infected cells revealed an association of M2-1 with Rel A protein. Furthermore, the activation of NF-{kappa}B depends on the C-terminus of the RSV M2-1 protein, as shown by NF-{kappa}B-induced gene expression of a reporter gene construct.

  6. Comparison of amino acid sequence of bovine coagulation Factor IX (Christmas Factor) with that of other vitamin K-dependent plasma proteins.

    PubMed

    Katayama, K; Ericsson, L H; Enfield, D L; Walsh, K A; Neurath, H; Davie, E W; Titani, K

    1979-10-01

    The amino acid sequence of bovine blood coagulation Factor IX (Christmas Factor) is presented and compared with the sequences of other vitamin K-dependent plasma proteins and pancreatic trypsinogen. The 416-residue sequence of Factor IX was determined largely by automated Edman degradation of two large segments, containing 181 and 235 residues, isolated after activating Factor IX with a protease from Russell's viper venom. Subfragments of the two segments were produced by enzymatic digestion and by chemical cleavage of methionyl, tryptophyl, and asparaginyl-glycyl bonds. Comparison of the amino acid sequences of Factor IX, Factor X, and Protein C demonstrates that they are homologous throughout. Their homology with prothrombin, however, is restricted to the amino-terminal region, which is rich in gamma-carboxyglutamic acid, and the carboxyl-terminal region, which represents the catalytic domain of these proteins and corresponds to that of pancreatic serine proteases.

  7. Factor H-related protein 5 interacts with pentraxin 3 and the extracellular matrix and modulates complement activation.

    PubMed

    Csincsi, Ádám I; Kopp, Anne; Zöldi, Miklós; Bánlaki, Zsófia; Uzonyi, Barbara; Hebecker, Mario; Caesar, Joseph J E; Pickering, Matthew C; Daigo, Kenji; Hamakubo, Takao; Lea, Susan M; Goicoechea de Jorge, Elena; Józsi, Mihály

    2015-05-15

    The physiological roles of the factor H (FH)-related proteins are controversial and poorly understood. Based on genetic studies, FH-related protein 5 (CFHR5) is implicated in glomerular diseases, such as atypical hemolytic uremic syndrome, dense deposit disease, and CFHR5 nephropathy. CFHR5 was also identified in glomerular immune deposits at the protein level. For CFHR5, weak complement regulatory activity and competition for C3b binding with the plasma complement inhibitor FH have been reported, but its function remains elusive. In this study, we identify pentraxin 3 (PTX3) as a novel ligand of CFHR5. Binding of native CFHR5 to PTX3 was detected in human plasma and the interaction was characterized using recombinant proteins. The binding of PTX3 to CFHR5 is of ∼2-fold higher affinity compared with that of FH. CFHR5 dose-dependently inhibited FH binding to PTX3 and also to the monomeric, denatured form of the short pentraxin C-reactive protein. Binding of PTX3 to CFHR5 resulted in increased C1q binding. Additionally, CFHR5 bound to extracellular matrix in vitro in a dose-dependent manner and competed with FH for binding. Altogether, CFHR5 reduced FH binding and its cofactor activity on pentraxins and the extracellular matrix, while at the same time allowed for enhanced C1q binding. Furthermore, CFHR5 allowed formation of the alternative pathway C3 convertase and supported complement activation. Thus, CFHR5 may locally enhance complement activation via interference with the complement-inhibiting function of FH, by enhancement of C1q binding, and by activating complement, thereby contributing to glomerular disease.

  8. A Mutant Library Approach to Identify Improved Meningococcal Factor H Binding Protein Vaccine Antigens

    PubMed Central

    Konar, Monica; Rossi, Raffaella; Walter, Helen; Pajon, Rolando; Beernink, Peter T.

    2015-01-01

    Factor H binding protein (FHbp) is a virulence factor used by meningococci to evade the host complement system. FHbp elicits bactericidal antibodies in humans and is part of two recently licensed vaccines. Using human complement Factor H (FH) transgenic mice, we previously showed that binding of FH decreased the protective antibody responses to FHbp vaccination. Therefore, in the present study we devised a library-based method to identify mutant FHbp antigens with very low binding of FH. Using an FHbp sequence variant in one of the two licensed vaccines, we displayed an error-prone PCR mutant FHbp library on the surface of Escherichia coli. We used fluorescence-activated cell sorting to isolate FHbp mutants with very low binding of human FH and preserved binding of control anti-FHbp monoclonal antibodies. We sequenced the gene encoding FHbp from selected clones and introduced the mutations into a soluble FHbp construct. Using this approach, we identified several new mutant FHbp vaccine antigens that had very low binding of FH as measured by ELISA and surface plasmon resonance. The new mutant FHbp antigens elicited protective antibody responses in human FH transgenic mice that were up to 20-fold higher than those elicited by the wild-type FHbp antigen. This approach offers the potential to discover mutant antigens that might not be predictable even with protein structural information and potentially can be applied to other microbial vaccine antigens that bind host proteins. PMID:26057742

  9. Vascular endothelial growth factor A protein level and gene expression in intracranial meningiomas with brain edema.

    PubMed

    Nassehi, Damoun; Dyrbye, Henrik; Andresen, Morten; Thomsen, Carsten; Juhler, Marianne; Laursen, Henning; Broholm, Helle

    2011-12-01

    Meningiomas are the second most common primary intracranial tumors in adults. Although meningiomas are mostly benign, more than 50% of patients with meningioma develop peritumoral brain edema (PTBE), which may be fatal because of increased intracranial pressure. Vascular endothelial growth factor (VEGF) is an endothelial cell-specific mitogen and angiogen. VEGF-A protein, which is identical to vascular permeability factor, is a regulator of angiogenesis. In this study, 101 patients with meningiomas, and possible co-factors to PTBE, such as meningioma subtypes and tumor location, were examined. Forty-three patients had primary, solitary, supratentorial meningiomas with PTBE. In these, correlations in PTBE, edema index, VEGF-A protein, VEGF gene expression, capillary length, and tumor water content were investigated. DNA-branched hybridization was used for measuring VEGF gene expression in tissue homogenates prepared from frozen tissue samples. The method for VEGF-A analysis resembled an ELISA assay, but was based on chemiluminescence. The edema index was positively correlated to VEGF-A protein (p = 0.014) and VEGF gene expression (p < 0.05). The capillary length in the meningiomas was positively correlated to the PTBE (p = 0.038). If VEGF is responsible for the formation of PTBE, the edema may be treated with the anti-VEGF drug Bevacizumab (Avastin), which has been shown to reduce PTBE in patients with glioblastoma multiforme. PMID:22085359

  10. The Clk/Sty protein kinase phosphorylates SR splicing factors and regulates their intranuclear distribution.

    PubMed Central

    Colwill, K; Pawson, T; Andrews, B; Prasad, J; Manley, J L; Bell, J C; Duncan, P I

    1996-01-01

    Mammalian Clk/Sty is the prototype for a family of dual specificity kinases (termed LAMMER kinases) that have been conserved in evolution, but whose physiological substrates are unknown. In a yeast two-hybrid screen, the Clk/Sty kinase specifically interacted with RNA binding proteins, particularly members of the serine/arginine-rich (SR) family of splicing factors. Clk/Sty itself has an serine/arginine-rich non-catalytic N-terminal region which is important for its association with SR splicing factors. In vitro, Clk/Sty efficiently phosphorylated the SR family member ASF/SF2 on serine residues located within its serine/arginine-rich region (the RS domain). Tryptic phosphopeptide mapping demonstrated that the sites on ASF/SF2 phosphorylated in vitro overlap with those phosphorylated in vivo. Immunofluorescence studies showed that a catalytically inactive form of Clk/Sty co-localized with SR proteins in nuclear speckles. Overexpression of the active Clk/Sty kinase caused a redistribution of SR proteins within the nucleus. These results suggest that Clk/Sty kinase directly regulates the activity and compartmentalization of SR splicing factors. Images PMID:8617202

  11. Improvement of texture and sensory properties of cakes by addition of potato peel powder with high level of dietary fiber and protein.

    PubMed

    Ben Jeddou, Khawla; Bouaziz, Fatma; Zouari-Ellouzi, Soumaya; Chaari, Fatma; Ellouz-Chaabouni, Semia; Ellouz-Ghorbel, Raoudha; Nouri-Ellouz, Oumèma

    2017-02-15

    Demand for health oriented products such as low calories and high fiber product is increasing. The aim of the present work was to determine the effect of the addition of potato peel powders as protein and dietary fiber source on the quality of the dough and the cake. Powders obtained from the two types of peel flour showed interesting water binding capacity and fat absorption capacity. Potato peel flours were incorporated in wheat flours at different concentration. The results showed that peel powders additionally considerably improved the Alveograph profile of dough and the texture of the prepared cakes. In addition color measurements showed a significant difference between the control dough and the dough containing potato peels. The replacement of wheat flour with the potato powders reduced the cake hardness significantly and the L(*) and b(*) dough color values. The increased consumption of cake enriched with potato peel fiber is proposed for health reasons. The study demonstrated that protein/fiber-enriched cake with good sensory quality could be produced by the substitution of wheat flour by 5% of potato peel powder. In addition and technological point of view, the incorporation of potato peel powder at 5% increase the dough strength and elasticity-to-extensibility ratio (P/L). PMID:27664685

  12. Improvement of texture and sensory properties of cakes by addition of potato peel powder with high level of dietary fiber and protein.

    PubMed

    Ben Jeddou, Khawla; Bouaziz, Fatma; Zouari-Ellouzi, Soumaya; Chaari, Fatma; Ellouz-Chaabouni, Semia; Ellouz-Ghorbel, Raoudha; Nouri-Ellouz, Oumèma

    2017-02-15

    Demand for health oriented products such as low calories and high fiber product is increasing. The aim of the present work was to determine the effect of the addition of potato peel powders as protein and dietary fiber source on the quality of the dough and the cake. Powders obtained from the two types of peel flour showed interesting water binding capacity and fat absorption capacity. Potato peel flours were incorporated in wheat flours at different concentration. The results showed that peel powders additionally considerably improved the Alveograph profile of dough and the texture of the prepared cakes. In addition color measurements showed a significant difference between the control dough and the dough containing potato peels. The replacement of wheat flour with the potato powders reduced the cake hardness significantly and the L(*) and b(*) dough color values. The increased consumption of cake enriched with potato peel fiber is proposed for health reasons. The study demonstrated that protein/fiber-enriched cake with good sensory quality could be produced by the substitution of wheat flour by 5% of potato peel powder. In addition and technological point of view, the incorporation of potato peel powder at 5% increase the dough strength and elasticity-to-extensibility ratio (P/L).

  13. The transcription factor ATF2 promotes melanoma metastasis by suppressing protein fucosylation

    PubMed Central

    Lau, Eric; Feng, Yongmei; Claps, Giuseppina; Fukuda, Michiko N.; Perlina, Ally; Donn, Dylan; Jilaveanu, Lucia; Kluger, Harriet; Freeze, Hudson H.; Ronai, Ze’ev A.

    2016-01-01

    Melanoma is one of the most lethal skin cancers worldwide, primarily because of its propensity to metastasize. Thus, the elucidation of mechanisms that govern metastatic propensity is urgently needed. We found that protein kinase Cε (PKCε)–mediated activation of activating transcription factor 2 (ATF2) controls the migratory and invasive behaviors of melanoma cells. PKCε-dependent phosphorylation of ATF2 promoted its transcriptional repression of the gene encoding fucokinase (FUK), which mediates the fucose salvage pathway and thus global cellular protein fucosylation. In primary melanocytes and cell lines representing early-stage melanoma, the abundance of PKCε-phosphorylated ATF2 was low, thereby enabling the expression of FUK and cellular protein fucosylation, which promoted cellular adhesion and reduced motility. In contrast, increased expression of the gene encoding PKCε and abundance of phosphorylated, transcriptionally active ATF2 were observed in advanced-stage melanomas and correlated with decreased FUK expression, decreased cellular protein fucosylation, attenuated cell adhesion, and increased cell motility. Restoring fucosylation in mice either by dietary fucose supplementation or by genetic manipulation of murine Fuk expression attenuated primary melanoma growth, increased the number of intratumoral natural killer cells, and decreased distal metastasis in murine isograft models. Tumor microarray analysis of human melanoma specimens confirmed reduced fucosylation in metastatic tumors and a better prognosis for primary melanomas that had high abundance of fucosylation. Thus, inhibiting PKCε or ATF2 or increasing protein fucosylation in tumor cells may improve clinical outcome in melanoma patients. PMID:26645581

  14. Phosphorylation of protein synthesis initiation factor 2 (elF-2) in the yeast Saccharomyces cerevisiae

    SciTech Connect

    Romero, D.P.

    1986-01-01

    Initiation Factor 2 (elF-2) in the yeast Saccharomyces cerevisiae is comprised of 3 subunits. The control of protein synthesis in mammalian cells have been shown to involve the phosphorylation of the small (alpha) subunit by a specific protein kinase. Phosphorylation results in an inhibition of protein synthesis. In order to determine whether or not an analogous system is operative in yeast, the phosphorylation state of the alpha subunit of elF-2 in Saccharomyces was determined during various growth and nongrowth conditions. Cells were radiolabelled with /sup 32/P and /sup 35/S, and the whole cell lysates were analyzed by two dimensional gel electrophoresis. These experiments revealed that the smallest subunit (alpha, M/sub r/ = 31,000) is a phosphoprotein in vivo under a variety of growth and nongrowth conditions. This is in direct contrast to the pattern exhibited in mammalian cells. The fact that the small subunit of elF-2 in yeast is phosphorylated under a variety of physiological conditions indicates that such a covalent modification is important for some aspects of elF-2 function. In order to investigate this problem further, a protein kinase that specifically labels the alpha subunit of elF-2 in vitro was isolated. The kinase is not autophosphorylating, utilizes ATP as a phosphate donor, phosphorylates an exogenous protein, casein, modifies serine residues in elF-2, is cyclic nucleotide-independent, and is strongly inhibited by heparin.

  15. Storage stability of keratinocyte growth factor-2 in lyophilized formulations: effects of formulation physical properties and protein fraction at the solid-air interface.

    PubMed

    Devineni, Dilip; Gonschorek, Christoph; Cicerone, Marcus T; Xu, Yemin; Carpenter, John F; Randolph, Theodore W

    2014-10-01

    Lyophilized formulations of keratinocyte growth factor-2 (KGF-2) were prepared with a range of disaccharide (sucrose or trehalose) and hydroxyethyl starch (HES) mass ratios. Protein degradation was assessed as a function of time of storage of the dried formulations at 40, 50 and 60°C. Lyophilized and stored samples were rehydrated, and protein degradation was quantified by measuring loss of monomeric protein with size exclusion chromatography and by determining chemical degradation in the soluble fraction with reverse-phase chromatography. The secondary structure of the protein in the lyophilized formulations was studied with infrared spectroscopy. The magnitudes of degradation were compared the key physical properties of the formulations including retention of protein native secondary structure, glass transition temperature (Tg), inverse mean square displacements 〈u(2)〉(-1) for hydrogen atoms (fast β relaxation), and the relaxation time τ(β), which correlates with relaxation due to fast Johari-Goldstein motions in the glass (Xu et al., 2013) [1]. In addition, specific surface areas of the lyophilized formulations were determined by Brunauer-Emmet-Teller analysis of krypton adsorption isotherms and used to estimate the fraction of the KGF-2 molecules residing at the solid-air interface. KGF-2 degradation rates were highest in formulations wherein the protein's structure was most perturbed, and wherein β relaxations were fastest, but the dominant factor governing KGF-2 degradation in freeze-dried formulations was the fraction of the protein found at the glass solid-air interface. PMID:24859390

  16. The gene for human TATA-binding-protein-associated factor (TAFII) 170: structure, promoter and chromosomal localization.

    PubMed Central

    Van Der Knaap, J A; Van Den Boom, V; Kuipers, J; Van Eijk, M J; Van Der Vliet, P C; Timmers, H T

    2000-01-01

    The TATA-binding protein (TBP) plays a central role in eukaryotic transcription and forms protein complexes with TBP-associated factors (TAFs). The genes encoding TAF(II) proteins frequently map to chromosomal regions altered in human neoplasias. TAF(II)170 of B-TFIID is a member of the SF2 superfamily of putative helicases. Members of this superfamily have also been implicated in several human genetic disorders. In this study we have isolated human genomic clones encoding TAF(II)170 and we show that the gene contains 37 introns. Ribonuclease-protection experiments revealed that TAF(II)170 has multiple transcription start sites, consistent with the observation that the promoter lacks a canonical TATA box and initiator element. Deletion analysis of the promoter region showed that a fragment of 264 bp is sufficient to direct transcription. In addition, we determined the chromosomal localization by two independent methods which mapped the gene to human chromosome 10q22-q23 between the markers D10S185 and WI-1183. The region surrounding these markers has been implicated in several human disorders. PMID:10642510

  17. Tumour necrosis factor-alpha induces translocation of protein kinase C in tumour necrosis factor-sensitive cell lines.

    PubMed Central

    Matsubara, N; Fuchimoto, S; Orita, K

    1991-01-01

    In this study we investigated whether the anti-proliferative effect of tumour necrosis factor-alpha (TNF-alpha) was associated with the activation of protein kinase C (PKC), using PANC-1 cells (TNF-alpha sensitive) and LoVo cells (TNF-alpha resistant). In combination with 12-0-tetradecanoylphorbol-13-acetate (TPA), a potent activator of PKC, TNF-alpha caused marked inhibition of the growth of LoVo cells. Inhibition of PANC-1 cell growth by TNF-alpha was blocked by pretreatment with TPA for 24 hr, along with down-regulation of PKC activity. Intracellular translocation of PKC from cytosol to membrane was induced by TNF-alpha treatment in PANC-1 cells but not in LoVo cells. PMID:1916896

  18. Protein complex detection via weighted ensemble clustering based on Bayesian nonnegative matrix factorization.

    PubMed

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

    2013-01-01

    Detecting protein complexes from protein-protein interaction (PPI) networks is a challenging task in computational biology. A vast number of computational methods have been proposed to undertake this task. However, each computational method is developed to capture one aspect of the network. The performance of different methods on the same network can differ substantially, even the same method may have different performance on networks with different topological characteristic. The clustering result of each computational method can be regarded as a feature that describes the PPI network from one aspect. It is therefore desirable to utilize these features to produce a more accurate and reliable clustering. In this paper, a novel Bayesian Nonnegative Matrix Factorization (NMF)-based weighted Ensemble Clustering algorithm (EC-BNMF) is proposed to detect protein complexes from PPI networks. We first apply different computational algorithms on a PPI network to generate some base clustering results. Then we integrate these base clustering results into an ensemble PPI network, in the form of weighted combination. Finally, we identify overlapping protein complexes from this network by employing Bayesian NMF model. When generating an ensemble PPI network, EC-BNMF can automatically optimize the values of weights such that the ensemble algorithm can deliver better results. Experimental results on four PPI networks of Saccharomyces cerevisiae well verify the effectiveness of EC-BNMF in detecting protein complexes. EC-BNMF provides an effective way to integrate different clustering results for more accurate and reliable complex detection. Furthermore, EC-BNMF has a high degree of flexibility in the choice of base clustering results. It can be coupled with existing clustering methods to identify protein complexes.

  19. Protein Composition of Infectious Spores Reveals Novel Sexual Development and Germination Factors in Cryptococcus.

    PubMed

    Huang, Mingwei; Hebert, Alexander S; Coon, Joshua J; Hull, Christina M

    2015-08-01

    Spores are an essential cell type required for long-term survival across diverse organisms in the tree of life and are a hallmark of fungal reproduction, persistence, and dispersal. Among human fungal pathogens, spores are presumed infectious particles, but relatively little is known about this robust cell type. Here we used the meningitis-causing fungus Cryptococcus neoformans to determine the roles of spore-resident proteins in spore biology. Using highly sensitive nanoscale liquid chromatography/mass spectrometry, we compared the proteomes of spores and vegetative cells (yeast) and identified eighteen proteins specifically enriched in spores. The genes encoding these proteins were deleted, and the resulting strains were evaluated for discernable phenotypes. We hypothesized that spore-enriched proteins would be preferentially involved in spore-specific processes such as dormancy, stress resistance, and germination. Surprisingly, however, the majority of the mutants harbored defects in sexual development, the process by which spores are formed. One mutant in the cohort was defective in the spore-specific process of germination, showing a delay specifically in the initiation of vegetative growth. Thus, by using this in-depth proteomics approach as a screening tool for cell type-specific proteins and combining it with molecular genetics, we successfully identified the first germination factor in C. neoformans. We also identified numerous proteins with previously unknown functions in both sexual development and spore composition. Our findings provide the first insights into the basic protein components of infectious spores and reveal unexpected molecular connections between infectious particle production and spore composition in a pathogenic eukaryote.

  20. Protein Composition of Infectious Spores Reveals Novel Sexual Development and Germination Factors in Cryptococcus

    PubMed Central

    Huang, Mingwei; Hebert, Alexander S.; Coon, Joshua J.; Hull, Christina M.

    2015-01-01

    Spores are an essential cell type required for long-term survival across diverse organisms in the tree of life and are a hallmark of fungal reproduction, persistence, and dispersal. Among human fungal pathogens, spores are presumed infectious particles, but relatively little is known about this robust cell type. Here we used the meningitis-causing fungus Cryptococcus neoformans to determine the roles of spore-resident proteins in spore biology. Using highly sensitive nanoscale liquid chromatography/mass spectrometry, we compared the proteomes of spores and vegetative cells (yeast) and identified eighteen proteins specifically enriched in spores. The genes encoding these proteins were deleted, and the resulting strains were evaluated for discernable phenotypes. We hypothesized that spore-enriched proteins would be preferentially involved in spore-specific processes such as dormancy, stress resistance, and germination. Surprisingly, however, the majority of the mutants harbored defects in sexual development, the process by which spores are formed. One mutant in the cohort was defective in the spore-specific process of germination, showing a delay specifically in the initiation of vegetative growth. Thus, by using this in-depth proteomics approach as a screening tool for cell type-specific proteins and combining it with molecular genetics, we successfully identified the first germination factor in C. neoformans. We also identified numerous proteins with previously unknown functions in both sexual development and spore composition. Our findings provide the first insights into the basic protein components of infectious spores and reveal unexpected molecular connections between infectious particle production and spore composition in a pathogenic eukaryote. PMID:26313153

  1. ZBP-89, a Krüppel-like zinc finger protein, inhibits epidermal growth factor induction of the gastrin promoter.

    PubMed Central

    Merchant, J L; Iyer, G R; Taylor, B R; Kitchen, J R; Mortensen, E R; Wang, Z; Flintoft, R J; Michel, J B; Bassel-Duby, R

    1996-01-01

    We have shown previously that a GC-rich element (GGGGCGGGGTGGGGGG) conferring epidermal growth factor (EGF) responsiveness to the human gastrin promoter binds Sp1 and additional undefined complexes. A rat GH4 cell line expression library was screened by using a multimer of the gastrin EGF response element, and three overlapping cDNA clones were identified. The full-length rat cDNA encoded an 89-kDa zinc finger protein (ZBP-89) that was 89% identical to a 49-kDa human factor, ht(beta), that binds a GTGGG/CACCC element in T-cell receptor promoters. The conservation of amino acids between the zinc fingers indicates that ZBP-89 is a member of the C2H2 zinc finger family subclass typified by the Drosophila Krüppel protein. ZBP-89 is ubiquitously expressed in normal adult tissues. It binds specifically to the gastrin EGF response element and inhibits EGF induction of the gastrin promoter. Collectively, these results demonstrate that ZBP-89 functions as a repressor of basal and inducible expression of the gastrin gene. PMID:8943318

  2. Regulation of the germinal center gene program by interferon (IFN) regulatory factor 8/IFN consensus sequence-binding protein

    PubMed Central

    Lee, Chang Hoon; Melchers, Mark; Wang, Hongsheng; Torrey, Ted A.; Slota, Rebecca; Qi, Chen-Feng; Kim, Ji Young; Lugar, Patricia; Kong, Hee Jeong; Farrington, Lila; van der Zouwen, Boris; Zhou, Jeff X.; Lougaris, Vassilios; Lipsky, Peter E.; Grammer, Amrie C.; Morse, Herbert C.

    2006-01-01

    Interferon (IFN) consensus sequence-binding protein/IFN regulatory factor 8 (IRF8) is a transcription factor that regulates the differentiation and function of macrophages, granulocytes, and dendritic cells through activation or repression of target genes. Although IRF8 is also expressed in lymphocytes, its roles in B cell and T cell maturation or function are ill defined, and few transcriptional targets are known. Gene expression profiling of human tonsillar B cells and mouse B cell lymphomas showed that IRF8 transcripts were expressed at highest levels in centroblasts, either from secondary lymphoid tissue or transformed cells. In addition, staining for IRF8 was most intense in tonsillar germinal center (GC) dark-zone centroblasts. To discover B cell genes regulated by IRF8, we transfected purified primary tonsillar B cells with enhanced green fluorescent protein–tagged IRF8, generated small interfering RNA knockdowns of IRF8 expression in a mouse B cell lymphoma cell line, and examined the effects of a null mutation of IRF8 on B cells. Each approach identified activation-induced cytidine deaminase (AICDA) and BCL6 as targets of transcriptional activation. Chromatin immunoprecipitation studies demonstrated in vivo occupancy of 5′ sequences of both genes by IRF8 protein. These results suggest previously unappreciated roles for IRF8 in the transcriptional regulation of B cell GC reactions that include direct regulation of AICDA and BCL6. PMID:16380510

  3. Melatonin reversed tumor necrosis factor-alpha-inhibited osteogenesis of human mesenchymal stem cells by stabilizing SMAD1 protein.

    PubMed

    Lian, Chengjie; Wu, Zizhao; Gao, Bo; Peng, Yan; Liang, Anjing; Xu, Caixia; Liu, Lei; Qiu, Xianjian; Huang, Junjun; Zhou, Hang; Cai, Yifeng; Su, Peiqiang; Huang, Dongsheng

    2016-10-01

    Tumor necrosis factor-alpha (TNFα) plays a pivotal role in inflammation-related osteoporosis through the promotion of bone resorption and suppression of bone formation. Numerous drugs have been produced to treat osteoporosis by inhibiting bone resorption, but they offer few benefits to bone formation, which is what is needed by patients with severe bone loss. Melatonin, which can exert both anti-inflammatory and pro-osteogenic effects, shows promise in overcoming TNFα-inhibited osteogenesis and deserves further research. This study demonstrated that melatonin rescued TNFα-inhibited osteogenesis of human mesenchymal stem cells and that the interactions between SMURF1 and SMAD1 mediated the crosstalk between melatonin signaling and TNFα signaling. Additionally, melatonin treatment was found to downregulate TNFα-induced SMURF1 expression and then decrease SMURF1-mediated ubiquitination and degradation of SMAD1 protein, leading to steady bone morphogenetic protein-SMAD1 signaling activity and restoration of TNFα-impaired osteogenesis. Thus, melatonin has prospects for treating osteoporosis caused by inflammatory factors due to its multifaceted functions on regulation of bone formation, bone resorption, and inflammation. Further studies will focus on unveiling the specific mechanisms by which melatonin downregulates SMURF1 expression and confirming the clinical therapeutic value of melatonin in the prevention and therapy of bone loss associated with inflammation. PMID:27265199

  4. Transcriptional repression of Kruppel like factor-2 by the adaptor protein p66shc

    PubMed Central

    Kumar, Ajay; Hoffman, Timothy A.; DeRicco, Jeremy; Naqvi, Asma; Jain, Mukesh K.; Irani, Kaikobad

    2009-01-01

    The adaptor protein p66shc promotes cellular oxidative stress and apoptosis. Here, we demonstrate a novel mechanistic relationship between p66shc and the kruppel like factor-2 (KLF2) transcription factor and show that this relationship has biological relevance to p66shc-regulated cellular oxidant level, as well as KLF2-induced target gene expression. Genetic knockout of p66shc in mouse embryonic fibroblasts (MEFs) stimulates activity of the core KLF2 promoter and increases KLF2 mRNA and protein expression. Similarly, shRNA-induced knockdown of p66shc increases KLF2-promoter activity in HeLa cells. The increase in KLF2-promoter activity in p66shc-knockout MEFs is dependent on a myocyte enhancing factor-2A (MEF2A)-binding sequence in the core KLF2 promoter. Short-hairpin RNA-induced knockdown of p66shc in endothelial cells also stimulates KLF2 mRNA and protein expression, as well as expression of the endothelial KLF2 target gene thrombomodulin. MEF2A protein and mRNA are more abundant in p66shc-knockout MEFs, resulting in greater occupancy of the KLF2 promoter by MEF2A. In endothelial cells, the increase in KLF2 and thrombomodulin protein by shRNA-induced decrease in p66shc expression is partly abrogated by knockdown of MEF2A. Finally, knockdown of KLF2 abolishes the decrease in the cellular reactive oxygen species hydrogen peroxide observed with knockdown of p66shc, and KLF2 overexpression suppresses cellular hydrogen peroxide levels, independent of p66shc expression. These findings illustrate a novel mechanism by which p66shc promotes cellular oxidative stress, through suppression of MEF2A expression and consequent repression of KLF2 transcription.—Kumar, A., Hoffman, T. A., DeRicco, J., Naqvi, A., Jain, M. K., Irani, K. Transcriptional repression of Kruppel like factor-2 by the adaptor protein p66shc. PMID:19696221

  5. Comparative protein profiling identifies elongation factor-1beta and tryparedoxin peroxidase as factors associated with metastasis in Leishmania guyanensis.

    PubMed

    Walker, John; Acestor, Nathalie; Gongora, Rafael; Quadroni, Manfredo; Segura, Iris; Fasel, Nicolas; Saravia, Nancy G

    2006-02-01

    Parasites of the Leishmania Viannia subgenus are major causative agents of mucocutaneous leishmaniasis (MCL), a disease characterised by parasite dissemination (metastasis) from the original cutaneous lesion to form debilitating secondary lesions in the nasopharyngeal mucosa. We employed a protein profiling approach to identify potential metastasis factors in laboratory clones of L. (V.) guyanensis with stable phenotypes ranging from highly metastatic (M+) through infrequently metastatic (M+/M-) to non-metastatic (M-). Comparison of the soluble proteomes of promastigotes by two-dimensional electrophoresis revealed two abundant protein spots specifically associated with M+ and M+/M- clones (Met2 and Met3) and two others exclusively expressed in M- parasites (Met1 and Met4). The association between clinical disease phenotype and differential expression of Met1-Met4 was less clear in L. Viannia strains from mucosal (M+) or cutaneous (M-) lesions of patients. Identification of Met1-Met4 by biological mass spectrometry (LC-ES-MS/MS) and bioinformatics revealed that M+ and M- clones express distinct acidic and neutral isoforms of both elongation factor-1 subunit beta (EF-1beta) and cytosolic tryparedoxin peroxidase (TXNPx). This interchange of isoforms may relate to the mechanisms by which the activities of EF-1beta and TXNPx are modulated, and/or differential post-translational modification of the gene product(s). The multiple metabolic functions of EF-1 and TXNPx support the plausibility of their participation in parasite survival and persistence and thereby, metastatic disease. Both polypeptides are active in resistance to chemical and oxidant stress, providing a basis for further elucidation of the importance of antioxidant defence in the pathogenesis underlying MCL. PMID:16325936

  6. Insulin-like growth factors and their binding proteins in human colonocytes: preferential degradation of insulin-like growth factor binding protein 2 in colonic cancers.

    PubMed Central

    Michell, N. P.; Langman, M. J.; Eggo, M. C.

    1997-01-01

    We have compared the expression of insulin-like growth factors (IGFs) and IGF binding proteins (IGFBPs) in ten paired samples of normal and tumour colonic tissue with regard to both mRNA and protein. We have compared sensitivity of these tissues to IGF-I using primary cultures of epithelial cells of colonic mucosa, and we have examined the production of IGFs and IGFBPs by these cells. In the tissues, IGFBP-2 mRNA was expressed in all normal and cancer samples but other IGFBPs showed variable expression. mRNAs for IGF-I were expressed in all normal and cancer tissues but IGF-II mRNA was only detected in cancer tissue (3 out of 10). Immunostaining of sections of normal and cancer tissue was negative for IGF-I and IGF-II; IGFBP-2 was positive in 2 out of 10 cancer tissues and 7 out of 10 normal tissues; IGFBP-3 was positive in 7 out of 10 cancer tissues and 7 out of 10 normal tissues; and IGFBP-4 was positive in 5 out of 10 cancer tissues and 6 out of 10 normal tissues. In the cells in culture, cancer cells showed increased incorporation of [35S]methionine into protein and [3H]thymidine into DNA (P < 0.02) when treated with IGF-I. Western blotting of serum-free conditioned media from cells in culture showed that 8 out of 10 normal and 3 out of 10 cancer cultures produced a 32-kDa immunoreactive IGFBP-2. No IGFBP-3 was secreted by any culture but 24-kDa IGFBP-4 was found in 3 out of 10 normal and 5 out of 10 cancer tissues. Because of the discrepancy between mRNA and protein expression for IGFBP-2, degradation of native IGFBPs was assessed using tissue extracts. Colon cancer extracts were able to degrade exogenous IGFBP-2, IGFBP-3 and IGFBP-4, whereas normal tissue extracts were without effect on IGFBP-2. We conclude that IGFBPs are synthesized and secreted by cells of the colonic mucosa but that proteolysis of secreted IGFBP-2 occurs in colon cancer tissue. This selective degradation may confer a growth advantage. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5

  7. Enhanced protective antibody to a mutant meningococcal factor H-binding protein with low-factor H binding.

    PubMed

    Granoff, Dan M; Giuntini, Serena; Gowans, Flor A; Lujan, Eduardo; Sharkey, Kelsey; Beernink, Peter T

    2016-01-01

    Meningococcal factor H-binding protein (FHbp) is an antigen in 2 serogroup B meningococcal vaccines. FHbp specifically binds human and some nonhuman primate complement FH. To investigate the effect of binding of FH to FHbp on protective antibody responses, we immunized infant rhesus macaques with either a control recombinant FHbp antigen that bound macaque FH or a mutant antigen with 2 amino acid substitutions and >250-fold lower affinity for FH. The mutant antigen elicited 3-fold higher serum IgG anti-FHbp titers and up to 15-fold higher serum bactericidal titers than the control FHbp vaccine. When comparing sera with similar IgG anti-FHbp titers, the antibodies elicited by the mutant antigen gave greater deposition of complement component C4b on live meningococci (classical complement pathway) and inhibited binding of FH, while the anti-FHbp antibodies elicited by the control vaccine enhanced FH binding. Thus, the mutant FHbp vaccine elicited an anti-FHbp antibody repertoire directed at FHbp epitopes within the FH binding site, which resulted in greater protective activity than the antibodies elicited by the control vaccine, which targeted FHbp epitopes outside of the FH combining site. Binding of a host protein to a vaccine antigen impairs protective antibody responses, which can be overcome with low-binding mutant antigens. PMID:27668287

  8. Enhanced protective antibody to a mutant meningococcal factor H-binding protein with low-factor H binding

    PubMed Central

    Granoff, Dan M.; Giuntini, Serena; Gowans, Flor A.; Lujan, Eduardo; Sharkey, Kelsey; Beernink, Peter T.

    2016-01-01

    Meningococcal factor H-binding protein (FHbp) is an antigen in 2 serogroup B meningococcal vaccines. FHbp specifically binds human and some nonhuman primate complement FH. To investigate the effect of binding of FH to FHbp on protective antibody responses, we immunized infant rhesus macaques with either a control recombinant FHbp antigen that bound macaque FH or a mutant antigen with 2 amino acid substitutions and >250-fold lower affinity for FH. The mutant antigen elicited 3-fold higher serum IgG anti-FHbp titers and up to 15-fold higher serum bactericidal titers than the control FHbp vaccine. When comparing sera with similar IgG anti-FHbp titers, the antibodies elicited by the mutant antigen gave greater deposition of complement component C4b on live meningococci (classical complement pathway) and inhibited binding of FH, while the anti-FHbp antibodies elicited by the control vaccine enhanced FH binding. Thus, the mutant FHbp vaccine elicited an anti-FHbp antibody repertoire directed at FHbp epitopes within the FH binding site, which resulted in greater protective activity than the antibodies elicited by the control vaccine, which targeted FHbp epitopes outside of the FH combining site. Binding of a host protein to a vaccine antigen impairs protective antibody responses, which can be overcome with low-binding mutant antigens. PMID:27668287

  9. Enhanced protective antibody to a mutant meningococcal factor H-binding protein with low-factor H binding

    PubMed Central

    Granoff, Dan M.; Giuntini, Serena; Gowans, Flor A.; Lujan, Eduardo; Sharkey, Kelsey; Beernink, Peter T.

    2016-01-01

    Meningococcal factor H-binding protein (FHbp) is an antigen in 2 serogroup B meningococcal vaccines. FHbp specifically binds human and some nonhuman primate complement FH. To investigate the effect of binding of FH to FHbp on protective antibody responses, we immunized infant rhesus macaques with either a control recombinant FHbp antigen that bound macaque FH or a mutant antigen with 2 amino acid substitutions and >250-fold lower affinity for FH. The mutant antigen elicited 3-fold higher serum IgG anti-FHbp titers and up to 15-fold higher serum bactericidal titers than the control FHbp vaccine. When comparing sera with similar IgG anti-FHbp titers, the antibodies elicited by the mutant antigen gave greater deposition of complement component C4b on live meningococci (classical complement pathway) and inhibited binding of FH, while the anti-FHbp antibodies elicited by the control vaccine enhanced FH binding. Thus, the mutant FHbp vaccine elicited an anti-FHbp antibody repertoire directed at FHbp epitopes within the FH binding site, which resulted in greater protective activity than the antibodies elicited by the control vaccine, which targeted FHbp epitopes outside of the FH combining site. Binding of a host protein to a vaccine antigen impairs protective antibody responses, which can be overcome with low-binding mutant antigens.

  10. Transcription factor Hes1 modulates osteoarthritis development in cooperation with calcium/calmodulin-dependent protein kinase 2

    PubMed Central

    Sugita, Shurei; Hosaka, Yoko; Okada, Keita; Mori, Daisuke; Yano, Fumiko; Kobayashi, Hiroshi; Taniguchi, Yuki; Mori, Yoshifumi; Okuma, Tomotake; Chang, Song Ho; Kawata, Manabu; Taketomi, Shuji; Chikuda, Hirotaka; Akiyama, Haruhiko; Kageyama, Ryoichiro; Chung, Ung-il; Tanaka, Sakae; Kawaguchi, Hiroshi; Ohba, Shinsuke; Saito, Taku

    2015-01-01

    Notch signaling modulates skeletal formation and pathogenesis of osteoarthritis (OA) through induction of catabolic factors. Here we examined roles of Hes1, a transcription factor and important target of Notch signaling, in these processes. SRY-box containing gene 9 (Sox9)-Cre mice were mated with Hes1fl/fl mice to generate tissue-specific deletion of Hes1 from chondroprogenitor cells; this deletion caused no obvious abnormality in the perinatal period. Notably, OA development was suppressed when Hes1 was deleted from articular cartilage after skeletal growth in type II collagen (Col2a1)-CreERT;Hes1fl/fl mice. In cultured chondrocytes, Hes1 induced metallopeptidase with thrombospondin type 1 motif, 5 (Adamts5) and matrix metalloproteinase-13 (Mmp13), which are catabolic enzymes that break down cartilage matrix. ChIP-seq and luciferase assays identified Hes1-responsive regions in intronic sites of both genes; the region in the ADAMTS5 gene contained a typical consensus sequence for Hes1 binding, whereas that in the MMP13 gene did not. Additionally, microarray analysis, together with the ChIP-seq, revealed novel Hes1 target genes, including Il6 and Il1rl1, coding a receptor for IL-33. We further identified calcium/calmodulin-dependent protein kinase 2δ (CaMK2δ) as a cofactor of Hes1; CaMK2δ was activated during OA development, formed a protein complex with Hes1, and switched it from a transcriptional repressor to a transcriptional activator to induce cartilage catabolic factors. Therefore, Hes1 cooperated with CaMK2δ to modulate OA pathogenesis through induction of catabolic factors, including Adamts5, Mmp13, Il6, and Il1rl1. Our findings have contributed to further understanding of the molecular pathophysiology of OA, and may provide the basis for development of novel treatments for joint disorders. PMID:25733872

  11. 42 CFR 136.408 - What are other factors, in addition to the minimum standards of character, that may be considered...

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 42 Public Health 1 2012-10-01 2012-10-01 false What are other factors, in addition to the minimum standards of character, that may be considered in determining placement of an individual in a position that involves regular contact with or control over Indian children? 136.408 Section 136.408 Public Health PUBLIC HEALTH SERVICE, DEPARTMENT...

  12. 42 CFR 136.408 - What are other factors, in addition to the minimum standards of character, that may be considered...

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 42 Public Health 1 2014-10-01 2014-10-01 false What are other factors, in addition to the minimum standards of character, that may be considered in determining placement of an individual in a position that involves regular contact with or control over Indian children? 136.408 Section 136.408 Public Health PUBLIC HEALTH SERVICE, DEPARTMENT...

  13. Ameliorative effects of telmisartan on the inflammatory response and impaired spatial memory in a rat model of Alzheimer's disease incorporating additional cerebrovascular disease factors.

    PubMed

    Shindo, Taro; Takasaki, Kotaro; Uchida, Kanako; Onimura, Rika; Kubota, Kaori; Uchida, Naoki; Irie, Keiichi; Katsurabayashi, Shutaro; Mishima, Kenichi; Nishimura, Ryoji; Fujiwara, Michihiro; Iwasaki, Katsunori

    2012-01-01

    Telmisartan, an angiotensin type 1 receptor blocker, is used in the management of hypertension to control blood pressure. In addition, telmisartan has a partial agonistic effect on peroxisome proliferator activated receptor γ (PPARγ). Recently, the effects of telmisartan on spatial memory or the inflammatory response were monitored in a mouse model of Alzheimer's disease (AD). However, to date, no studies have investigated the ameliorative effects of telmisartan on impaired spatial memory and the inflammatory response in an AD animal model incorporating additional cerebrovascular disease factors. In this study, we examined the effect of telmisartan on spatial memory impairment and the inflammatory response in a rat model of AD incorporating additional cerebrovascular disease factors. Rats were subjected to cerebral ischemia and an intracerebroventricular injection of oligomeric or aggregated amyloid-β (Aβ). Oral administration of telmisartan (0.3, 1, 3 mg/kg/d) seven days after ischemia and Aβ treatment resulted in better performance in the eight arm radial maze task in a dose-dependent manner. Telmisartan also reduced tumor necrosis factor α mRNA expression in the hippocampal region of rats with impaired spatial memory. These effects of telmisartan were antagonized by GW9662, an antagonist of PPARγ. These results suggest that telmisartan has ameliorative effects on the impairment of spatial memory in a rat model of AD incorporating additional cerebrovascular disease factors via its anti-inflammatory effect.

  14. Breeding site selection by coho salmon (Oncorhynchus kisutch) in relation to large wood additions and factors that influence reproductive success

    USGS Publications Warehouse

    Clark, Steven M.; Dunham, Jason B.; McEnroe, Jeffery R.; Lightcap, Scott W.

    2014-01-01

    The fitness of female Pacific salmon (Oncorhynchus spp.) with respect to breeding behavior can be partitioned into at least four fitness components: survival to reproduction, competition for breeding sites, success of egg incubation, and suitability of the local environment near breeding sites for early rearing of juveniles. We evaluated the relative influences of habitat features linked to these fitness components with respect to selection of breeding sites by coho salmon (Oncorhynchus kisutch). We also evaluated associations between breeding site selection and additions of large wood, as the latter were introduced into the study system as a means of restoring habitat conditions to benefit coho salmon. We used a model selection approach to organize specific habitat features into groupings reflecting fitness components and influences of large wood. Results of this work suggest that female coho salmon likely select breeding sites based on a wide range of habitat features linked to all four hypothesized fitness components. More specifically, model parameter estimates indicated that breeding site selection was most strongly influenced by proximity to pool-tail crests and deeper water (mean and maximum depths). Linkages between large wood and breeding site selection were less clear. Overall, our findings suggest that breeding site selection by coho salmon is influenced by a suite of fitness components in addition to the egg incubation environment, which has been the emphasis of much work in the past.

  15. Pluripotency factors and Polycomb Group proteins repress aryl hydrocarbon receptor expression in murine embryonic stem cells.

    PubMed

    Ko, Chia-I; Wang, Qin; Fan, Yunxia; Xia, Ying; Puga, Alvaro

    2014-01-01

    The aryl hydrocarbon receptor (AHR) is a transcription factor and environmental sensor that regulates expression of genes involved in drug-metabolism and cell cycle regulation. Chromatin immunoprecipitation analyses, Ahr ablation in mice and studies with orthologous genes in invertebrates suggest that AHR may also play a significant role in embryonic development. To address this hypothesis, we studied the regulation of Ahr expression in mouse embryonic stem cells and their differentiated progeny. In ES cells, interactions between OCT3/4, NANOG, SOX2 and Polycomb Group proteins at the Ahr promoter repress AHR expression, which can also be repressed by ectopic expression of reprogramming factors in hepatoma cells. In ES cells, unproductive RNA polymerase II binds at the Ahr transcription start site and drives the synthesis of short abortive transcripts. Activation of Ahr expression during differentiation follows from reversal of repressive marks in Ahr promoter chromatin, release of pluripotency factors and PcG proteins, binding of Sp factors, establishment of histone marks of open chromatin, and engagement of active RNAPII to drive full-length RNA transcript elongation. Our results suggest that reversible Ahr repression in ES cells holds the gene poised for expression and allows for a quick switch to activation during embryonic development.

  16. Electrical protein array chips for the detection of staphylococcal virulence factors.

    PubMed

    Quiel, Annett; Jürgen, Britta; Piechotta, Gundula; Le Foll, Anne-Pascale; Ziebandt, Anne-Kathrin; Kohler, Christian; Köster, Daniela; Engelmann, Susanne; Erck, Christian; Hintsche, Rainer; Wehland, Jürgen; Hecker, Michael; Schweder, Thomas

    2010-02-01

    A new approach for the detection of virulence factors of Staphylococcus aureus and Staphylococcus epidermidis using an electrical protein array chip technology is presented. The procedure is based on an enzyme-linked sandwich immunoassay, which includes recognition and binding of virulence factors by specific capture and detection antibodies. Detection of antibody-bound virulence factors is achieved by measuring the electrical current generated by redox recycling of an enzymatically released substance. The current (measured in nanoampere) corresponds to the amount of the target molecule in the analyzed sample. The electrical protein chip allows for a fast detection of Staphylococcus enterotoxin B (SEB) of S. aureus and immunodominant antigen A homologue (IsaA homologue) of S. epidermidis in different liquid matrices. The S. aureus SEB virulence factor could be detected in minimal medium, milk, and urine in a concentration of 1 ng/ml within less than 23 min. Furthermore, a simultaneous detection of SEB of S. aureus and IsaA homologue of S. epidermidis in a single assay could be demonstrated.

  17. Chemical synthesis and X-ray structure of a heterochiral {D-protein antagonist plus vascular endothelial growth factor} protein complex by racemic crystallography

    SciTech Connect

    Mandal, Kalyaneswar; Uppalapati, Maruti; Ault-Riché, Dana; Kenney, John; Lowitz, Joshua; Sidhu, Sachdev S.; Kent, Stephen B.H.

    2012-10-23

    Total chemical synthesis was used to prepare the mirror image (D-protein) form of the angiogenic protein vascular endothelial growth factor (VEGF-A). Phage display against D-VEGF-A was used to screen designed libraries based on a unique small protein scaffold in order to identify a high affinity ligand. Chemically synthesized D- and L- forms of the protein ligand showed reciprocal chiral specificity in surface plasmon resonance binding experiments: The L-protein ligand bound only to D-VEGF-A, whereas the D-protein ligand bound only to L-VEGF-A. The D-protein ligand, but not the L-protein ligand, inhibited the binding of natural VEGF{sub 165} to the VEGFR1 receptor. Racemic protein crystallography was used to determine the high resolution X-ray structure of the heterochiral complex consisting of {l_brace}D-protein antagonist + L-protein form of VEGF-A{r_brace}. Crystallization of a racemic mixture of these synthetic proteins in appropriate stoichiometry gave a racemic protein complex of more than 73 kDa containing six synthetic protein molecules. The structure of the complex was determined to a resolution of 1.6 {angstrom}. Detailed analysis of the interaction between the D-protein antagonist and the VEGF-A protein molecule showed that the binding interface comprised a contact surface area of approximately 800 {angstrom}{sup 2} in accord with our design objectives, and that the D-protein antagonist binds to the same region of VEGF-A that interacts with VEGFR1-domain 2.

  18. Expression of neu protein, epidermal growth factor receptor, and transforming growth factor alpha in breast cancer. Correlation with clinicopathologic parameters.

    PubMed Central

    Lundy, J.; Schuss, A.; Stanick, D.; McCormack, E. S.; Kramer, S.; Sorvillo, J. M.

    1991-01-01

    The major objectives of this study were twofold: to determine 1) if growth factors or growth factor receptors were expressed similarly or differently in a clinically well-characterized group of breast cancer patients and 2) if these phenotypic characteristics were associated with any of the commonly used prognostic parameters. Formalin-fixed paraffin-embedded tumor tissue from 51 node-positive breast cancer patients were analyzed for the expression of neu, epidermal growth factor-receptor (EGF-R), and transforming growth factor alpha (TGF alpha) using immunoperoxidase staining. Positive membranous staining for neu was observed in 15 (29%) tumors. Over-expression of neu was observed in high-grade, estrogen-receptor-negative tumors (P less than 0.05). Epidermal growth factor receptor was expressed in 22 (43%) of the tumors analyzed and found to a greater degree in estrogen-receptor-negative and high-grade tumors (P less than 0.025). A significant correlation between neu and EGF-R expression was also noted. Tumors expressing membranous staining of neu had a greater than 70% chance of expressing EGF-R (P less than 0.01). Expression of TGF alpha was found in 68% of tumors and TGF alpha was detected in grade 1 and 2 tumor to a greater degree than EGF-R. The authors conclude that assaying tumors for these antigens may give additional phenotypic characteristics that can give further insight into the biology of breast cancer. Images Figure 1 Figure 2 Figure 3 PMID:1711294

  19. Insulin-like growth factor-I, soy protein intake, and breast cancer risk.

    PubMed

    Sanderson, Maureen; Shu, Xiao Ou; Yu, Herbert; Dai, Qi; Malin, Alecia S; Gao, Yu-Tang; Zheng, Wei

    2004-01-01

    Previous studies have found that estrogen enhances the effect of insulin-like growth factor-I (IGF-I) levels on breast cancer cell growth. Participants in the Shanghai Breast Cancer Study (SBCS) consumed large amounts of soy that was high in isoflavones, which act as weak estrogens and as anti-estrogens. We assessed whether soy protein intake modified the effect of IGF-I levels on breast cancer risk. The SBCS is a population-based case-control study of breast cancer among women aged 25-64 conducted between 1996 and 1998 in urban Shanghai. In-person interviews were completed with 1,459 incident breast cancer cases ascertained through a population-based cancer registry and 1,556 controls randomly selected from the general population (with respective response rates of 91% and 90%). This analysis is restricted to the 397 cases and 397 matched controls for whom information on IGF-I levels was available. For premenopausal breast cancer, we found nearly significant interactions between soy protein intake and IGF-I levels (P = 0.080) and insulin-like growth factor-binding protein-3 (IGFBP-3) levels (P = 0.057). The direction of the interaction appeared to be negative for IGF-I levels but was positive for IGFBP-3 levels. No interaction was evident between soy protein intake and IGF-I or IGFBP-3 levels among postmenopausal women. Our results suggest that soy protein intake may negatively modulate the effect of IGF-I and may positively modulate the effect of IGFBP-3 levels on premenopausal breast cancer risk. Further studies are needed to confirm our finding and to understand the biological mechanisms of these potential interactions.

  20. Additively Manufactured 3D Porous Ti-6Al-4V Constructs Mimic Trabecular Bone Structure and Regulate Osteoblast Proliferation, Differentiation and Local Factor Production in a Porosity and Surface Roughness Dependent Manner

    PubMed Central

    Cheng, Alice; Humayun, Aiza; Cohen, David J.; Boyan, Barbara D.; Schwartz, Zvi

    2014-01-01

    Additive manufacturing by laser sintering is able to produce high resolution metal constructs for orthopaedic and dental implants. In this study, we used a human trabecular bone template to design and manufacture Ti-6Al-4V constructs with varying porosity via laser sintering. Characterization of constructs revealed interconnected porosities ranging from 15–70% with compressive moduli of 2063–2954 MPa. These constructs with macro porosity were further surface-treated to create a desirable multi-scale micro-/nano-roughness, which has been shown to enhance the osseointegration process. Osteoblasts (MG63 cells) exhibited high viability when grown on the constructs. Proliferation (DNA) and alkaline phosphatase specific activity (ALP), an early differentiation marker, decreased as porosity increased, while osteocalcin (OCN), a late differentiation marker, as well as osteoprotegerin (OPG), vascular endothelial growth factor (VEGF) and bone morphogenetic proteins 2 and 4 (BMP2, BMP4) increased with increasing porosity. 3D constructs with the highest porosity and surface modification supported the greatest osteoblast differentiation and local factor production. These results indicate that additively manufactured 3D porous constructs mimicking human trabecular bone and produced with additional surface treatment can be customized for increased osteoblast response. Increased factors for osteoblast maturation and differentiation on high porosity constructs suggest the enhanced performance of these surfaces for increasing osseointegration in vivo. PMID:25287305

  1. Colocalization of insulin-like growth factor-binding protein with insulin-like growth factor I.

    PubMed

    Kobayashi, S; Clemmons, D R; Venkatachalam, M A

    1991-07-01

    We report the localization of insulin-like growth factor I (IGF-I) and a 25-kDa form of insulin-like growth factor-binding protein (IGF-BP-1) in adult rat kidney. The antigens were localized using a rabbit anti-human IGF-I antibody, and a rabbit anti-human IGF-BP-1 antibody raised against human 25-kDa IGF-BP-1 purified from amniotic fluid. Immunohistochemistry by the avidin-biotin peroxidase conjugate technique showed that both peptides are located in the same nephron segments, in the same cell types. The most intense staining was in papillary collecting ducts. There was moderate staining also in cortical collecting ducts and medullary thick ascending limbs of Henle's loop. In collecting ducts the antigens were shown to be present in principal cells but not in intercalated cells. In distal convoluted tubules, cortical thick ascending limbs, and in structures presumptively identified as thin limbs of Henle's loops there was only modest staining. The macula densa, however, lacked immunoreactivity. Colocalization of IGF-I and IGF-BP-1 in the same cells supports the notion, derived from studies on cultured cells, that the actions of IGF-I may be modified by IGF-BPs that are present in the same location.

  2. Purification of scatter factor, a fibroblast-derived basic protein that modulates epithelial interactions and movement.

    PubMed Central

    Gherardi, E; Gray, J; Stoker, M; Perryman, M; Furlong, R

    1989-01-01

    Scatter factor is a fibroblast-derived protein that causes separation of contiguous epithelial cells and increased local mobility of unanchored cells. Highly purified scatter factor has been obtained by a combination of ion-exchange and reverse-phase chromatography from serum-free medium conditioned by a ras-transformed clone (D4) of mouse NIH 3T3 fibroblasts. Under nonreducing conditions scatter factor has a pI of approximately 9.5 and migrates in SDS/polyacrylamide gels as a single band at approximately 62 kDa from which epithelial scatter activity can be recovered. Treatment with reducing agents destroys biological activity and is associated with the appearance of two major bands at approximately 57 and approximately 30 kDa. Whether both the 57-kDa and 30-kDa polypeptides are required for biological activity remains to be established. All the activities observed in crude medium conditioned by cells producing scatter factor are retained by highly purified preparations of scatter factor. These include (i) increased local movement, modulation of morphology, and inhibition of junction formation by single epithelial cells and (ii) disruption of epithelial interactions and cell scattering from preformed epithelial sheets. These changes occur with picomolar concentrations of purified scatter factor and without an effect on cell growth. Images PMID:2527367

  3. Iron-responsive Transcription Factor Aft1 Interacts with Kinetochore Protein Iml3 and Promotes Pericentromeric Cohesin*

    PubMed Central

    Hamza, Akil; Baetz, Kristin

    2012-01-01

    The Saccharomyces cerevisiae iron-responsive transcription factor, Aft1, has a well established role in regulating iron homeostasis through the transcriptional induction of iron-regulon genes. However, recent studies have implicated Aft1 in other cellular processes independent of iron regulation such as chromosome stability. In addition, chromosome spreads and two-hybrid data suggest that Aft1 interacts with and co-localizes with kinetochore proteins; however, the cellular implications of this have not been established. Here, we demonstrate that Aft1 associates with the kinetochore complex through Iml3. Furthermore, like Iml3, Aft1 is required for the increased association of cohesin with pericentric chromatin, which is required to resist microtubule tension, and aft1Δ cells display chromosome segregation defects in meiosis. Our work defines a new role for Aft1 in chromosome stability and transmission. PMID:22157760

  4. Identification of RNA Binding Proteins Associated with Dengue Virus RNA in Infected Cells Reveals Temporally Distinct Host Factor Requirements

    PubMed Central

    Viktorovskaya, Olga V.; Greco, Todd M.; Cristea, Ileana M.; Thompson, Sunnie R.

    2016-01-01

    Background There are currently no vaccines or antivirals available for dengue virus infection, which can cause dengue hemorrhagic fever and death. A better understanding of the host pathogen interaction is required to develop effective therapies to treat DENV. In particular, very little is known about how cellular RNA binding proteins interact with viral RNAs. RNAs within cells are not naked; rather they are coated with proteins that affect localization, stability, translation and (for viruses) replication. Methodology/Principal Findings Seventy-nine novel RNA binding proteins for dengue virus (DENV) were identified by cross-linking proteins to dengue viral RNA during a live infection in human cells. These cellular proteins were specific and distinct from those previously identified for poliovirus, suggesting a specialized role for these factors in DENV amplification. Knockdown of these proteins demonstrated their function as viral host factors, with evidence for some factors acting early, while others late in infection. Their requirement by DENV for efficient amplification is likely specific, since protein knockdown did not impair the cell fitness for viral amplification of an unrelated virus. The protein abundances of these host factors were not significantly altered during DENV infection, suggesting their interaction with DENV RNA was due to specific recruitment mechanisms. However, at the global proteome level, DENV altered the abundances of proteins in particular classes, including transporter proteins, which were down regulated, and proteins in the ubiquitin proteasome pathway, which were up regulated. Conclusions/Significance The method for identification of host factors described here is robust and broadly applicable to all RNA viruses, providing an avenue to determine the conserved or distinct mechanisms through which diverse viruses manage the viral RNA within cells. This study significantly increases the number of cellular factors known to interact with

  5. Identification of Arabidopsis Cyclase-associated Protein 1 as the First Nucleotide Exchange Factor for Plant Actin

    PubMed Central

    Chaudhry, Faisal; Guérin, Christophe; von Witsch, Matthias

    2007-01-01

    The actin cytoskeleton powers organelle movements, orchestrates responses to abiotic stresses, and generates an amazing array of cell shapes. Underpinning these diverse functions of the actin cytoskeleton are several dozen accessory proteins that coordinate actin filament dynamics and construct higher-order assemblies. Many actin-binding proteins from the plant kingdom have been characterized and their function is often surprisingly distinct from mammalian and fungal counterparts. The adenylyl cyclase-associated protein (CAP) has recently been shown to be an important regulator of actin dynamics in vivo and in vitro. The disruption of actin organization in cap mutant plants indicates defects in actin dynamics or the regulated assembly and disassembly of actin subunits into filaments. Current models for actin dynamics maintain that actin-depolymerizing factor (ADF)/cofilin removes ADP–actin subunits from filament ends and that profilin recharges these monomers with ATP by enhancing nucleotide exchange and delivery of subunits onto filament barbed ends. Plant profilins, however, lack the essential ability to stimulate nucleotide exchange on actin, suggesting that there might be a missing link yet to be discovered from plants. Here, we show that Arabidopsis thaliana CAP1 (AtCAP1) is an abundant cytoplasmic protein; it is present at a 1:3 M ratio with total actin in suspension cells. AtCAP1 has equivalent affinities for ADP– and ATP–monomeric actin (Kd ∼ 1.3 μM). Binding of AtCAP1 to ATP–actin monomers inhibits polymerization, consistent with AtCAP1 being an actin sequestering protein. However, we demonstrate that AtCAP1 is the first plant protein to increase the rate of nucleotide exchange on actin. Even in the presence of ADF/cofilin, AtCAP1 can recharge actin monomers and presumably provide a polymerizable pool of subunits to profilin for addition onto filament ends. In turnover assays, plant profilin, ADF, and CAP act cooperatively to promote flux of

  6. Transfection of influenza A virus nuclear export protein induces the expression of tumor necrosis factor alpha.

    PubMed

    Lara-Sampablo, Alejandra; Flores-Alonso, Juan Carlos; De Jesús-Ortega, Nereyda; Santos-López, Gerardo; Vallejo-Ruiz, Verónica; Rosas-Murrieta, Nora; Reyes-Carmona, Sandra; Herrera-Camacho, Irma; Reyes-Leyva, Julio

    2014-06-24

    Influenza A virus genomic segments eight codes for non-structural 1 (NS1) protein that is involved in evasion of innate antiviral response, and nuclear export protein (NEP) that participates in the export of viral ribonucleoprotein (RNP) complexes, transcription and replication. Tumor necrosis factor alpha (TNF-α) is highly expressed during influenza virus infections and is considered an anti-infective cytokine. NS1 and NEP proteins were overexpressed and their role on TNF-α expression was evaluated. Both TNF-α mRNA and protein increased in cells transfected with NEP but not with NS1. We further investigate if NS1 or NEP regulates the activity of TNF-α promoter. In the presence of NEP the activity of TNF-α promoter increased significantly compared with the control (83.5±2.9 vs. 30.9±2.8, respectively; p=0.001). This effect decreased 15-fold when the TNF-α promoter distal region was deleted, suggesting the involvement of mitogen-activated protein kinases (MAPK) and NF-kB response elements. This was corroborated by testing the effect produced on TNF-α promoter by the treatment with Raf/MEK/ERK (U0126), NF-kB (Bay-11-7082) and PI3K (Ly294-002) cell signaling inhibitors. Treatment with U0126 and Bay-117082 reduced the activity of TNF-α promoter mediated by NEP (41.5±3.2, 70% inhibition; and 80.6±7.4, 35% inhibition, respectively) compared to mock-treated control. The results suggest a new role for NEP protein that participates in the transcriptional regulation of human TNF-α expression.

  7. The Trigger Factor Chaperone Encapsulates and Stabilizes Partial Folds of Substrate Proteins

    PubMed Central

    Singhal, Kushagra; Vreede, Jocelyne; Mashaghi, Alireza; Tans, Sander J.; Bolhuis, Peter G.

    2015-01-01

    How chaperones interact with protein chains to assist in their folding is a central open question in biology. Obtaining atomistic insight is challenging in particular, given the transient nature of the chaperone-substrate complexes and the large system sizes. Recent single-molecule experiments have shown that the chaperone Trigger Factor (TF) not only binds unfolded protein chains, but can also guide protein chains to their native state by interacting with partially folded structures. Here, we used all-atom MD simulations to provide atomistic insights into how Trigger Factor achieves this chaperone function. Our results indicate a crucial role for the tips of the finger-like appendages of TF in the early interactions with both unfolded chains and partially folded structures. Unfolded chains are kinetically trapped when bound to TF, which suppresses the formation of transient, non-native end-to-end contacts. Mechanical flexibility allows TF to hold partially folded structures with two tips (in a pinching configuration), and to stabilize them by wrapping around its appendages. This encapsulation mechanism is distinct from that of chaperones such as GroEL, and allows folded structures of diverse size and composition to be protected from aggregation and misfolding interactions. The results suggest that an ATP cycle is not required to enable both encapsulation and liberation. PMID:26512985

  8. The use of additive and subtractive approaches to examine the nuclear localization sequence of the polyomavirus major capsid protein VP1

    NASA Technical Reports Server (NTRS)

    Chang, D.; Haynes, J. I. 2nd; Brady, J. N.; Consigli, R. A.; Spooner, B. S. (Principal Investigator)

    1992-01-01

    A nuclear localization signal (NLS) has been identified in the N-terminal (Ala1-Pro-Lys-Arg-Lys-Ser-Gly-Val-Ser-Lys-Cys11) amino acid sequence of the polyomavirus major capsid protein VP1. The importance of this amino acid sequence for nuclear transport of VP1 protein was demonstrated by a genetic "subtractive" study using the constructs pSG5VP1 (full-length VP1) and pSG5 delta 5'VP1 (truncated VP1, lacking amino acids Ala1-Cys11). These constructs were used to transfect COS-7 cells, and expression and intracellular localization of the VP1 protein was visualized by indirect immunofluorescence. These studies revealed that the full-length VP1 was expressed and localized in the nucleus, while the truncated VP1 protein was localized in the cytoplasm and not transported to the nucleus. These findings were substantiated by an "additive" approach using FITC-labeled conjugates of synthetic peptides homologous to the NLS of VP1 cross-linked to bovine serum albumin or immunoglobulin G. Both conjugates localized in the nucleus after microinjection into the cytoplasm of 3T6 cells. The importance of individual amino acids found in the basic sequence (Lys3-Arg-Lys5) of the NLS was also investigated. This was accomplished by synthesizing three additional peptides in which lysine-3 was substituted with threonine, arginine-4 was substituted with threonine, or lysine-5 was substituted with threonine. It was found that lysine-3 was crucial for nuclear transport, since substitution of this amino acid with threonine prevented nuclear localization of the microinjected, FITC-labeled conjugate.

  9. Influence of physico-chemical factors on leaching of chemical additives from aluminium foils used for packaging of food materials.

    PubMed

    Ojha, Priyanka; Ojha, C S; Sharma, V P

    2007-01-01

    In recent years, the use of aluminium foils to wrap foodstuff and commodities has been increased to a great extent. Aluminium was found to leach out from the foil in different simulants particularly in distilled water, acidic and alkaline medium at 60 +/- 2 degrees C for 2 hours and 40 +/- 2 degrees C for 24 hours. The migration was found to be above the permissible limit as laid down by WHO guidelines, that is of 0.2 mg/L of water. The protocol used for this study was based on the recommendation of Bureau of Indian Standard regarding the migration of chemical additives from packaging materials used to pack food items. Migration of the aluminium metal was found significantly higher in acidic and aqueous medium in comparison to alcoholic and saline medium. Higher temperature conditions also enhanced the rate of migration of aluminium in acidic and aqueous medium. Leaching of aluminium metal occurred in double distilled water, acetic acid 3%, normal saline and sodium carbonate, except ethanol 8%, in which aluminium migration was below the detection limit of the instrument where three brands of the aluminium foil samples studied.

  10. Easy determination of the addition of soybean proteins to heat-processed meat products prepared with turkey meat or pork-turkey meat blends that could also contain milk proteins.

    PubMed

    Castro, F; Marina, M L; Rodríguez, J; García, M C

    2005-12-01

    The addition of non-meat proteins to processed meat products is limited by regulations. Therefore, this work has investigated the determination of added soybean proteins in commercial heat-processed meat products prepared with turkey meat or pork-turkey meat blends that could also contain milk proteins. The method consisted of extracting proteins from the meat products in a Tris-HCl buffer (pH 8) and analysing the extract by high-performance liquid chromatography with a linear gradient water-acetonitrile containing 0.05% (v/v) TFA. This method enabled the detection and quantitation of up to 0.08 and 0.28% (w/w), respectively, of soybean proteins (related to 6 g initial product) in these products. Satisfactory precision and recovery data were established. Accuracy was evaluated by a comparison of soybean protein contents determined by the proposed method and the existing AOAC official method based on an enzyme-linked immunosorbent assay (ELISA) from which no statistically significant differences were observed.

  11. Effects of a Dissostichus mawsoni-CaM recombinant proteins feed additive on the juvenile orange-spotted grouper (Epinephelus coioides) under the acute low temperature challenge.

    PubMed

    Luo, Sheng-Wei; Wang, Wei-Na; Cai, Luo; Qi, Zeng-Hua; Wang, Cong; Liu, Yuan; Peng, Chang-Lian; Chen, Liang-Biao

    2015-10-01

    The effects of Dissostichus mawsoni-Calmodulin (Dm-CaM) on growth performance, enzyme activities, respiratory burst, MDA level and immune-related gene expressions of the orange-spotted grouper (Epinephelus coioides) exposed to the acute low temperature stress were evaluated. The commercial diet supplemented with Dm-CaM protein was fed to the groupers for 6 weeks. No significant difference was observed in the specific growth rates, weight gains and survivals. After the feeding trial, the groupers were exposed to acute low temperature challenge. The groupers fed with Dm-CaM additive diet showed a significant decrease in the respiratory burst activity, while the blood cell number increased significantly at 25 °C by comparing with the control and additive control group. The enzymatic activity of SOD, ACP and ALP increased significantly in Dm-CaM additive group, while MDA level maintained stable with the lowest value. qRT-PCR analysis indicated that the up-regulated transcript expressions of CaM, C3, SOD2, LysC and HSPA4 were observed in Dm-CaM additive group. These results indicated that Dm-CaM additive diet may regulate the grouper immune response to the acute low temperature challenge. PMID:26122279

  12. Effects of a Dissostichus mawsoni-CaM recombinant proteins feed additive on the juvenile orange-spotted grouper (Epinephelus coioides) under the acute low temperature challenge.

    PubMed

    Luo, Sheng-Wei; Wang, Wei-Na; Cai, Luo; Qi, Zeng-Hua; Wang, Cong; Liu, Yuan; Peng, Chang-Lian; Chen, Liang-Biao

    2015-10-01

    The effects of Dissostichus mawsoni-Calmodulin (Dm-CaM) on growth performance, enzyme activities, respiratory burst, MDA level and immune-related gene expressions of the orange-spotted grouper (Epinephelus coioides) exposed to the acute low temperature stress were evaluated. The commercial diet supplemented with Dm-CaM protein was fed to the groupers for 6 weeks. No significant difference was observed in the specific growth rates, weight gains and survivals. After the feeding trial, the groupers were exposed to acute low temperature challenge. The groupers fed with Dm-CaM additive diet showed a significant decrease in the respiratory burst activity, while the blood cell number increased significantly at 25 °C by comparing with the control and additive control group. The enzymatic activity of SOD, ACP and ALP increased significantly in Dm-CaM additive group, while MDA level maintained stable with the lowest value. qRT-PCR analysis indicated that the up-regulated transcript expressions of CaM, C3, SOD2, LysC and HSPA4 were observed in Dm-CaM additive group. These results indicated that Dm-CaM additive diet may regulate the grouper immune response to the acute low temperature challenge.

  13. Signalling pathways involved in the synergistic effects of human growth differentiation factor 9 and bone morphogenetic protein 15.

    PubMed

    Reader, Karen L; Mottershead, David G; Martin, Georgia A; Gilchrist, Robert B; Heath, Derek A; McNatty, Kenneth P; Juengel, Jennifer L

    2016-03-01

    Growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) act synergistically to regulate granulosa cell proliferation and steroid production in several species. Several non-Sma and mothers against decapentaplegic (SMAD) signalling pathways are involved in the action of murine and ovine GDF9 and BMP15 in combination, with the pathways utilised differing between the two species. The aims of this research were to determine if human GDF9 and BMP15 also act in a synergistic manner to stimulate granulosa cell proliferation and to identify which non-SMAD signalling pathways are activated. Human GDF9 with BMP15 (GDF9+BMP15) stimulated an increase in (3)H-thymidine incorporation (P<0.001), which was greater than the increase with BMP15 alone, while GDF9 alone had no effect. The stimulation of (3)H-thymidine incorporation by GDF9+BMP15 was reduced by the addition of inhibitors to the SMAD2/3, nuclear factor-KB (NF-KB) and c-Jun N-terminal kinase (JNK) signalling pathways. Inhibitors to the SMAD1/5/8, extracellular signal-regulated kinase mitogen-activated protein kinase (ERK-MAPK) or p38-MAPK pathways had no effect. The addition of the BMP receptor 2 (BMPR2) extracellular domain also inhibited stimulation of (3)H-thymidine incorporation by GDF9+BMP15. In conclusion, human GDF9 and BMP15 act synergistically to stimulate granulosa cell proliferation, a response that also involves species-specific non-SMAD signalling pathways.

  14. Differential phosphorylation of the progesterone receptor by insulin, epidermal growth factor, and platelet-derived growth factor receptor tyrosine protein kinases.

    PubMed

    Woo, D D; Fay, S P; Griest, R; Coty, W; Goldfine, I; Fox, C F

    1986-01-01

    Purified preparations of insulin, epidermal growth factor (EGF), and platelet-derived growth factor (PDGF) receptors were compared for their abilities to phosphorylate purified hen oviduct progesterone receptors. The specific activities of all three peptide hormone-induced receptor kinases were first defined using a synthetic tridecapeptide tyrosine protein kinase substrate. Next, equivalent ligand-activated activities of the three receptor kinases were tested for their abilities to phosphorylate hen oviduct progesterone receptor. Both the insulin and EGF receptors phosphorylated progesterone receptor at high affinity, exclusively at tyrosine residues and with maximal stoichiometries that were near unity. In contrast, the PDGF receptor did not recognize progesterone receptor as a substrate. Insulin decreased the Km of the insulin receptor for progesterone receptor subunits as substrates, but had no significant effect on Vmax values. On the other hand, EGF increased the Vmax of the EGF receptor for progesterone receptor subunits as substrates. Phosphorylation of progesterone receptor by the insulin and EGF receptor kinases differed in two additional ways. 1) EGF-activated receptor phosphorylated the 80- and 105-kDa progesterone receptor subunits to an equal extent, whereas insulin-activated receptor preferentially phosphorylated the 80-kDa subunit. 2) Phosphopeptide fingerprinting analyses revealed that while insulin and EGF receptors phosphorylated one identical major site on both progesterone receptor subunits, they differed in their specificities for other sites. PMID:3001059

  15. Tumor Necrosis Factor-alpha Induced Protein 3 Interacting Protein 1 Gene Polymorphisms and Pustular Psoriasis in Chinese Han Population

    PubMed Central

    Han, Jian-Wen; Wang, Yong; Alateng, Chulu; Li, Hong-Bin; Bai, Yun-Hua; Lyu, Xin-Xiang; Wu, Rina

    2016-01-01

    Background: Psoriasis is a common immune-mediated inflammatory dermatosis. Generalized pustular psoriasis (GPP) is the severe and rare type of psoriasis. The association between tumor necrosis factor-alpha induced protein 3 interacting protein 1 (TNIP1) gene and psoriasis was confirmed in people with multiple ethnicities. This study was to investigate the association between TNIP1 gene polymorphisms and pustular psoriasis in Chinese Han population. Methods: Seventy-three patients with GPP, 67 patients with palmoplantar pustulosis (PPP), and 476 healthy controls were collected from Chinese Han population. Six single nucleotide polymorphisms (SNPs) of the TNIP1 gene, namely rs3805435, rs3792798, rs3792797, rs869976, rs17728338, and rs999011 were genotyped by using polymerase chain reaction-ligase detection reaction. Statistical analyses were performed using the PLINK 1.07 package. Allele frequencies and genotyping frequencies for six SNPs were compared by using Chi-square test, odd ratio (OR) (including 95% confidence interval) were calculated. The haplotype analysis was conducted by Haploview software. Results: The frequencies of alleles of five SNPs were significantly different between the GPP group and the control group (P ≤ 7.22 × 10−3), especially in the GPP patients without psoriasis vulgaris (PsV). In the haplotype analysis, the most significantly different haplotype was H4: ACGAAC, with 13.1% frequency in the GPP group but only 3.4% in the control group (OR = 4.16, P = 4.459 × 10−7). However, no significant difference in the allele frequencies was found between the PPP group and control group for each of the six SNPs (P > 0.05). Conclusions: Polymorphisms in TNIP1 are associated with GPP in Chinese Han population. However, no association with PPP was found. These findings suggest that TNIP1 might be a susceptibility gene for GPP. PMID:27364786

  16. The severity of retinal pathology in homozygous Crb1rd8/rd8 mice is dependent on additional genetic factors.

    PubMed

    Luhmann, Ulrich F O; Carvalho, Livia S; Holthaus, Sophia-Martha Kleine; Cowing, Jill A; Greenaway, Simon; Chu, Colin J; Herrmann, Philipp; Smith, Alexander J; Munro, Peter M G; Potter, Paul; Bainbridge, James W B; Ali, Robin R

    2015-01-01

    Understanding phenotype-genotype correlations in retinal degeneration is a major challenge. Mutations in CRB1 lead to a spectrum of autosomal recessive retinal dystrophies with variable phenotypes suggesting the influence of modifying factors. To establish the contribution of the genetic background to phenotypic variability associated with the Crb1(rd8/rd8) mutation, we compared the retinal pathology of Crb1(rd8/rd8)/J inbred mice with that of two Crb1(rd8/rd8) lines backcrossed with C57BL/6JOlaHsd mice. Topical endoscopic fundal imaging and scanning laser ophthalmoscopy fundus images of all three Crb1(rd8/rd8) lines showed a significant increase in the number of inferior retinal lesions that was strikingly variable between the lines. Optical coherence tomography, semithin, ultrastructural morphology and assessment of inflammatory and vascular marker by immunohistochemistry and quantitative reverse transcriptase-polymerase chain reaction revealed that the lesions were associated with photoreceptor death, Müller and microglia activation and telangiectasia-like vascular remodelling-features that were stable in the inbred, variable in the second, but virtually absent in the third Crb1(rd8/rd8) line, even at 12 months of age. This suggests that the Crb1(rd8/rd8) mutation is necessary, but not sufficient for the development of these degenerative features. By whole-genome SNP analysis of the genotype-phenotype correlation, a candidate region on chromosome 15 was identified. This may carry one or more genetic modifiers for the manifestation of the retinal pathology associated with mutations in Crb1. This study also provides insight into the nature of the retinal vascular lesions that likely represent a clinical correlate for the formation of retinal telangiectasia or Coats-like vasculopathy in patients with CRB1 mutations that are thought to depend on such genetic modifiers.

  17. The T protein encoded by Brachyury is a tissue-specific transcription factor.

    PubMed Central

    Kispert, A; Koschorz, B; Herrmann, B G

    1995-01-01

    The mouse Brachyury (T) gene is required for differentiation of the notochord and formation of mesoderm during posterior development. Homozygous embryos lacking T activity do not develop a trunk and tail and die in utero. The T gene is specifically expressed in notochord and early mesoderm cells in the embryo. recent data have demonstrated that the T protein is localized in the cell nucleus and specifically binds to a palindrome of 20 bp (the T site) in vitro. We show that the T protein activates expression of a reporter gene in HeLa cells through binding to the T site. Thus T is a novel tissue-specific transcription factor. It consists of a large N-terminal DNA binding domain (amino acids 1-229) and two pairs of transactivation and repression domains in the C-terminal protein half. T can also transactivate transcription through variously oriented and spaced T sites, a fact that may be relevant in the search for genes controlled by T protein and important in mesoderm development. Images PMID:7588606

  18. Regulation of WRKY46 Transcription Factor Function by Mitogen-Activated Protein Kinases in Arabidopsis thaliana

    PubMed Central

    Sheikh, Arsheed H.; Eschen-Lippold, Lennart; Pecher, Pascal; Hoehenwarter, Wolfgang; Sinha, Alok K.; Scheel, Dierk; Lee, Justin

    2016-01-01

    Mitogen-activated protein kinase (MAPK) cascades are central signaling pathways activated in plants after sensing internal developmental and external stress cues. Knowledge about the downstream substrate proteins of MAPKs is still limited in plants. We screened Arabidopsis WRKY transcription factors as potential targets downstream of MAPKs, and concentrated on characterizing WRKY46 as a substrate of the MAPK, MPK3. Mass spectrometry revealed in vitro phosphorylation of WRKY46 at amino acid position S168 by MPK3. However, mutagenesis studies showed that a second phosphosite, S250, can also be phosphorylated. Elicitation with pathogen-associated molecular patterns (PAMPs), such as the bacterial flagellin-derived flg22 peptide led to in vivo destabilization of WRKY46 in Arabidopsis protoplasts. Mutation of either phosphorylation site reduced the PAMP-induced degradation of WRKY46. Furthermore, the protein for the double phosphosite mutant is expressed at higher levels compared to wild-type proteins or single phosphosite mutants. In line with its nuclear localization and predicted function as a transcriptional activator, overexpression of WRKY46 in protoplasts raised basal plant defense as reflected by the increase in promoter activity of the PAMP-responsive gene, NHL10, in a MAPK-dependent manner. Thus, MAPK-mediated regulation of WRKY46 is a mechanism to control plant defense. PMID:26870073

  19. ADP ribosylation factor like 2 (Arl2) protein influences microtubule dynamics in breast cancer cells

    SciTech Connect

    Beghin, Anne . E-mail: anne.beghin@recherche.univ-lyon1.fr; Honore, Stephane; Messana, Celine; Matera, Eva-Laure; Aim, Jennifer; Burlinchon, Sandrine; Braguer, Diane; Dumontet, Charles

    2007-02-01

    ADP ribosylation factor like 2 (Arl2) protein is involved in the folding of tubulin peptides. Variants of the human adenocarcinoma line MCF7 cells with increased or reduced content of Arl2 protein were produced and characterized. Western blot analysis performed after separation of the different fractions of tubulins showed that the content in polymerizable soluble heterodimers was significantly increased in cells with the highest Arl2 expression level (MA+) and reduced in cells with the lowest Arl2 expression level (MA-) in comparison to control cells (MP). Microtubule dynamic instability, measured after microinjection of rhodamine-labelled tubulin in living cells, was significantly enhanced in MA+ cells and reduced in MA- cells. These alterations involved modifications of the microtubule growth and shortening rates, duration of attenuation phases, percentage of time spent in each phase (growth, shortening and attenuation) and catastrophe frequency. We also observed modifications in the expression level of the tumor suppressor protein phosphatase 2Ac, which has been shown to form a complex with Arl2. Finally, cell cycle progression was modified in these cells, particularly in regard to duration of telophase. In summary, alterations in Arl2 protein content were found to be associated with modifications in tubulin pools, microtubule dynamics as well as cell cycle progression.

  20. Pathogenic Leptospira Species Acquire Factor H and Vitronectin via the Surface Protein LcpA

    PubMed Central

    da Silva, Ludmila Bezerra; Miragaia, Lidia dos Santos; Breda, Leandro Carvalho Dantas; Abe, Cecilia Mari; Schmidt, Mariana Costa Braga; Moro, Ana Maria; Monaris, Denize; Conde, Jonas Nascimento; Józsi, Mihály; Isaac, Lourdes; Abreu, Patrícia Antônia Estima

    2014-01-01

    Upon infection, pathogenic Leptospira species bind several complement regulators in order to overcome host innate immunity. We previously characterized a 20-kDa leptospiral surface protein which interacts with C4b binding protein (C4BP): leptospiral complement regulator-acquiring protein A (LcpA). Here we show that LcpA also interacts with human factor H (FH), which remains functionally active once bound to the protein. Antibodies directed against short consensus repeat 20 (SCR20) inhibited binding of FH to LcpA by approximately 90%, thus confirming that this particular domain is involved in the interaction. We have also shown for the first time that leptospires bind human vitronectin and that the interaction is mediated by LcpA. Coincubation with heparin blocked LcpA-vitronectin interaction in a dose-dependent manner, strongly suggesting that binding may occur through the heparin binding domains of vitronectin. LcpA also bound to the terminal pathway component C9 and inhibited Zn2+-induced polymerization and membrane attack complex (MAC) formation. Competitive binding assays indicated that LcpA interacts with C4BP, FH, and vitronectin through distinct sites. Taken together, our findings indicate that LcpA may play a role in leptospiral immune evasion. PMID:25534939

  1. C4 protein of Beet severe curly top virus is a pathomorphogenetic factor in Arabidopsis.

    PubMed

    Park, Jungan; Hwang, Hyun-Sik; Buckley, Kenneth J; Park, Jong-Bum; Auh, Chung-Kyun; Kim, Dong-Giun; Lee, Sukchan; Davis, Keith R

    2010-12-01

    The Curtovirus C4 protein is required for symptom development during infection of Arabidopsis. Transgenic Arabidopsis plants expressing C4 from either Beet curly top virus or Beet severe curly top virus produced phenotypes that were similar to symptoms seen during infection with wild-type viruses. The pseudosymptoms caused by C4 protein alone were novel to transgenic Arabidopsis and included bumpy trichomes, severe enations, disorientation of vascular bundles and stomata, swelling, callus-like structure formation, and twisted siliques. C4 induced abnormal cell division and altered cell fate in a variety of tissues depending on the C4 expression level. C4 protein expression increased the expression levels of cell-cycle-related genes CYCs, CDKs and PCNA, and suppressed ICK1 and the retinoblastoma-related gene RBR1, resulting in activation of host cell division. These results suggest that the Curtovirus C4 proteins are involved actively in host cell-cycle regulation to recruit host factors for virus replication and symptom development. PMID:20960205

  2. Pentapeptide-repeat proteins that act as topoisomerase poison resistance factors have a common dimer interface

    PubMed Central

    Vetting, Matthew W.; Hegde, Subray S.; Zhang, Yong; Blanchard, John S.

    2011-01-01

    The protein AlbG is a self-resistance factor against albicidin, a nonribosomally encoded hybrid polyketide-peptide with antibiotic and phytotoxic properties produced by Xanthomonas albilineans. Primary-sequence analysis indicates that AlbG is a member of the pentapeptide-repeat family of proteins (PRP). The structure of AlbG from X. albilineans was determined at 2.0 Å resolution by SAD phasing using data collected from a single trimethyllead acetate derivative on a home source. AlbG folds into a right-handed quadrilateral β-helix composed of approximately eight semi-regular coils. The regularity of the β-­helix is blemished by a large loop/deviation in the β-helix between coils 4 and 5. The C-terminus of the β-helix is capped by a dimerization module, yielding a dimer with a 110 Å semi-collinear β-helical axis. This method of dimer formation appears to be common to all PRP proteins that confer resistance to topoisomerase poisons and contrasts with most PRP proteins, which are typically monomeric. PMID:21393830

  3. Synthesis of aberrant decay-accelerating factor proteins by affected paroxysmal nocturnal hemoglobinuria leukocytes.

    PubMed Central

    Carothers, D J; Hazra, S V; Andreson, S W; Medof, M E

    1990-01-01

    Paroxysmal nocturnal hemoglobinuria (PNH) leukocytes fail to express decay-accelerating factor (DAF) but contain DAF mRNA transcripts resembling those in normal cells. To further investigate the nature of the DAF defect in affected cells, patients' polymorphonuclear and mononuclear leukocytes (PMN and MNC) were biosynthetically labeled and newly synthesized DAF proteins examined. Analyses of greater than 98% surface DAF-negative PMN and MNC from a patient with PNH III erythrocytes showed precursor DAF protein approximately 3 kD smaller in each cell type than in normal cells. The proportion of precursor to mature (O-glycosylated) DAF protein was increased and soluble DAF protein was detected in the medium. Studies of 70-80% surface DAF-negative PMN and MNC from four patients with type II erythrocytes showed mixtures of the 3 kD smaller and normal DAF precursors. Partitioning with Triton X-114 detergent and biosynthetic labeling with the anchor precursor [3H]ethanolamine indicated that the abnormal peptides lacked glycosyl-inositolphospholipid membrane-anchoring structures. Thus, in PNH cells nascent DAF polypeptides are synthesized. Some of the abnormal pro-DAF molecules are processed in the Golgi and some are released extracellularly. Images PMID:1688570

  4. Physiological basis of tolerance to complete submergence in rice involves genetic factors in addition to the SUB1 gene.

    PubMed

    Singh, Sudhanshu; Mackill, David J; Ismail, Abdelbagi M

    2014-01-01

    1 lines. This suggests the possibility of further improvements in submergence tolerance by incorporating additional traits present in FR13A or other similar landraces. PMID:25281725

  5. The reduction of Na/H exchanger-3 protein and transcript expression in acute ischemia–reperfusion injury is mediated by extractable tissue factor(s)

    PubMed Central

    Zhang, Jianning; Babich, Victor; Bobulescu, I. Alexandru; Shi, Mingjun; McLeroy, Paul; Rogers, Thomas E.; Moe, Orson W.

    2014-01-01

    Ischemic renal injury is a formidable clinical problem, the pathophysiology of which is incompletely understood. As the Na/H exchanger-3 (NHE3) mediates the bulk of apical sodium transport and a significant fraction of oxygen consumption in the proximal tubule, we examined mechanisms by which ischemia–reperfusion affects the expression of NHE3. Ischemia–reperfusion dramatically decreased NHE3 protein and mRNA (immunohistochemistry, immunoblot, and RNA blot) in rat kidney cortex and medulla. The decrease in NHE3 protein was uniform throughout all tubules, including those appearing morphologically intact. In the kidney cortex, a decrease in NHE3 surface protein preceded that of NHE3 total protein and mRNA. Kidney homogenates from rats exposed to mild renal ischemia-reduced cell surface NHE3 protein expression in opossum kidney cells in vitro, whereas homogenates from animals with moderate-to-severe ischemia reduced both total NHE3 protein and mRNA. The decrease in total NHE3 protein was dependent on the proteasomal degradation associated with NHE3 ubiquitylation measured by coimmunoprecipitation. The transferable factor(s) from the ischemic homogenate that reduce NHE3 expression were found to be heat sensitive and to be associated with a lipid-enriched fraction, and did not include regulatory RNAs. Thus, transferable factor(s) mediate the ischemia–reperfusion injury-induced decrease in NHE3 of the kidney. PMID:21814178

  6. SR splicing factors serve as adapter proteins for TAP-dependent mRNA export.

    PubMed

    Huang, Yingqun; Gattoni, Renata; Stévenin, James; Steitz, Joan A

    2003-03-01

    The only mammalian RNA binding adapter proteins known to partner with TAP/NXF1, the primary receptor for general mRNA export, are members of the REF family. We demonstrate that at least three shuttling SR (serine/arginine-rich) proteins interact with the same domain of TAP/NXF1 that binds REFs. Included are 9G8 and SRp20, previously shown to promote the export of intronless RNAs. A peptide derived from the N terminus of 9G8 inhibits the binding of both REF and SR proteins to TAP/NXF1 in vitro, and this finding argues for competitive interactions. In Xenopus oocytes, the N terminus of 9G8 exhibits a dominant-negative effect on mRNA export from the nucleus, while addition of excess TAP/NXF1 overcomes this inhibition. Thus, multiple adapters including SR proteins most likely cooperate to recruit multiple copies of TAP/NXF1 for efficient mRNA export. PMID:12667464

  7. Heat shock protein-70 neutralizes apoptosis inducing factor in Bcr/Abl expressing cells.

    PubMed

    Wang, Fang; Dai, An-Ya; Tao, Kun; Xiao, Qing; Huang, Zheng-Lan; Gao, Miao; Li, Hui; Wang, Xin; Cao, Wei-Xi; Feng, Wen-Li

    2015-10-01

    Bcr/Abl fusion protein is a hallmark of human chronic myeloid leukemia (CML). The protein can activate various signaling pathways to make normal cells transform malignantly and thus to facilitate tumorigenesis. It has been reported that heat shock protein-70 (HSP-70) can be served as an anti-apoptotic protein that suppresses Bax and Apo-2L/TRAIL. But it is unclear whether HSP-70 affects AIF-initiated apoptosis in Bcr/Abl expressing cells considering that HSP-70 is coincidentally over-regulated in these cells. Our findings supported that abundant HSP-70 in Bcr/Abl cells neutralizes AIF by segregating it from nucleus via direct interaction, leading to the failure of AIF initiating cell death and the silence of caspase-independent apoptotic pathway upon apoptotic induction. Moderate inhibition of HSP-70 expression by siRNA leads to Vp-16 triggered re-distribution of AIF in nucleus. In addition, AIF bears a HSP-70 binding domain allowing association with HSP-70. Therefore, disruption of the association using an AIF mutant lacking this domain can restore the potential of AIF importing into nucleus, and finally triggers cell death in a time dependent manner.

  8. A common set of nuclear factors bind to promoter elements regulated by the retinoblastoma protein.

    PubMed

    Udvadia, A J; Rogers, K T; Horowitz, J M

    1992-09-01

    A 30-base pair element within the c-fos promoter, termed the RCE (retinoblastoma control element), has previously been shown to be the target of transcriptional regulation by the product of the retinoblastoma (Rb) gene. We have identified three nuclear proteins [retinoblastoma control proteins (RCPs)] that complex with this promoter element in vitro. The Rb gene does not appear to encode the RCPs as the expression of Rb in vivo does not correlate with RCE-RCP complex formation in vitro. A single binding site for the RCPs within the c-fos RCE was identified, and the nucleotides required for protein-DNA complex formation were defined. Similar sequences are found in the promoters of two additional genes that are regulated by Rb (c-myc and TGF-beta 1), and binding assays demonstrate that the RCPs also interact with these elements. Linkage of the c-fos RCE to the herpes simplex virus thymidine kinase promoter led to a 4-fold stimulation of expression in transient transfection assays. Mutations within the RCP binding site that abrogate stable interaction of the RCPs with the RCE in vitro block RCE transcriptional activity in vivo. Our results suggest a role for the RCPs in RCE-dependent transcription and the regulation of transcription by the Rb protein. PMID:1419910

  9. pH modulates the binding of early growth response protein 1 transcription factor to DNA.

    PubMed

    Mikles, David C; Bhat, Vikas; Schuchardt, Brett J; Deegan, Brian J; Seldeen, Kenneth L; McDonald, Caleb B; Farooq, Amjad

    2013-08-01

    The transcription factor early growth response protein (EGR)1 orchestrates a plethora of signaling cascades involved in cellular homeostasis, and its downregulation has been implicated in the development of prostate cancer. Herein, using a battery of biophysical tools, we show that the binding of EGR1 to DNA is tightly regulated by solution pH. Importantly, the binding affinity undergoes an enhancement of more than an order of magnitude with an increase in pH from 5 to 8, implying that the deprotonation of an ionizable residue accounts for such behavior. This ionizable residue is identified as His382 by virtue of the fact that its replacement by nonionizable residues abolishes the pH dependence of the binding of EGR1 to DNA. Notably, His382 inserts into the major groove of DNA, and stabilizes the EGR1-DNA interaction via both hydrogen bonding and van der Waals contacts. Remarkably, His382 is mainly conserved across other members of the EGR family, implying that histidine protonation-deprotonation may serve as a molecular switch for modulating the protein-DNA interactions that are central to this family of transcription factors. Collectively, our findings reveal an unexpected but a key step in the molecular recognition of the EGR family of transcription factors, and suggest that they may act as sensors of pH within the intracellular environment. PMID:23718776

  10. Plant NAC-type transcription factor proteins contain a NARD domain for repression of transcriptional activation.

    PubMed

    Hao, Yu-Jun; Song, Qing-Xin; Chen, Hao-Wei; Zou, Hong-Feng; Wei, Wei; Kang, Xu-Sheng; Ma, Biao; Zhang, Wan-Ke; Zhang, Jin-Song; Chen, Shou-Yi

    2010-10-01

    Plant-specific transcription factor NAC proteins play essential roles in many biological processes such as development, senescence, morphogenesis, and stress signal transduction pathways. In the NAC family, some members function as transcription activators while others act as repressors. In the present study we found that though the full-length GmNAC20 from soybean did not have transcriptional activation activity, the carboxy-terminal activation domain of GmNAC20 had high transcriptional activation activity in the yeast assay system. Deletion experiments revealed an active repression domain with 35 amino acids, named NARD (NAC Repression Domain), in the d subdomain of NAC DNA-binding domain. NARD can reduce the transcriptional activation ability of diverse transcription factors when fused to either the amino-terminal or the carboxy-terminal of the transcription factors. NARD-like sequences are also present in other NAC family members and they are functional repression domain when fused to VP16 in plant protoplast assay system. Mutation analysis of conserved amino acid residues in NARD showed that the hydrophobic LVFY motif may partially contribute to the repression function. It is hypothesized that the interactions between the repression domain NARD and the carboxy-terminal activation domain may finally determine the ability of NAC family proteins to regulate downstream gene expressions.

  11. Orthogonal matrix factorization enables integrative analysis of multiple RNA binding proteins

    PubMed Central

    Stražar, Martin; Žitnik, Marinka; Zupan, Blaž; Ule, Jernej; Curk, Tomaž

    2016-01-01

    Motivation: RNA binding proteins (RBPs) play important roles in post-transcriptional control of gene expression, including splicing, transport, polyadenylation and RNA stability. To model protein–RNA interactions by considering all available sources of information, it is necessary to integrate the rapidly growing RBP experimental data with the latest genome annotation, gene function, RNA sequence and structure. Such integration is possible by matrix factorization, where current approaches have an undesired tendency to identify only a small number of the strongest patterns with overlapping features. Because protein–RNA interactions are orchestrated by multiple factors, methods that identify discriminative patterns of varying strengths are needed. Results: We have developed an integrative orthogonality-regularized nonnegative matrix factorization (iONMF) to integrate multiple data sources and discover non-overlapping, class-specific RNA binding patterns of varying strengths. The orthogonality constraint halves the effective size of the factor model and outperforms other NMF models in predicting RBP interaction sites on RNA. We have integrated the largest data compendium to date, which includes 31 CLIP experiments on 19 RBPs involved in splicing (such as hnRNPs, U2AF2, ELAVL1, TDP-43 and FUS) and processing of 3’UTR (Ago, IGF2BP). We show that the integration of multiple data sources improves the predictive accuracy of retrieval of RNA binding sites. In our study the key predictive factors of protein–RNA interactions were the position of RNA structure and sequence motifs, RBP co-binding and gene region type. We report on a number of protein-specific patterns, many of which are consistent with experimentally determined properties of RBPs. Availability and implementation: The iONMF implementation and example datasets are available at https://github.com/mstrazar/ionmf. Contact: tomaz.curk@fri.uni-lj.si Supplementary information: Supplementary data are available

  12. Interleukin-18 binding protein reduces b16 melanoma hepatic metastasis by neutralizing adhesiveness and growth factors of sinusoidal endothelium.

    PubMed

    Carrascal, Maria Teresa; Mendoza, Lorea; Valcárcel, Maria; Salado, Clarisa; Egilegor, Eider; Tellería, Naiara; Vidal-Vanaclocha, Fernando; Dinarello, Charles A

    2003-01-15

    We studied the role of endogenous interleukin (IL)-18 in hepatic metastasis by blocking this cytokine using the naturally occurring IL-18 binding protein (IL-18BP). A single i.p. dose of IL-18BP given 30 min before intrasplenic injection of murine B16 melanoma (B16M) cells reduced the number of hepatic metastatic foci by 75% and metastatic volume by 80%. Same treatment reduced the intrahepatic retention of luciferase-transfected B16M by 50% and abolished VCAM-1 up-regulation in the hepatic microvasculature, as assessed by reverse transcription-PCR, Western blot, and immunohistochemistry. Twelve hours after IL-18BP, hepatic sinusoidal endothelium (HSE) cells were isolated, and adhesion of B16M cells to these cultured HSE cells was reduced to the level of vehicle-treated mice. IL-18BP treatment of mice with established micrometastases resulted in a 25% decrease in metastasis number and 40% decrease in metastasis volume, suggesting inhibition of endogenous growth factors. Indeed, the addition of IL-18BP to normal HSE abolished the release of melanoma cell growth factor(s) induced by B16M. IL-18 promoted the in vitro growth of B16M and human melanoma cells, which was IL-1 dependent. These data demonstrate a significant role of endogenous IL-18 on hepatic metastasis by up-regulating melanoma cell adhesion to HSE cells and tumor growth, implicating a possible antimetastatic benefit of neutralizing IL-18. PMID:12543807

  13. Symbolic integration of a product of two spherical Bessel functions with an additional exponential and polynomial factor

    NASA Astrophysics Data System (ADS)

    Gebremariam, B.; Duguet, T.; Bogner, S. K.

    2010-06-01

    We present a Mathematica package that performs the symbolic calculation of integrals of the form ∫0∞exj(x)j(x)dx where j(x) and j(x) denote spherical Bessel functions of integer orders, with ν⩾0 and μ⩾0. With the real parameter u>0 and the integer n, convergence of the integral requires that n+ν+μ⩾0. The package provides analytical result for the integral in its most simplified form. In cases where direct Mathematica implementations succeed in evaluating these integrals, the novel symbolic method implemented in this work obtains the same result and in general, it takes a fraction of the ti