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Sample records for acriflavine resistance protein

  1. Acriflavine-Resistant Mutant of Streptococcus cremoris†

    PubMed Central

    Sinha, R.P.

    1977-01-01

    Selection for resistance to acriflavine in Streptococcus cremoris resulted in cross-resistance to the drugs neomycin, streptomycin, ethidium bromide, mitomycin C, and proflavine. Furthermore, the mutants showed resistance to lytic bacteriophages to which the parental strain was sensitive, and, unlike the parent, the mutants grew well at higher temperatures (40°C). Revertants selected independently either for temperature sensitivity or for acriflavine sensitivity lost resistance to all the drugs and dyes but retained the bacteriophage resistance phenotype. The acriflavine-resistant mutation resulted in an increase in resistance by the bacterial cells to sodium dodecyl sulfate, a potent solvent of lipopolysaccharide and lipoprotein. It is suggested that the acriflavine resistance mutation determines the synthesis of a membrane substance resistant to higher temperatures. PMID:907329

  2. Isolation of acriflavine resistant Erysipelothrix rhusiopathiae from slaughter pigs in Japan.

    PubMed

    Makino, S; Ishizaki, H; Shirahata, T; Fujiwara, S; Sawada, T

    1998-09-01

    Erysipelothrix rhusiopathiae is the causative agent of swine erysipelas. Although an attenuated vaccine is used in Japan, recent increases in disease occurrence have cast doubts on its efficacy. We investigated the similarity between the vaccine strain and E. rhusiopathiae field isolates by the analysis of acriflavine resistance (the vaccine strain marker), serotype, DNA fingerprinting and pathogenicity to mice. Although 7 acriflavine resistant E. rhusiopathiae isolates were separated from arthritic lesions of slaughter pigs, we were unable to prove that they were identical to the vaccine strain.

  3. Potent Antimalarial Activity of Acriflavine In Vitro and In Vivo

    PubMed Central

    2015-01-01

    Malaria continues to be a major health problem globally. There is an urgent need to find new antimalarials. Acriflavine (ACF) is known as an antibacterial agent and more recently as an anticancer agent. Here, we report that ACF inhibits the growth of asexual stages of both chloroquine (CQ) sensitive and resistant strains of human malarial parasite, Plasmodium falciparum in vitro at nanomolar concentration. ACF clears the malaria infection in vivo from the bloodstreams of mice infected with Plasmodium berghei. Interestingly, ACF is accumulated only in the parasitized red blood cells (RBCs) and parasite specific transporters may have role in this specific drug accumulation. We further show that ACF impairs DNA replication foci formation in the parasites and affects the enzymatic activities of apicoplast specific Gyrase protein. We thus establish ACF as a potential antimalarial amidst the widespread incidences of drug resistant Plasmodium strains. PMID:25089658

  4. Structural and functional alterations of catalase induced by acriflavine, a compound causing apoptosis and necrosis.

    PubMed

    Attar, Farnoosh; Khavari-Nejad, Sarah; Keyhani, Jacqueline; Keyhani, Ezzatollah

    2009-08-01

    Acriflavine is an antiseptic agent causing both apoptosis and necrosis in yeast. In this work, its effect on the structure and function of catalase, a vital enzyme actively involved in protection against oxidative stress, was investigated. In vitro kinetic studies showed that acriflavine inhibited the enzymatic activity in a competitive manner. The residual activity detectable after preincubation of catalase (1.5 nmol/L) with various concentrations of acriflavine went from 50% to 20% of the control value as the acriflavine concentration increased from 30 to 90 micromol/L. Correlatively with the decrease in activity, alterations in the enzyme's conformation were observed as indicated by fluorescence spectroscopy, circular dichroism spectroscopy, and electronic absorption spectroscopy. The enzyme's intrinsic fluorescence obtained upon excitation at either 297 nm (tryptophan residues) or 280 nm (tyrosine and tryptophan residues) decreased as a function of acriflavine concentration. Circular dichroism studies showed alterations of the protein structure by acriflavine with up to 13% decrease in alpha helix, 16% increase in beta-sheet content, 17% increase in random coil, and 4% increase in beta turns. Spectrophotometric studies showed a blueshift and modifications in the chromicity of catalase at 405 nm, corresponding to an absorbance band due to the enzyme's prosthetic group. Thus, acriflavine induced in vitro a profound change in the structure of catalase so that the enzyme could no longer function. Our results showed that acriflavine, a compound producing apoptosis and necrosis, can have a direct effect on vital functions in cells by disabling key enzymes.

  5. Cytotoxic effect of acriflavine against clinical isolates of Acanthamoeba spp.

    PubMed

    Polat, Zubeyda Akin; Karakus, Gulderen

    2013-02-01

    Acanthamoeba keratitis (AK) is a potentially devastating and sight-threatening infection of the cornea caused by the ubiquitous free-living amoebae, Acanthamoeba species. Its eradication is difficult because the amoebas encyst, making it highly resistant to anti-amoebic drugs. Acriflavine neutral (ACF) has been used for treatment of microbial infections for humans and fishes. The aim of our study was to evaluate the time-dependent cytotoxicities of ACF against Acanthamoeba spp. Trophozoites and cysts of three different strains (strain PAT06 Acanthamoeba castellanii, strain 2HH Acanthamoeba hatchetti, and strain 11DS A. hatchetti) of Acanthamoeba spp. were tested. All strains had been isolated from patients suffering from a severe AK. The effects of the ACF with the concentrations ranging from 15 to 500 mg mL(-1) on the cytotoxicity of Acanthamoeba strains were examined. ACF showed a time- and dose-dependent amebicidal action on the trophozoites and cysts. Pat06 (A. castellanii) was the most resistant, while strain 11DS (A. hatchetti) was the most sensitive. As a result, ACF could be concluded as a new agent for the treatment of Acanthamoeba infections. On the other hand, it still needs to be further evaluated by in vivo test systems to confirm the efficiency of its biological effect.

  6. Inhibition of hypoxia inducible factor 1 and topoisomerase with acriflavine sensitizes perihilar cholangiocarcinomas to photodynamic therapy

    PubMed Central

    Krekorian, Massis; Alles, Lindy K.; van Wijk, Albert C.; Mackaaij, Claire; Verheij, Joanne; van der Wal, Allard C.; van Gulik, Thomas M.; Storm, Gert; Heger, Michal

    2016-01-01

    Background: Photodynamic therapy (PDT) induces tumor cell death by oxidative stress and hypoxia but also survival signaling through activation of hypoxia-inducible factor 1 (HIF-1). Since perihilar cholangiocarcinomas are relatively recalcitrant to PDT, the aims were to (1) determine the expression levels of HIF-1-associated proteins in human perihilar cholangiocarcinomas, (2) investigate the role of HIF-1 in PDT-treated human perihilar cholangiocarcinoma cells, and (3) determine whether HIF-1 inhibition reduces survival signaling and enhances PDT efficacy. Results: Increased expression of VEGF, CD105, CD31/Ki-67, and GLUT-1 was confirmed in human perihilar cholangiocarcinomas. PDT with liposome-delivered zinc phthalocyanine caused HIF-1α stabilization in SK-ChA-1 cells and increased transcription of HIF-1α downstream genes. Acriflavine was taken up by SK-ChA-1 cells and translocated to the nucleus under hypoxic conditions. Importantly, pretreatment of SK-ChA-1 cells with acriflavine enhanced PDT efficacy via inhibition of HIF-1 and topoisomerases I and II. Methods: The expression of VEGF, CD105, CD31/Ki-67, and GLUT-1 was determined by immunohistochemistry in human perihilar cholangiocarcinomas. In addition, the response of human perihilar cholangiocarcinoma (SK-ChA-1) cells to PDT with liposome-delivered zinc phthalocyanine was investigated under both normoxic and hypoxic conditions. Acriflavine, a HIF-1α/HIF-1β dimerization inhibitor and a potential dual topoisomerase I/II inhibitor, was evaluated for its adjuvant effect on PDT efficacy. Conclusions: HIF-1, which is activated in human hilar cholangiocarcinomas, contributes to tumor cell survival following PDT in vitro. Combining PDT with acriflavine pretreatment improves PDT efficacy in cultured cells and therefore warrants further preclinical validation for therapy-recalcitrant perihilar cholangiocarcinomas. PMID:26657503

  7. mmr, a Mycobacterium tuberculosis Gene Conferring Resistance to Small Cationic Dyes and Inhibitors

    PubMed Central

    De Rossi, Edda; Branzoni, Manuela; Cantoni, Rita; Milano, Anna; Riccardi, Giovanna; Ciferri, Orio

    1998-01-01

    The mmr gene, cloned from Mycobacterium tuberculosis, was shown to confer to Mycobacterium smegmatis resistance to tetraphenylphosphonium (TPP), erythromycin, ethidium bromide, acriflavine, safranin O, and pyronin Y. The gene appears to code for a protein containing four transmembrane domains. Studies of [3H]TPP intracellular accumulation strongly suggest that the resistance mediated by the Mmr protein involves active extrusion of TPP. PMID:9811672

  8. The HIF-1 antagonist acriflavine: visualization in retina and suppression of ocular neovascularization.

    PubMed

    Zeng, Mingbing; Shen, Jikui; Liu, Yuanyuan; Lu, Lucy Yang; Ding, Kun; Fortmann, Seth D; Khan, Mahmood; Wang, Jiangxia; Hackett, Sean F; Semenza, Gregg L; Campochiaro, Peter A

    2017-04-01

    Acriflavine, a fluorescent drug previously used for bacterial and trypanosomal infections, reduces hypoxia-inducible factor-1 (HIF-1) and HIF-2 transcriptional activity. In mice with oxygen-induced ischemic retinopathy, intraocular or intraperitoneal injections of acriflavine caused dose-dependent suppression of retinal neovascularization (NV) and significantly reduced expression of HIF-1-responsive genes. Intraocular injection of 100 ng caused inner retina fluorescence within 1 h that was seen throughout the entire retina between 1 and 5 days, and at 7 days after injection, strongly suppressed choroidal NV at Bruch's membrane rupture sites. After suprachoroidal injection of 300 ng in rats, there was retinal fluorescence in the quadrant of the injection at 1 h that spread throughout the entire retina and choroid by 1 day, was detectable for 5 days, and dramatically reduced choroidal NV 14 days after rupture of Bruch's membrane. After topical administration of acriflavine in mice, fluorescence was seen in the retina and retinal pigmented epithelium within 5 min and was detectable for 6-12 h. Administration of 0.5% drops to the cornea twice a day significantly reduced choroidal NV in mice. Electroretinographic b-wave amplitudes were normal 7 days after intravitreous injection of 100 ng of acriflavine in mice, showed mild threshold reductions at highest stimulus intensities after injection of 250 ng, and more extensive changes after injection of 500 ng. These data provide additional evidence for an important role for HIF-1 in retinal and choroidal NV and suggest that acriflavine can target HIF-1 through a variety of modes of administration and has good potential to provide a novel therapy for retinal and choroidal vascular diseases.

  9. Liquid chromatographic determination of acriflavine and proflavine residues in channel catfish muscle.

    PubMed

    Plakas, S M; el Said, K R; Jester, E L; Bencsath, F A; Hayton, W L

    1997-01-01

    A liquid chromatographic (LC) method was developed for determination of acriflavine (ACR) and proflavine (PRO) residues in channel catfish muscle. Residues were extracted with acidified methanol solution, and extracts were cleaned up with C18 solid-phase extraction columns. Residue concentrations were determined on an LC cyano column, with spectrophotometric detection at 454 nm. Catfish muscle was individually fortified with ACR (purified from commercial product) and PRO at concentrations of 5, 10, 20, 40, and 80 ppb (5 replicates per level). Mean recoveries from fortified muscle at each level ranged from 86 to 95%, with relative standard deviations (RSDs) of 2.5 to 5.7%. The method was applied to incurred residues of ACR and PRO in muscle after waterborne exposure of channel catfish to commercial acriflavine (10 ppm total dye for 4 h). RSDs for incurred residues of ACR and PRO were in the same range as those for fortified muscle. Low residue concentrations (< 1% of exposure water concentration) suggested poor absorption of ACR and PRO in catfish.

  10. Resistive Switching Memory Devices Based on Proteins.

    PubMed

    Wang, Hong; Meng, Fanben; Zhu, Bowen; Leow, Wan Ru; Liu, Yaqing; Chen, Xiaodong

    2015-12-09

    Resistive switching memory constitutes a prospective candidate for next-generation data storage devices. Meanwhile, naturally occurring biomaterials are promising building blocks for a new generation of environmentally friendly, biocompatible, and biodegradable electronic devices. Recent progress in using proteins to construct resistive switching memory devices is highlighted. The protein materials selection, device engineering, and mechanism of such protein-based resistive switching memory are discussed in detail. Finally, the critical challenges associated with protein-based resistive switching memory devices are presented, as well as insights into the future development of resistive switching memory based on natural biomaterials.

  11. Aptamer-based electrochemical biosensor by using Au-Pt nanoparticles, carbon nanotubes and acriflavine platform.

    PubMed

    Beiranvand, Zeinab Sahraei; Abbasi, Amir Reza; Dehdashtian, Sara; Karimi, Ziba; Azadbakht, Azadeh

    2017-02-01

    Herein, an ultrasensitive electrochemical aptasensor for quantitative detection of bisphenol A (BPA) was fabricated based on a novel signal amplification strategy. This aptasensor was developed by electrodeposition of gold-platinum nanoparticles (Au-PtNPs) on glassy carbon (GC) electrode modified with acid-oxidized carbon nanotubes (CNTs-COOH). In this protocol, acriflavine (ACF) was covalently immobilized at the surface of glassy carbon electrode modified with Au-PtNPs/CNTs-COOH nanocomposite. Attachment of BPA-aptamer at the surface of modified electrode was performed through the formation of phosphoramidate bonds between the amino group of ACF and phosphate group of the aptamer at 5'end. By interaction of BPA with the aptamer, the conformational of aptamer was changed which lead to retarding the interfacial electron transfer of ACF as a probe. Sensitive quantitative detection of BPA was carried out by monitoring the decrease of differential pulse voltammetric (DPV) responses of ACF peak current with increasing the BPA concentration. The resultant aptasensor exhibited good specificity, stability and reproducibility, indicating that the present strategy was promising for broad potential application.

  12. Investigation on the inclusion and toxicity of acriflavine with cyclodextrins: A spectroscopic approach

    NASA Astrophysics Data System (ADS)

    Manivannan, C.; Meenakshi Sundaram, K.; Sundararaman, M.; Renganathan, R.

    2014-03-01

    Acriflavine hydrochloride (AFN) is a prospective drug worn in the eradication of HIV1 infection. The toxicity and adverse side effects renders the potent drug to limits its usage. However, to overcome the dilemma we have aimed to select carriers with great complexation efficiencies in different cyclodextrins (CDs) of varying cavity size. The interaction of AFN with α, β and γ-CDs were investigated using absorption and steady state as well as lifetime measurements. From the obtained data it was found that AFN fits in the cavity of α and β-CDs but unable to form inclusion complex with γ-CD. The effect of quencher molecules during the inclusion phenomena of AFN with CDs was explored via steady state measurements. The nature of binding forces responsible for the inclusion of AFN with CDs was discussed by using thermodynamic parameters. Using Benesi-Hildebrand equation the stoichiometry of AFN with CDs was predominantly found to be 1:1. To get deeper in situ, the in vitro toxicity of AFN and its complexation product were probed by Artemia salina sp. The toxicity of AFN was reduced when complexed with α and β-CDs.

  13. Pharmacokinetics, tissue distribution and metabolism of acriflavine and proflavine in the channel catfish (Ictalurus punctatus).

    PubMed

    Plakas, S M; el Said, K R; Bencsath, F A; Musser, S M; Hayton, W L

    1998-06-01

    1. The disposition of proflavine (PRO) and acriflavine (ACR) were examined in channel catfish after intravascular (i.v.) dosing (1 mg/kg) or waterborne exposure (10 mg/l for 4 h). 2. After i.v. dosing, plasma concentration-time profiles of parent PRO and ACR were best described by two- and three-compartment pharmacokinetic models respectively. Terminal elimination half-lives of PRO and ACR in plasma were 8.7 and 11.4 h respectively. 3. In animals dosed with 14C-PRO or 14C-ACR, total drug equivalent concentrations were highest in the excretory organs and lowest in muscle, fat and plasma. In PRO-dosed animals, residues in the liver and trunk kidney were composed primarily of glucuronosyl and acetyl conjugates of PRO; residues in muscle were composed mostly (> 95%) of the parent drug. In ACR-dosed animals, the parent compound comprised > 90% of the total residues in all tissues examined. 4. PRO and ACR were poorly absorbed in catfish during waterborne exposure. At the end of a 4-h exposure, parent PRO and ACR concentrations in muscle were 0.064 and 0.020 microgram/g respectively. Levels in muscle declined below the limit of determination (0.005 microgram/g) within 1-2 weeks.

  14. Predicting Resistance Mutations Using Protein Design Algorithms

    SciTech Connect

    Frey, K.; Georgiev, I; Donald, B; Anderson, A

    2010-01-01

    Drug resistance resulting from mutations to the target is an unfortunate common phenomenon that limits the lifetime of many of the most successful drugs. In contrast to the investigation of mutations after clinical exposure, it would be powerful to be able to incorporate strategies early in the development process to predict and overcome the effects of possible resistance mutations. Here we present a unique prospective application of an ensemble-based protein design algorithm, K*, to predict potential resistance mutations in dihydrofolate reductase from Staphylococcus aureus using positive design to maintain catalytic function and negative design to interfere with binding of a lead inhibitor. Enzyme inhibition assays show that three of the four highly-ranked predicted mutants are active yet display lower affinity (18-, 9-, and 13-fold) for the inhibitor. A crystal structure of the top-ranked mutant enzyme validates the predicted conformations of the mutated residues and the structural basis of the loss of potency. The use of protein design algorithms to predict resistance mutations could be incorporated in a lead design strategy against any target that is susceptible to mutational resistance.

  15. Resisting protein adsorption on biodegradable polyester brushes.

    PubMed

    Hu, Xinfang; Gorman, Christopher B

    2014-08-01

    The protein adsorption and degradation behaviors of poly(lactic acid), poly(glycolic acid) (PGA) and poly(ε-caprolactone) (PCL) brushes and their co-polymer brushes with oligo(ethylene glycol) (OEG) were studied. Both brush structure and relative amount of OEG and polyester were found to be important to the protein resistance of the brushes. A protein-resisting surface can be fabricated either by using OEG as the top layer of a copolymer brush or by increasing the amount of OEG relative to polyester when using a hydroxyl terminated OEG (OEG-OH) and a methoxy terminated OEG (OEG-OMe) mixture as the substrate layer. The degradation of single polyester brushes and their co-polymer brushes using OEG-OH as a substrate layer or using OEG as a top layer was hindered. This phenomenon was rationalized by the inhibition of the proposed back-biting process as the hydroxy end groups of polyester were blocked by OEG molecules. Among these brushes tested, PGA co-polymer brushes using the methoxy/hydroxyl OEG mixture as the substrate layer proved to be both protein-resistant and degradable due to the relatively large amount of OEG moieties and the good biodegradability of PGA.

  16. ABC-F Proteins Mediate Antibiotic Resistance through Ribosomal Protection.

    PubMed

    Sharkey, Liam K R; Edwards, Thomas A; O'Neill, Alex J

    2016-03-22

    Members of the ABC-F subfamily of ATP-binding cassette proteins mediate resistance to a broad array of clinically important antibiotic classes that target the ribosome of Gram-positive pathogens. The mechanism by which these proteins act has been a subject of long-standing controversy, with two competing hypotheses each having gained considerable support: antibiotic efflux versus ribosomal protection. Here, we report on studies employing a combination of bacteriological and biochemical techniques to unravel the mechanism of resistance of these proteins, and provide several lines of evidence that together offer clear support to the ribosomal protection hypothesis. Of particular note, we show that addition of purified ABC-F proteins to anin vitrotranslation assay prompts dose-dependent rescue of translation, and demonstrate that such proteins are capable of displacing antibiotic from the ribosomein vitro To our knowledge, these experiments constitute the first direct evidence that ABC-F proteins mediate antibiotic resistance through ribosomal protection.IMPORTANCEAntimicrobial resistance ranks among the greatest threats currently facing human health. Elucidation of the mechanisms by which microorganisms resist the effect of antibiotics is central to understanding the biology of this phenomenon and has the potential to inform the development of new drugs capable of blocking or circumventing resistance. Members of the ABC-F family, which includelsa(A),msr(A),optr(A), andvga(A), collectively yield resistance to a broader range of clinically significant antibiotic classes than any other family of resistance determinants, although their mechanism of action has been controversial since their discovery 25 years ago. Here we present the first direct evidence that proteins of the ABC-F family act to protect the bacterial ribosome from antibiotic-mediated inhibition.

  17. Characterization of Cercospora nicotianae Hypothetical Proteins in Cercosporin Resistance

    PubMed Central

    Beseli, Aydin; Noar, Roslyn; Daub, Margaret E.

    2015-01-01

    The photoactivated toxin, cercosporin, produced by Cercospora species, plays an important role in pathogenesis of this fungus to host plants. Cercosporin has almost universal toxicity to cells due to its production of reactive oxygen species including singlet oxygen. For that reason, Cercospora species, which are highly resistant to their own toxin, are good candidates to identify genes for resistance to cercosporin and to the reactive oxygen species it produces. In previous research, the zinc cluster transcription factor CRG1 (cercosporin resistance gene 1) was found to be crucial for Cercospora species’ resistance against cercosporin, and subtractive hybridization analysis identified 185 genes differentially expressed between Cercospora nicotianae wild type (wt) and a crg1 mutant. The focus of this work was to identify and characterize the hypothetical proteins that were identified in the Cercospora nicotianae subtractive library as potential resistance factors. Quantitative RT-PCR analysis of the 20 genes encoding hypothetical proteins showed that two, 24cF and 71cR, were induced under conditions of cercosporin toxicity, suggesting a role in resistance. Transformation and expression of 24cF and 71cR in the cercosporin-sensitive fungus, Neurospora crassa, showed that 71cR provided increased resistance to cercosporin toxicity, whereas no significant increase was observed in 24cF transformants. Gene disruption was used to generate C. nicotianae 71cR mutants; these mutants did not differ from wt C. nicotianae in cercosporin resistance or production. Quantitative RT-PCR analysis showed induction of other resistance genes in the 71cR mutant that may compensate for the loss of 71cR. Analysis of 71cR conserved domains and secondary and tertiary structure identify the protein as having an NTF2-like superfamily DUF1348 domain with unknown function, to be intracellular and localized in the cytosol, and to have similarities to proteins in the steroid delta

  18. Characterization of Cercospora nicotianae Hypothetical Proteins in Cercosporin Resistance.

    PubMed

    Beseli, Aydin; Noar, Roslyn; Daub, Margaret E

    2015-01-01

    The photoactivated toxin, cercosporin, produced by Cercospora species, plays an important role in pathogenesis of this fungus to host plants. Cercosporin has almost universal toxicity to cells due to its production of reactive oxygen species including singlet oxygen. For that reason, Cercospora species, which are highly resistant to their own toxin, are good candidates to identify genes for resistance to cercosporin and to the reactive oxygen species it produces. In previous research, the zinc cluster transcription factor CRG1 (cercosporin resistance gene 1) was found to be crucial for Cercospora species' resistance against cercosporin, and subtractive hybridization analysis identified 185 genes differentially expressed between Cercospora nicotianae wild type (wt) and a crg1 mutant. The focus of this work was to identify and characterize the hypothetical proteins that were identified in the Cercospora nicotianae subtractive library as potential resistance factors. Quantitative RT-PCR analysis of the 20 genes encoding hypothetical proteins showed that two, 24cF and 71cR, were induced under conditions of cercosporin toxicity, suggesting a role in resistance. Transformation and expression of 24cF and 71cR in the cercosporin-sensitive fungus, Neurospora crassa, showed that 71cR provided increased resistance to cercosporin toxicity, whereas no significant increase was observed in 24cF transformants. Gene disruption was used to generate C. nicotianae 71cR mutants; these mutants did not differ from wt C. nicotianae in cercosporin resistance or production. Quantitative RT-PCR analysis showed induction of other resistance genes in the 71cR mutant that may compensate for the loss of 71cR. Analysis of 71cR conserved domains and secondary and tertiary structure identify the protein as having an NTF2-like superfamily DUF1348 domain with unknown function, to be intracellular and localized in the cytosol, and to have similarities to proteins in the steroid delta-isomerase family.

  19. Synergistic effects of resistance training and protein intake: practical aspects.

    PubMed

    Guimarães-Ferreira, Lucas; Cholewa, Jason Michael; Naimo, Marshall Alan; Zhi, X I A; Magagnin, Daiane; de Sá, Rafaele Bis Dal Ponte; Streck, Emilio Luiz; Teixeira, Tamiris da Silva; Zanchi, Nelo Eidy

    2014-10-01

    Resistance training is a potent stimulus to increase skeletal muscle mass. The muscle protein accretion process depends on a robust synergistic action between protein intake and overload. The intake of protein after resistance training increases plasma amino acids, which results in the activation of signaling molecules leading to increased muscle protein synthesis (MPS) and muscle hypertrophy. Although both essential and non-essential amino acids are necessary for hypertrophy, the intake of free L-leucine or high-leucine whole proteins has been specifically shown to increase the initiation of translation that is essential for elevated MPS. The literature supports the use of protein intake following resistance-training sessions to enhance MPS; however, less understood are the effects of different protein sources and timing protocols on MPS. The sum of the adaptions from each individual training session is essential to muscle hypertrophy, and thus highlights the importance of an optimal supplementation protocol. The aim of this review is to present recent findings reported in the literature and to discuss the practical application of these results. In that light, new speculations and questions will arise that may direct future investigations. The information and recommendations generated in this review should be of benefit to clinical dietitians as well as those engaged in sports.

  20. Tween surfactants: Adsorption, self-organization, and protein resistance

    NASA Astrophysics Data System (ADS)

    Shen, Lei; Guo, Athena; Zhu, Xiaoyang

    2011-03-01

    Tween surfactants, each containing hydrophilic ethylene glycol head groups and a hydrophobic alkyl tail, are being actively explored as protein-resistant surface coatings, but little is known about how they adsorb on surfaces. We carry out a comparative study of the adsorption of two Tween molecules (same hydrophilic head group, but a shorter dodecyl tail for Tween 20 and a longer octadecyl tail for Tween 40) on Au and polystyrene surfaces. Despite the similarity between these two molecules, there is a drastic difference in their protein resistance: a monolayer of Tween 20 on a hydrophobic surface is repulsive against protein adsorption but that of Tween 40 is not. The difference in protein resistance can be attributed to two distinctly different adsorption mechanisms. While the adsorption of Tween 40 is described by a simple first-order mechanism, that of Tween 20 consists of a fast adsorption step and a slower reorganization process at a high surface coverage. The latter leads to the formation of a high-density and self-organized monolayer, which is responsible for the enhanced stability and resistance against non-specific protein adsorption.

  1. [ABC transporter proteins in multidrug resistance of microorganisms].

    PubMed

    Balková, K; Gbelská, Y

    2007-08-01

    The ABC (ATP binding cassette) transporter family includes membrane proteins that can transport a wide variety of substrates across biological membranes. These proteins play an essential role in the protection of cells from toxic compounds/metabolites. Their overexpression which leads to the development of multidrug resistance (MDR) in pathogens and enables cancer cells to survive chemotherapy is of major concern for human health. Mutations in ABC transporters are implicated in a number of Mendelian disorders such as cystic fibrosis, adrenoleukodystrophy and cholesterol and bile transport defects. In microbial cells, several homologues of human ABC transporters were identified. Their further molecular biological study can contribute to better understanding and treatment of MDR or diseases caused by dysfunction of ABC transporter proteins. A review is presented of the state of the art in ABC transporter proteins in both prokaryotic and eucaryotic cells. The role of microbial ABC transporters in the development of drug resistance is analyzed.

  2. ABC-F Proteins Mediate Antibiotic Resistance through Ribosomal Protection

    PubMed Central

    Sharkey, Liam K. R.; Edwards, Thomas A.

    2016-01-01

    ABSTRACT Members of the ABC-F subfamily of ATP-binding cassette proteins mediate resistance to a broad array of clinically important antibiotic classes that target the ribosome of Gram-positive pathogens. The mechanism by which these proteins act has been a subject of long-standing controversy, with two competing hypotheses each having gained considerable support: antibiotic efflux versus ribosomal protection. Here, we report on studies employing a combination of bacteriological and biochemical techniques to unravel the mechanism of resistance of these proteins, and provide several lines of evidence that together offer clear support to the ribosomal protection hypothesis. Of particular note, we show that addition of purified ABC-F proteins to an in vitro translation assay prompts dose-dependent rescue of translation, and demonstrate that such proteins are capable of displacing antibiotic from the ribosome in vitro. To our knowledge, these experiments constitute the first direct evidence that ABC-F proteins mediate antibiotic resistance through ribosomal protection. PMID:27006457

  3. Interferon-induced human protein with homology to protein Mx of influenza virus-resistant mice.

    PubMed Central

    Staeheli, P; Haller, O

    1985-01-01

    Polyclonal and monoclonal antibodies with specificity for protein Mx (a karyophilic 75,000-dalton protein induced by interferon [IFN] in mouse cells carrying the influenza virus resistance allele Mx+) detected an IFN-induced 80,000-dalton protein in peripheral blood lymphocytes and in fibroblasts of healthy human donors. The human protein, like protein Mx, was induced by IFN-alpha but not by IFN-gamma. Unlike the mouse protein, it was predominantly localized in the cell cytoplasm. Images PMID:3939324

  4. Targeting Protein Kinases to Reverse Multidrug Resistance in Sarcoma

    PubMed Central

    Chen, Hua; Shen, Jacson; Choy, Edwin; Hornicek, Francis J.; Duan, Zhenfeng

    2015-01-01

    Sarcomas are a group of cancers that arise from transformed cells of mesenchymal origin. They can be classified into over 50 subtypes, accounting for approximately 1% of adult and 15% of pediatric cancers. Wide surgical resection, radiotherapy, and chemotherapy are the most common treatments for the majority of sarcomas. Among these therapies, chemotherapy can palliate symptoms and prolong life for some sarcoma patients. However, sarcoma cells can have intrinsic or acquired resistance after treatment with chemotherapeutics drugs, leading to the development of multidrug resistance (MDR). MDR attenuates the efficacy of anticancer drugs and results in treatment failure for sarcomas. Therefore, overcoming MDR is an unmet need for sarcoma therapy. Certain protein kinases demonstrate aberrant expression and/or activity in sarcoma cells, which have been found to be involved in the regulation of sarcoma cell progression, such as cell cycle, apoptosis, and survival. Inhibiting these protein kinases may not only decrease the proliferation and growth of sarcoma cells, but also reverse their resistance to chemotherapeutic drugs to subsequently reduce the doses of anticancer drugs and decrease drug side-effects. The discovery of novel strategies targeting protein kinases opens a door to a new area of sarcoma research and provides insight into the mechanisms of MDR in chemotherapy. This review will focus on the recent studies in targeting protein kinase to reverse chemotherapeutic drug resistance in sarcoma. PMID:26827688

  5. M. tuberculosis Hypothetical Proteins and Proteins of Unknown Function: Hope for Exploring Novel Resistance Mechanisms as well as Future Target of Drug Resistance

    PubMed Central

    Sharma, Divakar; Bisht, Deepa

    2017-01-01

    Drug resistance in tuberculosis predominantly, mono-resistance, multi drug resistance, extensively drug resistance and totally drug resistance have emerged as a major problem in the chemotherapy of tuberculosis. Failures of first and second line anti-tuberculosis drugs treatment leads to emergence of resistant Mycobacterium tuberculosis. Few genes are reported as the principal targets of the resistance and apart from the primary targets many explanations have been proposed for drug resistance but still some resistance mechanisms are unknown. As proteins involved in most of the biological processes, these are potentially explored the unknown mechanism of drug resistance and attractive targets for diagnostics/future therapeutics against drug resistance. In last decade a panel of studies on expression proteomics of drug resistant M. tuberculosis isolates reported the differential expression of uncharacterized proteins and suggested these might be involved in resistance. Here we emphasize that detailed bioinformatics analysis (like molecular docking, pupylation, and proteins-proteins interaction) of these uncharacterized and hypothetical proteins might predict their interactive partners (other proteins) which are involved in various pathways of M. tuberculosis system biology and might give a clue for novel mechanism of drug resistance or future drug targets. In future these uncharacterized targets might be open the new resistance mechanism and used as potential drug targets against drug resistant tuberculosis. PMID:28377758

  6. Resistance of platelet proteins to effects of ionizing radiation

    SciTech Connect

    Prodouz, K.N.; Habraken, J.W.; Moroff, G. )

    1990-12-01

    Gamma irradiation of blood components prevents lymphocyte-induced graft-versus-host disease after transfusion in immunocompromised individuals. In this report we demonstrate the resistance of blood platelet proteins to gamma radiation-induced protein cleavage and aggregate formation when platelet concentrates were treated with a dose of 5000 rad. Results of one- and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis of total platelet protein and cytoskeletal protein preparations indicate that platelet proteins are neither cleaved nor cross-linked under these conditions of irradiation. These results support those of a previous study that documented the lack of any adverse effect of 5000 rad gamma radiation on in vitro platelet properties.

  7. Protein design algorithms predict viable resistance to an experimental antifolate.

    PubMed

    Reeve, Stephanie M; Gainza, Pablo; Frey, Kathleen M; Georgiev, Ivelin; Donald, Bruce R; Anderson, Amy C

    2015-01-20

    Methods to accurately predict potential drug target mutations in response to early-stage leads could drive the design of more resilient first generation drug candidates. In this study, a structure-based protein design algorithm (K* in the OSPREY suite) was used to prospectively identify single-nucleotide polymorphisms that confer resistance to an experimental inhibitor effective against dihydrofolate reductase (DHFR) from Staphylococcus aureus. Four of the top-ranked mutations in DHFR were found to be catalytically competent and resistant to the inhibitor. Selection of resistant bacteria in vitro reveals that two of the predicted mutations arise in the background of a compensatory mutation. Using enzyme kinetics, microbiology, and crystal structures of the complexes, we determined the fitness of the mutant enzymes and strains, the structural basis of resistance, and the compensatory relationship of the mutations. To our knowledge, this work illustrates the first application of protein design algorithms to prospectively predict viable resistance mutations that arise in bacteria under antibiotic pressure.

  8. Marine Natural Products as Breast Cancer Resistance Protein Inhibitors

    PubMed Central

    Cherigo, Lilia; Lopez, Dioxelis; Martinez-Luis, Sergio

    2015-01-01

    Breast cancer resistance protein (BCRP) is a protein belonging to the ATP-binding cassette (ABC) transporter superfamily that has clinical relevance due to its multi-drug resistance properties in cancer. BCRP can be associated with clinical cancer drug resistance, in particular acute myelogenous or acute lymphocytic leukemias. The overexpression of BCRP contributes to the resistance of several chemotherapeutic drugs, such as topotecan, methotrexate, mitoxantrone, doxorubicin and daunorubicin. The Food and Drugs Administration has already recognized that BCRP is clinically one of the most important drug transporters, mainly because it leads to a reduction of clinical efficacy of various anticancer drugs through its ATP-dependent drug efflux pump function as well as its apparent participation in drug resistance. This review article aims to summarize the different research findings on marine natural products with BCRP inhibiting activity. In this sense, the potential modulation of physiological targets of BCRP by natural or synthetic compounds offers a great possibility for the discovery of new drugs and valuable research tools to recognize the function of the complex ABC-transporters. PMID:25854646

  9. Mosaic tetracycline resistance genes encoding ribosomal protection proteins.

    PubMed

    Warburton, Philip J; Amodeo, Nina; Roberts, Adam P

    2016-12-01

    First reported in 2003, mosaic tetracycline resistance genes are a subgroup of the genes encoding ribosomal protection proteins (RPPs). They are formed when two or more RPP-encoding genes recombine resulting in a functional chimera. To date, the majority of mosaic genes are derived from sections of three RPP genes, tet(O), tet(W) and tet(32), with others comprising tet(M) and tet(S). In this first review of mosaic genes, we report on their structure, diversity and prevalence, and suggest that these genes may be responsible for an under-reported contribution to tetracycline resistance in bacteria.

  10. Human APOBEC3 proteins, retrovirus restriction, and HIV drug resistance.

    PubMed

    Haché, Guylaine; Mansky, Louis M; Harris, Reuben S

    2006-01-01

    Over 40 million people worldwide currently have HIV/AIDS. Many antiretroviral drugs have proven effective, but drug-resistant HIV variants frequently emerge to thwart treatment efforts. Reverse transcription errors undoubtedly contribute to drug resistance, but additional significant sources of viral genetic variation are debatable. The human APOBEC3F and APOBEC3G proteins can potently inhibit retrovirus infection by a mechanism that involves retroviral cDNA cytosine deamination. Here we review the current knowledge on the mechanism of APOBEC3-dependent retrovirus restriction and discuss whether this innate host-defense system actively contributes to HIV genetic variation.

  11. Mosaic tetracycline resistance genes encoding ribosomal protection proteins

    PubMed Central

    Warburton, Philip J.; Amodeo, Nina; Roberts, Adam P.

    2016-01-01

    First reported in 2003, mosaic tetracycline resistance genes are a subgroup of the genes encoding ribosomal protection proteins (RPPs). They are formed when two or more RPP-encoding genes recombine resulting in a functional chimera. To date, the majority of mosaic genes are derived from sections of three RPP genes, tet(O), tet(W) and tet(32), with others comprising tet(M) and tet(S). In this first review of mosaic genes, we report on their structure, diversity and prevalence, and suggest that these genes may be responsible for an under-reported contribution to tetracycline resistance in bacteria. PMID:27494928

  12. Multidrug resistance protein gene expression in Trichoplusia ni caterpillars.

    PubMed

    Simmons, Jason; D'Souza, Olivia; Rheault, Mark; Donly, Cam

    2013-02-01

    Many insect species exhibit pesticide-resistant phenotypes. One of the mechanisms capable of contributing to resistance is the overexpression of multidrug resistance (MDR) transporter proteins. Here we describe the cloning of three genes encoding MDR proteins from Trichoplusia ni: trnMDR1, trnMDR2 and trnMDR3. Real-time quantitative PCR (qPCR) detected trnMDR mRNA in the whole nervous system, midgut and Malpighian tubules of final instar T. ni caterpillars. To test whether these genes are upregulated in response to chemical challenge in this insect, qPCR was used to compare trnMDR mRNA levels in unchallenged insects with those of insects fed the synthetic pyrethroid, deltamethrin. Only limited increases were detected in a single gene, trnMDR2, which is the most weakly expressed of the three MDR genes, suggesting that increased multidrug resistance of this type is not a significant part of the response to deltamethrin exposure.

  13. The Assembly Motif of a Bacterial Small Multidrug Resistance Protein*

    PubMed Central

    Poulsen, Bradley E.; Rath, Arianna; Deber, Charles M.

    2009-01-01

    Multidrug transporters such as the small multidrug resistance (SMR) family of bacterial integral membrane proteins are capable of conferring clinically significant resistance to a variety of common therapeutics. As antiporter proteins of ∼100 amino acids, SMRs must self-assemble into homo-oligomeric structures for efflux of drug molecules. Oligomerization centered at transmembrane helix four (TM4) has been implicated in SMR assembly, but the full complement of residues required to mediate its self-interaction remains to be characterized. Here, we use Hsmr, the 110-residue SMR family member of the archaebacterium Halobacterium salinarum, to determine the TM4 residue motif required to mediate drug resistance and SMR self-association. Twelve single point mutants that scan the central portion of the TM4 helix (residues 85–104) were constructed and were tested for their ability to confer resistance to the cytotoxic compound ethidium bromide. Six residues were found to be individually essential for drug resistance activity (Gly90, Leu91, Leu93, Ile94, Gly97, and Val98), defining a minimum activity motif of 90GLXLIXXGV98 within TM4. When the propensity of these mutants to dimerize on SDS-PAGE was examined, replacements of all but Ile resulted in ∼2-fold reduction of dimerization versus the wild-type antiporter. Our work defines a minimum activity motif of 90GLXLIXXGV98 within TM4 and suggests that this sequence mediates TM4-based SMR dimerization along a single helix surface, stabilized by a small residue heptad repeat sequence. These TM4-TM4 interactions likely constitute the highest affinity locus for disruption of SMR function by directly targeting its self-assembly mechanism. PMID:19224913

  14. Molecular basis of glyphosate resistance: Different approaches through protein engineering

    PubMed Central

    Pollegioni, Loredano; Schonbrunn, Ernst; Siehl, Daniel

    2011-01-01

    Glyphosate (N-phosphonomethyl-glycine) is the most-used herbicide in the world: glyphosate-based formulations exhibit broad-spectrum herbicidal activity with minimal human and environmental toxicity. The extraordinary success of this simple small molecule is mainly due to the high specificity of glyphosate towards the plant enzyme enolpyruvylshikimate-3-phosphate synthase in the shikimate pathway leading to biosynthesis of aromatic amino acids. Starting in 1996, transgenic glyphosate-resistant plants were introduced thus allowing the application of the herbicide to the crop (post-emergence) to remove emerged weeds without crop damage. This review focuses on the evolution of mechanisms of resistance to glyphosate as obtained through natural diversity, the gene shuffling approach to molecular evolution, and a rational, structure-based approach to protein engineering. In addition, we offer rationale for the means by which the modifications made have had their intended effect. PMID:21668647

  15. Blast resistance of CC-NB-LRR protein Pb1 is mediated by WRKY45 through protein-protein interaction.

    PubMed

    Inoue, Haruhiko; Hayashi, Nagao; Matsushita, Akane; Xinqiong, Liu; Nakayama, Akira; Sugano, Shoji; Jiang, Chang-Jie; Takatsuji, Hiroshi

    2013-06-04

    Panicle blast 1 (Pb1) is a panicle blast resistance gene derived from the indica rice cultivar "Modan." Pb1 encodes a coiled-coil-nucleotide-binding site-leucine-rich repeat (CC-NB-LRR) protein and confers durable, broad-spectrum resistance to Magnaporthe oryzae races. Here, we investigated the molecular mechanisms underlying Pb1-mediated blast resistance. The Pb1 protein interacted with WRKY45, a transcription factor involved in induced resistance via the salicylic acid signaling pathway that is regulated by the ubiquitin proteasome system. Pb1-mediated panicle blast resistance was largely compromised when WRKY45 was knocked down in a Pb1-containing rice cultivar. Leaf-blast resistance by Pb1 overexpression (Pb1-ox) was also compromised in WRKY45 knockdown/Pb1-ox rice. Blast infection induced higher accumulation of WRKY45 in Pb1-ox than in control Nipponbare rice. Overexpression of Pb1-Quad, a coiled-coil domain mutant that had weak interaction with WRKY45, resulted in significantly weaker blast resistance than that of wild-type Pb1. Overexpression of Pb1 with a nuclear export sequence failed to confer blast resistance to rice. These results suggest that the blast resistance of Pb1 depends on its interaction with WRKY45 in the nucleus. In a transient system using rice protoplasts, coexpression of Pb1 enhanced WRKY45 accumulation and increased WRKY45-dependent transactivation activity, suggesting that protection of WRKY45 from ubiquitin proteasome system degradation is possibly involved in Pb1-dependent blast resistance.

  16. Listeria phage and phage tail induction triggered by components of bacterial growth media (phosphate, LiCl, nalidixic acid, and acriflavine).

    PubMed

    Lemaître, Jean-Paul; Duroux, Amandine; Pimpie, Romain; Duez, Jean-Marie; Milat, Marie-Louise

    2015-03-01

    The detection of Listeria monocytogenes from food is currently carried out using a double enrichment. For the ISO methodology, this double enrichment is performed using half-Fraser and Fraser broths, in which the overgrowth of L. innocua can occur in samples where both species are present. In this study, we analyzed the induction of phages and phage tails of Listeria spp. in these media and in two brain heart infusion (BHI) broths (BHIM [bioMérieux] and BHIK [Biokar]) to identify putative effectors. It appears that Na2HPO4 at concentrations ranging from 1 to 40 g/liter with an initial pH of 7.5 can induce phage or phage tail production of Listeria spp., especially with 10 g/liter of Na2HPO4 and a pH of 7.5, conditions present in half-Fraser and Fraser broths. Exposure to LiCl in BHIM (18 to 21 g/liter) can also induce phage and phage tail release, but in half-Fraser and Fraser broths, the concentration of LiCl is much lower (3 g/liter). Low phage titers were induced by acriflavine and/or nalidixic acid. We also show that the production of phages and phage tails can occur in half-Fraser and Fraser broths. This study points out that induction of phages and phage tails could be triggered by compounds present in enrichment media. This could lead to a false-negative result for the detection of L. monocytogenes in food products.

  17. Uncoupling Proteins: Role in Insulin Resistance and Insulin Insufficiency

    PubMed Central

    Chan, Catherine B.; Harper, Mary-Ellen

    2010-01-01

    Uncoupling proteins (UCPs) are modulators of mitochondrial metabolism that have been implicated in the development of both insulin resistance and insulin insufficiency, the two major pathophysiological events associated with type 2 diabetes. UCP2 mRNA is expressed in a wide range of tissues; however UCP2 protein expression is restricted to fewer tissues, including the endocrine pancreas, spleen, stomach, brain and the lung. To date, its role in the pathophysiology of diabetes has been most strongly associated with impaired glucose-stimulated insulin secretion from the β-cell, particularly after its induction by free fatty acids. The physiological role of UCP2 remains controversial, but it may act as a downstream signal transducer of superoxide. UCP3 mRNA and protein are expressed in relatively few tissues, predominately skeletal muscle, brown adipose tissue and heart. Increased expression of UCP3 in skeletal muscle is associated with protection from diet-induced insulin resistance in mice. In patients with type 2 diabetes UCP3 protein in muscle is reduced by 50% compared to healthy controls. The primary physiological role of the novel UCPs does not appear to be protection against positive energy balance and obesity; this is based largely on findings from studies of UCP2 and UCP3 knockout mice and from observed increases in UCP3 expression with fasting. The mechanism(s) of action of UCP2 and UCP3 are poorly understood. However, findings support roles for UCP2 and UCP3 as modifiers of fatty acid metabolism and in mitigating damage from reactive oxygen species. PMID:18220632

  18. Protein Anabolic Resistance in Cancer: Does it really exist?

    PubMed Central

    Engelen, Mariëlle P. K. J.; van der Meij, Barbara S.; Deutz, Nicolaas E. P.

    2016-01-01

    Purpose of review Preventing unintentional weight and muscle loss is of crucial importance to maintain the condition and well-being of patients with cancer, improve treatment response and tolerance, and prolong survival. Anabolic resistance might explain why some cancer patients do not respond to nutritional intervention but does recent evidence actually support this? We will discuss recent literature that cast doubt on attenuated anabolic potential in cancer. Recent findings Although anabolic resistance was observed in the past, more recent studies have shown that advanced cancer patients have an anabolic potential after intake of high-quality proteins. Furthermore a consistent linear relationship is observed in cancer between (essential) amino acid availability from the diet and net protein gain. The studied cancer patients however were often characterized by a normal or obese body weight, following the trend in the general population, and mild systemic inflammation. Factors like recent chemotherapy, surgery or cachexia do not seem to attenuate the anabolic potential to feeding. Summary Cancer patients have a normal anabolic potential which relates to the amount of essential amino acids in the meal. It remains to be determined if this is also the case in weak cancer patients with a short life expectancy and high systemic inflammation. PMID:26560520

  19. Role of Breast Cancer Resistance Protein (BCRP/ABCG2) in Cancer Drug Resistance

    PubMed Central

    Natarajan, Karthika; Xie, Yi; Baer, Maria R.; Ross, Douglas D.

    2012-01-01

    Since cloning of the ATP-binding cassette (ABC) family member breast cancer resistance protein (BCRP/ABCG2) and its characterization as a multidrug resistance efflux transporter in 1998, BCRP has been the subject of more than two thousand scholarly articles. In normal tissues, BCRP functions as a defense mechanism against toxins and xenobiotics, with expression in the gut, bile canaliculi, placenta, blood-testis and blood-brain barriers facilitating excretion and limiting absorption of potentially toxic substrate molecules, including many cancer chemotherapeutic drugs. BCRP also plays a key role in heme and folate homeostasis, which may help normal cells survive under conditions of hypoxia. BCRP expression appears to be a characteristic of certain normal tissue stem cells termed “side population cells,” which are identified on flow cytometric analysis by their ability to exclude Hoechst 33342, a BCRP substrate fluorescent dye. Hence, BCRP expression may contribute to the natural resistance and longevity of these normal stem cells. Malignant tissues can exploit the properties of BCRP to survive hypoxia and to evade exposure to chemotherapeutic drugs. Evidence is mounting that many cancers display subpopulations of stem cells that are responsible for tumor self-renewal. Such stem cells frequently manifest the “side population” phenotype characterized by expression of BCRP and other ABC transporters. Along with other factors, these transporters may contribute to the inherent resistance of these neoplasms and their failure to be cured. PMID:22248732

  20. Studies on pyrrolopyrimidines as selective inhibitors of multidrug-resistance-associated protein in multidrug resistance.

    PubMed

    Wang, Shouming; Folkes, Adrian; Chuckowree, Irina; Cockcroft, Xiaoling; Sohal, Sukhjit; Miller, Warren; Milton, John; Wren, Stephen P; Vicker, Nigel; Depledge, Paul; Scott, John; Smith, Lyndsay; Jones, Hazel; Mistry, Prakash; Faint, Richard; Thompson, Deanne; Cocks, Simon

    2004-03-11

    Multidrug resistance mediated by P-glycoprotein (Pgp) or multidrug-resistance-associated protein (MRP) remains a major obstacle for successful treatment of cancer. Inhibition of Pgp and MRP transport is important for high efficacy of anticancer drugs. While several Pgp inhibitors have entered clinical trials, the development of specific MRP1 inhibitors is still in its infancy. In our screening program, we have identified a pyrrolopyrimidine (4) as a novel and selective MRP1 inhibitor. Subsequent SAR work on the 4-position of the template revealed the phenethylpiperazine side chain as a potent replacement of the benzylthio group of the lead molecule. Introduction of groups at the 2-position seems to have no detrimental effect on activity. Modifications to the nitrile group at the 7-position resulted in the identification of analogues with groups, such as amides, with superior pharmacokinetic profiles. In vivo efficacy has been demonstrated by xenograft studies on selected compounds.

  1. Extracellular Proteins: Novel Key Components of Metal Resistance in Cyanobacteria?

    PubMed Central

    Giner-Lamia, Joaquín; Pereira, Sara B.; Bovea-Marco, Miquel; Futschik, Matthias E.; Tamagnini, Paula; Oliveira, Paulo

    2016-01-01

    Metals are essential for all living organisms and required for fundamental biochemical processes. However, when in excess, metals can turn into highly-toxic agents able to disrupt cell membranes, alter enzymatic activities, and damage DNA. Metal concentrations are therefore tightly controlled inside cells, particularly in cyanobacteria. Cyanobacteria are ecologically relevant prokaryotes that perform oxygenic photosynthesis and can be found in many different marine and freshwater ecosystems, including environments contaminated with heavy metals. As their photosynthetic machinery imposes high demands for metals, homeostasis of these micronutrients has been widely studied in cyanobacteria. So far, most studies have focused on how cells are capable of controlling their internal metal pools, with a strong bias toward the analysis of intracellular processes. Ultrastructure, modulation of physiology, dynamic changes in transcription and protein levels have been studied, but what takes place in the extracellular environment when cells are exposed to an unbalanced metal availability remains largely unknown. The interest in studying the subset of proteins present in the extracellular space has only recently begun and the identification and functional analysis of the cyanobacterial exoproteomes are just emerging. Remarkably, metal-related proteins such as the copper-chaperone CopM or the iron-binding protein FutA2 have already been identified outside the cell. With this perspective, we aim to raise the awareness that metal-resistance mechanisms are not yet fully known and hope to motivate future studies assessing the role of extracellular proteins on bacterial metal homeostasis, with a special focus on cyanobacteria. PMID:27375598

  2. The TolC Homologue of Brucella suis Is Involved in Resistance to Antimicrobial Compounds and Virulence▿

    PubMed Central

    Posadas, Diana M.; Martín, Fernando A.; Sabio y García, Julia V.; Spera, Juan M.; Delpino, M. Victoria; Baldi, Pablo; Campos, Eleonora; Cravero, Silvio L.; Zorreguieta, Angeles

    2007-01-01

    Brucella spp., like other pathogens, must cope with the environment of diverse host niches during the infection process. In doing this, pathogens evolved different type of transport systems to help them survive and disseminate within the host. Members of the TolC family have been shown to be involved in the export of chemically diverse molecules ranging from large protein toxins to small toxic compounds. The role of proteins from the TolC family in Brucella and other α-2-proteobacteria has been explored little. The gene encoding the unique member of the TolC family from Brucella suis (BepC) was cloned and expressed in an Escherichia coli mutant disrupted in the gene encoding TolC, which has the peculiarity of being involved in diverse transport functions. BepC fully complemented the resistance to drugs such as chloramphenicol and acriflavine but was incapable of restoring hemolysin secretion in the tolC mutant of E. coli. An insertional mutation in the bepC gene strongly affected the resistance phenotype of B. suis to bile salts and toxic chemicals such as ethidium bromide and rhodamine and significantly decreased the resistance to antibiotics such as erythromycin, ampicillin, tetracycline, and norfloxacin. Moreover, the B. suis bepC mutant was attenuated in the mouse model of infection. Taken together, these results suggest that BepC-dependent efflux processes of toxic compounds contribute to B. suis survival inside the host. PMID:17088356

  3. Role of the Mmr Efflux Pump in Drug Resistance in Mycobacterium tuberculosis

    PubMed Central

    Rodrigues, Liliana; Villellas, Cristina; Bailo, Rebeca; Viveiros, Miguel

    2013-01-01

    Efflux pumps are membrane proteins capable of actively transporting a broad range of substrates from the cytoplasm to the exterior of the cell. Increased efflux activity in response to drug treatment may be the first step in the development of bacterial drug resistance. Previous studies showed that the efflux pump Mmr was significantly overexpressed in strains exposed to isoniazid. In the work to be described, we constructed mutants lacking or overexpressing Mmr in order to clarify the role of this efflux pump in the development of resistance to isoniazid and other drugs in M. tuberculosis. The mmr knockout mutant showed an increased susceptibility to ethidium bromide, tetraphenylphosphonium, and cetyltrimethylammonium bromide (CTAB). Overexpression of mmr caused a decreased susceptibility to ethidium bromide, acriflavine, and safranin O that was obliterated in the presence of the efflux inhibitors verapamil and carbonyl cyanide m-chlorophenylhydrazone. Isoniazid susceptibility was not affected by the absence or overexpression of mmr. The fluorometric method allowed the detection of a decreased efflux of ethidium bromide in the knockout mutant, whereas the overexpressed strain showed increased efflux of this dye. This increased efflux activity was inhibited in the presence of efflux inhibitors. Under our experimental conditions, we have found that efflux pump Mmr is mainly involved in the susceptibility to quaternary compounds such as ethidium bromide and disinfectants such as CTAB. The contribution of this efflux pump to isoniazid resistance in Mycobacterium tuberculosis still needs to be further elucidated. PMID:23165464

  4. Modelling proteins' hidden conformations to predict antibiotic resistance

    NASA Astrophysics Data System (ADS)

    Hart, Kathryn M.; Ho, Chris M. W.; Dutta, Supratik; Gross, Michael L.; Bowman, Gregory R.

    2016-10-01

    TEM β-lactamase confers bacteria with resistance to many antibiotics and rapidly evolves activity against new drugs. However, functional changes are not easily explained by differences in crystal structures. We employ Markov state models to identify hidden conformations and explore their role in determining TEM's specificity. We integrate these models with existing drug-design tools to create a new technique, called Boltzmann docking, which better predicts TEM specificity by accounting for conformational heterogeneity. Using our MSMs, we identify hidden states whose populations correlate with activity against cefotaxime. To experimentally detect our predicted hidden states, we use rapid mass spectrometric footprinting and confirm our models' prediction that increased cefotaxime activity correlates with reduced Ω-loop flexibility. Finally, we design novel variants to stabilize the hidden cefotaximase states, and find their populations predict activity against cefotaxime in vitro and in vivo. Therefore, we expect this framework to have numerous applications in drug and protein design.

  5. Role of C reactive protein (CRP) in leptin resistance.

    PubMed

    Hribal, Marta Letizia; Fiorentino, Teresa Vanessa; Sesti, Giorgio

    2014-01-01

    Increased plasma levels of both leptin and C reactive protein (CRP) have been reported in a number of conditions, including obesity, and have been linked to cardiovascular pathophysiological processes and increased cardiovascular risk; interestingly these two biomarkers appear to be able to reciprocally regulate their bioavailability, through complex mechanisms that have not been completely clarified yet. Here we first review clinical evidence suggesting not only that the circulatory levels of CRP and leptin show an independent correlation, but also that assessing them in tandem may result in an increased ability to predict cardiovascular disease. We summarize also molecular studies showing that leptin is able to promote CRP production from hepatocytes and endothelial cells in vitro and discuss the studies addressing the possibility that in vivo leptin administration may be able to modulate plasma CRP levels. Furthermore, we describe two studies demonstrating that CRP directly binds leptin in extra-cellular settings, thus impairing its biological actions. Finally we report genetic evidence that common variations at the leptin receptor locus are associated with CRP blood levels. Overall, the data reviewed here show that the chronic elevation of CRP observed in obese subjects may worsen leptin resistance, contributing to the pathogenesis of cardiovascular disease, and highlight a potential link between conditions, such as leptin resistance and endothelial dysfunction, that may be amenable of pharmacological treatment targeted to the disruption of leptin-CRP interaction.

  6. Exocytosis of polyubiquitinated proteins in bortezomib-resistant leukemia cells: a role for MARCKS in acquired resistance to proteasome inhibitors

    PubMed Central

    Franke, Niels E.; Kaspers, Gertjan L.; Assaraf, Yehuda G.; van Meerloo, Johan; Niewerth, Denise; Kessler, Floortje L.; Poddighe, Pino J.; Kole, Jeroen; Smeets, Serge J.; Ylstra, Bauke; Bi, Chonglei; Chng, Wee Joo; Horton, Terzah M.; Menezes, Rene X.; Musters, Renée J.P.; Zweegman, Sonja; Jansen, Gerrit; Cloos, Jacqueline

    2016-01-01

    PSMB5 mutations and upregulation of the β5 subunit of the proteasome represent key determinants of acquired resistance to the proteasome inhibitor bortezomib (BTZ) in leukemic cells in vitro. We here undertook a multi-modality (DNA, mRNA, miRNA) array-based analysis of human CCRF-CEM leukemia cells and BTZ-resistant subclones to determine whether or not complementary mechanisms contribute to BTZ resistance. These studies revealed signatures of markedly reduced expression of proteolytic stress related genes in drug resistant cells over a broad range of BTZ concentrations along with a high upregulation of myristoylated alanine-rich C-kinase substrate (MARCKS) gene expression. MARCKS upregulation was confirmed on protein level and also observed in other BTZ-resistant tumor cell lines as well as in leukemia cells with acquired resistance to other proteasome inhibitors. Moreover, when MARCKS protein expression was demonstrated in specimens derived from therapy-refractory pediatric leukemia patients (n = 44), higher MARCKS protein expression trended (p = 0.073) towards a dismal response to BTZ-containing chemotherapy. Mechanistically, we show a BTZ concentration-dependent association of MARCKS protein levels with the emergence of ubiquitin-containing vesicles in BTZ-resistant CEM cells. These vesicles were found to be extruded and taken up in co-cultures with proteasome-proficient acceptor cells. Consistent with these observations, MARCKS protein associated with ubiquitin-containing vesicles was also more prominent in clinical leukemic specimen with ex vivo BTZ resistance compared to BTZ-sensitive leukemia cells. Collectively, we propose a role for MARCKS in a novel mechanism of BTZ resistance via exocytosis of ubiquitinated proteins in BTZ-resistant cells leading to quenching of proteolytic stress. PMID:27542283

  7. Biased random walk model for the prioritization of drug resistance associated proteins

    PubMed Central

    Guo, Hao; Dong, Jiaqiang; Hu, Sijun; Cai, Xiqiang; Tang, Guangbo; Dou, Jianhua; Tian, Miaomiao; He, Fuchu; Nie, Yongzhan; Fan, Daiming

    2015-01-01

    Multi-drug resistance is the main cause of treatment failure in cancer patients. How to identify molecules underlying drug resistance from multi-omics data remains a great challenge. Here, we introduce a data biased strategy, ProteinRank, to prioritize drug-resistance associated proteins in cancer cells. First, we identified differentially expressed proteins in Adriamycin and Vincristine resistant gastric cancer cells compared to their parental cells using iTRAQ combined with LC-MS/MS experiments, and then mapped them to human protein-protein interaction network; second, we applied ProteinRank to analyze the whole network and rank proteins similar to known drug resistance related proteins. Cross validations demonstrated a better performance of ProteinRank compared to the method without usage of MS data. Further validations confirmed the altered expressions or activities of several top ranked proteins. Functional study showed PIM3 or CAV1 silencing was sufficient to reverse the drug resistance phenotype. These results indicated ProteinRank could prioritize key proteins related to drug resistance in gastric cancer and provided important clues for cancer research. PMID:26039373

  8. [National evaluation of the diagnosis of activated protein C resistance].

    PubMed

    Montiel-Manzano, Guadalupe; de la Peña-Díaz, Aurora; Majluf-Cruz, Abraham; Cesarman-Maus, Gabriela; Corona-de la Peña, Norma; Cruz-Cruz, Donají; Gaminio, Elizabeth; Martínez-Murillo, Carlos; Mayagoitia, Teresa; Miranda-Peralta, Enrique; Poblete, Teresita; Quintana-Martínez, Sandra; Ramírez, Raúl; Razo, Daniel; Ruiz de Chávez-Ochoa, Adriana; Reyes-Núñez, Virginia Adriana; Salazar, Rosario; Vicencio-Santiago, Guadalupe Virginia; Villa, Rosario; Reyes-Núñez, Aurelia Virginia

    2003-01-01

    Thrombophilia or prothrombotic state appears when activation of blood hemostatic mechanisms overcomes the physiological anticoagulant capacity allowing a thrombotic event. Thrombosis is the leading worldwide mortality cause and due to its high associated morbidity and mortality, it should be insisted in the opportune identification of a thrombophilic state. The study of thrombophilia identifies individuals at high risk for thrombosis. This meeting was conceived first to analyze the current status of the diagnosis of thrombophilia in Mexico and second to create the base for a national consensus for thrombophilia screening and for the establishment of a national center for laboratory reference and quality control for thrombophilia. Since searching of activated protein C resistance (APCR) and FV Leiden seem to have priority either in the clinical setting and in public health services, it was decided to start with these two abnormalities as a model to analyze the current status of thrombophilia diagnosis in the clinical laboratory. At this time, several thrombophilic abnormalities have been described however, APCR remains the most important cause of thrombophilia, accounting for as much as 20% to 60% of all venous thrombosis. APCR is a consequence of the resistance of activated FV to be inactivated by activated protein C. Procoagulant activity of activated FV increases the risk of thrombosis. Hereditary APCR is almost always due to a point mutation at the nucleotide 1691 of the FV gen inducing an Arg506Glu substitution in FV molecule. This mutation is better known as FV Leiden. Heterocygous carriers of FV Leiden have a thrombotic risk 5 to 10 times higher than general population while the risk for the homocygote state is increased 50 to 100-fold. When activated PC is added to plasma from patients with FV Leiden, this last resists the anticoagulant effect of activated PC. Therefore, thrombin production is not inhibited. This phenomenon is called APCR. The functional

  9. Protein kinase PKR mutants resistant to the poxvirus pseudosubstrate K3L protein.

    PubMed

    Seo, Eun Joo; Liu, Furong; Kawagishi-Kobayashi, Makiko; Ung, Tekly L; Cao, Chune; Dar, Arvin C; Sicheri, Frank; Dever, Thomas E

    2008-11-04

    As part of the mammalian cell innate immune response, the double-stranded RNA activated protein kinase PKR phosphorylates the translation initiation factor eIF2alpha to inhibit protein synthesis and thus block viral replication. Poxviruses including vaccinia and smallpox viruses express PKR inhibitors such as the vaccinia virus K3L protein that resembles the N-terminal substrate-targeting domain of eIF2alpha. Whereas high-level expression of human PKR was toxic in yeast, this growth inhibition was suppressed by coexpression of the K3L protein. We used this yeast assay to screen for PKR mutants that are resistant to K3L inhibition, and we identified 12 mutations mapping to the C-terminal lobe of the PKR kinase domain. The PKR mutations specifically conferred resistance to the K3L protein both in yeast and in vitro. Consistently, the PKR-D486V mutation led to nearly a 15-fold decrease in K3L binding affinity yet did not impair eIF2alpha phosphorylation. Our results support the identification of the eIF2alpha-binding site on an extensive face of the C-terminal lobe of the kinase domain, and they indicate that subtle changes to the PKR kinase domain can drastically impact pseudosubstrate inhibition while leaving substrate phosphorylation intact. We propose that these paradoxical effects of the PKR mutations on pseudosubstrate vs. substrate interactions reflect differences between the rigid K3L protein and the plastic nature of eIF2alpha around the Ser-51 phosphorylation site.

  10. Overcoming Resistance to Inhibitors of the Akt Protein Kinase by Modulation of the Pim Kinase Pathway

    DTIC Science & Technology

    2014-10-01

    kinase . This grant proposal will explore the resistance to small molecule AKT protein kinase inhibitors mediated by the... molecule AKT protein kinase inhibitors is potentially mediated by the Pim-1 protein kinase , and that unique Pim protein kinase inhibitors that can in...application is essential for the development of this combined chemotherapeutic strategy. 15. SUBJECT TERMS Small Molecule AKT Inhibitors ,

  11. Physical association of the NB-LRR resistance protein Rx with a Ran GTPase-activating protein is required for extreme resistance to Potato virus X.

    PubMed

    Tameling, Wladimir I L; Baulcombe, David C

    2007-05-01

    Nucleotide binding leucine-rich repeat (NB-LRR) proteins play an important role in plant and mammalian innate immunity. In plants, these resistance proteins recognize specific pathogen-derived effector proteins. Recognition subsequently triggers a rapid and efficient defense response often associated with the hypersensitive response and other poorly understood processes that suppress the pathogen. To investigate mechanisms associated with the activation of disease resistance responses, we investigated proteins binding to the potato (Solanum tuberosum) NB-LRR protein Rx that confers extreme resistance to Potato virus X (PVX) in potato and Nicotiana benthamiana. By affinity purification experiments, we identified an endogenous N. benthamiana Ran GTPase-Activating Protein2 (RanGAP2) as an Rx-associated protein in vivo. Further characterization confirmed the specificity of this interaction and showed that the association occurs through their N-terminal domains. By specific virus-induced gene silencing of RanGAP2 in N. benthamiana carrying Rx, we demonstrated that this interaction is required for extreme resistance to PVX and suggest that RanGAP2 is part of the Rx signaling complex. These results implicate RanGAP-mediated cellular mechanisms, including nucleocytoplasmic trafficking, in the activation of disease resistance.

  12. Outer Membrane Proteins form Specific Patterns in Antibiotic-Resistant Edwardsiella tarda

    PubMed Central

    Peng, Bo; Wang, Chao; Li, Hui; Su, Yu-bin; Ye, Jin-zhou; Yang, Man-jun; Jiang, Ming; Peng, Xuan-xian

    2017-01-01

    Outer membrane proteins of Gram-negative bacteria play key roles in antibiotic resistance. However, it is unknown whether outer membrane proteins that respond to antibiotics behave in a specific manner. The present study specifically investigated the differentially expressed outer membrane proteins of an antibiotic-resistant bacterium, Edwardsiella tarda, a Gram-negative pathogen that can lead to unnecessary mass medication of antimicrobials and consequently resistance development in aquaculture and a spectrum of intestinal and extraintestinal diseases in humans. The comparison of a clinically isolated strain to the laboratory derived kanamycin-, tetracycline-, or chloramphenicol-resistant strains identified their respective outer membrane proteins expression patterns, which are distinct to each other. Similarly, the same approach was utilized to profile the patterns in double antibiotic-resistant bacteria. Surprisingly, one pattern is always dominant over the other as to these three antibiotics; the pattern of chloramphenicol is over tetracycline, which is over kanamycin. This type of pattern was also confirmed in clinically relevant multidrug-resistant bacteria. In addition, the presence of plasmid encoding antibiotic-resistant genes also alters the outer membrane protein profile in a similar manner. Our results demonstrate that bacteria adapt the antibiotic stress through the regulation of outer membrane proteins expression. And more importantly, different outer membrane protein profiles were required to cope with different antibiotics. This type of specific pattern provides the rationale for the development of novel strategy to design outer membrane protein arrays to identify diverse multidrug resistance profiles as biomarkers for clinical medication. PMID:28210241

  13. Outer Membrane Proteins form Specific Patterns in Antibiotic-Resistant Edwardsiella tarda.

    PubMed

    Peng, Bo; Wang, Chao; Li, Hui; Su, Yu-Bin; Ye, Jin-Zhou; Yang, Man-Jun; Jiang, Ming; Peng, Xuan-Xian

    2017-01-01

    Outer membrane proteins of Gram-negative bacteria play key roles in antibiotic resistance. However, it is unknown whether outer membrane proteins that respond to antibiotics behave in a specific manner. The present study specifically investigated the differentially expressed outer membrane proteins of an antibiotic-resistant bacterium, Edwardsiella tarda, a Gram-negative pathogen that can lead to unnecessary mass medication of antimicrobials and consequently resistance development in aquaculture and a spectrum of intestinal and extraintestinal diseases in humans. The comparison of a clinically isolated strain to the laboratory derived kanamycin-, tetracycline-, or chloramphenicol-resistant strains identified their respective outer membrane proteins expression patterns, which are distinct to each other. Similarly, the same approach was utilized to profile the patterns in double antibiotic-resistant bacteria. Surprisingly, one pattern is always dominant over the other as to these three antibiotics; the pattern of chloramphenicol is over tetracycline, which is over kanamycin. This type of pattern was also confirmed in clinically relevant multidrug-resistant bacteria. In addition, the presence of plasmid encoding antibiotic-resistant genes also alters the outer membrane protein profile in a similar manner. Our results demonstrate that bacteria adapt the antibiotic stress through the regulation of outer membrane proteins expression. And more importantly, different outer membrane protein profiles were required to cope with different antibiotics. This type of specific pattern provides the rationale for the development of novel strategy to design outer membrane protein arrays to identify diverse multidrug resistance profiles as biomarkers for clinical medication.

  14. Comparative proteomic analysis of horseweed (Conyza canadensis) biotypes identifies candidate proteins for glyphosate resistance

    PubMed Central

    González-Torralva, Fidel; Brown, Adrian P.; Chivasa, Stephen

    2017-01-01

    Emergence of glyphosate-resistant horseweed (Conyza canadensis) biotypes is an example of how unrelenting use of a single mode of action herbicide in agricultural weed control drives genetic adaptation in targeted species. While in other weeds glyphosate resistance arose from target site mutation or target gene amplification, the resistance mechanism in horseweed uses neither of these, being instead linked to reduced herbicide uptake and/or translocation. The molecular components underpinning horseweed glyphosate-resistance remain unknown. Here, we used an in vitro leaf disc system for comparative analysis of proteins extracted from control and glyphosate-treated tissues of glyphosate-resistant and glyphosate-susceptible biotypes. Analysis of shikimic acid accumulation, ABC-transporter gene expression, and cell death were used to select a suitable glyphosate concentration and sampling time for enriching proteins pivotal to glyphosate resistance. Protein gel analysis and mass spectrometry identified mainly chloroplast proteins differentially expressed between the biotypes before and after glyphosate treatment. Chloroplasts are the organelles in which the shikimate pathway, which is targeted by glyphosate, is located. Calvin cycle enzymes and proteins of unknown function were among the proteins identified. Our study provides candidate proteins that could be pivotal in engendering resistance and implicates chloroplasts as the primary sites driving glyphosate-resistance in horseweed. PMID:28198407

  15. A Novel ATP-Binding Cassette Transporter Involved in Multidrug Resistance in the Phytopathogenic Fungus Penicillium digitatum

    PubMed Central

    Nakaune, Ryoji; Adachi, Kiichi; Nawata, Osamu; Tomiyama, Masamitsu; Akutsu, Katsumi; Hibi, Tadaaki

    1998-01-01

    Demethylation inhibitor (DMI)-resistant strains of the plant pathogenic fungus Penicillium digitatum were shown to be simultaneously resistant to cycloheximide, 4-nitroquinoline-N-oxide (4NQO), and acriflavine. A PMR1 (Penicillium multidrug resistance) gene encoding an ATP-binding cassette (ABC) transporter (P-glycoprotein) was cloned from a genomic DNA library of a DMI-resistant strain (LC2) of Penicillium digitatum by heterologous hybridization with a DNA fragment containing an ABC-encoding region from Botrytis cinerea. Sequence analysis revealed significant amino acid homology to the primary structures of PMR1 (protein encoded by the PMR1 gene) and ABC transporters of Saccharomyces cerevisiae (PDR5 and SNQ2), Schizosaccharomyces pombe (HBA2), Candida albicans (CDR1), and Aspergillus nidulans (AtrA and AtrB). Disruption of the PMR1 gene of P. digitatum DMI-resistant strain LC2 demonstrated that PMR1 was an important determinant of resistance to DMIs. The effective concentrations inhibiting radial growth by 50% (EC50s) and the MICs of fenarimol and bitertanol for the PMR1 disruptants (Δpmr1 mutants) were equivalent to those for DMI-sensitive strains. Northern blot analysis indicated that severalfold more PMR1 transcript accumulated in the DMI-resistant strains compared with those in DMI-sensitive strains in the absence of fungicide. In both DMI-resistant and -sensitive strains, transcription of PMR1 was strongly enhanced within 10 min after treatment with the DMI fungicide triflumizole. These results suggested that the toxicant efflux system comprised of PMR1 participates directly in the DMI resistance of the fungus. PMID:9758830

  16. Ribosomal protein alterations in thiostrepton- and Micrococcin-resistant mutants of Bacillus subtilis.

    PubMed

    Wienen, B; Ehrlich, R; Stöffler-Meilicke, M; Stöffler, G; Smith, I; Weiss, D; Vince, R; Pestka, S

    1979-08-25

    Ribosomal proteins of parental thiostrepton- and micrococcin-sensitive Bacillus subtilis cysA14 and thiostrepton-and micrococcin-resistant mutants were compared. Several electrophoretic and immunochemical techniques showed unambiguously that BS-L11 was not present on 50 S ribosomal subunits from the six thiostrepton-resistant mutants. Protein BS-L11 reappeared in all six revertants from thiostrepton resistance to thiostrepton sensitivity. No definitive protein alteration could be ascribed to the mutation from micrococcin sensitivity to resistance. It was also demonstrated that B. subtilis protein BS-L11 is homologous to Escherichia coli ribosomal protein L11. The finding that ribosomes from thiostrepton-resistant mutants do not contain protein L11 suggests that L11 not only is involved in binding of thiostrepton, but also, when mutationally altered, confers resistance to this antibiotic. Although the ribosomes of these strains do not contain protein L11, all thiostrepton-resistant mutants showed the same viability as the parental strain. Thus protein L11 cannot be obligatory for the structure and function of the ribosome.

  17. Heat-resistant protein expression during germination of maize seeds under water stress.

    PubMed

    Abreu, V M; Silva Neta, I C; Von Pinho, E V R; Naves, G M F; Guimarães, R M; Santos, H O; Von Pinho, R G

    2016-08-12

    Low water availability is one of the factors that limit agricultural crop development, and hence the development of genotypes with increased water stress tolerance is a challenge in plant breeding programs. Heat-resistant proteins have been widely studied, and are reported to participate in various developmental processes and to accumulate in response to stress. This study aimed to evaluate heat-resistant protein expression under water stress conditions during the germination of maize seed inbreed lines differing in their water stress tolerance. Maize seed lines 91 and 64 were soaked in 0, -0.3, -0.6, and -0.9 MPa water potential for 0, 6, 12, 18, and 24 h. Line 91 is considered more water stress-tolerant than line 64. The analysis of heat-resistant protein expression was made by gel electrophoresis and spectrophotometry. In general, higher expression of heat-resistant proteins was observed in seeds from line 64 subjected to shorter soaking periods and lower water potentials. However, in the water stress-tolerant line 91, a higher expression was observed in seeds that were subjected to -0.3 and -0.6 MPa water potentials. In the absence of water stress, heat-resistant protein expression was reduced with increasing soaking period. Thus, there was a difference in heat-resistant protein expression among the seed lines differing in water stress tolerance. Increased heat-resistant protein expression was observed in seeds from line 91 when subjected to water stress conditions for longer soaking periods.

  18. Differentially expressed proteins in fluconazole-susceptible and fluconazole-resistant isolates of Candida glabrata.

    PubMed

    Shen, Yinzhong; Zhang, Lijun; Jia, Xiaofang; Zhang, Yongxin; Lu, Hongzhou

    2015-06-01

    The current study aimed to identify the differences presented in the proteome of fluconazole-susceptible isolates of Candida glabrata compared to those with fluconazole-resistant ones. Two-dimensional differential gel electrophoresis was applied to identify proteins that were differentially expressed in fluconazole-susceptible and fluconazole-resistant isolates of C. glabrata. Eight proteins including aspartyl-tRNA synthetase, translation elongation factor 3, 3-phosphoglycerate kinase, ribosomal protein L5, coproporphyrinogen III oxidase, pyruvate kinase, G-beta like protein, and F1F0-ATPase alpha subunit were found to be more abundantly represented, while four proteins including vitamin B12-(cobalamin)-independent isozyme of methionine synthase, microtubule-associated protein, adenylosuccinate synthetase, and aldose reductase were found to be less abundantly represented in fluconazole-resistant strains versus those with fluconazole-susceptible ones. These differentially expressed proteins were primarily associated with energy metabolism, stress response, and macromolecule synthesis. Proteins associated with energy metabolism, stress response, and macromolecule synthesis may play a role in the development of fluconazole resistance in the clinical isolates of C. glabrata. Multiple different mechanisms are involved in the development of fluconazole resistance in C. glabrata. These findings provide a scientific basis for discovering new genes and mechanisms associated with fluconazole resistance in C. glabrata.

  19. Protein cyclization enhanced thermostability and exopeptidase-resistance of green fluorescent protein.

    PubMed

    Zhao, Zhonglin; Ma, Xin; Li, Liang; Zhang, Wei; Ping, Shuzhen; Xu, Ming-Qun; Lin, Min

    2010-03-01

    A mutant of green fluorescent protein (GFPmut3*) from the jellyfish Aequorea victoria was cyclized in vitro and in vivo by the use of a naturally split intein from the dnaE gene of Synechocystis species PCC6803 (Ssp). Cyclization of GFPmut3* was confirmed by amino acid sequencing and resulted in an increased electrophoretic mobility compared with the linear GFPmut3*. The circular GFPmut3* was 5 degrees C more thermostable than the linear form and significantly more resistant to proteolysis of exopeptidase. The circular GFPmut3* also displayed increased relative fluorescence intensity. In addition, chemical stability of GFPmut3* against GdnHCl revealed more stability of the circular form compared with the linear form.

  20. Crystal structure of the carbapenem intrinsic resistance protein CarG.

    PubMed

    Tichy, E M; Luisi, B F; Salmond, G P C

    2014-05-01

    In the Gram-negative enterobacterium Erwinia (Pectobacterium) and Serratia sp. ATCC 39006, intrinsic resistance to the carbapenem antibiotic 1-carbapen-2-em-3-carboxylic acid is mediated by the CarF and CarG proteins, by an unknown mechanism. Here, we report a high-resolution crystal structure for the Serratia sp. ATCC 39006 carbapenem resistance protein CarG. This structure of CarG is the first in the carbapenem intrinsic resistance (CIR) family of resistance proteins from carbapenem-producing bacteria. The crystal structure shows the protein to form a homodimer, in agreement with results from analytical gel filtration. The structure of CarG does not show homology with any known antibiotic resistance proteins nor does it belong to any well-characterised protein structural family. However, it is a close structural homologue of the bacterial inhibitor of invertebrate lysozyme, PliI-Ah, with some interesting structural variations, including the absence of the catalytic site responsible for lysozyme inhibition. Both proteins show a unique β-sandwich fold with short terminal α-helices. The core of the protein is formed by stacked anti-parallel sheets that are individually very similar in the two proteins but differ in their packing interface, causing the splaying of the two sheets in CarG. Furthermore, a conserved cation binding site identified in CarG is absent from the homologue.

  1. Differential expression of hemolymph proteins between susceptible and insecticide-resistant Blattella germanica (Blattodea: Blattellidae).

    PubMed

    Zhang, F; Wang, X J; Huang, Y H; Zhao, Z G; Zhang, S S; Gong, X S; Xie, L; Kang, D M; Jing, X

    2014-08-01

    A proteomic approach combining two-dimensional polyacrylamide gel electrophoresis and tandem mass spectrometry was used to compare hemolymph expression profiles of a beta-cypermethrin-resistant Blattella germanica L. strain and a beta-cypermethrin-susceptible strain. Twenty-eight hemolymph proteins were differentially expressed in the resistant cockroach strain; 19 proteins were upregulated and 9 proteins were downregulated compared with the susceptible strain. Protein identification indicated that expression of putative cuticular protein, nitric oxide synthase, triosephosphate isomerase, alpha-amylase, ABC transporter, and Per a 3 allergen was elevated, and expression of arginine kinase and glycosidase was reduced. The differential expression of these proteins reflects the overall change in cellular structure and metabolism related to the resistance of pyrethroid insecticides.

  2. High throughput atmospheric pressure plasma-induced graft polymerization for identifying protein-resistant surfaces.

    PubMed

    Gu, Minghao; Kilduff, James E; Belfort, Georges

    2012-02-01

    Three critical aspects of searching for and understanding how to find highly resistant surfaces to protein adhesion are addressed here with specific application to synthetic membrane filtration. They include the (i) discovery of a series of previously unreported monomers from a large library of monomers with high protein resistance and subsequent low fouling characteristics for membrane ultrafiltration of protein-containing fluids, (ii) development of a new approach to investigate protein-resistant mechanisms from structure-property relationships, and (iii) adaptation of a new surface modification method, called atmospheric pressure plasma-induced graft polymerization (APP), together with a high throughput platform (HTP), for low cost vacuum-free synthesis of anti-fouling membranes. Several new high-performing chemistries comprising two polyethylene glycol (PEG), two amines and one zwitterionic monomers were identified from a library (44 commercial monomers) of five different classes of monomers as strong protein-resistant monomers. Combining our analysis here, using the Hansen solubility parameters (HSP) approach, and data from the literature, we conclude that strong interactions with water (hydrogen bonding) and surface flexibility are necessary for producing the highest protein resistance. Superior protein-resistant surfaces and subsequent anti-fouling performance was obtained with the HTP-APP as compared with our earlier HTP-photo graft-induced polymerization (PGP).

  3. Comparative proteomic analysis reveals mite (Varroa destructor) resistance-related proteins in Eastern honeybees (Apis cerana).

    PubMed

    Ji, T; Shen, F; Liu, Z; Yin, L; Shen, J; Liang, Q; Luo, Y X

    2015-08-21

    The mite (Varroa destructor) has become the greatest threat to apiculture worldwide. As the original host of the mite, Apis cerana can effectively resist the mite. An increased understanding of the resistance mechanisms of Eastern honeybees against V. destructor may help researchers to protect other species against these parasites. In this study, the proteomes of 4 Apis cerana colonies were analyzed using an isobaric tag for relative and absolute quantitation technology. We determined the differences in gene and protein expression between susceptible and resistant colonies that were either unchallenged or challenged by V. destructor. The results showed that a total of 1532 proteins were identified. Gene Ontology enrichment analysis suggested that the transcription factors and basic metabolic and respiratory processes were efficient and feasible factors controlling this resistance, and 12 differentially expressed proteins were identified in Venn analysis. The results were validated by quantitative polymerase chain reaction. This study may provide insight into the genetic mechanisms underlying the resistance of honeybee to mites.

  4. There is a baby in the bath water: AcrB contamination is a major problem in membrane-protein crystallization.

    PubMed

    Veesler, David; Blangy, Stéphanie; Cambillau, Christian; Sciara, Giuliano

    2008-10-01

    In the course of a crystallographic study of the Methanosarcina mazei CorA transporter, the membrane protein was obtained with at least 95% purity and was submitted to crystallization trials. Small crystals (<100 microm) were grown that diffracted to 3.42 A resolution and belonged to space group R32, with unit-cell parameters a = b = 145.74, c = 514.0 A. After molecular-replacement attempts using available CorA structures as search models failed to yield a solution, it was discovered that the crystals consisted of an Escherichia coli contaminating protein, acriflavine resistance protein B (AcrB), that was present at less than 5% in the protein preparations. AcrB contamination is a major problem when expressing membrane proteins in E. coli since it binds naturally to immobilized metal-ion affinity chromatography (IMAC) resins. Here, the structure is compared with previously deposited AcrB structures and strategies are proposed to avoid this contamination.

  5. Ontogeny, aging, and gender-related changes in hepatic multidrug resistant protein genes in rats.

    PubMed

    Zhu, Qiong-Ni; Hou, Wei-Yu; Xu, Shang-Fu; Lu, Yuan-Fu; Liu, Jie

    2017-02-01

    Multidrug resistance proteins (Mrps) are efflux transporters playing important roles in endogenous substances and xenobiotics transport out of the liver. Children, elderly, gender and physio-pathological conditions could influence their expression and result in changes in drug disposition.

  6. Identification of a putative protein profile associating with tamoxifen therapy resistance in breast cancer

    SciTech Connect

    Umar, Arzu; Kang, Hyuk; Timmermans, A. M.; Look, Maxime P.; Meijer-van Gelder, M. E.; den Bakker, Michael A.; Jaitly, Navdeep; Martens, John W.; Luider, Theo M.; Foekens, John A.; Pasa-Tolic, Ljiljana

    2009-06-01

    Tamoxifen-resistance is a major cause of death in patients with recurrent breast cancer. Current clinical factors can correctly predict therapy response in only half of the treated patients. Identification of proteins that associate with tamoxifen-resistance is a first step towards better response prediction and tailored treatment of patients. In the present study we intended to identify putative protein biomarkers indicative of tamoxifen therapy-resistance in breast cancer, using nanoLC coupled with FTICR MS. Comparative proteome analysis was performed on ~5,500 pooled tumor cells (corresponding to ~550 ng protein lysate/analysis) obtained through laser capture microdissection (LCM) from two independently processed data sets (n=24 and n=27) containing both tamoxifen therapy-sensitive and therapy-resistant tumors. Peptides and proteins were identified by matching mass and elution time of newly acquired LC-MS features to information in previously generated accurate mass and time tag (AMT) reference databases.

  7. Contemporary Issues in Protein Requirements and Consumption for Resistance Trained Athletes

    PubMed Central

    Wilson, Jacob; Wilson, Gabriel J

    2006-01-01

    In recent years an explosion of research papers concerning protein consumption has been published. The need to consolidate this information has become critical from both practical and future research standpoints. For this reason, the following paper presents an in depth analysis of contemporary issues in protein requirements and consumption for resistance trained athletes. Specifically, the paper covers: 1.) protein requirements for resistance trained athletes; 2.) the effect of the digestion rate of protein on muscular protein balance; 3.) the optimal timing of protein intake relative to exercise; 4.) the optimal pattern of protein ingestion, relative to how an individual should consume their protein throughout a 24 hour period, and what sources are utilized during this time frame; 5.) protein composition and its interaction with measures of protein balance and strength performance; 6.) the combination of protein and carbohydrates on plasma insulin levels and protein balance; 7.) the efficacy of protein supplements and whole food protein sources. Our goal is to provide the reader with practical information in optimizing protein intake as well as for provision of sound advice to their clients. Finally, special care was taken to provide future research implications. PMID:18500966

  8. Activated protein C resistance in patients with central retinal vein occlusion

    PubMed Central

    Larsson, J; Sellman, A; Bauer, B

    1997-01-01

    AIM/BACKGROUND—A new defect in the anticoagulant system has recently been discovered—activated protein C resistance. The frequency of this disorder has been shown to be increased in young patients (<50 years of age) with central retinal vein occlusion. This study was carried out to determine if there was any overrepresentation of activated protein C resistance in patients >50 years of age with central retinal vein occlusion.
METHODS—Blood samples were obtained from 83 patients >50 years of age and with a history of central retinal vein occlusion. The blood samples were analysed for activated protein C resistance with standard clinical laboratory methods.
RESULTS—In this material 11% of the patients were resistant to activated protein C. The normal incidence of activated protein C resistance in the same geographical area is 10-11%.
CONCLUSION—Activated protein C resistance does not seem to be a cause of central retinal vein occlusion in people older than 50 years.

 PMID:9486021

  9. Polyglycerol coatings of glass vials for protein resistance.

    PubMed

    Höger, Kerstin; Becherer, Tobias; Qiang, Wei; Haag, Rainer; Friess, Wolfgang; Küchler, Sarah

    2013-11-01

    Proteins are surface active molecules which undergo non-specific adsorption when getting in contact with surfaces such as the primary packaging material. This process is critical as it may cause a loss of protein content or protein aggregation. To prevent unspecific adsorption, protein repellent coatings are of high interest. We describe the coating of industrial relevant borosilicate glass vials with linear methoxylated polyglycerol, hyperbranched polyglycerol, and hyperbranched methoxylated polyglycerol. All coatings provide excellent protein repellent effects. The hyperbranched, non-methoxylated coating performed best. The protein repellent properties were maintained also after applying industrial relevant sterilization methods (≥200 °C). Marginal differences in antibody stability between formulations stored in bare glass vials and coated vials were detected after 3 months storage; the protein repellent effect remained largely stable. Here, we describe a new material suitable for the coating of primary packaging material of proteins which significantly reduces the protein adsorption and thus could present an interesting new possibility for biomedical applications.

  10. Trehalose glycopolymer resists allow direct writing of protein patterns by electron-beam lithography.

    PubMed

    Bat, Erhan; Lee, Juneyoung; Lau, Uland Y; Maynard, Heather D

    2015-03-20

    Direct-write patterning of multiple proteins on surfaces is of tremendous interest for a myriad of applications. Precise arrangement of different proteins at increasingly smaller dimensions is a fundamental challenge to apply the materials in tissue engineering, diagnostics, proteomics and biosensors. Herein, we present a new resist that protects proteins during electron-beam exposure and its application in direct-write patterning of multiple proteins. Polymers with pendant trehalose units are shown to effectively crosslink to surfaces as negative resists, while at the same time providing stabilization to proteins during the vacuum and electron-beam irradiation steps. In this manner, arbitrary patterns of several different classes of proteins such as enzymes, growth factors and immunoglobulins are realized. Utilizing the high-precision alignment capability of electron-beam lithography, surfaces with complex patterns of multiple proteins are successfully generated at the micrometre and nanometre scale without requiring cleanroom conditions.

  11. Trehalose Glycopolymer Resists Allow Direct Writing of Protein Patterns by Electron-Beam Lithography

    PubMed Central

    Bat, Erhan; Lee, Juneyoung; Lau, Uland Y.; Maynard, Heather D.

    2015-01-01

    Direct-write patterning of multiple proteins on surfaces is of tremendous interest for a myriad of applications. Precise arrangement of different proteins at increasingly smaller dimensions is a fundamental challenge to apply the materials in tissue engineering, diagnostics, proteomics and biosensors. Herein we present a new resist that protects proteins during electron beam exposure and its application in direct-write patterning of multiple proteins. Polymers with pendant trehalose units are shown to effectively cross-link to surfaces as negative resists, while at the same time providing stabilization to proteins during the vacuum and electron beam irradiation steps. In this manner, arbitrary patterns of several different classes of proteins such as enzymes, growth factors and immunoglobulins are realized. Utilizing the high precision alignment capability of electron-beam lithography, surfaces with complex patterns of multiple proteins are successfully generated at the micrometer and nanometer scale without requiring cleanroom conditions. PMID:25791943

  12. Expression, detergent solubilization, and purification of a membrane transporter, the MexB multidrug resistance protein.

    PubMed

    Bhatt, Forum H; Jeffery, Constance J

    2010-12-03

    Multidrug resistance (MDR), the ability of a cancer cell or pathogen to be resistant to a wide range of structurally and functionally unrelated anti-cancer drugs or antibiotics, is a current serious problem in public health. This multidrug resistance is largely due to energy-dependent drug efflux pumps. The pumps expel anti-cancer drugs or antibiotics into the external medium, lowering their intracellular concentration below a toxic threshold. We are studying multidrug resistance in Pseudomonas aeruginosa, an opportunistic bacterial pathogen that causes infections in patients with many types of injuries or illness, for example, burns or cystic fibrosis, and also in immuno-compromised cancer, dialysis, and transplantation patients. The major MDR efflux pumps in P. aeruginosa are tripartite complexes comprised of an inner membrane proton-drug antiporter (RND), an outer membrane channel (OMF), and a periplasmic linker protein (MFP). The RND and OMF proteins are transmembrane proteins. Transmembrane proteins make up more than 30% of all proteins and are 65% of current drug targets. The hydrophobic transmembrane domains make the proteins insoluble in aqueous buffer. Before a transmembrane protein can be purified, it is necessary to find buffer conditions containing a mild detergent that enable the protein to be solubilized as a protein detergent complex (PDC). In this example, we use an RND protein, the P. aeruginosa MexB transmembrane transporter, to demonstrate how to express a recombinant form of a transmembrane protein, solubilize it using detergents, and then purify the protein detergent complexes. This general method can be applied to the expression, purification, and solubilization of many other recombinantly expressed membrane proteins. The protein detergent complexes can later be used for biochemical or biophysical characterization including X-ray crystal structure determination or crosslinking studies.

  13. Crystal structure of the TLDc domain of oxidation resistance protein 2 from zebrafish.

    PubMed

    Blaise, Mickaël; Alsarraf, Husam M A B; Wong, Jaslyn E M M; Midtgaard, Søren Roi; Laroche, Fabrice; Schack, Lotte; Spaink, Herman; Stougaard, Jens; Thirup, Søren

    2012-06-01

    The oxidation resistance proteins (OXR) help to protect eukaryotes from reactive oxygen species. The sole C-terminal domain of the OXR, named TLDc is sufficient to perform this function. However, the mechanism by which oxidation resistance occurs is poorly understood. We present here the crystal structure of the TLDc domain of the oxidation resistance protein 2 from zebrafish. The structure was determined by X-ray crystallography to atomic resolution (0.97Å) and adopts an overall globular shape. Two antiparallel β-sheets form a central β-sandwich, surrounded by two helices and two one-turn helices. The fold shares low structural similarity to known structures.

  14. Identifying the Proteins that Mediate the Ionizing Radiation Resistance of Deinococcus Radiodurans R1

    SciTech Connect

    Battista, John R

    2010-02-22

    The primary objectives of this proposal was to define the subset of proteins required for the ionizing radiation (IR) resistance of Deinococcus radiodurans R1, characterize the activities of those proteins, and apply what was learned to problems of interest to the Department of Energy.

  15. Lipid transfer protein-mediated resistance to a trichothecene mycotoxin – Novel players in FHB resistance

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Lipid transfer proteins are a class of basic cysteine rich proteins characterized by an eight cysteine motif backbone with intrinsic antimicrobial activities against bacterial and fungal pathogens. Previously, we identified two type IV nonspecific lipid transfer protein (nsLTP) genes (LTP4.4 and LTP...

  16. Nuclear inheritance of erythromycin resistance in human cells: New class of mitochondrial protein synthesis mutants

    SciTech Connect

    Doersen, C.J.; Stanbridge, E.J.

    1982-06-01

    The characterization of two new erythromycin-resistant mutants of HeLa cells is described. The strains ERY2305 and ERY2309 both exhibited resistance to erythromycin in growth assays and cell-free mitochondrial protein synthesis assays. The erythromycin resistance phenotype could not be transferred by cybridization. The mutation appeared to be encoded in the nucleus and inherited as a recessive trait. These two mutants, therefore, represent a new class of erythromycin-resistant mutants in human cells that is distinct from the cytoplasmically inherited mutation in strain ERY2301 described previously.

  17. Multidrug resistance protein 1 (ABCC1) confers resistance to arsenic compounds in human myeloid leukemic HL-60 cells.

    PubMed

    Xu, Shi; Zhang, Yan Fang; Carew, Micheal W; Hao, Wen Hui; Loo, Jacky Fong Chuen; Naranmandura, Hua; Le, X Chris

    2013-06-01

    Arsenic trioxide (As(2)O(3)) is established as one of the most effective drugs for treatment of patients with acute promyelocytic leukemia, as well as other types of malignant tumors. However, HL-60 cells are resistant to As(2)O(3), and little is known about the underlying resistance mechanism for As(2)O(3) and its biomethylation products, namely, monomethylarsonous acid (MMA(III)) on the treatment of tumors. In the present study, we investigated the molecular mechanisms underlying iAs(III) and its intermediate metabolite MMA(III)-induced anticancer effects in the HL-60 cells. Here, we show that the HL-60 cells exhibit resistance to inorganic iAs(III) (IC(50) = 10 μM), but are relatively sensitive to its intermediate MMA(III) (IC(50) = 3.5 μM). Moreover, we found that the multidrug resistance protein 1 (MRP1), but not MRP2, is expressed in HL-60 cells, which reduced the intracellular arsenic accumulation, and conferred resistance to inorganic iAs(III) and MMA(III). Pretreatment of HL-60 with MK571, an inhibitor of MRP1, significantly increased iAs(III) and MMA(III)-induced cytotoxicity and arsenic accumulations, suggesting that the expression of MRP1/4 may lead to HL-60 cells resistance to trivalent arsenic compounds.

  18. Azide-resistant mutants in Acinetobacter calcoaceticus A2 are defective in protein secretion.

    PubMed

    Elkeles, A; Rosenberg, E; Ron, E Z

    1994-02-15

    Azide, an inhibitor of ATPase, and a specific inhibitor of protein export was used in order to select for protein secretion mutants in Acinetobacter calcoaceticus A2. Two such mutants were isolated that were azide-resistant and defective in the general protein transport system. The mutation also conferred additional phenotypic changes, including an inability to grow on minimal media or at 40 degrees C. The existence of protein secretion mutants with a selectable phenotype may be useful for the genetic study of protein export.

  19. Comparative proteomic analysis of paclitaxel resistance-related proteins in human breast cancer cell lines

    PubMed Central

    Fujioka, Hiroya; Sakai, Akiko; Tanaka, Satoru; Kimura, Kosei; Miyamoto, Akiko; Iwamoto, Mitsuhiko; Uchiyama, Kazuhisa

    2017-01-01

    Paclitaxel is widely used to treat various cancers; however, resistance to this drug is a major obstacle to breast cancer chemotherapy. To identify the proteins involved in paclitaxel resistance, the present study compared the proteomes of MCF-7 human breast cancer cells and its paclitaxel-resistant subclone MCF-7/PTX. Using two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization time of flight mass spectrometry, 11 upregulated and 12 downregulated proteins were identified in MCF-7/PTX cells compared with the parental cell line. These 23 proteins were functionally classified as stress-induced chaperones, metabolic enzymes and cytoskeletal proteins. The anti-apoptotic proteins, stress-70 protein, 78-kD glucose-regulated protein, peptidyl-prolyl cis-trans isomerase A (PPIA) and heterogeneous nuclear ribonucleoprotein H3, were also upregulated in MCF-7/PTX cells. Notably, knockdown of the stress-response chaperone PPIA using small interfering RNA in MCF-7/PTX cells restored their sensitivity to paclitaxel. These findings indicated that PPIA may have an important role in paclitaxel resistance in MCF-7/PTX cells. PMID:28123557

  20. Possibly similar genetic basis of resistance to Bacillus thuringiensis Cry1Ab protein in 3 resistant colonies of the sugarcane borer collected from Louisiana, USA.

    PubMed

    Yang, Fei; Chen, Mao; Gowda, Anilkumar; Kerns, David L; Huang, Fangneng

    2016-11-08

    The sugarcane borer, Diatraea saccharalis (F.), is a major maize borer pest and a target of transgenic maize expressing Bacillus thuringiensis (Bt) proteins in South America and the mid-southern region of the United States. Evolution of resistance in target pest populations is a great threat to the long-term efficacy of Bt crops. In this study, we compared the genetic basis of resistance to Cry1Ab protein in 3 resistant colonies of sugarcane borer established from field populations in Louisiana, USA. Responses of larvae to the Cry1Ab protein for the parental and 10 other cross colonies were assayed in a diet-incorporated bioassay. All 3 resistant colonies were highly resistant to the Cry1Ab protein with a resistance ratio of >555.6 fold. No maternal effect or sex linkage was evident for the resistance in the 3 colonies; and the resistance was functionally nonrecessive at the Cry1Ab concentrations of ≤ 3.16 μg/g, but it became recessive at ≥10 μg/g. In an interstrain complementation test for allelism, the F1 progeny from crosses between any 2 of the 3 resistant colonies exhibited the similar resistance levels as their parental colonies, indicating that the 3 colonies most likely shared a locus of Cry1Ab resistance. Results generated from this study should provide useful information in developing effective strategies for managing Bt resistance in the insect.

  1. OSPREY Predicts Resistance Mutations Using Positive and Negative Computational Protein Design.

    PubMed

    Ojewole, Adegoke; Lowegard, Anna; Gainza, Pablo; Reeve, Stephanie M; Georgiev, Ivelin; Anderson, Amy C; Donald, Bruce R

    2017-01-01

    Drug resistance in protein targets is an increasingly common phenomenon that reduces the efficacy of both existing and new antibiotics. However, knowledge of future resistance mutations during pre-clinical phases of drug development would enable the design of novel antibiotics that are robust against not only known resistant mutants, but also against those that have not yet been clinically observed. Computational structure-based protein design (CSPD) is a transformative field that enables the prediction of protein sequences with desired biochemical properties such as binding affinity and specificity to a target. The use of CSPD to predict previously unseen resistance mutations represents one of the frontiers of computational protein design. In a recent study (Reeve et al. Proc Natl Acad Sci U S A 112(3):749-754, 2015), we used our OSPREY (Open Source Protein REdesign for You) suite of CSPD algorithms to prospectively predict resistance mutations that arise in the active site of the dihydrofolate reductase enzyme from methicillin-resistant Staphylococcus aureus (SaDHFR) in response to selective pressure from an experimental competitive inhibitor. We demonstrated that our top predicted candidates are indeed viable resistant mutants. Since that study, we have significantly enhanced the capabilities of OSPREY with not only improved modeling of backbone flexibility, but also efficient multi-state design, fast sparse approximations, partitioned continuous rotamers for more accurate energy bounds, and a computationally efficient representation of molecular-mechanics and quantum-mechanical energy functions. Here, using SaDHFR as an example, we present a protocol for resistance prediction using the latest version of OSPREY. Specifically, we show how to use a combination of positive and negative design to predict active site escape mutations that maintain the enzyme's catalytic function but selectively ablate binding of an inhibitor.

  2. OSPREY Predicts Resistance Mutations using Positive and Negative Computational Protein Design

    PubMed Central

    Ojewole, Adegoke; Lowegard, Anna; Gainza, Pablo; Reeve, Stephanie M.; Georgiev, Ivelin; Anderson, Amy C.; Donald, Bruce R.

    2016-01-01

    Summary Drug resistance in protein targets is an increasingly common phenomenon that reduces the efficacy of both existing and new antibiotics. However, knowledge of future resistance mutations during pre-clinical phases of drug development would enable the design of novel antibiotics that are robust against not only known resistant mutants, but also against those that have not yet been clinically observed. Computational structure-based protein design (CSPD) is a transformative field that enables the prediction of protein sequences with desired biochemical properties such as binding affinity and specificity to a target. The use of CSPD to predict previously unseen resistance mutations represents one of the frontiers of computational protein design. In a recent study (1), we used our OSPREY (Open Source Protein REdesign for You) suite of CSPD algorithms to prospectively predict resistance mutations that arise in the active site of the dihydrofolate reductase enzyme from methicillin-resistant Staphylococcus aureus (SaDHFR) in response to selective pressure from an experimental competitive inhibitor. We demonstrated that our top predicted candidates are indeed viable resistant mutants. Since that study, we have significantly enhanced the capabilities of OSPREY with not only improved modeling of backbone flexibility, but also efficient multi-state design, fast sparse approximations, partitioned rotamers for more accurate energy bounds, and a computationally efficient representation of molecular-mechanics and quantum-mechanical energy functions. Here, using SaDHFR as an example, we present a protocol for resistance prediction using the latest version of OSPREY. Specifically, we show how to use a combination of positive and negative design to predict active site escape mutations that maintain the enzyme’s catalytic function but selectively ablate binding of an inhibitor. PMID:27914058

  3. Widespread genetic switches and toxicity resistance proteins for fluoride.

    PubMed

    Baker, Jenny L; Sudarsan, Narasimhan; Weinberg, Zasha; Roth, Adam; Stockbridge, Randy B; Breaker, Ronald R

    2012-01-13

    Most riboswitches are metabolite-binding RNA structures located in bacterial messenger RNAs where they control gene expression. We have discovered a riboswitch class in many bacterial and archaeal species whose members are selectively triggered by fluoride but reject other small anions, including chloride. These fluoride riboswitches activate expression of genes that encode putative fluoride transporters, enzymes that are known to be inhibited by fluoride, and additional proteins of unknown function. Our findings indicate that most organisms are naturally exposed to toxic levels of fluoride and that many species use fluoride-sensing RNAs to control the expression of proteins that alleviate the deleterious effects of this anion.

  4. Expression of xeroderma pigmentosum complementation group C protein predicts cisplatin resistance in lung adenocarcinoma patients.

    PubMed

    Lai, Tan-Chen; Chow, Kuan-Chih; Fang, Hsin-Yuan; Cho, Hsin-Ching; Chen, Chih-Yi; Lin, Tze-Yi; Chiang, I-Ping; Ho, Shu-Peng

    2011-05-01

    DNA repair has been suggested to be a major cause of spontaneous drug resistance in patients with lung adenocarcinomas (LADC). Among the DNA repair-related proteins, excision repair cross-complementation group 1 (ERCC1) has been shown to be essential for repairing cisplatin-induced interstrand cross-linkage. However, the role of other DNA repair-related proteins in drug resistance has not been clearly elucidated. In this study, we used suppression subtractive hybridization and microarray analysis to identify the DNA repair-related genes associated with cisplatin resistance. We focused on the association of XPC protein expression, which plays a pivotal role in the earliest response to global genomic repair, with the survival of LADC patients. Using suppression subtractive hybridization and a microarray analysis to identify drug resistance-associated DNA repair-related genes, we found that the mRNA levels of ERCC1, MSH-3, MSH-6 and XPC were significantly increased in LADC patients. Since the results of ERCC1 mRNA expression corresponded well with those in previous reports, in this study we focused on the clinical correlation between XPC expression and patient survival. The level of XPC protein was determined by immunohistochemical and immunoblotting analyses. We detected the XPC protein in 46 (43%) of 107 pathological LADC samples. XPC protein expression correlated with tumor stage, cigarette smoking and poor survival. In the in vitro experiments with LADC cell lines, increased XPC expression was associated with elevated drug resistance, and silencing of XPC expression reduced cisplatin resistance. Our results suggest that XPC expression predicts drug resistance in LADC.

  5. IMP3 protein promotes chemoresistance in breast cancer cells by regulating breast cancer resistance protein (ABCG2) expression.

    PubMed

    Samanta, Sanjoy; Pursell, Bryan; Mercurio, Arthur M

    2013-05-03

    IMP3, a member of a family of insulin-like growth factor II (IGF-II) mRNA-binding proteins (IMPs), is expressed preferentially in triple-negative breast cancers, which are resistant to many chemotherapeutics. However, the mechanisms by which it impacts breast cancer have not been elucidated. We hypothesized a role for IMP3 in chemoresistance based on these observations. Depletion of IMP3 expression in triple-negative breast cancer cells increased their sensitivity to doxorubicin and mitoxantrone significantly but not to taxol. Given that doxorubicin and mitoxantrone are effluxed by breast cancer resistance protein (BCRP), we assessed whether IMP3 regulates BCRP. The data obtained demonstrate that IMP3 binds to BCRP mRNA and regulates BCRP expression. These findings are significant because they provide insight into the mechanism by which IMP3 contributes to aggressive cancers, and they highlight the potential for targeting this mRNA-binding protein for the clinical management of cancer.

  6. MRP proteins as potential mediators of heavy metal resistance in zebrafish cells.

    PubMed

    Long, Yong; Li, Qing; Wang, Youhui; Cui, Zongbin

    2011-04-01

    Acquired resistance of mammalian cells to heavy metals is closely relevant to enhanced expression of several multidrug resistance-associated proteins (MRP), but it remains unclear whether MRP proteins confer resistance to heavy metals in zebrafish. In this study, we obtained zebrafish (Danio rerio) fibroblast-like ZF4 cells with resistance to toxic heavy metals after chronic cadmium exposure and selection for 6months. These cadmium-resistant cells (ZF4-Cd) were maintained in 5μM cadmium and displayed cross-resistance to cadmium, mercury, arsenite and arsenate. ZF4-Cd cells remained the resistance to heavy metals after protracted culture in cadmium-free medium. In comparison with ZF4-WT cells, ZF4-Cd cells exhibited accelerated rate of cadmium excretion, enhanced activity of MRP-like transport, elevated expression of abcc2, abcc4 and mt2 genes, and increased content of cellular GSH. Inhibition of MRP-like transport activity, GSH biosynthesis and GST activity significantly attenuated the resistance of ZF4-Cd cells to heavy metals. The results indicate that some of MRP transporters are involved in the efflux of heavy metals conjugated with cellular GSH and thus play crucial roles in heavy metal detoxification of zebrafish cells.

  7. Protein promiscuity: drug resistance and native functions--HIV-1 case.

    PubMed

    Fernández, Ariel; Tawfik, Dan S; Berkhout, Ben; Sanders, Rogier; Kloczkowski, Andrzej; Sen, Taner; Jernigan, Bob

    2005-06-01

    The association of a drug with its target protein has the effect of blocking the protein activity and is termed a promiscuous function to distinguish from the protein's native function (Tawfik and associates, Nat. Genet. 37, 73-6, 2005). Obviously, a protein has not evolved naturally for drug association or drug resistance. Promiscuous protein functions exhibit unique traits of evolutionary adaptability, or evolvability, which is dependent on the induction of novel phenotypic traits by a small number of mutations. These mutations might have small effects on native functions, but large effects on promiscuous function; for example, an evolving protein could become increasingly drug resistant while maintaining its original function. Ariel Fernandez, in his opinion piece, notes that drug-binding "promiscuity" can hardly be dissociated from native functions; a dominant approach to drug discovery is the protein-native-substrate transition-state mimetic strategy. Thus, man-made ligands (e.g. drugs) have been successfully crafted to restrain enzymatic activity by focusing on the very same structural features that determine the native function. Using the successful inhibition of HIV-1 protease as an example, Fernandez illustrates how drug designers have employed naturally evolved features of the protein to suppress its activity. Based on these arguments, he dismisses the notion that drug binding is quintessentially promiscuous, even though in principle, proteins did not evolve to associate with man made ligands. In short, Fernandez argues that there may not be separate protein domains that one could term promiscuous domains. While acknowledging that drugs may bind promiscuously or in a native-like manner a la Fernandez, Tawfik maintains the role of evolutionary adaptation, even when a drug binds native-like. In the case of HIV-1 protease, drugs bind natively, and the initial onset of mutations results in drug resistance in addition to a dramatic decline in enzymatic

  8. CCND1 mutations increase protein stability and promote ibrutinib resistance in mantle cell lymphoma.

    PubMed

    Mohanty, Atish; Sandoval, Natalie; Das, Manasi; Pillai, Raju; Chen, Lu; Chen, Robert W; Amin, Hesham M; Wang, Michael; Marcucci, Guido; Weisenburger, Dennis D; Rosen, Steven T; Pham, Lan V; Ngo, Vu N

    2016-11-08

    Mantle cell lymphoma (MCL) is characterized by the t(11;14) translocation, which leads to deregulated expression of the cell cycle regulatory protein cyclin D1 (CCND1). Genomic studies of MCL have also identified recurrent mutations in the coding region of CCND1. However, the functional consequence of these mutations is not known. Here, we showed that, compared to wild type (WT), single E36K, Y44D or C47S CCND1 mutations increased CCND1 protein levels in MCL cell lines. Mechanistically, these mutations stabilized CCND1 protein through attenuation of threonine-286 phosphorylation, which is important for proteolysis through the ubiquitin-proteasome pathway. In addition, the mutant proteins preferentially localized to the nucleus. Interestingly, forced expression of WT or mutant CCND1 increased resistance of MCL cell lines to ibrutinib, an FDA-approved Bruton tyrosine kinase inhibitor for MCL treatment. The Y44D mutant sustained the resistance to ibrutinib even at supraphysiologic concentrations (5-10 μM). Furthermore, primary MCL tumors with CCND1 mutations also expressed stable CCND1 protein and were resistant to ibrutinib. These findings uncover a new mechanism that is critical for the regulation of CCND1 protein levels, and is directly relevant to primary ibrutinib resistance in MCL.

  9. CCND1 mutations increase protein stability and promote ibrutinib resistance in mantle cell lymphoma

    PubMed Central

    Mohanty, Atish; Sandoval, Natalie; Das, Manasi; Pillai, Raju; Chen, Lu; Chen, Robert W.; Amin, Hesham M.; Wang, Michael; Marcucci, Guido; Weisenburger, Dennis D.; Rosen, Steven T.; Pham, Lan V.; Ngo, Vu N.

    2016-01-01

    Mantle cell lymphoma (MCL) is characterized by the t(11;14) translocation, which leads to deregulated expression of the cell cycle regulatory protein cyclin D1 (CCND1). Genomic studies of MCL have also identified recurrent mutations in the coding region of CCND1. However, the functional consequence of these mutations is not known. Here, we showed that, compared to wild type (WT), single E36K, Y44D or C47S CCND1 mutations increased CCND1 protein levels in MCL cell lines. Mechanistically, these mutations stabilized CCND1 protein through attenuation of threonine-286 phosphorylation, which is important for proteolysis through the ubiquitin-proteasome pathway. In addition, the mutant proteins preferentially localized to the nucleus. Interestingly, forced expression of WT or mutant CCND1 increased resistance of MCL cell lines to ibrutinib, an FDA-approved Bruton tyrosine kinase inhibitor for MCL treatment. The Y44D mutant sustained the resistance to ibrutinib even at supraphysiologic concentrations (5–10 μM). Furthermore, primary MCL tumors with CCND1 mutations also expressed stable CCND1 protein and were resistant to ibrutinib. These findings uncover a new mechanism that is critical for the regulation of CCND1 protein levels, and is directly relevant to primary ibrutinib resistance in MCL. PMID:27713153

  10. Anti-protozoal and anti-bacterial antibiotics that inhibit protein synthesis kill cancer subtypes enriched for stem cell-like properties.

    PubMed

    Cuyàs, Elisabet; Martin-Castillo, Begoña; Corominas-Faja, Bruna; Massaguer, Anna; Bosch-Barrera, Joaquim; Menendez, Javier A

    2015-01-01

    Key players in translational regulation such as ribosomes might represent powerful, but hitherto largely unexplored, targets to eliminate drug-refractory cancer stem cells (CSCs). A recent study by the Lisanti group has documented how puromycin, an old antibiotic derived from Streptomyces alboniger that inhibits ribosomal protein translation, can efficiently suppress CSC states in tumorspheres and monolayer cultures. We have used a closely related approach based on Biolog Phenotype Microarrays (PM), which contain tens of lyophilized antimicrobial drugs, to assess the chemosensitivity profiles of breast cancer cell lines enriched for stem cell-like properties. Antibiotics directly targeting active sites of the ribosome including emetine, puromycin and cycloheximide, inhibitors of ribosome biogenesis such as dactinomycin, ribotoxic stress agents such as daunorubicin, and indirect inhibitors of protein synthesis such as acriflavine, had the largest cytotoxic impact against claudin-low and basal-like breast cancer cells. Thus, biologically aggressive, treatment-resistant breast cancer subtypes enriched for stem cell-like properties exhibit exacerbated chemosensitivities to anti-protozoal and anti-bacterial antibiotics targeting protein synthesis. These results suggest that old/existing microbicides might be repurposed not only as new cancer therapeutics, but also might provide the tools and molecular understanding needed to develop second-generation inhibitors of ribosomal translation to eradicate CSC traits in tumor tissues.

  11. Artificial TALE as a Convenient Protein Platform for Engineering Broad-Spectrum Resistance to Begomoviruses

    PubMed Central

    Cheng, Xiaofei; Li, Fangfang; Cai, Jianyu; Chen, Wei; Zhao, Nan; Sun, Yuqiang; Guo, Yushuang; Yang, Xiuling; Wu, Xiaoyun

    2015-01-01

    Transcription activator–like effectors (TALEs) are a class of sequence-specific DNA-binding proteins that utilize a simple and predictable modality to recognize target DNA. This unique characteristic allows for the rapid assembly of artificial TALEs, with high DNA binding specificity, to any target DNA sequences for the creation of customizable sequence-specific nucleases used in genome engineering. Here, we report the use of an artificial TALE protein as a convenient platform for designing broad-spectrum resistance to begomoviruses, one of the most destructive plant virus groups, which cause tremendous losses worldwide. We showed that artificial TALEs, which were assembled based on conserved sequence motifs within begomovirus genomes, could confer partial resistance in transgenic Nicotiana benthamiana to all three begomoviruses tested. Furthermore, the resistance was maintained even in the presence of their betasatellite. These results shed new light on the development of broad-spectrum resistance against DNA viruses, such as begomoviruses. PMID:26308041

  12. Signalling functions and biochemical properties of pertussis toxin-resistant G-proteins.

    PubMed Central

    Fields, T A; Casey, P J

    1997-01-01

    Pertussis toxin (PTX) has been widely used as a reagent to characterize the involvement of heterotrimeric G-proteins in signalling. This toxin catalyses the ADP-ribosylation of specific G-protein alpha subunits of the Gi family, and this modification prevents the occurrence of the receptor-G-protein interaction. This review focuses on the biochemical properties and signalling of those G-proteins historically classified as 'PTX-resistant' due to the inability of the toxin to influence signalling through them. These G-proteins include members of the Gq and G12 families and one Gi family member, i.e. Gz. Signalling pathways controlled by these G-proteins are well characterized only for Gq family members, which activate specific isoforms of phospholipase C, resulting in increases in intracellular calcium and activation of protein kinase C (PKC), among other responses. While members of the G12 family have been implicated in processes that regulate cell growth, and Gz has been shown to inhibit adenylate cyclase, the specific downstream targets to these G-proteins in vivo have not been clearly established. Since two of these proteins, G12 alpha and Gz alpha, are excellent substrates for PKC, there is the potential for cross-talk between their signalling and Gq-dependent processes leading to activation of PKC. In tissues that express these G-proteins, a number of guanine-nucleotide-dependent, PTX-resistant, signalling pathways have been defined for which the G-protein involved has not been identified. This review summarizes these pathways and discusses the evidence both for the participation of specific PTX-resistant G-proteins in them and for the regulation of these processes by PKC. PMID:9032437

  13. Heparin-induced thrombocytopenia and fatal thrombosis in a patient with activated protein C resistance.

    PubMed

    Gardyn, J; Sorkin, P; Kluger, Y; Kabili, S; Klausner, J M; Zivelin, A; Eldor, A

    1995-12-01

    We report a 20-year-old woman who developed heparin-induced thrombocytopenia (HIT) associated with devastating and fatal multiorgan thrombosis. The patient, her mother, and her brother were found to have resistance to activated protein C (APC), and the congenital thrombophilia in this family was verified by the finding of the Arg506 Gln mutation in factor V. This is the first case of HIT and APC resistance. The consequences of this association are discussed.

  14. Reduced resting skeletal muscle protein synthesis is rescued by resistance exercise and protein ingestion following short-term energy deficit.

    PubMed

    Areta, José L; Burke, Louise M; Camera, Donny M; West, Daniel W D; Crawshay, Siobhan; Moore, Daniel R; Stellingwerff, Trent; Phillips, Stuart M; Hawley, John A; Coffey, Vernon G

    2014-04-15

    The myofibrillar protein synthesis (MPS) response to resistance exercise (REX) and protein ingestion during energy deficit (ED) is unknown. In young men (n = 8) and women (n = 7), we determined protein signaling and resting postabsorptive MPS during energy balance [EB; 45 kcal·kg fat-free mass (FFM)(-1)·day(-1)] and after 5 days of ED (30 kcal·kg FFM(-1)·day(-1)) as well as MPS while in ED after acute REX in the fasted state and with the ingestion of whey protein (15 and 30 g). Postabsorptive rates of MPS were 27% lower in ED than EB (P < 0.001), but REX stimulated MPS to rates equal to EB. Ingestion of 15 and 30 g of protein after REX in ED increased MPS ~16 and ~34% above resting EB (P < 0.02). p70 S6K Thr(389) phosphorylation increased above EB only with combined exercise and protein intake (~2-7 fold, P < 0.05). In conclusion, short-term ED reduces postabsorptive MPS; however, a bout of REX in ED restores MPS to values observed at rest in EB. The ingestion of protein after REX further increases MPS above resting EB in a dose-dependent manner. We conclude that combining REX with increased protein availability after exercise enhances rates of skeletal muscle protein synthesis during short-term ED and could in the long term preserve muscle mass.

  15. The outer membrane protein TolC from Sinorhizobium meliloti affects protein secretion, polysaccharide biosynthesis, antimicrobial resistance, and symbiosis.

    PubMed

    Cosme, Ana M; Becker, Anke; Santos, Mário R; Sharypova, Larissa A; Santos, Pedro M; Moreira, Leonilde M

    2008-07-01

    Sinorhizobium meliloti is capable of establishing a symbiotic nitrogen fixation relationship with Medicago sativa. During this process, it must cope with diverse environments and has evolved different types of transport systems that help its propagation in the plant roots. TolC protein family members are the outer-membrane components of several transport systems involved in the export of diverse molecules, playing an important role in bacterial survival. In this work, we have characterized the protein TolC from S. meliloti 2011. An insertional mutation in the tolC gene strongly affected the resistance phenotype to antimicrobial agents and induced higher susceptibility to osmotic and oxidative stresses. Immunodetection experiments and comparison of the extracellular proteins present in the supernatant of the wild-type versus tolC mutant strains showed that the calcium-binding protein ExpE1, the endoglycanase ExsH, and the product of open reading frame SMc04171, a putative hemolysin-type calcium-binding protein, are secreted by a TolC-dependent secretion system. In the absence of TolC, neither succinoglycan nor galactoglucan were detected in the culture supernatant. Moreover, S. meliloti tolC mutant induced a reduced number of nonfixing nitrogen nodules in M. sativa roots. Taken together, our results confirm the importance of TolC in protein secretion, exopolysaccharide biosynthesis, antimicrobials resistance, and symbiosis.

  16. DBC2 resistance is achieved by enhancing 26S proteasome-mediated protein degradation.

    PubMed

    Collado, Denise; Yoshihara, Takashi; Hamaguchi, Masaaki

    2007-08-31

    Tumor suppressor gene DBC2 stops growth of tumor cells through regulation of CCND1. Interference of CCND1 down-regulation prevented growth arrest caused by DBC2 [T. Yoshihara, D. Collado, M. Hamaguchi, Cyclin D1 down-regulation is essential for DBC2's tumor suppressor function, Biochemical and biophysical research communications 358 (2007) 1076-1079]. It was also noted that DBC2 resistant cells eventually arose after repeated induction of DBC2 with muristerone A treatment [M. Hamaguchi, J.L. Meth, C. Von Klitzing, W. Wei, D. Esposito, L. Rodgers, T. Walsh, P. Welcsh, M.C. King, M.H. Wigler, DBC2, a candidate for a tumor suppressor gene involved in breast cancer, Proc. Natl. Acad. Sci. USA 99 (2002) 13647-13652]. In order to elucidate the mechanism of resistance acquisition, we analyzed DBC2 sensitive and resistant cells derived from the same progenitor cells (T-47D). We discovered that DBC2 protein was abundantly expressed in the sensitive cells when DBC2 was induced. In contrast, it was undetectable by western blot analysis in the resistant cells. We confirmed that the inducible gene expression system was responsive in both cells by detecting induced GFP. Additionally, inhibition of 26S proteasome by MG132 revealed production of DBC2 protein in the resistant cells. These findings indicate that the resistant T-47D cells survive DBC2 induction by rapid destruction of DBC2 through 26S proteasome-mediated protein degradation.

  17. Prediction of HIV drug resistance from genotype with encoded three-dimensional protein structure

    PubMed Central

    2014-01-01

    Background Drug resistance has become a severe challenge for treatment of HIV infections. Mutations accumulate in the HIV genome and make certain drugs ineffective. Prediction of resistance from genotype data is a valuable guide in choice of drugs for effective therapy. Results In order to improve the computational prediction of resistance from genotype data we have developed a unified encoding of the protein sequence and three-dimensional protein structure of the drug target for classification and regression analysis. The method was tested on genotype-resistance data for mutants of HIV protease and reverse transcriptase. Our graph based sequence-structure approach gives high accuracy with a new sparse dictionary classification method, as well as support vector machine and artificial neural networks classifiers. Cross-validated regression analysis with the sparse dictionary gave excellent correlation between predicted and observed resistance. Conclusion The approach of encoding the protein structure and sequence as a 210-dimensional vector, based on Delaunay triangulation, has promise as an accurate method for predicting resistance from sequence for drugs inhibiting HIV protease and reverse transcriptase. PMID:25081370

  18. Correlation between penicillin-binding protein 2 mutations and carbapenem resistance in Escherichia coli.

    PubMed

    Yamachika, Shinichiro; Sugihara, Chika; Kamai, Yasuki; Yamashita, Makoto

    2013-03-01

    It is well known that carbapenem-resistant mutations in penicillin-binding proteins (PBPs) are not observed in most Gram-negative bacteria under either clinical or experimental conditions. To understand the mechanisms involved in carbapenem resistance, this study constructed a mutS- and tolC-deficient Escherichia coli strain, which was expected to have elevated mutation frequencies and to lack drug efflux. Using this mutant, carbapenem-resistant strains with target mutations were successfully and efficiently isolated. The mutations T547I/A, M574I and G601D were identified in the PBP2 gene. Meropenem (MEPM)-resistant strains with the PBP2 T547I mutation showed fourfold increased resistance to 1-β-methyl-substituted carbapenems, such as doripenem, MEPM and biapenem, but not to non-substituted carbapenems such as imipenem and panipenem and other β-lactams. In addition, resistance resulting from the G601D mutation was limited to MEPM, whilst the M574I mutation conferred resistance to MEPM, imipenem and panipenem. This is the first report, to the best of our knowledge, that E. coli also has a carbapenem-resistance mechanism as a result of PBP2 mutations, and it provides insight into the resistance profiles of PBP2 mutations to carbapenems with and without the 1-β-methyl group.

  19. Structures of replication initiation proteins from staphylococcal antibiotic resistance plasmids reveal protein asymmetry and flexibility are necessary for replication

    PubMed Central

    Carr, Stephen B.; Phillips, Simon E.V.; Thomas, Christopher D.

    2016-01-01

    Antibiotic resistance in pathogenic bacteria is a continual threat to human health, often residing in extrachromosomal plasmid DNA. Plasmids of the pT181 family are widespread and confer various antibiotic resistances to Staphylococcus aureus. They replicate via a rolling circle mechanism that requires a multi-functional, plasmid-encoded replication protein to initiate replication, recruit a helicase to the site of initiation and terminate replication after DNA synthesis is complete. We present the first atomic resolution structures of three such replication proteins that reveal distinct, functionally relevant conformations. The proteins possess a unique active site and have been shown to contain a catalytically essential metal ion that is bound in a manner distinct from that of any other rolling circle replication proteins. These structures are the first examples of the Rep_trans Pfam family providing insights into the replication of numerous antibiotic resistance plasmids from Gram-positive bacteria, Gram-negative phage and the mobilisation of DNA by conjugative transposons. PMID:26792891

  20. Protein-Adsorbed Magnetic-Nanoparticle-Mediated Assay for Rapid Detection of Bacterial Antibiotic Resistance.

    PubMed

    Cowger, Taku A; Yang, Yaping; Rink, David E; Todd, Trever; Chen, Hongmin; Shen, Ye; Yan, Yajun; Xie, Jin

    2017-02-17

    Antibiotic susceptibility tests have been used for years as a crucial diagnostic tool against antibiotic-resistant bacteria. However, due to a lack of biomarkers specific to resistant types, these approaches are often time-consuming, inaccurate, and inflexible in drug selections. Here, we present a novel susceptibility test method named protein-adsorbed nanoparticle-mediated matrix-assisted laser desorption-ionization mass spectrometry, or PANMS. Briefly, we adsorb five different proteins (β-casein, α-lactalbumin, human serum albumin, fibrinogen, and avidin) onto the surface of Fe3O4. Upon interaction with bacteria surface, proteins were displaced from the nanoparticle surface, the amounts of which were quantified by matrix-assisted laser desorption ionization mass spectrometry. We find that the protein displacement profile was different distinctive among different bacteria strains and, in particular, between wild-type and drug-resistant strains. More excitingly, we observe bacteria resistant to drugs of the same mechanisms share similar displacement profiles on a linear discriminant analysis (LDA) map. This suggests the possibility of using PANMS to identify the type of mechanism behind antibiotic resistance, which was confirmed in a blind test. Given that PANMS is free of drug incubation and the whole procedure takes less than 50 min, it holds great potential as a high-throughput, low-cost, and accurate drug susceptibility test in the clinic.

  1. Proteomics Analysis of Ovarian Cancer Cell Lines and Tissues Reveals Drug Resistance-associated Proteins

    PubMed Central

    CRUZ*, ISA N.; COLEY*, HELEN M.; KRAMER, HOLGER B.; MADHURI, THUMULURU KAVITAH; SAFUWAN, NUR A.M.; ANGELINO, ANA RITA; YANG, MIN

    2016-01-01

    Background: Carboplatin and paclitaxel form the cornerstone of chemotherapy for epithelial ovarian cancer, however, drug resistance to these agents continues to present challenges. Despite extensive research, the mechanisms underlying this resistance remain unclear. Materials and Methods: A 2D-gel proteomics method was used to analyze protein expression levels of three human ovarian cancer cell lines and five biopsy samples. Representative proteins identified were validated via western immunoblotting. Ingenuity pathway analysis revealed metabolomic pathway changes. Results: A total of 189 proteins were identified with restricted criteria. Combined treatment targeting the proteasome-ubiquitin pathway resulted in re-sensitisation of drug-resistant cells. In addition, examination of five surgical biopsies of ovarian tissues revealed α-enolase (ENOA), elongation factor Tu, mitochondrial (EFTU), glyceraldehyde-3-phosphate dehydrogenase (G3P), stress-70 protein, mitochondrial (GRP75), apolipoprotein A-1 (APOA1), peroxiredoxin (PRDX2) and annexin A (ANXA) as candidate biomarkers of drug-resistant disease. Conclusion: Proteomics combined with pathway analysis provided information for an effective combined treatment approach overcoming drug resistance. Analysis of cell lines and tissues revealed potential prognostic biomarkers for ovarian cancer. *These Authors contributed equally to this study. PMID:28031236

  2. Antiviral Resistance Protein Tm-22 Functions on the Plasma Membrane1[OPEN

    PubMed Central

    Chen, Tianyuan; Liu, Dan; Niu, Xiaolin; Qian, Lichao; Han, Lu; Liu, Na; Zhao, Jinping

    2017-01-01

    The tomato Tobacco mosaic virus resistance-22 (Tm-22) gene encodes a coiled-coil-nucleotide binding site-Leu-rich repeat protein lacking a conventional plasma membrane (PM) localization motif. Tm-22 confers plant extreme resistance against tobamoviruses including Tobacco mosaic virus (TMV) by recognizing the avirulence (Avr) viral movement protein (MP). However, the subcellular compartment where Tm-22 functions is unclear. Here, we demonstrate that Tm-22 interacts with TMV MP to form a protein complex at the PM. We show that both inactive and active Tm-22 proteins are localized to the PM. When restricted to PM by fusing Tm-22 to the S-acylated PM association motif, the Tm-22 fusion protein can still induce a hypersensitive response cell death, consistent with its activation at the PM. Through analyses of viral MP mutants, we find that the plasmodesmata (PD) localization of the Avr protein MP is not required for Tm-22 function. These results suggest that Tm-22-mediated resistance takes place on PM without requirement of its Avr protein to be located to PD. PMID:28258211

  3. Identification of differentially expressed proteins in the cervical mucosa of HIV-1-resistant sex workers.

    PubMed

    Burgener, Adam; Boutilier, Julie; Wachihi, Charles; Kimani, Joshua; Carpenter, Michael; Westmacott, Garrett; Cheng, Keding; Ball, Terry B; Plummer, Francis

    2008-10-01

    Novel tools are necessary to understand mechanisms of altered susceptibility to HIV-1 infection in women of the Pumwani Sex Worker cohort, Kenya. In this cohort, more than 140 of the 2000 participants have been characterized to be relatively resistant to HIV-1 infection. Given that sexual transmission of HIV-1 occurs through mucosal surfaces such as that in the cervicovaginal environment, our hypothesis is that innate immune factors in the genital tract may play a role in HIV-1 infection resistance. Understanding this mechanism may help develop microbicides and/or vaccines against HIV-1. A quantitative proteomics technique (2D-DIGE: two-dimensional difference in-gel electrophoresis) was used to examine cervical mucosa of HIV-1 resistant women ( n = 10) for biomarkers of HIV-1 resistance. Over 15 proteins were found to be differentially expressed between HIV-1-resistant women and control groups ( n = 29), some which show a greater than 8-fold change. HIV-1-resistant women overexpressed several antiproteases, including those from the serpin B family, and also cystatin A, a known anti-HIV-1 factor. Immunoblotting for a selection of the identified proteins confirmed the DIGE volume differences. Validation of these results on a larger sample of individuals will provide further evidence these biomarkers are associated with HIV-1 resistance and could help aid in the development of effective microbicides against HIV-1.

  4. Annotated differentially expressed salivary proteins of susceptible and insecticide-resistant mosquitoes of Anopheles stephensi.

    PubMed

    Vijay, Sonam; Rawal, Ritu; Kadian, Kavita; Raghavendra, Kamaraju; Sharma, Arun

    2015-01-01

    Vector control is one of the major global strategies for control of malaria. However, the major obstacle for vector control is the development of multiple resistances to organochlorine, organophosphorus insecticides and pyrethroids that are currently being used in public health for spraying and in bednets. Salivary glands of vectors are the first target organ for human-vector contact during biting and parasite-vector contact prior to parasite development in the mosquito midguts. The salivary glands secrete anti-haemostatic, anti-inflammatory biologically active molecules to facilitate blood feeding from the host and also inadvertently inject malaria parasites into the vertebrate host. The Anopheles stephensi mosquito, an urban vector of malaria to both human and rodent species has been identified as a reference laboratory model to study mosquito-parasite interactions. In this study, we adopted a conventional proteomic approach of 2D-electrophoresis coupled with MALDI-TOF mass spectrometry and bioinformatics to identify putative differentially expressed annotated functional salivary proteins between An. stephensi susceptible and multiresistant strains with same genetic background. Our results show 2D gel profile and MALDI-TOF comparisons that identified 31 differentially expressed putative modulated proteins in deltamethrin/DDT resistant strains of An. stephensi. Among these 15 proteins were found to be upregulated and 16 proteins were downregulated. Our studies interpret that An. stephensi (multiresistant) caused an upregulated expression of proteins and enzymes like cytochrome 450, short chain dehyrdogenase reductase, phosphodiesterase etc that may have an impact in insecticide resistance and xenobiotic detoxification. Our study elucidates a proteomic response of salivary glands differentially regulated proteins in response to insecticide resistance development which include structural, redox and regulatory enzymes of several pathways. These identified proteins

  5. Identification of a Putative Protein Profile Associated with Tamoxifen Therapy Resistance in Breast Cancer*S⃞

    PubMed Central

    Umar, Arzu; Kang, Hyuk; Timmermans, Annemieke M.; Look, Maxime P.; Meijer-van Gelder, Marion E.; den Bakker, Michael A.; Jaitly, Navdeep; Martens, John W. M.; Luider, Theo M.; Foekens, John A.; Paša-Tolić, Ljiljana

    2009-01-01

    Tamoxifen resistance is a major cause of death in patients with recurrent breast cancer. Current clinical factors can correctly predict therapy response in only half of the treated patients. Identification of proteins that are associated with tamoxifen resistance is a first step toward better response prediction and tailored treatment of patients. In the present study we intended to identify putative protein biomarkers indicative of tamoxifen therapy resistance in breast cancer using nano-LC coupled with FTICR MS. Comparative proteome analysis was performed on ∼5,500 pooled tumor cells (corresponding to ∼550 ng of protein lysate/analysis) obtained through laser capture microdissection (LCM) from two independently processed data sets (n = 24 and n = 27) containing both tamoxifen therapy-sensitive and therapy-resistant tumors. Peptides and proteins were identified by matching mass and elution time of newly acquired LC-MS features to information in previously generated accurate mass and time tag reference databases. A total of 17,263 unique peptides were identified that corresponded to 2,556 non-redundant proteins identified with ≥2 peptides. 1,713 overlapping proteins between the two data sets were used for further analysis. Comparative proteome analysis revealed 100 putatively differentially abundant proteins between tamoxifen-sensitive and tamoxifen-resistant tumors. The presence and relative abundance for 47 differentially abundant proteins were verified by targeted nano-LC-MS/MS in a selection of unpooled, non-microdissected discovery set tumor tissue extracts. ENPP1, EIF3E, and GNB4 were significantly associated with progression-free survival upon tamoxifen treatment for recurrent disease. Differential abundance of our top discriminating protein, extracellular matrix metalloproteinase inducer, was validated by tissue microarray in an independent patient cohort (n = 156). Extracellular matrix metalloproteinase inducer levels were higher in therapy-resistant

  6. Sodium Orthovanadate-Resistant Mutants of Saccharomyces Cerevisiae Show Defects in Golgi-Mediated Protein Glycosylation, Sporulation and Detergent Resistance

    PubMed Central

    Kanik-Ennulat, C.; Montalvo, E.; Neff, N.

    1995-01-01

    Orthovanadate is a small toxic molecule that competes with the biologically important oxyanion orthophosphate. Orthovanadate resistance arises spontaneously in Saccharomyces cerevisiae haploid cells by mutation in a number of genes. Mutations selected at 3 mM sodium orthovanadate have different degrees of vanadate resistance, hygromycin sensitivity, detergent sensitivity and sporulation defects. Recessive vanadate-resistant mutants belong to at least six genetic loci. Most mutants are defective in outer chain glycosylation of secreted invertase (van1, van2, van4, van5, van6, VAN7-116 and others), a phenotype found in some MNN or VRG mutants. The phenotypes of these vanadate-resistant mutants are consistent with an alteration in the permeability or specificity of the Golgi apparatus. The previously published VAN1 gene product has a 200 amino acid domain with 40% identity with the MNN9 gene product and 70% identity with the ANP1 gene product. Cells containing the van1-18, mnn9 (vrg6) or anp1 mutations have some phenotypic similarities. The VAN2 gene was isolated and its coding region is identified and reported. It is an essential gene on chromosome XV and its translated amino acid sequence predicts a unique 337 amino acid protein with multiple transmembrane domains. PMID:7672592

  7. Protein detection using tunable pores: resistive pulses and current rectification.

    PubMed

    Blundell, Emma L C J; Mayne, Laura J; Lickorish, Michael; Christie, Steven D R; Platt, Mark

    2016-12-12

    We present the first comparison between assays that use resistive pulses or rectification ratios on a tunable pore platform. We compare their ability to quantify the cancer biomarker Vascular Endothelial Growth Factor (VEGF). The first assay measures the electrophoretic mobility of aptamer modified nanoparticles as they traverse the pore. By controlling the aptamer loading on the particle surface, and measuring the speed of each translocation event we are able to observe a change in velocity as low as 18 pM. A second non-particle assay exploits the current rectification properties of conical pores. We report the first use of Layer-by-Layer (LbL) assembly of polyelectrolytes onto the surface of the polyurethane pore. The current rectification ratios demonstrate the presence of the polymers, producing pH and ionic strength-dependent currents. The LbL assembly allows the facile immobilisation of DNA aptamers onto the pore allowing a specific dose response to VEGF. Monitoring changes to the current rectification allows for a rapid detection of 5 pM VEGF. Each assay format offers advantages in their setup and ease of preparation but comparable sensitivities.

  8. Iron Regulatory Proteins Mediate Host Resistance to Salmonella Infection.

    PubMed

    Nairz, Manfred; Ferring-Appel, Dunja; Casarrubea, Daniela; Sonnweber, Thomas; Viatte, Lydie; Schroll, Andrea; Haschka, David; Fang, Ferric C; Hentze, Matthias W; Weiss, Guenter; Galy, Bruno

    2015-08-12

    Macrophages are essential for systemic iron recycling, and also control iron availability to pathogens. Iron metabolism in mammalian cells is orchestrated posttranscriptionally by iron-regulatory proteins (IRP)-1 and -2. Here, we generated mice with selective and combined ablation of both IRPs in macrophages to investigate the role of IRPs in controlling iron availability. These animals are hyperferritinemic but otherwise display normal clinical iron parameters. However, mutant mice rapidly succumb to systemic infection with Salmonella Typhimurium, a pathogenic bacterium that multiplies within macrophages, with increased bacterial burdens in liver and spleen. Ex vivo infection experiments indicate that IRP function restricts bacterial access to iron via the EntC and Feo bacterial iron-acquisition systems. Further, IRPs contain Salmonella by promoting the induction of lipocalin 2, a host antimicrobial factor that inhibits bacterial uptake of iron-laden siderophores, and by suppressing the ferritin iron pool. This work reveals the importance of the IRPs in innate immunity.

  9. DRPPP: A machine learning based tool for prediction of disease resistance proteins in plants.

    PubMed

    Pal, Tarun; Jaiswal, Varun; Chauhan, Rajinder S

    2016-11-01

    Plant disease outbreak is increasing rapidly around the globe and is a major cause for crop loss worldwide. Plants, in turn, have developed diverse defense mechanisms to identify and evade different pathogenic microorganisms. Early identification of plant disease resistance genes (R genes) can be exploited for crop improvement programs. The present prediction methods are either based on sequence similarity/domain-based methods or electronically annotated sequences, which might miss existing unrecognized proteins or low similarity proteins. Therefore, there is an urgent need to devise a novel machine learning technique to address this problem. In the current study, a SVM-based tool was developed for prediction of disease resistance proteins in plants. All known disease resistance (R) proteins (112) were taken as a positive set, whereas manually curated negative dataset consisted of 119 non-R proteins. Feature extraction generated 10,270 features using 16 different methods. The ten-fold cross validation was performed to optimize SVM parameters using radial basis function. The model was derived using libSVM and achieved an overall accuracy of 91.11% on the test dataset. The tool was found to be robust and can be used for high-throughput datasets. The current study provides instant identification of R proteins using machine learning approach, in addition to the similarity or domain prediction methods.

  10. Severe Injury Is Associated With Insulin Resistance, Endoplasmic Reticulum Stress Response, and Unfolded Protein Response

    PubMed Central

    Jeschke, Marc G.; Finnerty, Celeste C.; Herndon, David N.; Song, Juquan; Boehning, Darren; Tompkins, Ronald G.; Baker, Henry V.; Gauglitz, Gerd G.

    2012-01-01

    Objective We determined whether postburn hyperglycemia and insulin resistance are associated with endoplasmic reticulum (ER) stress/unfolded protein response (UPR) activation leading to impaired insulin receptor signaling. Background Inflammation and cellular stress, hallmarks of severely burned and critically ill patients, have been causally linked to insulin resistance in type 2 diabetes via induction of ER stress and the UPR. Methods Twenty severely burned pediatric patients were compared with 36 nonburned children. Clinical markers, protein, and GeneChip analysis were used to identify transcriptional changes in ER stress and UPR and insulin resistance–related signaling cascades in peripheral blood leukocytes, fat, and muscle at admission and up to 466 days postburn. Results Burn-induced inflammatory and stress responses are accompanied by profound insulin resistance and hyperglycemia. Genomic and protein analysis revealed that burn injury was associated with alterations in the signaling pathways that affect insulin resistance, ER/sarcoplasmic reticulum stress, inflammation, and cell growth/apoptosis up to 466 days postburn. Conclusion Burn-induced insulin resistance is associated with persistent ER/sarcoplasmic reticulum stress/UPR and subsequent suppressed insulin receptor signaling over a prolonged period of time. PMID:22241293

  11. dc-Sheet resistance as sensitive monitoring tool of protein immobilization on thin metal films.

    PubMed

    Neff, H; Beeby, T; Lima, A M N; Borre, M; Thirstrup, C; Zong, W; de Almeida, L A L

    2006-03-15

    The suitability of high resolution, in situ dc-sheet resistance monitoring (SRM) as a simplified and reliable sensing technique towards detection and tracking of protein immobilization has been explored. Non-specific adsorption of bovine serum albumin (BSA) onto a very thin gold film, acting as the sensing resistor, has been employed as a model system. For comparison, the novel sensing method was combined with surface plasmon resonance (SPR) spectroscopy, using the same flow cell and sensing surface. Two different, well known adsorption states, involving a composite layer of irreversibly and reversibly bound BSA, were clearly resolved by both methods. Clearly structured, pronounced and fully reproducible film resistance modulations have been resolved in the associated SRM data. The transition from reversibly bound BSA to the diluted protein phase is associated with an unusually large decrease in the dc-sheet resistance. The observed resistance modulation magnitude for an adsorbed BSA monolayer corresponds to approximately 1%, and up to 100 mOmega at a 10 Omega sensing resistor. The sheet resistance of irreversibly bound BSA was determined to 0.24 kOmega/cm2, and the associated specific resistivity estimated to 1-2x10(4) Omega cm.

  12. Enhanced pest resistance of maize leaves expressing monocot crop plant derived ribosome inactivating protein and agglutinin

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Although many insect resistance genes have been identified, the number of studies examining their effects in combination using transgenic systems is limited. We introduced a construct into maize containing the coding sequence for maize ribosome inactivating protein (MRIP), wheat germ agglutinin (WGA...

  13. L-Alanylglutamine inhibits signaling proteins that activate protein degradation, but does not affect proteins that activate protein synthesis after an acute resistance exercise.

    PubMed

    Wang, Wanyi; Choi, Ran Hee; Solares, Geoffrey J; Tseng, Hung-Min; Ding, Zhenping; Kim, Kyoungrae; Ivy, John L

    2015-07-01

    Sustamine™ (SUS) is a dipeptide composed of alanine and glutamine (AlaGln). Glutamine has been suggested to increase muscle protein accretion; however, the underlying molecular mechanisms of glutamine on muscle protein metabolism following resistance exercise have not been fully addressed. In the present study, 2-month-old rats climbed a ladder 10 times with a weight equal to 75 % of their body mass attached at the tail. Rats were then orally administered one of four solutions: placebo (PLA-glycine = 0.52 g/kg), whey protein (WP = 0.4 g/kg), low dose of SUS (LSUS = 0.1 g/kg), or high dose of SUS (HSUS = 0.5 g/kg). An additional group of sedentary (SED) rats was intubated with glycine (0.52 g/kg) at the same time as the ladder-climbing rats. Blood samples were collected immediately after exercise and at either 20 or 40 min after recovery. The flexor hallucis longus (FHL), a muscle used for climbing, was excised at 20 or 40 min post exercise and analyzed for proteins regulating protein synthesis and degradation. All supplements elevated the phosphorylation of FOXO3A above SED at 20 min post exercise, but only the SUS supplements significantly reduced the phosphorylation of AMPK and NF-kB p65. SUS supplements had no effect on mTOR signaling, but WP supplementation yielded a greater phosphorylation of mTOR, p70S6k, and rpS6 compared with PLA at 20 min post exercise. However, by 40 min post exercise, phosphorylation of mTOR and rpS6 in PLA had risen to levels not different than WP. These results suggest that SUS blocks the activation of intracellular signals for MPB, whereas WP accelerates mRNA translation.

  14. Revisiting the mechanism of macrolide-antibiotic resistance mediated by ribosomal protein L22

    PubMed Central

    Moore, Sean D.; Sauer, Robert T.

    2008-01-01

    Bacterial antibiotic resistance can occur by many mechanisms. An intriguing class of mutants is resistant to macrolide antibiotics even though these drugs still bind to their targets. For example, a 3-residue deletion (ΔMKR) in ribosomal protein L22 distorts a loop that forms a constriction in the ribosome exit tunnel, apparently allowing nascent-chain egress and translation in the presence of bound macrolides. Here, however, we demonstrate that ΔMKR and wild-type ribosomes show comparable macrolide sensitivity in vitro. In Escherichia coli, we find that this mutation reduces antibiotic occupancy of the target site on ribosomes in a manner largely dependent on the AcrAB-TolC efflux system. We propose a model for antibiotic resistance in which ΔMKR ribosomes alter the translation of specific proteins, possibly via changes in programmed stalling, and modify the cell envelope in a manner that lowers steady-state macrolide levels. PMID:19015512

  15. Chromate Efflux by Means of the ChrA Chromate Resistance Protein from Pseudomonas aeruginosa

    PubMed Central

    Alvarez, Angel H.; Moreno-Sánchez, Rafael; Cervantes, Carlos

    1999-01-01

    Everted membrane vesicles of Pseudomonas aeruginosa PAO1 harboring plasmid pCRO616, expressing the ChrA chromate resistance protein, accumulated four times more 51CrO42− than vesicles from plasmidless cells, indicating that a chromate efflux system functions in the resistant strain. Chromate uptake showed saturation kinetics with an apparent Km of 0.12 mM chromate and a Vmax of 0.5 nmol of chromate/min per mg of protein. Uptake of chromate by vesicles was dependent on NADH oxidation and was abolished by energy inhibitors and by the chromate analog sulfate. The mechanism of resistance to chromate determined by ChrA appears to be based on the active efflux of chromate driven by the membrane potential. PMID:10572148

  16. Molecular mechanism of ATP-dependent solute transport by multidrug resistance-associated protein 1.

    PubMed

    Chang, Xiu-bao

    2010-01-01

    Millions of new cancer patients are diagnosed each year and over half of these patients die from this devastating disease. Thus, cancer causes a major public health problem worldwide. Chemotherapy remains the principal mode to treat many metastatic cancers. However, occurrence of cellular multidrug resistance (MDR) prevents efficient killing of cancer cells, leading to chemotherapeutic treatment failure. Over-expression of ATP-binding cassette transporters, such as P-glycoprotein, breast cancer resistance protein and/or multidrug resistance-associated protein 1 (MRP1), confers an acquired MDR due to their capabilities of transporting a broad range of chemically diverse anticancer drugs across the cell membrane barrier. In this review, the molecular mechanism of ATP-dependent solute transport by MRP1 will be addressed.

  17. Revisiting the mechanism of macrolide-antibiotic resistance mediated by ribosomal protein L22.

    PubMed

    Moore, Sean D; Sauer, Robert T

    2008-11-25

    Bacterial antibiotic resistance can occur by many mechanisms. An intriguing class of mutants is resistant to macrolide antibiotics even though these drugs still bind to their targets. For example, a 3-residue deletion (DeltaMKR) in ribosomal protein L22 distorts a loop that forms a constriction in the ribosome exit tunnel, apparently allowing nascent-chain egress and translation in the presence of bound macrolides. Here, however, we demonstrate that DeltaMKR and wild-type ribosomes show comparable macrolide sensitivity in vitro. In Escherichia coli, we find that this mutation reduces antibiotic occupancy of the target site on ribosomes in a manner largely dependent on the AcrAB-TolC efflux system. We propose a model for antibiotic resistance in which DeltaMKR ribosomes alter the translation of specific proteins, possibly via changes in programmed stalling, and modify the cell envelope in a manner that lowers steady-state macrolide levels.

  18. Role of Ingested Amino Acids and Protein in the Promotion of Resistance Exercise–Induced Muscle Protein Anabolism123

    PubMed Central

    Rasmussen, Blake B

    2016-01-01

    The goal of this critical review is to comprehensively assess the evidence for the molecular, physiologic, and phenotypic skeletal muscle responses to resistance exercise (RE) combined with the nutritional intervention of protein and/or amino acid (AA) ingestion in young adults. We gathered the literature regarding the translational response in human skeletal muscle to acute exposure to RE and protein/AA supplements and the literature describing the phenotypic skeletal muscle adaptation to RE and nutritional interventions. Supplementation of protein/AAs with RE exhibited clear protein dose–dependent effects on translational regulation (protein synthesis) through mammalian target of rapamycin complex 1 (mTORC1) signaling, which was most apparent through increases in p70 ribosomal protein S6 kinase 1 (S6K1) phosphorylation, compared with postexercise recovery in the fasted or carbohydrate-fed state. These acute findings were critically tested via long-term exposure to RE training (RET) and protein/AA supplementation, and it was determined that a diminishing protein/AA supplement effect occurs over a prolonged exposure stimulus after exercise training. Furthermore, we found that protein/AA supplements, combined with RET, produced a positive, albeit minor, effect on the promotion of lean mass growth (when assessed in >20 participants/treatment); a negligible effect on muscle mass; and a negligible to no additional effect on strength. A potential concern we discovered was that the majority of the exercise training studies were underpowered in their ability to discern effects of protein/AA supplementation. Regardless, even when using optimal methodology and large sample sizes, it is clear that the effect size for protein/AA supplementation is low and likely limited to a subset of individuals because the individual variability is high. With regard to nutritional intakes, total protein intake per day, rather than protein timing or quality, appears to be more of a factor

  19. High Levels of Expression of P-glycoprotein/Multidrug Resistance Protein Result in Resistance to Vintafolide.

    PubMed

    Guertin, Amy D; O'Neil, Jennifer; Stoeck, Alexander; Reddy, Joseph A; Cristescu, Razvan; Haines, Brian B; Hinton, Marlene C; Dorton, Ryan; Bloomfield, Alicia; Nelson, Melissa; Vetzel, Marilynn; Lejnine, Serguei; Nebozhyn, Michael; Zhang, Theresa; Loboda, Andrey; Picard, Kristen L; Schmidt, Emmett V; Dussault, Isabelle; Leamon, Christopher P

    2016-08-01

    Targeting surface receptors overexpressed on cancer cells is one way to specifically treat cancer versus normal cells. Vintafolide (EC145), which consists of folate linked to a cytotoxic small molecule, desacetylvinblastine hydrazide (DAVLBH), takes advantage of the overexpression of folate receptor (FR) on cancer cells. Once bound to FR, vintafolide enters the cell by endocytosis, and the reducing environment of the endosome cleaves the linker, releasing DAVLBH to destabilize microtubules. Vintafolide has shown efficacy and improved tolerability compared with DAVLBH in FR-positive preclinical models. As the first FR-targeting drug to reach the clinic, vintafolide has achieved favorable responses in phase II clinical trials in FR-positive ovarian and lung cancer. However, some FR-positive patients in these clinical trials do not respond to vintafolide. We sought to identify potential biomarkers of resistance to aid in the future development of this and other FR-targeting drugs. Here, we confirm that high P-glycoprotein (P-gp) expression was the strongest predictor of resistance to DAVLBH in a panel of 359 cancer cell lines. Furthermore, targeted delivery of DAVLBH via the FR, as in vintafolide, fails to overcome P-gp-mediated efflux of DAVLBH in both in vitro and in vivo preclinical models. Therefore, we suggest that patients whose tumors express high levels of P-gp be excluded from future clinical trials for vintafolide as well as other FR-targeted therapeutics bearing a P-gp substrate. Mol Cancer Ther; 15(8); 1998-2008. ©2016 AACR.

  20. Improving protein resistance of α-Al 2O 3 membranes by modification with POEGMA brushes

    NASA Astrophysics Data System (ADS)

    He, Huating; Jing, Wenheng; Xing, Weihong; Fan, Yiqun

    2011-11-01

    A kind of protein-resistant ceramic membrane is prepared by grafting poly(oligo (ethylene glycol) methyl ether methacrylate) (POEGMA) brushes onto the surfaces and pore walls of α-Al2O3 membrane (AM) by surface-initiated atom-transfer radical polymerization (SI-ATRP). Contact-angle, Fourier-transform infrared spectroscopy (FTIR), thermogravimetric analysis (TGA) and field-emission scanning electron microscopy (FESEM) were measured to confirm that the surfaces and pore walls of the ceramic porous membranes have been modified by the brushes with this method successfully. The protein interaction behavior with the POEGMA modified membranes (AM-POEGMA) was studied by the model protein of bovine serum albumin (BSA). A protein-resistant mechanism of AM-POEGMA was proposed to describe an interesting phenomenon discovered in the filtration experiment, in which the initial flux filtrating BSA solution is higher than the pure water flux. The fouling of AM-POEGMA was easier to remove than AM for the action of POEGMA brushes, indicated that the ceramic porous membranes modified with POEGMA brushes exhibit excellent protein resistance.

  1. The Role of Organic Proteins on the Crack Growth Resistance of Human Enamel

    PubMed Central

    Yahyazadehfar, Mobin; Arola, Dwayne

    2015-01-01

    With only 1% protein by weight, tooth enamel is the most highly mineralized tissue in mammals. The focus of this study was to evaluate contributions of the proteins on the fracture resistance of this unique structural material. Sections of enamel were obtained from the cusps of human molars and the crack growth resistance was quantified using a conventional fracture mechanics approach with complementary finite element analysis. In selected specimens the proteins were extracted using a potassium hydroxide treatment. Removal of the proteins resulted in approximately 40% decrease in the fracture toughness with respect to the fully proteinized control. The loss of organic content was most detrimental to the extrinsic toughening mechanisms, causing over 80% reduction in their contribution to the total energy to fracture. This degradation occurred by embrittlement of the unbroken bridging ligaments and consequent reduction in the crack closure stress. Although the organic content of tooth enamel is very small, it is essential to crack growth toughening by facilitating the formation of unbroken ligaments and in fortifying their potency. Replicating functions of the organic content will be critical to the successful development of bio-inspired materials that are designed for fracture resistance. PMID:25805107

  2. Arabidopsis dual resistance proteins, both RPS4 and RRS1, are required for resistance to bacterial wilt in transgenic Brassica crops.

    PubMed

    Narusaka, Mari; Hatakeyama, Katsunori; Shirasu, Ken; Narusaka, Yoshihiro

    2014-01-01

    Bacterial wilt phytopathogen Ralstonia solanacearum is a serious soil-borne disease that attacks several economically important plants worldwide, including Brassicaceae. Previous studies indicate that recognition of avirulence (Avr)-effector PopP2 by resistance (R) protein, RRS1-R, and physical interaction between RRS1-R and PopP2 in the nucleus are required for resistance. Of late, we showed that a pair of Arabidopsis thaliana TIR-NLR proteins, RRS1 and RPS4, function together in disease resistance against multiple pathogen isolates. Here, we report that dual R proteins, RRS1 and RPS4, from A. thaliana ecotype Wassilewskija confer resistance to bacterial wilt in transgenic Brassica crops. For practical applications, this finding may provide a new strategy for developing disease resistant plants that express R genes from other plants.

  3. Lipid A and resistance of Salmonella typhimurium to antimicrobial granule proteins of human neutrophil granulocytes.

    PubMed Central

    Shafer, W M; Casey, S G; Spitznagel, J K

    1984-01-01

    Granule extracts from human polymorphonuclear leukocytes were prepared and fractionated by chromatography on Sephadex G75-SF. One fraction exhibited potent antimicrobial activity against an Rd1 lipopolysaccharide (LPS) mutant of Salmonella typhimurium. Susceptibility of the mutant to antimicrobial activity appeared to be due to binding of granule proteins to lipid A because isolated native LPS succeeded in blocking the antimicrobial activity of granule extracts whereas base-hydrolyzed LPS failed to do so. Centrifugation of control and base-hydrolyzed LPS-protein mixtures in cesium chloride gradients suggested that only control LPS formed complexes with antimicrobial proteins. Further evidence that bactericidal proteins from polymorphonuclear leukocyte granules interact with lipid A was that sublethal concentrations of polymyxin B (an antibiotic known to bind to lipid A) rendered target bacteria phenotypically resistant to granule proteins. Moreover, a mutant of S. typhimurium which synthesized a lipid A with decreased electronegativity due to increased 4-amino-4-deoxy-L-arabinosylation at the 4'-phosphate exhibited increased resistance to both polymyxin B and granule proteins. These results suggest that polymyxin B and antimicrobial proteins derived from polymorphonuclear leukocyte granules interact with lipid A in an analogous manner. PMID:6199303

  4. Deciphering the protein translation inhibition and coping mechanism of trichothecene toxin in resistant fungi.

    PubMed

    Kumari, Indu; Ahmed, Mushtaq; Akhter, Yusuf

    2016-09-01

    In modern times for combating the deleterious soil microbes for improved sustainable agricultural practices, there is a need to have a proper understanding of the plant-microbe interactions present in the rhizospheric microbiome of the plant roots. In the present study, the interactions of trichodermin with petidyltransferase centre of ribosomal complex was studied by molecular dynamics and in silico interaction methods to demonstrate its mechanism of action and to decipher the possible reason how it may inhibit protein synthesis at the ribosomal complex. Further we have illustrated how trichodermin resistance protein (60S ribosomal protein L3) helps to overcome the deleterious effects of trichothecene compounds like trichodermin. Normal mode analysis of trichodermin resistance protein and 25S rRNA that constitutes the petidyltransferase centre showed that the W-finger region of the protein moved towards 25S rRNA. Further analysis of molecular dynamics simulation time frames showed that several intermediate states of large motions of the protein molecules towards the 25S rRNA which finally blocks the binding pocket of the trichodermin. It indicated that this protein not only changes the local environment and conformation of the petidyltransferase centre but also restrain trichodermin from binding to the 25S rRNA at the petidyltransferase centre.

  5. Prediction of HIV-1 protease inhibitor resistance using a protein-inhibitor flexible docking approach.

    PubMed

    Jenwitheesuk, Ekachai; Samudrala, Ram

    2005-01-01

    Emergence of drug resistance remains one of the most challenging issues in the treatment of HIV-1 infection. Here we focus on resistance to HIV-1 protease inhibitors (PIs) at a molecular level, which can be analysed genotypically or phenotypically. Genotypic assays are based on the analysis of mutations associated with reduced drug susceptibility, but are problematic because of the numerous mutations and mutational patterns that confer drug resistance. Phenotypic resistance or susceptibility can be experimentally evaluated by measuring the amount of free drug bound to HIV-1 protease molecules, but this procedure is expensive and time-consuming. To overcome these problems, we have developed a docking protocol that takes protein-inhibitor flexibility into account to predict phenotypic drug resistance. For six FDA-approved Pls and a total of 1792 HIV-1 protease sequence mutants, we used a combination of inhibitor flexible docking and molecular dynamics (MD) simulations to calculate protein-inhibitor binding energies. Prediction results were expressed as fold changes of the calculated inhibitory constant (Ki), and the samples predicted to have fold-increase in calculated Ki above the fixed cut-off were defined as drug resistant. Our combined docking and MD protocol achieved accuracies ranging from 72-83% in predicting resistance/susceptibility for five of the six drugs evaluated. Evaluating the method only on samples where our predictions concurred with established knowledge-based methods resulted in increased accuracies of 83-94% for the six drugs. The results suggest that a physics-based approach, which is readily applicable to any novel PI and/or mutant, can be used judiciously with knowledge-based approaches that require experimental training data to devise accurate models of HIV-1 Pl resistance prediction.

  6. Resistance of polysaccharide coatings to proteins, hematopoietic cells, and marine organisms.

    PubMed

    Cao, Xinyu; Pettit, Michala E; Conlan, Sheelagh L; Wagner, Wolfgang; Ho, Anthony D; Clare, Anthony S; Callow, James A; Callow, Maureen E; Grunze, Michael; Rosenhahn, Axel

    2009-04-13

    The interaction of covalently coupled hyaluronic acid, alginic acid, and pectic acid with proteins, cells (hematopoietic KG1a and Jurkat cells), and marine organisms (algal zoospores and barnacle cypris larvae) is compared. In contrast to cells and proteins for which such polysaccharide coatings are known for their antiadhesive properties, marine algal spores and barnacle cyprids were able to colonize the surfaces. Of the three polysaccharides, hyaluronic acid showed the lowest settlement of both Ulva zoopores and barnacles. Photoelectron spectroscopy reveals that the polysaccharide coatings tend to bind bivalent ions, such as calcium, from salt water. Such pretreatment with a high salinity medium significantly changes the protein and hematopoietic cell resistance of the surfaces. Complexation of bivalent ions is therefore considered as one reason for the decreased resistance of polysaccharide coatings when applied in the marine environment.

  7. Relative penicillin G resistance in Neisseria meningitidis and reduced affinity of penicillin-binding protein 3.

    PubMed Central

    Mendelman, P M; Campos, J; Chaffin, D O; Serfass, D A; Smith, A L; Sáez-Nieto, J A

    1988-01-01

    We examined clinical isolates of Neisseria meningitidis relatively resistant to penicillin G (mean MIC, 0.3 micrograms/ml; range, 0.1 to 0.7 micrograms/ml), which were isolated from blood and cerebrospinal fluid for resistance mechanisms, by using susceptible isolates (mean MIC, less than or equal to 0.06 micrograms/ml) for comparison. The resistant strains did not produce detectable beta-lactamase activity, otherwise modify penicillin G, or bind less total penicillin. Penicillin-binding protein (PBP) 3 of the six resistant isolates tested uniformly bound less penicillin G in comparison to the same PBP of four susceptible isolates. Reflecting the reduced binding affinity of PBP 3 of the two resistant strains tested, the amount of 3H-labeled penicillin G required for half-maximal binding was increased in comparison with that of PBP 3 of the two susceptible isolates. We conclude that the mechanism of resistance in these meningococci relatively resistant to penicillin G was decreased affinity of PBP 3. Images PMID:3134848

  8. TgPRELID, a Mitochondrial Protein Linked to Multidrug Resistance in the Parasite Toxoplasma gondii

    PubMed Central

    Kamau, Edwin T.; Srinivasan, Ananth R.; Harper, Jonathan; Sankaran, Preethi; Post, Sarah E.; Varberg, Joseph M.

    2017-01-01

    ABSTRACT New drugs to control infection with the protozoan parasite Toxoplasma gondii are needed as current treatments exert toxic side effects on patients. Approaches to develop novel compounds for drug development include screening of compound libraries and targeted inhibition of essential cellular pathways. We identified two distinct compounds that display inhibitory activity against the parasite’s replicative stage: F3215-0002, which we previously identified during a compound library screen, and I-BET151, an inhibitor of bromodomains, the “reader” module of acetylated lysines. In independent studies, we sought to determine the targets of these two compounds using forward genetics, generating resistant mutants and identifying the determinants of resistance with comparative genome sequencing. Despite the dissimilarity of the two compounds, we recovered resistant mutants with nonsynonymous mutations in the same domain of the same gene, TGGT1_254250, which we found encodes a protein that localizes to the parasite mitochondrion (designated TgPRELID after the name of said domain). We found that mutants selected with one compound were cross resistant to the other compound, suggesting a common mechanism of resistance. To further support our hypothesis that TgPRELID mutations facilitate resistance to both I-BET151 and F3215-0002, CRISPR (clustered regularly interspaced short palindromic repeat)/CAS9-mediated mutation of TgPRELID directly led to increased F3215-0002 resistance. Finally, all resistance mutations clustered in the same subdomain of TgPRELID. These findings suggest that TgPRELID may encode a multidrug resistance factor or that I-BET151 and F3215-0002 have the same target(s) despite their distinct chemical structures. IMPORTANCE We report the discovery of TgPRELID, a previously uncharacterized mitochondrial protein linked to multidrug resistance in the parasite Toxoplasma gondii. Drug resistance remains a major problem in the battle against parasitic

  9. TgPRELID, a Mitochondrial Protein Linked to Multidrug Resistance in the Parasite Toxoplasma gondii.

    PubMed

    Jeffers, Victoria; Kamau, Edwin T; Srinivasan, Ananth R; Harper, Jonathan; Sankaran, Preethi; Post, Sarah E; Varberg, Joseph M; Sullivan, William J; Boyle, Jon P

    2017-01-01

    New drugs to control infection with the protozoan parasite Toxoplasma gondii are needed as current treatments exert toxic side effects on patients. Approaches to develop novel compounds for drug development include screening of compound libraries and targeted inhibition of essential cellular pathways. We identified two distinct compounds that display inhibitory activity against the parasite's replicative stage: F3215-0002, which we previously identified during a compound library screen, and I-BET151, an inhibitor of bromodomains, the "reader" module of acetylated lysines. In independent studies, we sought to determine the targets of these two compounds using forward genetics, generating resistant mutants and identifying the determinants of resistance with comparative genome sequencing. Despite the dissimilarity of the two compounds, we recovered resistant mutants with nonsynonymous mutations in the same domain of the same gene, TGGT1_254250, which we found encodes a protein that localizes to the parasite mitochondrion (designated TgPRELID after the name of said domain). We found that mutants selected with one compound were cross resistant to the other compound, suggesting a common mechanism of resistance. To further support our hypothesis that TgPRELID mutations facilitate resistance to both I-BET151 and F3215-0002, CRISPR (clustered regularly interspaced short palindromic repeat)/CAS9-mediated mutation of TgPRELID directly led to increased F3215-0002 resistance. Finally, all resistance mutations clustered in the same subdomain of TgPRELID. These findings suggest that TgPRELID may encode a multidrug resistance factor or that I-BET151 and F3215-0002 have the same target(s) despite their distinct chemical structures. IMPORTANCE We report the discovery of TgPRELID, a previously uncharacterized mitochondrial protein linked to multidrug resistance in the parasite Toxoplasma gondii. Drug resistance remains a major problem in the battle against parasitic infection, and

  10. Intracellular accumulation and resistance to degradation of the Alzheimer amyloid A4/beta protein.

    PubMed Central

    Knauer, M F; Soreghan, B; Burdick, D; Kosmoski, J; Glabe, C G

    1992-01-01

    The A4 or beta protein is a peptide that constitutes the major protein component of senile plaques in Alzheimer disease. The A4/beta protein is derived from a larger, transmembrane amyloid precursor protein (APP). The putative abnormal processing events leading to amyloid accumulation are largely unknown. Here we report that a 42-residue synthetic peptide, beta 1-42, corresponding to one of the longer forms of the A4/beta protein, accumulates in cultured human skin fibroblasts and is stable for at least 3 days. The peptide appears to accumulate intracellularly, since it does not accumulate under conditions that prevent endocytosis and accumulation is correlated with the acquisition of resistance to removal by trypsin digestion. This intracellular accumulation is also correlated with the ability of the peptide to aggregate as determined by SDS/polyacrylamide gel electrophoresis. At low concentrations of the beta 1-42 peptide, which favor the nonaggregated state, no accumulation is observed. Shorter peptide analogs (28 or 39 residues) that are truncated at the C terminus, which lack the ability to aggregate in SDS gels, fail to accumulate. The accumulated intracellular beta 1-42 peptide is in an aggregated state and is contained in a dense organellar compartment that overlaps the distribution of late endosomes or secondary lysosomes. Immunofluorescence of the internalized peptide in permeabilized cells reveals that it is contained in granular deposits, consistent with localization in late endosomes or secondary lysosomes. Sequence analysis indicates that some of the internalized peptide is subject to N-terminal trimming. These results suggest that the aggregated A4/beta protein may be resistant to degradation and suggest that the A4/beta protein may arise, at least in part, by endosomal or lysosomal processing of APP. Our results also suggest that relatively nonspecific proteolysis may be sufficient to generate the A4/beta protein if this part of APP is selectively

  11. Coordinate regulation of proteins associated with radiation resistance in cultured insect cells

    SciTech Connect

    Rand, A.; Koval, T.M.

    1994-04-01

    Cultured TN-368 lepidopteran insect cells exhibit a pronounced resistance to the lethal effects of a variety of physical agents, including X rays and 254 nm UV light, as well as a large number of chemicals. The resistance to ionizing radiation has previously been associated with an inducible process which is not expressed in unirradiated cells or cells receiving less than some minimal amount of radiation necessary for activating the process. The studies in this paper were initiated in an attempt to identify and characterize the inducible proteins associated with the marked radiation resistance of the TN-368 cells. Cells were exposed to doses of 0, 25, 64 or 350 Gy of {sup 137}Cs {gamma} rays and incubated either for 3 h in medium containing [{sup 35}S]methionine or for 2 h without labeling. Labeled cells were separated into nuclear and cytoplasmic fractions and proteins were analyzed on two-dimensional polyacrylamide gels. Unlabeled cells were used to isolate total RNA which was translated in vitro in a rabbit reticulocyte lysate system with {sup 35}S label. These translation products were also analyzed by two-dimensional electrophoresis. Gamma irradiation of the TN-368 cells resulted in the de novo synthesis of several proteins as well as the complete inhibition of others. The number of such proteins identified was 19. These proteins ranged in size from 18-73 kDa, with a pI distribution of 4.7 to 6.1. In addition to the unique proteins, a large number of other proteins were also either up- or down-regulated. These observations were made in both nuclear and cytoplasmic fractions as well as in the translation products of RNA produced after irradiation. These studies indicate that RNA and protein synthesis in lepidopteran cells are coordinately regulated in response to ionizing radiation and may participate in the pronounced radioresistance of the TN-368 cells. 15 refs., 3 figs., 1 tab.

  12. Characterization of a Novel Endoplasmic Reticulum Protein Involved in Tubercidin Resistance in Leishmania major

    PubMed Central

    Aoki, Juliana Ide; Coelho, Adriano Cappellazzo; Muxel, Sandra Marcia; Zampieri, Ricardo Andrade; Sanchez, Eduardo Milton Ramos; Nerland, Audun Helge; Floeter-Winter, Lucile Maria; Cotrim, Paulo Cesar

    2016-01-01

    Background Tubercidin (TUB) is a toxic adenosine analog with potential antiparasitic activity against Leishmania, with mechanism of action and resistance that are not completely understood. For understanding the mechanisms of action and identifying the potential metabolic pathways affected by this drug, we employed in this study an overexpression/selection approach using TUB for the identification of potential targets, as well as, drug resistance genes in L. major. Although, TUB is toxic to the mammalian host, these findings can provide evidences for a rational drug design based on purine pathway against leishmaniasis. Methodology/Principal findings After transfection of a cosmid genomic library into L. major Friedlin (LmjF) parasites and application of the overexpression/selection method, we identified two cosmids (cosTUB1 and cosTU2) containing two different loci capable of conferring significant levels of TUB resistance. In the cosTUB1 contained a gene encoding NUPM1-like protein, which has been previously described as associated with TUB resistance in L. amazonensis. In the cosTUB2 we identified and characterized a gene encoding a 63 kDa protein that we denoted as tubercidin-resistance protein (TRP). Functional analysis revealed that the transfectants were less susceptible to TUB than LmjF parasites or those transfected with the control vector. In addition, the trp mRNA and protein levels in cosTUB2 transfectants were higher than LmjF. TRP immunolocalization revealed that it was co-localized to the endoplasmic reticulum (ER), a cellular compartment with many functions. In silico predictions indicated that TRP contains only a hypothetical transmembrane domain. Thus, it is likely that TRP is a lumen protein involved in multidrug efflux transport that may be involved in the purine metabolic pathway. Conclusions/Significance This study demonstrated for the first time that TRP is associated with TUB resistance in Leishmania. The next challenge is to determine how

  13. Combinatorial synthesis with high throughput discovery of protein-resistant membrane surfaces

    PubMed Central

    Gu, Minghao; Vegas, Arturo J.; Anderson, Daniel G.; Langer, Robert S.; Kilduff, James E.; Belfort, Georges

    2014-01-01

    Using combinatorial methods, we synthesized a series of new vinyl amide monomers and graft-polymerized them to light-sensitive poly(ether sulfone) (PES) porous films for protein resistance. To increase the discovery rate and statistical confidence, we developed high throughput surface modification methods (HTP) that allow synthesis, screening and selection of desirable monomers from a large library in a relatively short time (days). A series of amide monomers were synthesized by amidation of methacryloyl chloride with amines and grafted onto commercial poly(ether sulfone) (PES) membranes using irradiation from atmospheric pressure plasma (APP). The modified PES membrane surfaces were then tested and screened for static protein adhesion using HTP. Hydroxyl amide monomers N-(3-hydroxypropyl)methacrylamide (A3), N-(4-hydroxybutyl)methacrylamide (A4), and N-(4-hydroxybutyl) methacrylamide (A6), ethylene glycol (EG) monomer N-(3-methoxypropyl)methacrylamide (A7), and N-(2-(dimethylamino)ethyl)-N-methylmethacrylamide (A8), and N-(2-(diethylamino)ethyl)-N-methylmethacrylamide (A9) all terminated with tertiary amines and were shown to have protein resistance. The PES membranes modified with these monomers exhibited both low protein adhesion (i.e. membrane plugging or fouling) and high flux. Their performance is comparable with previously identified best performing PEG and zwitterionic monomers, i.e. the so-called gold-standard for protein resistance. Combining a Hansen solubility parameter (HSP) analysis of the amide monomers and the HTP filtration results, we conclude that monomer solubility in water correlates with protein-resistant surfaces, presumably through its effects on surface–water interactions. PMID:23706542

  14. Resistance training with soy vs whey protein supplements in hyperlipidemic males

    PubMed Central

    DeNysschen, Carol A; Burton, Harold W; Horvath, Peter J; Leddy, John J; Browne, Richard W

    2009-01-01

    Background Most individuals at risk for developing cardiovascular disease (CVD) can reduce risk factors through diet and exercise before resorting to drug treatment. The effect of a combination of resistance training with vegetable-based (soy) versus animal-based (whey) protein supplementation on CVD risk reduction has received little study. The study's purpose was to examine the effects of 12 weeks of resistance exercise training with soy versus whey protein supplementation on strength gains, body composition and serum lipid changes in overweight, hyperlipidemic men. Methods Twenty-eight overweight, male subjects (BMI 25–30) with serum cholesterol >200 mg/dl were randomly divided into 3 groups (placebo (n = 9), and soy (n = 9) or whey (n = 10) supplementation) and participated in supervised resistance training for 12 weeks. Supplements were provided in a double blind fashion. Results All 3 groups had significant gains in strength, averaging 47% in all major muscle groups and significant increases in fat free mass (2.6%), with no difference among groups. Percent body fat and waist-to-hip ratio decreased significantly in all 3 groups an average of 8% and 2%, respectively, with no difference among groups. Total serum cholesterol decreased significantly, again with no difference among groups. Conclusion Participation in a 12 week resistance exercise training program significantly increased strength and improved both body composition and serum cholesterol in overweight, hypercholesterolemic men with no added benefit from protein supplementation. PMID:19284589

  15. Functions of oligochitosan induced protein kinase in tobacco mosaic virus resistance and pathogenesis related proteins in tobacco.

    PubMed

    Yafei, Chen; Yong, Zhan; Xiaoming, Zhao; Peng, Guo; Hailong, An; Yuguang, Du; Yingrong, Han; Hui, Liu; Yuhong, Zhang

    2009-08-01

    Oligochitosan (OC) can regulate plant defense responses in many aspects, but the basic signal transduction pathway is still unclear. In this study, we used transgenic (TG) tobacco (Nicotiana Tabacum var. Samsun NN) as plant material whose oligochitosan induced protein kinase (OIPK) gene was inhibited by antisense transformation, to study the role of OIPK in tobacco defense reactions. The results showed that OIPK could increase tobacco resistance against tobacco mosaic virus (TMV), in that wild-type (WT) tobacco showed longer lesion appearance time, higher lesion inhibition ratio, smaller average final lesion diameter and lower average final lesion area percent to whole leaf area. It led us to analyze some pathogenesis related (PR) enzymes' activities and mRNA level, which played roles in tobacco resistance against TMV. We found that phenylalanine ammonia-lyase (PAL) and peroxidase (POD) activities were positively related to OIPK, but not polyphenol oxidase (PPO). It was also demonstrated that OIPK mRNA could be induced by OC, wound and TMV infection. In addition, OIPK could up-regulated three PR genes, PAL, chitinase (CHI) and beta-1, 3-glucanase (GLU) mRNA level to different extent. Taken together, these results implied that OIPK could function in tobacco resistance against both biotic and abiotic stress, possibly via various PR proteins.

  16. Rewiring mitogen-activated protein kinase cascade by positive feedback confers potato blight resistance.

    PubMed

    Yamamizo, Chihiro; Kuchimura, Kazuo; Kobayashi, Akira; Katou, Shinpei; Kawakita, Kazuhito; Jones, Jonathan D G; Doke, Noriyuki; Yoshioka, Hirofumi

    2006-02-01

    Late blight, caused by the notorious pathogen Phytophthora infestans, is a devastating disease of potato (Solanum tuberosum) and tomato (Solanum lycopersicum), and during the 1840s caused the Irish potato famine and over one million fatalities. Currently, grown potato cultivars lack adequate blight tolerance. Earlier cultivars bred for resistance used disease resistance genes that confer immunity only to some strains of the pathogen harboring corresponding avirulence gene. Specific resistance gene-mediated immunity and chemical controls are rapidly overcome in the field when new pathogen races arise through mutation, recombination, or migration from elsewhere. A mitogen-activated protein kinase (MAPK) cascade plays a pivotal role in plant innate immunity. Here we show that the transgenic potato plants that carry a constitutively active form of MAPK kinase driven by a pathogen-inducible promoter of potato showed high resistance to early blight pathogen Alternaria solani as well as P. infestans. The pathogen attack provoked defense-related MAPK activation followed by induction of NADPH oxidase gene expression, which is implicated in reactive oxygen species production, and resulted in hypersensitive response-like phenotype. We propose that enhancing disease resistance through altered regulation of plant defense mechanisms should be more durable and publicly acceptable than engineering overexpression of antimicrobial proteins.

  17. LEA proteins are involved in cyst desiccation resistance and other abiotic stresses in Azotobacter vinelandii.

    PubMed

    Rodriguez-Salazar, Julieta; Moreno, Soledad; Espín, Guadalupe

    2017-03-03

    Late embryogenesis abundant (LEA) proteins constitute a large protein family that is closely associated with resistance to abiotic stresses in multiple organisms and protect cells against drought and other stresses. Azotobacter vinelandii is a soil bacterium that forms desiccation-resistant cysts. This bacterium possesses two genes, here named lea1 and lea2, coding for avLEA1 and avLEA2 proteins, both containing 20-mer motifs characteristic of eukaryotic plant LEA proteins. In this study, we found that disruption of the lea1 gene caused a loss of the cysts' viability after 3 months of desiccation, whereas at 6 months, wild-type or lea2 mutant strain cysts remained viable. Vegetative cells of the lea1 mutant were more sensitive to osmotic stress; cysts developed by this mutant were also more sensitive to high temperatures than cysts or vegetative cells of the wild type or of the lea2 mutant. Expression of lea1 was induced several fold during encystment. In addition, the protective effects of these proteins were assessed in Escherichia coli cells. We found that E. coli cells overexpressing avLEA1 were more tolerant to salt stress than control cells; finally, in vitro analysis showed that avLEA1 protein was able to prevent the freeze thaw-induced inactivation of lactate dehydrogenase. In conclusion, avLEA1 is essential for the survival of A. vinelandii in dry conditions and for protection against hyper-osmolarity, two major stress factors that bacteria must cope with for survival in the environment. This is the first report on the role of bacterial LEA proteins on the resistance of cysts to desiccation.

  18. PVY-resistant transgenic potato plants expressing an anti-NIa protein scFv antibody.

    PubMed

    Gargouri-Bouzid, Radhia; Jaoua, Leïla; Rouis, Souad; Saïdi, Mohamed Najib; Bouaziz, Donia; Ellouz, Radhouane

    2006-06-01

    A synthetic gene encoding a single chain Fv fragment of an antibody directed against the nuclear inclusion a (NIa) protein of potato virus Y (PVY) was used to transform two commercial potato cultivars (Claustar and BF15). The NIa protease forms the nuclear inclusion body A and acts as the major protease in the cleavage of the viral polyprotein into functional proteins. Immunoblot analysis showed that most of the resulting transgenic plants accumulate high levels of the transgenic protein. Furthermore, a majority of the selected transgenic lines showed an efficient and complete protection against the challenge virus after mechanical inoculation with PVYO strain. Two transgenic lines showed an incomplete resistance with delayed appearance of symptoms accompanied by low virus titers, whereas one line developed symptoms during the first days after inoculation but recovered rapidly, leading to a low virus accumulation rate. These results confirm that expression of scFv antibody is able to inhibit a crucial step in the virus multiplication, such as polyprotein cleavage is a powerful strategy for engineered virus resistance. It can lead to a complete resistance that was not obtained previously by expression of scFv directed against the viral coat protein.

  19. Resistance to Ag(I) Cations in Bacteria: Environments, Genes and Proteins

    PubMed Central

    Gupta, Amit; Matsui, Kazuaki; Lo, Jeng-Fan

    1999-01-01

    Bacterial resistance to Ag(I) has been reported periodically with isolates from many environments where toxic levels of silver might be expected to occur, but initial reports were limited to the occurrence of resistant bacteria. The availability of silver-resistance conferring DNA sequences now allow genetic and mechanistic studies that had basically been missing. The genes determining Ag(I) resistance were sequenced from a plasmid found in a burn ward isolate. The 14.2 kb determinant contains seven recognized genes, arranged in three mRNA transcriptional units. The silE gene determines an extracellular (periplasmic space) metal-binding protein of 123 amino acids, including ten histidine residues implicated in Ag(I) binding. SilE is homologous to PcoE, of copper resistance. The next two genes, silR and silS, determine a two protein, histidine-kinase membrane sensor and aspartyl phosphate transcriptional responder, similar to other two component systems such as CzcR and CzcS (for cadmium, zinc and cobalt resistance) and PcoR and PcoS (for copper resistance). The remaining four genes, silCBAP, are co-transcribed and appear to determine Ag+ efflux, with SilCBA homologous to CzcCBA, a three component cation/proton antiporter, and SilP a novel P-type ATPase with a amino-terminal histidine-rich cation-specificity region. The effects of increasing Ag+ concentrations and growth medium halides (Cl-, Br- and I-) have been characterized, with lower Cl- concentrations facilitating resistance and higher concentrations toxicity. The properties of this unique Ag(I)-binding SilE protein are being characterized. Sequences similar to the silver-resistance DNA are being characterized by Southern blot DNA/DNA hybridization, PCR in vitro DNA synthesis and DNA sequencing. More than 25 additional closely related sequences have been identified in bacteria from diverse sources. Initial DNA sequencing results shows approximately 5-20% differences in DNA sequences. PMID:18475907

  20. Transgenic Sugarcane Resistant to Sorghum mosaic virus Based on Coat Protein Gene Silencing by RNA Interference

    PubMed Central

    Guo, Jinlong; Gao, Shiwu; Lin, Qinliang; Wang, Hengbo; Que, Youxiong; Xu, Liping

    2015-01-01

    As one of the critical diseases of sugarcane, sugarcane mosaic disease can lead to serious decline in stalk yield and sucrose content. It is mainly caused by Potyvirus sugarcane mosaic virus (SCMV) and/or Sorghum mosaic virus (SrMV), with additional differences in viral strains. RNA interference (RNAi) is a novel strategy for producing viral resistant plants. In this study, based on multiple sequence alignment conducted on genomic sequences of different strains and isolates of SrMV, the conserved region of coat protein (CP) genes was selected as the target gene and the interference sequence with size of 423 bp in length was obtained through PCR amplification. The RNAi vector pGII00-HACP with an expression cassette containing both hairpin interference sequence and cp4-epsps herbicide-tolerant gene was transferred to sugarcane cultivar ROC22 via Agrobacterium-mediated transformation. After herbicide screening, PCR molecular identification, and artificial inoculation challenge, anti-SrMV positive transgenic lines were successfully obtained. SrMV resistance rate of the transgenic lines with the interference sequence was 87.5% based on SrMV challenge by artificial inoculation. The genetically modified SrMV-resistant lines of cultivar ROC22 provide resistant germplasm for breeding lines and can also serve as resistant lines having the same genetic background for study of resistance mechanisms. PMID:25685813

  1. Expression and localization of p-glycoprotein, multidrug resistance protein 4, and breast cancer resistance protein in the female lower genital tract of human and pigtailed macaque.

    PubMed

    Zhou, Tian; Hu, Minlu; Pearlman, Andrew; Patton, Dorothy; Rohan, Lisa

    2014-11-01

    Antiretroviral drug absorption and disposition in cervicovaginal tissue is important for the effectiveness of vaginally or orally administered drug products in preexposure prophylaxis (PrEP) of HIV-1 sexual transmission to women. Therefore, it is imperative to understand critical determinants of cervicovaginal tissue pharmacokinetics. This study aimed to examine the mRNA expression and protein localization of three efflux transporters, P-glycoprotein (P-gp), multidrug resistance-associated protein 4 (MRP4), and breast cancer resistance protein (BCRP), in the lower genital tract of premenopausal women and pigtailed macaques. Along the human lower genital tract, the three transporters were moderately to highly expressed compared to colorectal tissue and liver, as revealed by real-time reverse transcriptase polymerase chain reaction (RT-PCR). In a given genital tract segment, the transporter with the highest expression level was either BCRP or P-gp, while MRP4 was always expressed at the lowest level among the three transporters tested. The immunohistochemical staining showed that P-gp and MRP4 were localized in multiple cell types including epithelial cells and vascular endothelial cells. BCRP was predominantly localized in the vascular endothelial cells. Differences in transporter mRNA level and localization were observed among endocervix, ectocervix, and vagina. Compared to human tissues, the macaque cervicovaginal tissues displayed comparable expression and localization patterns of the three transporters, although subtle differences were observed between the two species. The role of these cervicovaginal transporters in drug absorption and disposition warrants further studies. The resemblance between human and pigtailed macaque in transporter expression and localization suggests the utility of the macaque model in the studies of human cervicovaginal transporters.

  2. Covalently modified silicon and diamond surfaces: resistance to nonspecific protein adsorption and optimization for biosensing.

    PubMed

    Lasseter, Tami L; Clare, Brian H; Abbott, Nicholas L; Hamers, Robert J

    2004-08-25

    We report the direct covalent functionalization of silicon and diamond surfaces with short ethylene glycol (EG) oligomers via photochemical reaction of the hydrogen-terminated surfaces with terminal vinyl groups of the oligomers, and the use of these monolayers to control protein binding at surfaces. Photochemical modification of Si(111) and polycrystalline diamond surfaces produces EG monolayers linked via Si-C bond formation (silicon) or C-C bond formation (diamond). X-ray photoelectron spectroscopy was used to characterize the monolayer composition. Measurements using fluorescently labeled proteins show that the EG-functionalized surfaces effectively resist nonspecific adsorption of proteins. Additionally, we demonstrate the use of mixed monolayers on silicon and diamond and apply these surfaces to control specific versus nonspecific binding to optimize a model protein sensing assay.

  3. Importance of detecting multidrug resistance proteins in acute leukemia prognosis and therapy.

    PubMed

    de Moraes, Ana Carolina Rabello; Maranho, Caroline Klein; Rauber, Gabriela Schneider; Santos-Silva, Maria Cláudia

    2013-01-01

    Multidrug resistance (MDR) is a multifactorial phenomenon and the role of these proteins in generating the MDR phenotype is controversial. With this in mind, this review compiled the current data on the role of ABCB1, ABCC1, and LRP proteins in the prognosis of hematologic neoplasms and their influence on the choice of therapy. Literature showed that the detection of these proteins, mainly ABCB1, is important in the AL prognosis. However, there is controversy regarding the methodology used for their detection. In summary, the expression and activity profiles of ABCB1, ABCC1, and LRP, proteins capable of promoting the efflux of a variety of chemotherapeutic agents from the cell cytoplasm represent one of the greatest causes of failure in AL treatment.

  4. Structure and function of Toll/interleukin-1 receptor/resistance protein (TIR) domains.

    PubMed

    Ve, Thomas; Williams, Simon J; Kobe, Bostjan

    2015-02-01

    The Toll/interleukin-1 receptor/resistance protein (TIR) domain is a protein-protein interaction domain consisting of 125-200 residues, widely distributed in animals, plants and bacteria but absent from fungi, archea and viruses. In plants and animals, these domains are found in proteins with functions in innate immune pathways, while in bacteria, some TIR domain-containing proteins interfere with the innate immune pathways in the host. TIR domains function as protein scaffolds, mostly involving self-association and homotypic interactions with other TIR domains. In the last 15 years, the three-dimensional structures of TIR domains from several mammalian, plant and bacterial proteins have been reported. These structures, jointly with functional data including the identification of interacting proteins, have started to provide insight into the molecular basis of the assembly of animal and plant immune signaling complexes, and for host immunosuppression by bacterial pathogens. This review focuses on the current knowledge of the structures of the TIR domains and how the structure relates to function.

  5. Insulin resistance of protein metabolism in type 2 diabetes and impact on dietary needs: a review.

    PubMed

    Gougeon, Réjeanne

    2013-04-01

    Evidence shows that the metabolism of protein is altered in type 2 diabetes mellitus and insulin resistance not only applies to glucose and lipid but protein metabolism as well. Population surveys report greater susceptibility to loss of lean tissue and muscle strength with aging in diabetes. Prevention of sarcopenia requires that protein receives more attention in dietary prescriptions. Protein intake of 1-1.2 g/kg of body weight (with weight at a body mass index of 25 kg/m(2))/day may be distributed equally among 3 meals a day, including breakfast, to optimize anabolism. Adopting a dietary pattern that provides a high plant-to-animal ratio and greater food volume favouring consumption of vegetables, legumes, fruits, complemented with fish, low fat dairy and meat (preferably cooked slowly in moisture), soy and nuts may assist with metabolic and weight control. Depending on the magnitude of energy restriction, usual protein intake should be maintained or increased, and the caloric deficit taken from fat and carbohydrate foods. Exercise before protein-rich meals improves skeletal muscle protein anabolism. Because high levels of amino acids lower glucose uptake in individuals without diabetes, the challenge remains to define the optimal protein intake and exercise regimen to protect from losses of muscle mass and strength while maintaining adequate glucose control in type 2 diabetes.

  6. Teicoplanin-resistant Staphylococcus aureus expresses a novel membrane protein and increases expression of penicillin-binding protein 2 complex.

    PubMed Central

    Shlaes, D M; Shlaes, J H; Vincent, S; Etter, L; Fey, P D; Goering, R V

    1993-01-01

    In the recent clinical trials of teicoplanin therapy of endocarditis caused by Staphylococcus aureus, at least one instance of the emergence of teicoplanin-resistant strains during therapy has been reported (G.W. Kaatz, S. M. Seo, N. J. Dorman, and S. A. Lerner, J. Infect. Dis 162:103-108, 1990). We have confirmed, using conventional electrophoresis of EcoRI-digested chromosomal DNA and pulsed-field gel electrophoresis of SmaI-digested chromosomal DNA, that the resistant strain (12873) (MIC, 16 micrograms/ml) is genetically very similar to the susceptible parent (12871) (MIC, 4 micrograms/ml). Kaatz et al. were able to select spontaneous teicoplanin-resistant mutants (10(-9)), suggesting that a single gene might be involved. We have shown that the mutation is highly stable during growth in the absence of teicoplanin. Using Tn551, we have selected insertion mutants of 12873 that become teicoplanin susceptible. We have examined a number of aspects of cell wall physiology in strains 12871 and 12873 and the teicoplanin-susceptible Tn551 mutants of 12873. 12873 was more susceptible to lysostaphin lysis than 12871 and the susceptible Tn551 derivatives of 12873. Autolysis in phosphate buffer (pH 7.5) and cell wall turnover rates were similar in 12871 and 12873. An analysis of membrane proteins revealed the expression of a ca. 35-kDa protein and increased expression of both polypeptides of penicillin-binding protein (PBP) 2 (PBP2) in 12873 relative to 12871 and the Tn551 mutants of 12873. This increased expression was not related to PBP2', since both strains were susceptible to oxacillin in 2% NaCl (MIC, < or = 0.25 microgram/ml) and cellular DNA from neither strain hybridized with a specific mec gene probe. Two independent Tn551 inserts have been mapped to a ca. 117-kb SmaI fragment of the chromosome. These data suggest the possibility that the mutation resulting in resistance to teicoplanin involves the regulation of expression of both polypeptides of PBP2 and a 35-k

  7. Multidrug-resistance proteins are weak tumor associated antigens for colorectal carcinoma

    PubMed Central

    2011-01-01

    Background Multidrug resistance (MDR) is a clinically, highly relevant phenomenon. Under chemotherapy many tumors show an increasing resistance towards the applied substance(s) and to a certain extent also towards other agents. An important molecular cause of this phenomenon is an increased expression of transporter proteins. The functional relationship between high expression levels and chemotherapy resistance makes these MDR and MRP (MDR related protein) proteins to interesting therapeutic targets. We here wanted to systematically analyze, whether these proteins are tumor specific antigens which could be targeted immunologically. Results Using the reverse immunology approach, 30 HLA-A2.1 restricted MDR and MRP derived peptides (MDP) were selected. Stimulated T cell lines grew well and mainly contained activated CD8+ cells. Peptide specificity and HLA-A2.1 restriction were proven in IFN-γ-ELISpot analyses and in cytotoxicity tests against MDP loaded target cells for a total of twelve peptides derived from MDR-1, MDR-3, MRP-1, MRP-2, MRP-3 and MRP-5. Of note, two of these epitopes are shared between MDR-1 and MDR-3 as well as MRP-2 and MRP-3. However, comparably weak cytotoxic activities were additionally observed against HLA-A2.1+ tumor cells even after upregulation of MDR protein expression by in vitro chemotherapy. Conclusions Taken together, these data demonstrate that human T cells can be sensitised towards MDPs and hence, there is no absolute immunological tolerance. However, our data also hint towards rather low endogenous tumor cell processing and presentation of MDPs in the context of HLA-A2.1 molecules. Consequently, we conclude that MDR and MRP proteins must be considered as weak tumor specific antigens-at least for colorectal carcinoma. Their direct contribution to therapy-failure implies however, that it is worth to further pursue this approach. PMID:21740599

  8. Effect of protein source and resistance training on body composition and sex hormones

    PubMed Central

    Kalman, Douglas; Feldman, Samantha; Martinez, Michele; Krieger, Diane R; Tallon, Mark J

    2007-01-01

    Background Evidence suggests an inverse relationship between soy protein intake and serum concentrations of male sex hormones. Anecdotal evidence indicates that these alterations in serum sex hormones may attenuate changes in lean body mass following resistance training. However, little empirical data exists regarding the effects of soy and milk-based proteins on circulating androgens and exercise induced body composition changes. Methods For 12 weeks 20 subjects were supplemented with 50 g per day of one of four different protein sources (Soy concentrate; Soy isolate; Soy isolate and whey blend, and Whey blend only) in combination with a resistance-training program. Body composition, testosterone, estradiol and sex hormone binding globulin (SHBG) were measured at baseline and week 12. Results Protein supplementation resulted in a significant increase in lean body mass independent of protein source (0.5 ± 1.1 and 0.9 ± 1.4 kg, p = 0.006, p = 0.007). No significant differences were observed between groups for total and free testosterone, SHBG, percentage body fat, BMI or body weight. The Testosterone/Estradiol ratio increased across all groups (+13.4, p = 0.005) and estradiol decreased (p = 0.002). Within group analysis showed significant increases in the Testosterone/Estradiol ratio in soy isolate + whey blend group (+16.3, p = 0.030). Estradiol was significantly lower in the whey blend group (-9.1 ± 8.7 pg/ml, p = 0.033). Conclusion This investigation shows that 12 week supplementation with soy protein does not decrease serum testosterone or inhibit lean body mass changes in subjects engaged in a resistance exercise program. PMID:17908338

  9. Protein Ingestion Induces Muscle Insulin Resistance Independent of Leucine-Mediated mTOR Activation.

    PubMed

    Smith, Gordon I; Yoshino, Jun; Stromsdorfer, Kelly L; Klein, Seth J; Magkos, Faidon; Reeds, Dominic N; Klein, Samuel; Mittendorfer, Bettina

    2015-05-01

    Increased plasma branched-chain amino acid concentrations are associated with insulin resistance, and intravenous amino acid infusion blunts insulin-mediated glucose disposal. We tested the hypothesis that protein ingestion impairs insulin-mediated glucose disposal by leucine-mediated mTOR signaling, which can inhibit AKT. We measured glucose disposal and muscle p-mTOR(Ser2448), p-AKT(Ser473), and p-AKT(Thr308) in 22 women during a hyperinsulinemic-euglycemic clamp procedure with and without concomitant ingestion of whey protein (0.6 g/kg fat-free mass; n = 11) or leucine that matched the amount given with whey protein (n = 11). Both whey protein and leucine ingestion raised plasma leucine concentration by approximately twofold and muscle p-mTOR(Ser2448) by ∼30% above the values observed in the control (no amino acid ingestion) studies; p-AKT(Ser473) and p-AKT(Thr308) were not affected by whey protein or leucine ingestion. Whey protein ingestion decreased insulin-mediated glucose disposal (median 38.8 [quartiles 30.8, 61.8] vs. 51.9 [41.0, 77.3] µmol glucose/µU insulin · mL(-1) · min(-1); P < 0.01), whereas ingestion of leucine did not (52.3 [43.3, 65.4] vs. 52.3 [43.9, 73.2]). These results indicate that 1) protein ingestion causes insulin resistance and could be an important regulator of postprandial glucose homeostasis and 2) the insulin-desensitizing effect of protein ingestion is not due to inhibition of AKT by leucine-mediated mTOR signaling.

  10. Protein Ingestion Induces Muscle Insulin Resistance Independent of Leucine-Mediated mTOR Activation

    PubMed Central

    Smith, Gordon I.; Yoshino, Jun; Stromsdorfer, Kelly L.; Klein, Seth J.; Magkos, Faidon; Reeds, Dominic N.; Klein, Samuel

    2015-01-01

    Increased plasma branched-chain amino acid concentrations are associated with insulin resistance, and intravenous amino acid infusion blunts insulin-mediated glucose disposal. We tested the hypothesis that protein ingestion impairs insulin-mediated glucose disposal by leucine-mediated mTOR signaling, which can inhibit AKT. We measured glucose disposal and muscle p-mTORSer2448, p-AKTSer473, and p-AKTThr308 in 22 women during a hyperinsulinemic-euglycemic clamp procedure with and without concomitant ingestion of whey protein (0.6 g/kg fat-free mass; n = 11) or leucine that matched the amount given with whey protein (n = 11). Both whey protein and leucine ingestion raised plasma leucine concentration by approximately twofold and muscle p-mTORSer2448 by ∼30% above the values observed in the control (no amino acid ingestion) studies; p-AKTSer473 and p-AKTThr308 were not affected by whey protein or leucine ingestion. Whey protein ingestion decreased insulin-mediated glucose disposal (median 38.8 [quartiles 30.8, 61.8] vs. 51.9 [41.0, 77.3] µmol glucose/µU insulin · mL−1 · min−1; P < 0.01), whereas ingestion of leucine did not (52.3 [43.3, 65.4] vs. 52.3 [43.9, 73.2]). These results indicate that 1) protein ingestion causes insulin resistance and could be an important regulator of postprandial glucose homeostasis and 2) the insulin-desensitizing effect of protein ingestion is not due to inhibition of AKT by leucine-mediated mTOR signaling. PMID:25475435

  11. Multiple changes of penicillin-binding proteins in penicillin-resistant clinical isolates of Streptococcus pneumoniae.

    PubMed Central

    Hakenbeck, R; Tarpay, M; Tomasz, A

    1980-01-01

    Penicillin-binding properties and characteristics of penicillin-binding proteins (PBPs) were investigated in several clinical isolates of Streptococcus pneumoniae differing in their susceptibilities to penicillin (minimal inhibitory concentration [MIC], 0.03 to 0.5 microgram/ml) and compared with the penicillin-susceptible strain R36A (MIC, 0.07 microgram/ml). Several changes accompanied the development of resistance: the relative affinity to penicillin of whole cells, isolated membranes, and two major PBPs after in vivo or in vitro labeling decreased (with increasing resistance). Furthermore, one additional PBP (2') appeared in four of five relatively resistant strains with an MIC of 0.25 microgram/ml and higher. PBP 3 maintained the same high affinity toward penicillin in all strains under all labeling conditions. Images PMID:7425601

  12. Consequences of manganese replacement of copper for prion protein function and proteinase resistance

    PubMed Central

    Brown, David R.; Hafiz, Farida; Glasssmith, Leslie L.; Wong, Boon-Seng; Jones, Ian M.; Clive, Christine; Haswell, Stephen J.

    2000-01-01

    The prion protein (PrP) binds copper and has antioxidant activity enhancing the survival of neurones in culture. The ability of the PrP to bind other cations was tested and it was found that only manganese could substitute for copper. Although initially manganese-loaded PrP exhibited similar structure and activity to copper-loaded PrP, after aging, manganese-loaded PrP became proteinase resistant and lost function. It was also found that manganese could be incorporated into PrP expressed by astrocytes and that this PrP was partially proteinase resistant. These results show that it is possible to generate proteinase-resistant PrP from cells and suggest a possible mechanism for the formation of the scrapie isoform of the PrP as generated in sporadic prion disease. PMID:10716918

  13. Pokeweed Antiviral Protein: Its Cytotoxicity Mechanism and Applications in Plant Disease Resistance

    PubMed Central

    Di, Rong; Tumer, Nilgun E.

    2015-01-01

    Pokeweed antiviral protein (PAP) is a 29 kDa type I ribosome inactivating protein (RIP) found in pokeweed plants. Pokeweed produces different forms of PAP. This review focuses on the spring form of PAP isolated from Phytolacca americana leaves. PAP exerts its cytotoxicity by removing a specific adenine from the α-sarcin/ricin loop of the large ribosomal RNA. Besides depurination of the rRNA, PAP has additional activities that contribute to its cytotoxicity. The mechanism of PAP cytotoxicity is summarized based on evidence from the analysis of transgenic plants and the yeast model system. PAP was initially found to be anti-viral when it was co-inoculated with plant viruses onto plants. Transgenic plants expressing PAP and non-toxic PAP mutants have displayed broad-spectrum resistance to both viral and fungal infection. The mechanism of PAP-induced disease resistance in transgenic plants is summarized. PMID:25756953

  14. The impact of protein quality on the promotion of resistance exercise-induced changes in muscle mass.

    PubMed

    Phillips, Stuart M

    2016-01-01

    Protein supplementation during resistance exercise training augments hypertrophic gains. Protein ingestion and the resultant hyperaminoacidemia provides the building blocks (indispensable amino acids - IAA) for, and also triggers an increase in, muscle protein synthesis (MPS), suppression of muscle protein breakdown (MPB), and net positive protein balance (i.e., MPS > MPB). The key amino acid triggering the rise in MPS is leucine, which stimulates the mechanistic target of rapamycin complex-1, a key signalling protein, and triggers a rise in MPS. As such, ingested proteins with a high leucine content would be advantageous in triggering a rise in MPS. Thus, protein quality (reflected in IAA content and protein digestibility) has an impact on changes in MPS and could ultimately affect skeletal muscle mass. Protein quality has been measured by the protein digestibility-corrected amino acid score (PDCAAS); however, the digestible indispensable amino acid score (DIAAS) has been recommended as a better method for protein quality scoring. Under DIAAS there is the recognition that amino acids are individual nutrients and that protein quality is contingent on IAA content and ileal (as opposed to fecal) digestibility. Differences in protein quality may have important ramifications for exercise-induced changes in muscle mass gains made with resistance exercise as well as muscle remodelling. Thus, the purpose of this review is a critical appraisal of studies examining the effects of protein quality in supplementation on changes in muscle mass and strength as well as body composition during resistance training.

  15. Protein resistance efficacy of PEO-silane amphiphiles: Dependence on PEO-segment length and concentration

    PubMed Central

    Rufin, Marc A.; Barry, Mikayla E.; Adair, Paige A.; Hawkins, Melissa L.; Raymond, Jeffery E.; Grunlan, Melissa A.

    2016-01-01

    In contrast to modification with conventional PEO-silanes (i.e. no siloxane tether), silicones with dramatically enhanced protein resistance have been previously achieved via bulk-modification with poly (ethylene oxide) (PEO)-silane amphiphiles α-(EtO)3Si(CH2)2-oligodimethylsiloxane13-block-PEOn-OCH3 when n = 8 and 16 but not when n = 3. In this work, their efficacy was evaluated in terms of optimal PEO-segment length and minimum concentration required in silicone. For each PEO-silane amphiphile (n = 3, 8, and 16), five concentrations (5, 10, 25, 50, and 100 μmol per 1 g silicone) were evaluated. Efficacy was quantified in terms of the modified silicones’ abilities to undergo rapid, water-driven surface restructuring to form hydrophilic surfaces as well as resistance to fibrinogen adsorption. Only n = 8 and 16 were effective, with a lower minimum concentration in silicone required for n = 8 (10 μmol per 1 g silicone) versus n = 16 (25 μmol per 1 g silicone). Statement of Significance Silicone is commonly used for implantable medical devices, but its hydrophobic surface promotes protein adsorption which leads to thrombosis and infection. Typical methods to incorporate poly(ethylene oxide) (PEO) into silicones have not been effective due to the poor migration of PEO to the surface-biological interface. In this work, PEO-silane amphiphiles – comprised of a siloxane tether (m = 13) and variable PEO segment lengths (n = 3, 8, 16) – were blended into silicone to improve its protein resistance. The efficacy of the amphiphiles was determined to be dependent on PEO length. With the intermediate PEO length (n = 8), water-driven surface restructuring and resulting protein resistance was achieved with a concentration of only 1.7 wt%. PMID:27090588

  16. Steroidogenic acute regulatory protein (StAR) overexpression reduces inflammation and insulin resistance in obese mice.

    PubMed

    Qiu, Yanyan; Sui, Xianxian; Cao, Shengxuan; Li, Xiaobo; Ning, Yanxia; Wang, Songmei; Yin, Lianhua; Zhi, Xiuling

    2017-04-12

    Steroidogenic acute regulatory protein (StAR), a mitochondrial cholesterol delivery protein, plays a beneficial role in hyperlipidemia, NAFLD and endothelial inflammation. Elevated circulating fatty acids and low grade inflammation are known as key risk factors of insulin resistance and type 2 diabetes. In the present study, C57BL/6J mice were fed with a HFD and infected with recombinant adenovirus expressing StAR by tail-vein injection. Intraperitoneal glucose/insulin tolerance test was performed to assess the insulin sensitivity. Morphological analysis and intramuscular lipid determination were used to illustrate the adipose hypertrophy and ectopic fat accumulation in skeletal muscle. The levels of inflammatory factor and nitric oxide were determined by ELISA and classic Griess reagent methods respectively. The fatty acids composition was analysis using gas chromatography -mass spectrometry (GC-MS). The expression of genes associated with inflammation and insulin resistance were determined by Western blotting and qPCR to elucidate the underlying mechanism.We demonstrated that StAR overexpression ameliorated insulin resistance and systemic inflammatory response with the reduction of adipose hypertrophy and intramuscular lipid in HFD fed mice. In addition, StAR overexpression increased serum unsaturated fatty acids and PPARγ expression in muscle and adipose tissue of obese mice. In conclusion, StAR may activate PPARγ by increasing unsaturated fatty acids, which leads to a protective role in systemic inflammation and insulin resistance in obese mice. This article is protected by copyright. All rights reserved.

  17. Cellular prion protein acquires resistance to proteolytic degradation following copper ion binding.

    PubMed

    Kuczius, Thorsten; Buschmann, Anne; Zhang, Wenlan; Karch, Helge; Becker, Karsten; Peters, Georg; Groschup, Martin H

    2004-08-01

    The conversion of cellular prion protein (PrP(C)) into its pathological isoform (PrP(Sc)) conveys an increase in hydrophobicity and induces a partial resistance to proteinase K (PK). Interestingly, co-incubation with high copper ion concentrations also modifies the solubility of PrP(c) and induces a partial PK resistance which was reminiscent of PrP(Sc). However, concerns were raised whether this effect was not due to a copper-induced inhibition of the PK itself. We have therefore analyzed the kinetics of the formation of PK-resistant PrP(C) and excluded possible interference effects by removing unbound copper ions prior to the addition of PK by methanol precipitation or immobilization of PrP(C) followed by washing steps. We found that preincubation of PrPc with copper ions at concentrations as low as 50 microM indeed rendered these proteins completely PK resistant, while control substrates were proteolyzed. No other divalent cations induced a similar effect. However, in addition to this specific stabilizing effect on PrP(C), higher copper ion concentrations in solution (>200 microM) directly blocked the enzymatic activity of PK, possibly by replacing the Ca2+ ions in the active center of the enzyme. Therefore, as a result of this inhibition the proteolytic degradation of PrP(C) as well as PrP(Sc) molecules was suppressed.

  18. Towards predictive resistance models for agrochemicals by combining chemical and protein similarity via proteochemometric modelling.

    PubMed

    van Westen, Gerard J P; Bender, Andreas; Overington, John P

    2014-10-01

    Resistance to pesticides is an increasing problem in agriculture. Despite practices such as phased use and cycling of 'orthogonally resistant' agents, resistance remains a major risk to national and global food security. To combat this problem, there is a need for both new approaches for pesticide design, as well as for novel chemical entities themselves. As summarized in this opinion article, a technique termed 'proteochemometric modelling' (PCM), from the field of chemoinformatics, could aid in the quantification and prediction of resistance that acts via point mutations in the target proteins of an agent. The technique combines information from both the chemical and biological domain to generate bioactivity models across large numbers of ligands as well as protein targets. PCM has previously been validated in prospective, experimental work in the medicinal chemistry area, and it draws on the growing amount of bioactivity information available in the public domain. Here, two potential applications of proteochemometric modelling to agrochemical data are described, based on previously published examples from the medicinal chemistry literature.

  19. Toxicological relevance of the multidrug resistance protein 1, MRP1 (ABCC1) and related transporters.

    PubMed

    Leslie, E M; Deeley, R G; Cole, S P

    2001-10-05

    The 190 kDa multidrug resistance protein 1 (MRP1/ABCC1) is a founding member of a subfamily of the ATP binding cassette (ABC) superfamily of transport proteins and was originally identified on the basis of its elevated expression in multidrug resistant lung cancer cells. In addition to its ability to confer resistance in tumour cells, MRP1 is ubiquitously expressed in normal tissues and is a primary active transporter of GSH, glucuronate and sulfate conjugated and unconjugated organic anions of toxicological relevance. Substrates include lipid peroxidation products, herbicides, tobacco specific nitrosamines, mycotoxins, heavy metals, and natural product and antifolate anti-cancer agents. MRP1 also transports unmodified xenobiotics but often requires GSH to do so. Active efflux is generally an important aspect of cellular detoxification since it prevents the accumulation of conjugated and unconjugated compounds that have the potential to be directly toxic. The related transporters MRP2 and MRP3 have overlapping substrate specificities with MRP1 but different tissue distributions, and evidence that they also have chemoprotective functions are discussed. Finally, MRP homologues have been described in other species including yeast and nematodes. Those isolated from the vascular plant Arabidopsis thaliana (AtMRPs) decrease the cytoplasmic concentration of conjugated toxins through sequestration in vacuoles and are implicated in providing herbicide resistance to plants.

  20. Bactobolin Resistance Is Conferred by Mutations in the L2 Ribosomal Protein

    PubMed Central

    Chandler, Josephine R.; Truong, Thao T.; Silva, Patricia M.; Seyedsayamdost, Mohammad R.; Carr, Gavin; Radey, Matthew; Jacobs, Michael A.; Sims, Elizabeth H.; Clardy, Jon; Greenberg, E. Peter

    2012-01-01

    ABSTRACT Burkholderia thailandensis produces a family of polyketide-peptide molecules called bactobolins, some of which are potent antibiotics. We found that growth of B. thailandensis at 30°C versus that at 37°C resulted in increased production of bactobolins. We purified the three most abundant bactobolins and determined their activities against a battery of bacteria and mouse fibroblasts. Two of the three compounds showed strong activities against both bacteria and fibroblasts. The third analog was much less potent in both assays. These results suggested that the target of bactobolins might be conserved across bacteria and mammalian cells. To learn about the mechanism of bactobolin activity, we isolated four spontaneous bactobolin-resistant Bacillus subtilis mutants. We used genomic sequencing technology to show that each of the four resistant variants had mutations in rplB, which codes for the 50S ribosome-associated L2 protein. Ectopic expression of a mutant rplB gene in wild-type B. subtilis conferred bactobolin resistance. Finally, the L2 mutations did not confer resistance to other antibiotics known to interfere with ribosome function. Our data indicate that bactobolins target the L2 protein or a nearby site and that this is not the target of other antibiotics. We presume that the mammalian target of bactobolins involves the eukaryotic homolog of L2 (L8e). PMID:23249812

  1. Heat Resistant Characteristics of Major Royal Jelly Protein 1 (MRJP1) Oligomer.

    PubMed

    Moriyama, Takanori; Ito, Aimi; Omote, Sumire; Miura, Yuri; Tsumoto, Hiroki

    2015-01-01

    Soluble royal jelly protein is a candidate factor responsible for mammiferous cell proliferation. Major royal jelly protein 1 (MRJP1), which consists of oligomeric and monomeric forms, is an abundant proliferative protein in royal jelly. We previously reported that MRJP1 oligomer has biochemical heat resistance. Therefore, in the present study, we investigated the effects of several heat treatments (56, 65 and 96°C) on the proliferative activity of MRJP1 oligomer. Heat resistance studies showed that the oligomer molecular forms were slightly maintained until 56℃, but the molecular forms were converted to macromolecular heat-aggregated MRJP1 oligomer at 65℃ and 96℃. But, the growth activity of MRJP1 oligomer treated with 96°C was slightly attenuated when compared to unheated MRJP1 oligomer. On the other hand, the cell proliferation activity was preserved until 96℃ by the cell culture analysis of Jurkat cells. In contrast, those of IEC-6 cells were not preserved even at 56°C. The present observations suggest that the bioactive heat-resistance properties were different by the origin of the cells. The cell proliferation analysis showed that MRJP1 oligomer, but not MRJP2 and MRJP3, significantly increased cell numbers, suggesting that MRJP1 oligomer is the predominant proliferation factor for mammiferous cells.

  2. Physical Cross-Linking Starch-Based Zwitterionic Hydrogel Exhibiting Excellent Biocompatibility, Protein Resistance, and Biodegradability.

    PubMed

    Ye, Lei; Zhang, Yabin; Wang, Qiangsong; Zhou, Xin; Yang, Boguang; Ji, Feng; Dong, Dianyu; Gao, Lina; Cui, Yuanlu; Yao, Fanglian

    2016-06-22

    In this work, a novel starch-based zwitterionic copolymer, starch-graft-poly(sulfobetaine methacrylate) (ST-g-PSBMA), was synthesized via Atom Transfer Radical Polymerization. Starch, which formed the main chain, can be degraded completely in vivo, and the pendent segments of PSBMA endowed the copolymer with excellent protein resistance properties. This ST-g-PSBMA copolymer could self-assemble into a physical hydrogel in normal saline, and studies of the formation mechanism indicated that the generation of the physical hydrogel was driven by electrostatic interactions between PSBMA segments. The obtained hydrogels were subjected to detailed analysis by scanning electron microscopy, swelling ratio, protein resistance, and rheology tests. Toxicity and hemolysis analysis demonstrated that the ST-g-PSBMA hydrogels possess excellent biocompatibility and hemocompatibility. Moreover, the cytokine secretion assays (IL-6, TNF-α, and NO) confirmed that ST-g-PSBMA hydrogels had low potential to trigger the activation of macrophages and were suitable for in vivo biomedical applications. On the basis of these in vitro results, the ST-g-PSBMA hydrogels were implanted in SD rats. The tissue responses to hydrogel implantation and the hydrogel degradation in vivo were determined by histological analysis (Hematoxylin and eosin, Van Gieson, and Masson's Trichrome stains). The results presented in this study demonstrate that the physical cross-linking, starch-based zwitterionic hydrogels possess excellent protein resistance, low macrophage-activation properties, and good biocompatibility, and they are a promising candidate for an in vivo biomedical application platform.

  3. Characterization of putative multidrug resistance transporters of the major facilitator-superfamily expressed in Salmonella Typhi.

    PubMed

    Shaheen, Aqsa; Ismat, Fouzia; Iqbal, Mazhar; Haque, Abdul; De Zorzi, Rita; Mirza, Osman; Walz, Thomas; Rahman, Moazur

    2015-05-01

    Multidrug resistance mediated by efflux pumps is a well-known phenomenon in infectious bacteria. Although much work has been carried out to characterize multidrug efflux pumps in Gram-negative and Gram-positive bacteria, such information is still lacking for many deadly pathogens. The aim of this study was to gain insight into the substrate specificity of previously uncharacterized transporters of Salmonella Typhi to identify their role in the development of multidrug resistance. S. Typhi genes encoding putative members of the major facilitator superfamily were cloned and expressed in the drug-hypersensitive Escherichia coli strain KAM42, and tested for transport of 25 antibacterial compounds, including representative antibiotics of various classes, antiseptics, dyes and detergents. Of the 15 tested putative transporters, STY0901, STY2458 and STY4874 exhibited a drug-resistance phenotype. Among these, STY4874 conferred resistance to at least ten of the tested antimicrobials: ciprofloxacin, norfloxacin, levofloxacin, kanamycin, streptomycin, gentamycin, nalidixic acid, chloramphenicol, ethidium bromide, and acriflavine, including fluoroquinolone antibiotics, which were drugs of choice to treat S. Typhi infections. Cell-based functional studies using ethidium bromide and acriflavine showed that STY4874 functions as a H(+)-dependent exporter. These results suggest that STY4874 may be an important drug target, which can now be tested by studying the susceptibility of a STY4874-deficient S. Typhi strain to antimicrobials.

  4. Responses in the expression of extracellular proteins in methicillin-resistant Staphylococcus aureus treated with rhodomyrtone.

    PubMed

    Visutthi, Monton; Srimanote, Potjanee; Voravuthikunchai, Supayang Piyawan

    2011-12-01

    Rhodomyrtone from a medicinal plant species, Rhodomyrtus tomentosa, is a challenged effective agent against Gram-positive bacteria, especially methicillin-resistant Staphylococcus aureus (MRSA). The present study was undertaken to provide insight into MRSA extracellular protein expression following rhodomyrtone treatment. Secreteomic approach was performed on a representative clinical MRSA isolate exposing to subinhibitory concentration rhodomyrtone (0.174 μg/ml). The identified extracellular proteins of a response of MRSA to rhodomyrtone treated condition were both suppressed and overexpressed. Staphylococcal antigenic proteins, immunodominant antigen A (IsaA) and staphylococcal secretory antigen (SsaA) involved in cell wall hydrolysis were downregulated after the treatment. The results suggested that rhodomyrtone may interfere with WalK/WalR (YycG/YycF) system. Other enzymes such as lipase precursor and another lipase, glycerophosphoryl diester phosphodiesterase, were absent. In contrast, cytoplasmic proteins such as SpoVG and glycerol phosphate lipoteichoic acid synthase, and ribosomal proteins were found in the treated sample. Appearance of several cytoplasmic proteins in the treated culture supernatant revealed that the bacterial cell wall biosynthesis was disturbed. This finding provides a proteomic mapping of extracellular proteins after rhodomytone treatment. Extensive investigation is required for this natural compound as it has a great potency as an alternative anti-MRSA drug.

  5. Mechanosensitive Molecular Networks Involved in Transducing Resistance Exercise-Signals into Muscle Protein Accretion

    PubMed Central

    Rindom, Emil; Vissing, Kristian

    2016-01-01

    Loss of skeletal muscle myofibrillar protein with disease and/or inactivity can severely deteriorate muscle strength and function. Strategies to counteract wasting of muscle myofibrillar protein are therefore desirable and invite for considerations on the potential superiority of specific modes of resistance exercise and/or the adequacy of low load resistance exercise regimens as well as underlying mechanisms. In this regard, delineation of the potentially mechanosensitive molecular mechanisms underlying muscle protein synthesis (MPS), may contribute to an understanding on how differentiated resistance exercise can transduce a mechanical signal into stimulation of muscle accretion. Recent findings suggest specific upstream exercise-induced mechano-sensitive myocellular signaling pathways to converge on mammalian target of rapamycin complex 1 (mTORC1), to influence MPS. This may e.g. implicate mechanical activation of signaling through a diacylglycerol kinase (DGKζ)-phosphatidic acid (PA) axis or implicate integrin deformation to signal through a Focal adhesion kinase (FAK)-Tuberous Sclerosis Complex 2 (TSC2)-Ras homolog enriched in brain (Rheb) axis. Moreover, since initiation of translation is reliant on mRNA, it is also relevant to consider potentially mechanosensitive signaling pathways involved in muscle myofibrillar gene transcription and whether some of these pathways converge with those affecting mTORC1 activation for MPS. In this regard, recent findings suggest how mechanical stress may implicate integrin deformation and/or actin dynamics to signal through a Ras homolog gene family member A protein (RhoA)-striated muscle activator of Rho signaling (STARS) axis or implicate deformation of Notch to affect Bone Morphogenetic Protein (BMP) signaling through a small mother of decapentaplegic (Smad) axis. PMID:27909410

  6. Mechanosensitive Molecular Networks Involved in Transducing Resistance Exercise-Signals into Muscle Protein Accretion.

    PubMed

    Rindom, Emil; Vissing, Kristian

    2016-01-01

    Loss of skeletal muscle myofibrillar protein with disease and/or inactivity can severely deteriorate muscle strength and function. Strategies to counteract wasting of muscle myofibrillar protein are therefore desirable and invite for considerations on the potential superiority of specific modes of resistance exercise and/or the adequacy of low load resistance exercise regimens as well as underlying mechanisms. In this regard, delineation of the potentially mechanosensitive molecular mechanisms underlying muscle protein synthesis (MPS), may contribute to an understanding on how differentiated resistance exercise can transduce a mechanical signal into stimulation of muscle accretion. Recent findings suggest specific upstream exercise-induced mechano-sensitive myocellular signaling pathways to converge on mammalian target of rapamycin complex 1 (mTORC1), to influence MPS. This may e.g. implicate mechanical activation of signaling through a diacylglycerol kinase (DGKζ)-phosphatidic acid (PA) axis or implicate integrin deformation to signal through a Focal adhesion kinase (FAK)-Tuberous Sclerosis Complex 2 (TSC2)-Ras homolog enriched in brain (Rheb) axis. Moreover, since initiation of translation is reliant on mRNA, it is also relevant to consider potentially mechanosensitive signaling pathways involved in muscle myofibrillar gene transcription and whether some of these pathways converge with those affecting mTORC1 activation for MPS. In this regard, recent findings suggest how mechanical stress may implicate integrin deformation and/or actin dynamics to signal through a Ras homolog gene family member A protein (RhoA)-striated muscle activator of Rho signaling (STARS) axis or implicate deformation of Notch to affect Bone Morphogenetic Protein (BMP) signaling through a small mother of decapentaplegic (Smad) axis.

  7. Overexpression of uncoupling protein 3 in skeletal muscle protects against fat-induced insulin resistance

    PubMed Central

    Choi, Cheol Soo; Fillmore, Jonathan J.; Kim, Jason K.; Liu, Zhen-Xiang; Kim, Sheene; Collier, Emily F.; Kulkarni, Ameya; Distefano, Alberto; Hwang, Yu-Jin; Kahn, Mario; Chen, Yan; Yu, Chunli; Moore, Irene K.; Reznick, Richard M.; Higashimori, Takamasa; Shulman, Gerald I.

    2007-01-01

    Insulin resistance is a major factor in the pathogenesis of type 2 diabetes and is strongly associated with obesity. Increased concentrations of intracellular fatty acid metabolites have been postulated to interfere with insulin signaling by activation of a serine kinase cascade involving PKCθ in skeletal muscle. Uncoupling protein 3 (UCP3) has been postulated to dissipate the mitochondrial proton gradient and cause metabolic inefficiency. We therefore hypothesized that overexpression of UCP3 in skeletal muscle might protect against fat-induced insulin resistance in muscle by conversion of intramyocellular fat into thermal energy. Wild-type mice fed a high-fat diet were markedly insulin resistant, a result of defects in insulin-stimulated glucose uptake in skeletal muscle and hepatic insulin resistance. Insulin resistance in these tissues was associated with reduced insulin-stimulated insulin receptor substrate 1– (IRS-1–) and IRS-2–associated PI3K activity in muscle and liver, respectively. In contrast, UCP3-overexpressing mice were completely protected against fat-induced defects in insulin signaling and action in these tissues. Furthermore, these changes were associated with a lower membrane-to-cytosolic ratio of diacylglycerol and reduced PKCθ activity in whole-body fat–matched UCP3 transgenic mice. These results suggest that increasing mitochondrial uncoupling in skeletal muscle may be an excellent therapeutic target for type 2 diabetes mellitus. PMID:17571165

  8. Adaptive Resistance and Differential Protein Expression of Salmonella enterica Serovar Enteritidis Biofilms Exposed to Benzalkonium Chloride▿

    PubMed Central

    Mangalappalli-Illathu, Anil K.; Korber, Darren R.

    2006-01-01

    The development of adaptive resistance of Salmonella enterica serovar Enteritidis ATCC 4931 biofilms following exposure to benzalkonium chloride (BC) either continuously (1 μg ml−1) or intermittently (10 μg ml−1 for 10 min daily) was examined. Biofilms adapted to BC over a 144-h period could survive a normally lethal BC challenge (500 μg ml−1 for 10 min) and then regrow, as determined by increases in biofilm thickness, total biomass, and the ratio of the viable biomass to the nonviable biomass. Exposure of untreated control biofilms to the lethal BC challenge resulted in biofilm erosion and cell death. Proteins found to be up-regulated following BC adaptation were those involved in energy metabolism (TpiA and Eno), amino acid and protein biosynthesis (WrbA, TrxA, RplL, Tsf, Tuf, DsbA, and RpoZ), nutrient binding (FruB), adaptation (CspA), detoxification (Tpx, SodB, and a probable peroxidase), and degradation of 1,2-propanediol (PduJ and PduA). A putative universal stress protein (YnaF) was also found to be up-regulated. Proteins involved in proteolysis (DegQ), cell envelope formation (RfbH), adaptation (UspA), heat shock response (DnaK), and broad regulatory functions (Hns) were found to be down-regulated following adaptation. An overall increase in cellular protein biosynthesis was deduced from the significant up-regulation of ribosomal subunit proteins, translation elongation factors, and amino acid biosynthesis protein and down-regulation of serine endoprotease. The cold shock response, stress response, and detoxification are suggested to play roles in the adaptive resistance of Salmonella serovar Enteritidis biofilms to BC. PMID:16940079

  9. Effect of resistance exercise contraction mode and protein supplementation on members of the STARS signalling pathway.

    PubMed

    Vissing, Kristian; Rahbek, Stine K; Lamon, Severine; Farup, Jean; Stefanetti, Renae J; Wallace, Marita A; Vendelbo, Mikkel H; Russell, Aaron

    2013-08-01

    The striated muscle activator of Rho signalling (STARS) pathway is suggested to provide a link between external stress responses and transcriptional regulation in muscle. However, the sensitivity of STARS signalling to different mechanical stresses has not been investigated. In a comparative study, we examined the regulation of the STARS signalling pathway in response to unilateral resistance exercise performed as either eccentric (ECC) or concentric (CONC) contractions as well as prolonged training; with and without whey protein supplementation. Skeletal muscle STARS, myocardian-related transcription factor-A (MRTF-A) and serum response factor (SRF) mRNA and protein, as well as muscle cross-sectional area and maximal voluntary contraction, were measured. A single-bout of exercise produced increases in STARS and SRF mRNA and decreases in MRTF-A mRNA with both ECC and CONC exercise, but with an enhanced response occurring following ECC exercise. A 31% increase in STARS protein was observed exclusively after CONC exercise (P < 0.001), while pSRF protein levels increased similarly by 48% with both CONC and ECC exercise (P < 0.001). Prolonged ECC and CONC training equally stimulated muscle hypertrophy and produced increases in MRTF-A protein of 125% and 99%, respectively (P < 0.001). No changes occurred for total SRF protein. There was no effect of whey protein supplementation. These results show that resistance exercise provides an acute stimulation of the STARS pathway that is contraction mode dependent. The responses to acute exercise were more pronounced than responses to accumulated training, suggesting that STARS signalling is primarily involved in the initial phase of exercise-induced muscle adaptations.

  10. Adaptive resistance and differential protein expression of Salmonella enterica serovar Enteritidis biofilms exposed to benzalkonium chloride.

    PubMed

    Mangalappalli-Illathu, Anil K; Korber, Darren R

    2006-11-01

    The development of adaptive resistance of Salmonella enterica serovar Enteritidis ATCC 4931 biofilms following exposure to benzalkonium chloride (BC) either continuously (1 microg ml(-1)) or intermittently (10 microg ml(-1) for 10 min daily) was examined. Biofilms adapted to BC over a 144-h period could survive a normally lethal BC challenge (500 microg ml(-1) for 10 min) and then regrow, as determined by increases in biofilm thickness, total biomass, and the ratio of the viable biomass to the nonviable biomass. Exposure of untreated control biofilms to the lethal BC challenge resulted in biofilm erosion and cell death. Proteins found to be up-regulated following BC adaptation were those involved in energy metabolism (TpiA and Eno), amino acid and protein biosynthesis (WrbA, TrxA, RplL, Tsf, Tuf, DsbA, and RpoZ), nutrient binding (FruB), adaptation (CspA), detoxification (Tpx, SodB, and a probable peroxidase), and degradation of 1,2-propanediol (PduJ and PduA). A putative universal stress protein (YnaF) was also found to be up-regulated. Proteins involved in proteolysis (DegQ), cell envelope formation (RfbH), adaptation (UspA), heat shock response (DnaK), and broad regulatory functions (Hns) were found to be down-regulated following adaptation. An overall increase in cellular protein biosynthesis was deduced from the significant up-regulation of ribosomal subunit proteins, translation elongation factors, and amino acid biosynthesis protein and down-regulation of serine endoprotease. The cold shock response, stress response, and detoxification are suggested to play roles in the adaptive resistance of Salmonella serovar Enteritidis biofilms to BC.

  11. The plasmid-encoded chloramphenicol-resistance protein of Rhodococcus fascians is homologous to the transmembrane tetracycline efflux proteins.

    PubMed

    Desomer, J; Vereecke, D; Crespi, M; Van Montagu, M

    1992-08-01

    The nucleotide sequence of the chloramphenicol-resistance gene (cmr) of Rhodococcus fascians NCPPB 1675 (located on the conjugative plasmid pRF2) allowed the identification of two possible open reading frames (ORFs), of which ORF1 was consistent with the mutational analysis. Biochemical analysis of cmr revealed that it does not encode an antibiotic-modifying enzyme. The amino acid sequence of ORF1 predicted a hydrophobic protein, with 12 putative membrane-spanning domains, homologous to proteins involved in the efflux of tetracycline across the plasma membrane. Expression of the cmr gene was induced by addition of chloramphenicol to the growth media. The promoter of this gene was restricted to 50 bp upstream from a 200 bp 5'-untranslated mRNA region, the latter containing two inverted repeats. At the amino acid level, the cmr gene is 52% identical to a previously identified chloramphenicol-resistance determinant in Streptomyces lividans, indicating a wider dispersion of this type of cmr gene among the actinomycetes.

  12. Inhibition of neuraminidase inhibitor-resistant influenza virus by DAS181, a novel sialidase fusion protein.

    PubMed

    Triana-Baltzer, Gallen B; Gubareva, Larisa V; Klimov, Alexander I; Wurtman, David F; Moss, Ronald B; Hedlund, Maria; Larson, Jeffrey L; Belshe, Robert B; Fang, Fang

    2009-11-06

    Antiviral drug resistance for influenza therapies remains a concern due to the high prevalence of H1N1 2009 seasonal influenza isolates which display H274Y associated oseltamivir-resistance. Furthermore, the emergence of novel H1N1 raises the potential that additional reassortments can occur, resulting in drug resistant virus. Thus, additional antiviral approaches are urgently needed. DAS181 (Fludase), a sialidase fusion protein, has been shown to have inhibitory activity against a large number of seasonal influenza strains and a highly pathogenic avian influenza (HPAI) strain (H5N1). Here, we examine the in vitro activity of DAS181 against a panel of 2009 oseltamivir-resistant seasonal H1N1 clinical isolates. The activity of DAS181 against nine 2009, two 2007, and two 2004 clinical isolates of seasonal IFV H1N1 was examined using plaque number reduction assay on MDCK cells. DAS181 strongly inhibited all tested isolates. EC50 values remained constant against isolates from 2004, 2007, and 2009, suggesting that there was no change in DAS181 sensitivity over time. As expected, all 2007 and 2009 isolates were resistant to oseltamivir, consistent with the identification of the H274Y mutation in the NA gene of all these isolates. Interestingly, several of the 2007 and 2009 isolates also exhibited reduced sensitivity to zanamivir, and accompanying HA mutations near the sialic acid binding site were observed. DAS181 inhibits IFV that is resistant to NAIs. Thus, DAS181 may offer an alternative therapeutic option for seasonal or pandemic IFVs that become resistant to currently available antiviral drugs.

  13. Cross-talk between signalling pathways and the multidrug resistant protein MDR-1

    PubMed Central

    Ding, S; Chamberlain, M; McLaren, A; Goh, L-b; Duncan, I; Wolf, C R

    2001-01-01

    The multidrug resistant protein MDR-1 has been associated with the resistance to a wide range of anti-cancer drugs. Taxol is a substrate for this transporter system and is used in the treatment of a wide range of human malignancies including lung, breast and ovarian cancer. We have generated a series of ovarian cell lines resistant to this compound, all of which overexpress MDR-1 through gene amplification. We present novel evidence that a constitutive activation of the ERK1/2 MAP kinase pathway was also observed although the level of active JNK and p38 remained unchanged. Inhibition of the ERK1/2 MAP kinase pathway using UO126 or PD098059 re-sensitised the Taxol resistant cells at least 20-fold. Importantly, when Mdr-1 cDNA was stably expressed in the wild-type cell line to generate a highly Taxol-resistant sub-line, 1847/MDR5, ERK1/2 MAP kinases again became activated. This result demonstrated that the increased activity of the signalling pathway in the Taxol-resistant lines was directly attributable to MDR-1 overexpression and was not due to the effects of Taxol itself. Additionally, we demonstrated that inhibition of the P13K pathway with LY294002 sensitised the MDR-1-expressing 1847/TX0.5 cells and 1847/MDR5 cells at least 10-fold but had no effect in the wild-type cells. This finding suggests a possible role for this pathway, also, in the generation of resistance to Taxol. © 2001 Cancer Research Campaign  http://www.bjcancer.com PMID:11710832

  14. Effects of soy protein and isoflavones on insulin resistance and adiponectin in male monkeys.

    PubMed

    Wagner, Janice D; Zhang, Li; Shadoan, Melanie K; Kavanagh, Kylie; Chen, Haiying; Tresnasari, Kristianti; Kaplan, Jay R; Adams, Michael R

    2008-07-01

    Isoflavones may influence insulin action by means of their well-known receptor-mediated estrogenic activity. However, isoflavones also bind to peroxisome proliferator-activated receptors (PPARs) that are strongly associated with insulin action. Soy protein with its isoflavones has previously been shown to improve glycemic control in diabetic postmenopausal women and to improve insulin sensitivity in ovariectomized monkeys. The purpose of the current report was to extend our studies of dietary soy protein to male monkeys and determine effects of the soy isoflavones on insulin resistance. Two studies are reported here. Study one involved 91 male monkeys consuming 3 diets differing only by the source of protein (casein-lactalbumin, soy protein with a low isoflavone concentration, or soy protein with a high isoflavone concentration). Intravenous glucose tolerance tests were done, and plasma adiponectin and lipoprotein concentrations were determined after 25 months of study. Samples of visceral fat were obtained at 31 months for assessment of adiponectin and PPARgamma expression. The second study involved 8 monkeys in a Latin-square design that compared the effects of diets with casein/lactalbumin, soy protein with a high isoflavone concentration, or soy protein that was alcohol-washed to deplete the isoflavones. After 8 weeks of treatment, insulin sensitivity and plasma lipoproteins were assessed. At 10 weeks, a biopsy of the skeletal muscle was performed for determination of insulin receptor, PPARalpha, and PPARgamma content. The major findings were that consumption of isoflavone-containing soy protein dose-dependently increased insulin responses to the glucose challenge and decreased plasma adiponectin, whereas isoflavone-depleted soy protein decreased body weight and had no effect on plasma adiponectin concentrations. Muscle PPARalpha and gamma expression was also increased with the isoflavone-depleted soy relative to either casein or soy protein containing the

  15. Effect of electrical stimulation-induced resistance exercise on mitochondrial fission and fusion proteins in rat skeletal muscle.

    PubMed

    Kitaoka, Yu; Ogasawara, Riki; Tamura, Yuki; Fujita, Satoshi; Hatta, Hideo

    2015-11-01

    It is well known that resistance exercise increases muscle protein synthesis and muscle strength. However, little is known about the effect of resistance exercise on mitochondrial dynamics, which is coupled with mitochondrial function. In skeletal muscle, mitochondria exist as dynamic networks that are continuously remodeling through fusion and fission. The purpose of this study was to investigate the effect of acute and chronic resistance exercise, which induces muscle hypertrophy, on the expression of proteins related to mitochondrial dynamics in rat skeletal muscle. Resistance exercise consisted of maximum isometric contraction, which was induced by percutaneous electrical stimulation of the gastrocnemius muscle. Our results revealed no change in levels of proteins that regulate mitochondrial fission (Fis1 and Drp1) or fusion (Opa1, Mfn1, and Mfn2) over the 24-h period following acute resistance exercise. Phosphorylation of Drp1 at Ser616 was increased immediately after exercise (P < 0.01). Four weeks of resistance training (3 times/week) increased Mfn1 (P < 0.01), Mfn2 (P < 0.05), and Opa1 (P < 0.01) protein levels without altering mitochondrial oxidative phosphorylation proteins. These observations suggest that resistance exercise has little effect on mitochondrial biogenesis but alters the expression of proteins involved in mitochondrial fusion and fission, which may contribute to mitochondrial quality control and improved mitochondrial function.

  16. Anaerobiosis increases resistance of Neisseria gonorrhoeae to O2-independent antimicrobial proteins from human polymorphonuclear granulocytes.

    PubMed Central

    Casey, S G; Shafer, W M; Spitznagel, J K

    1985-01-01

    We investigated the in vitro resistance of Neisseria gonorrhoeae FA19 to the O2-independent antimicrobial systems of human polymorphonuclear leukocytes. Acid extracts of polymorphonuclear leukocyte granules (crude granule extracts) and a purified granule protein (57 kilodaltons) were, at low concentrations, bactericidal for gonococci under aerobic conditions that permitted growth. However, they were less effective under anaerobic conditions that imposed bacteriostasis. We found that adding sodium nitrite to reduced growth media permitted the growth of strain FA19 in an anaerobic environment. Under these conditions with nitrite, anaerobic cultures of strain FA19 were no more resistant to the crude granule extract and the 57-kilodalton protein than aerobic cultures. In contrast, Salmonella typhimurium SL-1004, a facultative anaerobe, was readily killed by both the crude granule extract and the 57-kilodalton antimicrobial protein regardless of the presence or absence of free molecular oxygen. This is the first demonstration that an isolated antimicrobial protein from polymorphonuclear leukocyte granules is active against bacteria under anaerobic conditions. Our results also indicated that the efficacy of human polymorphonuclear leukocyte O2-independent killing of N. gonorrhoeae may, in part, be inhibited by bacteriostatic conditions imposed by hypoxia. Images PMID:3917976

  17. Phenylarsine Oxide Can Induce the Arsenite-Resistance Mutant PML Protein Solubility Changes

    PubMed Central

    Jiang, Yu Han; Chen, Ye Jia; Wang, Chao; Lan, Yong Fei; Yang, Chang; Wang, Qian Qian; Hussain, Liaqat; Maimaitiying, Yasen; Islam, Khairul; Naranmandura, Hua

    2017-01-01

    Arsenic trioxide (As2O3) has recently become one of the most effective drugs for treatment of patient with acute promyelocytic leukemia (APL), and its molecular mechanism has also been largely investigated. However, it has been reported that As2O3 resistant patients are frequently found in relapsed APL after consolidation therapy, which is due to the point mutations in B-box type 2 motifs of promyelocytic leukemia (PML) gene. In the present study, we for the first time establish whether organic arsenic species phenylarsine oxide (PAO) could induce the mutant PML-IV (A216V) protein solubility changes and degradation. Here, three different PML protein variants (i.e., PML-IV, PML-V and mutant PML-A216V) were overexpressed in HEK293T cells and then exposed to PAO in time- and dose-dependent manners. Interestingly, PAO is found to have potential effect on induction of mutant PML-IV (A216V) protein solubility changes and degradation, but no appreciable effects were found following exposure to high concentrations of iAsIII, dimethylarsinous acid (DMAIII) and adriamycin (doxorubicin), even though they cause cell death. Our current data strongly indicate that PAO has good effects on the mutant PML protein solubility changes, and it may be helpful for improving the therapeutic strategies for arsenic-resistant APL treatments in the near future. PMID:28125064

  18. Fibroblasts From Longer-Lived Species of Primates, Rodents, Bats, Carnivores, and Birds Resist Protein Damage.

    PubMed

    Pickering, Andrew M; Lehr, Marcus; Kohler, William J; Han, Melissa L; Miller, Richard A

    2015-07-01

    Species differ greatly in their rates of aging. Among mammalian species life span ranges from 2 to over 60 years. Here, we test the hypothesis that skin-derived fibroblasts from long-lived species of animals differ from those of short-lived animals in their defenses against protein damage. In parallel studies of rodents, nonhuman primates, birds, and species from the Laurasiatheria superorder (bats, carnivores, shrews, and ungulates), we find associations between species longevity and resistance of proteins to oxidative stress after exposure to H(2)O(2) or paraquat. In addition, baseline levels of protein carbonyl appear to be higher in cells from shorter-lived mammals compared with longer-lived mammals. Thus, resistance to protein oxidation is associated with species maximal life span in independent clades of mammals, suggesting that this cellular property may be required for evolution of longevity. Evaluation of the properties of primary fibroblast cell lines can provide insights into the factors that regulate the pace of aging across species of mammals.

  19. Fibroblasts From Longer-Lived Species of Primates, Rodents, Bats, Carnivores, and Birds Resist Protein Damage

    PubMed Central

    Pickering, Andrew M.; Lehr, Marcus; Kohler, William J.; Han, Melissa L.

    2015-01-01

    Species differ greatly in their rates of aging. Among mammalian species life span ranges from 2 to over 60 years. Here, we test the hypothesis that skin-derived fibroblasts from long-lived species of animals differ from those of short-lived animals in their defenses against protein damage. In parallel studies of rodents, nonhuman primates, birds, and species from the Laurasiatheria superorder (bats, carnivores, shrews, and ungulates), we find associations between species longevity and resistance of proteins to oxidative stress after exposure to H2O2 or paraquat. In addition, baseline levels of protein carbonyl appear to be higher in cells from shorter-lived mammals compared with longer-lived mammals. Thus, resistance to protein oxidation is associated with species maximal life span in independent clades of mammals, suggesting that this cellular property may be required for evolution of longevity. Evaluation of the properties of primary fibroblast cell lines can provide insights into the factors that regulate the pace of aging across species of mammals. PMID:25070662

  20. 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

  1. Biological protein-resistance layer construction of recombinant hirudin on polymethyl methacrylate IOL surface.

    PubMed

    Zheng, Zhiwen; Jiao, Yan; Ren, Li; Wang, Yingjun

    2015-03-01

    In this article, the surface of intraocular len material PMMA was first aminated for activation on which some polar groups generated such as C-N, COO(-), -OH, NH3(+), etc. Then the anticoagulant drugs recombinant hirudin (rH) was grafted with amido bonds to look forward to resist the adsorption of nonspecific protein or cells in tear, even the cataract. The detailed analysis and discussion about the grafting quantity, molography, wettability, electric charges, chemical structure, and the dynamic adsorption of protein Fn on the material surface were carried on by the technology of ultraviolet photometric, contact angle, solid Zeta potential, X-ray photoelectron spectroscopy, and quartz crystal microbalance. The surface with a certain amount of rH modification existed more hydrophilic due to the amphiphilic structure than before, on which the protein adsorption was the most unstable. The results indicated that the rH modification improved the resistance of PMMA to nonspecific adsorption of protein Fn to achieve the expectative effect.

  2. Interaction of oxazaphosphorines with multidrug resistance-associated protein 4 (MRP4).

    PubMed

    Zhang, Jing; Ng, Ka-Yun; Ho, Paul C

    2010-09-01

    Multidrug resistance-associated protein 4 (MRP4) is an organic anion efflux pump capable of transporting nucleoside, nucleotide analogs, and cyclic nucleotide. MRP4 could have an influence on the resistance and transport of the two oxazaphosphorines, cyclophosphamide (CP) and ifosfamide (IF). V/HepG2 (HepG2, hepatoma cells stably transfected with an empty vehicle plasmid) and MRP4/HepG2 (HepG2 cells stably expressing MRP4) were exposed to CP and IF in the absence or presence of various MRP4 inhibitors. HepG2 and HEK293 human kidney cells were also used to investigate the inducing potency of oxazaphosphorines on the MRP4 expression. In this study, insertion of MRP4 gene in HepG2 cells was found to confer significant resistance to CP and IF in the 48-h drug-exposure assays. In the presence of various MRP4 inhibitors, the resistance to CP and IF was then partially reversed. These indicate that CP and IF are highly possible substrates of MRP4. In addition, CP and clofibrate (CFB), a reported MRP4 inducer, in vivo significantly increased the MRP4 expression at both protein level and mRNA level in HEK293 cells at higher concentrations, while IF significantly decreased the MRP4 expression at mRNA level at lower concentration and had no effect at higher concentrations. However, all tested compounds (CP, IF, and CFB) did not change the MRP4 protein expression in HepG2 cells. CP and CFB are cell-specific and concentration-dependent MRP4 inducers. The finding may have implications in the CP- or IF-based chemotherapy.

  3. High expression of intratumoral stromal proteins is associated with chemotherapy resistance in breast cancer

    PubMed Central

    Wang, Tingting; Srivastava, Supriya; Hartman, Mikael; Buhari, Shaik Ahmad; Chan, Ching-Wan; Iau, Philip; Khin, Lay Wai; Wong, Andrea; Tan, Sing-Huang; Goh, Boon-Cher; Lee, Soo-Chin

    2016-01-01

    We studied the changes of intratumoral stromal proteins including THBS1, TNC, FN, SPARC and α-SMA, following neoadjuvant chemotherapy. The underlying mechanisms by which THBS1 and TNC regulated resistance to docetaxel were further studied using functional studies. 100 patients with newly diagnosed breast cancer were treated with alternating sequential doxorubicin and docetaxel. Immunohistochemistry (IHC) staining for stromal proteins was performed on pre- and post-treatment core biopsies respectively. THBS1 and TNC were further validated with IHC in an independent cohort of 31 patients. A high baseline combined expression score of the 5 stromal proteins predicted independently for poor progression-free (HRadjusted 2.22, 95% CI 1.06–4.64) and overall survival (HRadjusted 5.94, 95% CI 2.25–15.71). After 1–2 cycles of chemotherapy, increased expression of THBS1, TNC, FN, SPARC and α-SMA was seen in patients with subsequent pathological lymph node involvement at surgery. Increased expression of THBS1 and TNC compared to baseline was also seen in intrinsically resistant tumors, but not in sensitive ones. Both THBS1 and TNC-associated chemoresistance were confirmed in an independent validation cohort. Exogenous THBS1 and TNC protected MCF-7 cells against proliferation inhibition induced by docetaxel through activating integrin β1/mTOR pathway. Thus, up-regulation of THBS1, TNC, FN, SPARC and α-SMA following neoadjuvant chemotherapy was associated with chemotherapy resistance in breast cancer patients. Functional studies showed THBS1 and TNC to mediate chemoresistance through the integrin β1/mTOR pathway, suggesting that therapies targeting integrin β1/mTOR pathway may be a promising strategy to overcome chemotherapy resistance. PMID:27487140

  4. Macrophage-specific Mycobacterium tuberculosis genes: identification by green fluorescent protein and kanamycin resistance selection.

    PubMed

    Srivastava, Vikas; Rouanet, Carine; Srivastava, Ranjana; Ramalingam, B; Locht, Camille; Srivastava, Brahm S

    2007-03-01

    Mycobacterium tuberculosis survives and multiplies inside macrophages of its host by modulating the expression of several genes essential for in vivo survival. An in vivo expression system has been developed, based on green fluorescent protein and kanamycin resistance, to identify M. tuberculosis genes which appear to be up-regulated in infected macrophages. A promoter-trap shuttle vector, pLL192, was constructed, containing a streptomycin resistance gene as selection marker and an artificial bicistronic operon composed of the promoterless green fluorescent protein (gfp) gene, followed by the kanamycin resistance gene. A unique BamHI site upstream of the gfp gene allowed for insertion of promoter libraries. The vector was validated by the use of known regulated or constitutive M. tuberculosis promoters. In addition, an M. tuberculosis genomic DNA library was inserted into pLL192 and then introduced into Mycobacterium bovis BCG. The recombinant BCG cells were then used to infect the J774A.1 murine macrophage-like cell line in the presence of kanamycin. Several recombinant BCG cells were thereby selected that were resistant to kanamycin within infected macrophages, but were sensitive to kanamycin when grown in vitro. The kanamycin resistance phenotype was paralleled by the fluorescence phenotype. After nucleotide sequencing, the corresponding genes were identified as mce1A, PE_PGRS63(RV3097c), Rv2232, Rv1026, Rv1635c, viuB, Rv2231(cobC) and Rv0997. Real-time PCR analysis using RNA isolated at various time points from M. tuberculosis and M. bovis BCG grown in vitro and within macrophages, confirmed the up-regulation of these genes. The level of up-regulation varied from 2- to 40-fold in macrophages compared to growth in vitro.

  5. Resistance exercise volume affects myofibrillar protein synthesis and anabolic signalling molecule phosphorylation in young men

    PubMed Central

    Burd, Nicholas A; Holwerda, Andrew M; Selby, Keegan C; West, Daniel W D; Staples, Aaron W; Cain, Nathan E; Cashaback, Joshua G A; Potvin, James R; Baker, Steven K; Phillips, Stuart M

    2010-01-01

    We aimed to determine if any mechanistic differences exist between a single set (1SET) and multiple sets (i.e. 3 sets; 3SET) of resistance exercise by utilizing a primed constant infusion of [ring-13C6]phenylalanine to determine myofibrillar protein synthesis (MPS) and Western blot analysis to examine anabolic signalling molecule phosphorylation following an acute bout of resistance exercise. Eight resistance-trained men (24 ± 5 years, BMI = 25 ± 4 kg m−2) were randomly assigned to perform unilateral leg extension exercise at 70% concentric one repetition maximum (1RM) until volitional fatigue for 1SET or 3SET. Biopsies from the vastus lateralis were taken in the fasted state (Fast) and fed state (Fed; 20 g of whey protein isolate) at rest, 5 h Fed, 24 h Fast and 29 h Fed post-exercise. Fed-state MPS was transiently elevated above rest at 5 h for 1SET (2.3-fold) and returned to resting levels by 29 h post-exercise. However, the exercise induced increase in MPS following 3SET was superior in amplitude and duration as compared to 1SET at both 5 h (3.1-fold above rest) and 29 h post-exercise (2.3-fold above rest). Phosphorylation of 70 kDa S6 protein kinase (p70S6K) demonstrated a coordinated increase with MPS at 5 h and 29 h post-exercise such that the extent of p70S6K phosphorylation was related to the MPS response (r = 0.338, P = 0.033). Phosphorylation of 90 kDa ribosomal S6 protein kinase (p90RSK) and ribosomal protein S6 (rps6) was similar for 1SET and 3SET at 24 h Fast and 29 h Fed, respectively. However, 3SET induced a greater activation of eukaryotic translation initiation factor 2Bɛ (eIF2Bɛ) and rpS6 at 5 h Fed. These data suggest that 3SET of resistance exercise is more anabolic than 1SET and may lead to greater increases in myofibrillar protein accretion over time. PMID:20581041

  6. The Arabidopsis Tellurite resistance C protein together with ALB3 is involved in photosystem II protein synthesis.

    PubMed

    Schneider, Anja; Steinberger, Iris; Strissel, Henning; Kunz, Hans-Henning; Manavski, Nikolay; Meurer, Jörg; Burkhard, Gabi; Jarzombski, Sabine; Schünemann, Danja; Geimer, Stefan; Flügge, Ulf-Ingo; Leister, Dario

    2014-04-01

    Assembly of photosystem II (PSII) occurs sequentially and requires several auxiliary proteins, such as ALB3 (ALBINO3). Here, we describe the role of the Arabidopsis thaliana thylakoid membrane protein Tellurite resistance C (AtTerC) in this process. Knockout of AtTerC was previously shown to be seedling-lethal. This phenotype was rescued by expressing TerC fused C-terminally to GFP in the terc-1 background, and the resulting terc-1TerC- GFP line and an artificial miRNA-based knockdown allele (amiR-TerC) were used to analyze the TerC function. The alterations in chlorophyll fluorescence and thylakoid ultrastructure observed in amiR-TerC plants and terc-1TerC- GFP were attributed to defects in PSII. We show that this phenotype resulted from a reduction in the rate of de novo synthesis of PSII core proteins, but later steps in PSII biogenesis appeared to be less affected. Yeast two-hybrid assays showed that TerC interacts with PSII proteins. In particular, its interaction with the PSII assembly factor ALB3 has been demonstrated by co-immunoprecipitation. ALB3 is thought to assist in incorporation of CP43 into PSII via interaction with Low PSII Accumulation2 (LPA2) Low PSII Accumulation3 (LPA3). Homozygous lpa2 mutants expressing amiR-TerC displayed markedly exacerbated phenotypes, leading to seedling lethality, indicating an additive effect. We propose a model in which TerC, together with ALB3, facilitates de novo synthesis of thylakoid membrane proteins, for instance CP43, at the membrane insertion step.

  7. Structure-function analysis of the NB-ARC domain of plant disease resistance proteins.

    PubMed

    van Ooijen, Gerben; Mayr, Gabriele; Kasiem, Mobien M A; Albrecht, Mario; Cornelissen, Ben J C; Takken, Frank L W

    2008-01-01

    Resistance (R) proteins in plants are involved in pathogen recognition and subsequent activation of innate immune responses. Most resistance proteins contain a central nucleotide-binding domain. This so-called NB-ARC domain consists of three subdomains: NB, ARC1, and ARC2. The NB-ARC domain is a functional ATPase domain, and its nucleotide-binding state is proposed to regulate activity of the R protein. A highly conserved methionine-histidine-aspartate (MHD) motif is present at the carboxy-terminus of ARC2. An extensive mutational analysis of the MHD motif in the R proteins I-2 and Mi-1 is reported. Several novel autoactivating mutations of the MHD invariant histidine and conserved aspartate were identified. The combination of MHD mutants with autoactivating hydrolysis mutants in the NB subdomain showed that the autoactivation phenotypes are not additive. This finding indicates an important regulatory role for the MHD motif in the control of R protein activity. To explain these observations, a three-dimensional model of the NB-ARC domain of I-2 was built, based on the APAF-1 template structure. The model was used to identify residues important for I-2 function. Substitution of the selected residues resulted in the expected distinct phenotypes. Based on the model, it is proposed that the MHD motif fulfils the same function as the sensor II motif found in AAA+ proteins (ATPases associated with diverse cellular activities)-co-ordination of the nucleotide and control of subdomain interactions. The presented 3D model provides a framework for the formulation of hypotheses on how mutations in the NB-ARC exert their effects.

  8. Prion protein is ubiquitinated after developing protease resistance in the brains of scrapie-infected mice.

    PubMed

    Kang, Shin-Chung; Brown, David R; Whiteman, Matthew; Li, Ruliang; Pan, Tao; Perry, George; Wisniewski, Thomas; Sy, Man-Sun; Wong, Boon-Seng

    2004-05-01

    Although the key event in the pathology of prion diseases is thought to be the conversion of cellular prion protein (PrP(C)) to the protease-resistant scrapie species termed PrP(Sc), the factors that contribute to neurodegeneration in scrapie-infected animals are poorly understood. One probable determinant could be when the accumulation of PrP(Sc) in infected brain overwhelms the ubiquitin-proteasome system and triggers the degenerative cascade. In the present study, it was found that in mouse brains infected with the ME7 scrapie strain, the level of ubiquitin protein conjugates increased significantly at approximately 144 days post-infection (pi) when clinical signs first become apparent. This elevation correlated with the detection of protease-resistant PrP(Sc) and a decline in two endopeptidase activities associated with proteasome function. However, ubiquitination of PrP was only detected at the terminal stage, 3 weeks after the development of clinical symptoms (approximately 165 days pi). These results suggest that ubiquitination of PrP is a late event phenomenon and this conjugation occurs after the formation of protease-resistant PrP(Sc). Whether this post-translational modification and the impairment of proteasome function are pivotal events in the pathogenesis of prion diseases remains to be determined.

  9. Identification of putative drug targets in Vancomycin-resistant Staphylococcus aureus (VRSA) using computer aided protein data analysis.

    PubMed

    Hasan, Md Anayet; Khan, Md Arif; Sharmin, Tahmina; Hasan Mazumder, Md Habibul; Chowdhury, Afrin Sultana

    2016-01-01

    Vancomycin-resistant Staphylococcus aureus (VRSA) is a Gram-positive, facultative aerobic bacterium which is evolved from the extensive exposure of Vancomycin to Methicillin resistant S. aureus (MRSA) that had become the most common cause of hospital and community-acquired infections. Due to the emergence of different antibiotic resistance strains, there is an exigency to develop novel drug targets to address the provocation of multidrug-resistant bacteria. In this study, in-silico genome subtraction methodology was used to design potential and pathogen specific drug targets against VRSA. Our study divulged 1987 proteins from the proteome of 34,549 proteins, which have no homologues in human genome after sequential analysis through CD-HIT and BLASTp. The high stringency analysis of the remaining proteins against database of essential genes (DEG) resulted in 169 proteins which are essential for S. aureus. Metabolic pathway analysis of human host and pathogen by KAAS at the KEGG server sorted out 19 proteins involved in unique metabolic pathways. 26 human non-homologous membrane-bound essential proteins including 4 which were also involved in unique metabolic pathway were deduced through PSORTb, CELLO v.2.5, ngLOC. Functional classification of uncharacterized proteins through SVMprot derived 7 human non-homologous membrane-bound hypothetical essential proteins. Study of potential drug target against Drug Bank revealed pbpA-penicillin-binding protein 1 and hypothetical protein MQW_01796 as the best drug target candidate. 2D structure was predicted by PRED-TMBB, 3D structure and functional analysis was also performed. Protein-protein interaction network of potential drug target proteins was analyzed by using STRING. The identified drug targets are expected to have great potential for designing novel drugs against VRSA infections and further screening of the compounds against these new targets may result in the discovery of novel therapeutic compounds that can be

  10. Undetectable bacterial resistance to phage lytic proteins from the Staphylococcus aureus bacteriophage vB_SauS-phiIPLA88

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The increase in antibiotic resistance world-wide revitalized the interest in the use of phage lysins to combat pathogenic bacteria. In this work, we tested for the emergence of resistant Staphylococcus aureus to any of three phage lytic proteins constructs. The investigated cell wall lytic enzymes w...

  11. Assessment of Relationship Between Bacterial Stripe Resistance And Leaf Protein Bands In Rice (Oryza sativa L.) Varieties.

    NASA Astrophysics Data System (ADS)

    Talei, D.; Fotokian, M. H.

    2008-01-01

    Bacterial stripe as a new rice disease in Iran is more frequent nowadays. The objective of this study was to assessment of resistance in rice varieties together with evaluating of zymogram bands resulted from SDS PAGE electrophoresis of leaf proteins. For this purpose, 30 lines were tested in a randomized complete block design with three replications. The analysis of variance showed that there was significant difference between genotypes for resistance. Mean compare based on field results revealed that Domsiyah had the lowest resistance while Nemat and 7162 demonstrated the highest resistance. Laboratory results showed that there were significant difference between protein bands resulted from sensitive and resistance verities. Twenty bands were observed through SDS PAGE electrophoresis of leaf proteins. The 9th and 12th bands were found in sensitive varieties while were not in resistance genotypes. According to the results of this study, 7162 variety can be considered as the sources of resistance in breeding programs. Meanwhile attending to existence of 9th and 12th bands in sensitive varieties, resistance against bacterial stripe of rice maybe influenced by absence of these proteins.

  12. Breast cancer resistance protein (BCRP/ABCG2): its role in multidrug resistance and regulation of its gene expression

    PubMed Central

    Nakanishi, Takeo; Ross, Douglas D.

    2012-01-01

    Breast cancer resistance protein (BCRP)/ATP-binding cassette subfamily G member 2 (ABCG2) is an ATP-binding cassette (ABC) transporter identified as a molecular cause of multidrug resistance (MDR) in diverse cancer cells. BCRP physiologically functions as a part of a self-defense mechanism for the organism; it enhances elimination of toxic xenobiotic substances and harmful agents in the gut and biliary tract, as well as through the blood-brain, placental, and possibly blood-testis barriers. BCRP recognizes and transports numerous anticancer drugs including conventional chemotherapeutic and targeted small therapeutic molecules relatively new in clinical use. Thus, BCRP expression in cancer cells directly causes MDR by active efflux of anticancer drugs. Because BCRP is also known to be a stem cell marker, its expression in cancer cells could be a manifestation of metabolic and signaling pathways that confer multiple mechanisms of drug resistance, self-renewal (sternness), and invasiveness (aggressiveness), and thereby impart a poor prognosis. Therefore, blocking BCRP-mediated active efflux may provide a therapeutic benefit for cancers. Delineating the precise molecular mechanisms for BCRP gene expression may lead to identification of a novel molecular target to modulate BCRP-mediated MDR. Current evidence suggests that BCRP gene transcription is regulated by a number of trans-acting elements including hypoxia inducible factor 1α, estrogen receptor, and peroxisome proliferator-activated receptor. Furthermore, alternative promoter usage, demethylation of the BCRP promoter, and histone modification are likely associated with drug-induced BCRP overexpression in cancer cells. Finally, PI3K/AKT signaling may play a critical role in modulating BCRP function under a variety of conditions. These biological events seem involved in a complicated manner. Untangling the events would be an essential first step to developing a method to modulate BCRP function to aid patients with

  13. Structure-guided optimization of protein kinase inhibitors reverses aminoglycoside antibiotic resistance

    PubMed Central

    Stogios, Peter J.; Spanogiannopoulos, Peter; Evdokimova, Elena; Egorova, Olga; Shakya, Tushar; Todorovic, Nick; Capretta, Alfredo; Wright, Gerard D.; Savchenko, Alexei

    2013-01-01

    SYNOPSIS Activity of the aminoglycoside phosphotransferase APH(3’)-Ia leads to resistance to aminoglycoside antibiotics in pathogenic Gram-negative bacteria, and contributes to the clinical obsolescence of this class of antibiotics. One strategy to rescue compromised antibiotics such as aminoglycosides is targeting the enzymes that confer resistance with small molecules. Previously we demonstrated that eukaryotic protein kinase (ePK) inhibitors could inhibit APH enzymes, due to the structural similarity between these two enzyme families. However, limited structural information of enzyme-inhibitor complexes hindered interpretation of the results. As well, cross-reactivity of compounds between APHs and ePKs represents an obstacle to their use as aminoglycoside adjuvants to rescue aminoglycoside antibiotic activity. Here, we structurally and functionally characterize inhibition of APH(3’)-Ia by three diverse chemical scaffolds – anthrapyrazolone, 4-anilinoquinazoline and pyrazolopyrimidine (PP) – and reveal distinctions in the binding mode of anthrapyrazolone and PP compounds to APH(3’)-Ia versus ePKs. Using this observation, we identify PP-derivatives that select against ePKs, attenuate APH(3’)-Ia activity and rescue aminoglycoside antibiotic activity against a resistant E. coli strain. The structures presented here and these inhibition studies provide an important opportunity for structure-based design of compounds to target aminoglycoside phosphotransferases for inhibition, potentially overcoming this form of antibiotic resistance. PMID:23758273

  14. Carbon nanotubes induce apoptosis resistance of human lung epithelial cells through FLICE-inhibitory protein.

    PubMed

    Pongrakhananon, Varisa; Luanpitpong, Sudjit; Stueckle, Todd A; Wang, Liying; Nimmannit, Ubonthip; Rojanasakul, Yon

    2015-02-01

    Chronic exposure to single-walled carbon nanotubes (SWCNT) has been reported to induce apoptosis resistance of human lung epithelial cells. As resistance to apoptosis is a foundation of neoplastic transformation and cancer development, we evaluated the apoptosis resistance characteristic of the exposed lung cells to understand the pathogenesis mechanism. Passage control and SWCNT-transformed human lung epithelial cells were treated with known inducers of apoptosis via the intrinsic (antimycin A and CDDP) or extrinsic (FasL and TNF-α) pathway and analyzed for apoptosis by DNA fragmentation, annexin-V expression, and caspase activation assays. Whole-genome microarray was performed to aid the analysis of apoptotic gene signaling network. The SWCNT-transformed cells exhibited defective death receptor pathway in association with cellular FLICE-inhibitory protein (c-FLIP) overexpression. Knockdown or chemical inhibition of c-FLIP abrogated the apoptosis resistance of SWCNT-transformed cells. Whole-genome expression signature analysis confirmed these findings. This study is the first to demonstrate carbon nanotube-induced defective death receptor pathway and the role of c-FLIP in the process.

  15. Protein phosphatase AP2C1 negatively regulates basal resistance and defense responses to Pseudomonas syringae.

    PubMed

    Shubchynskyy, Volodymyr; Boniecka, Justyna; Schweighofer, Alois; Simulis, Justinas; Kvederaviciute, Kotryna; Stumpe, Michael; Mauch, Felix; Balazadeh, Salma; Mueller-Roeber, Bernd; Boutrot, Freddy; Zipfel, Cyril; Meskiene, Irute

    2017-01-06

    Mitogen-activated protein kinases (MAPKs) mediate plant immune responses to pathogenic bacteria. However, less is known about the cell autonomous negative regulatory mechanism controlling basal plant immunity. We report the biological role of Arabidopsis thaliana MAPK phosphatase AP2C1 as a negative regulator of plant basal resistance and defense responses to Pseudomonas syringae AP2C2, a closely related MAPK phosphatase, also negatively controls plant resistance. Loss of AP2C1 leads to enhanced pathogen-induced MAPK activities, increased callose deposition in response to pathogen-associated molecular patterns or to P. syringae pv. tomato (Pto) DC3000, and enhanced resistance to bacterial infection with Pto. We also reveal the impact of AP2C1 on the global transcriptional reprogramming of transcription factors during Pto infection. Importantly, ap2c1 plants show salicylic acid-independent transcriptional reprogramming of several defense genes and enhanced ethylene production in response to Pto This study pinpoints the specificity of MAPK regulation by the different MAPK phosphatases AP2C1 and MKP1, which control the same MAPK substrates, nevertheless leading to different downstream events. We suggest that precise and specific control of defined MAPKs by MAPK phosphatases during plant challenge with pathogenic bacteria can strongly influence plant resistance.

  16. Structure-guided optimization of protein kinase inhibitors reverses aminoglycoside antibiotic resistance.

    PubMed

    Stogios, Peter J; Spanogiannopoulos, Peter; Evdokimova, Elena; Egorova, Olga; Shakya, Tushar; Todorovic, Nick; Capretta, Alfredo; Wright, Gerard D; Savchenko, Alexei

    2013-09-01

    Activity of the aminoglycoside phosphotransferase APH(3')-Ia leads to resistance to aminoglycoside antibiotics in pathogenic Gram-negative bacteria, and contributes to the clinical obsolescence of this class of antibiotics. One strategy to rescue compromised antibiotics such as aminoglycosides is targeting the enzymes that confer resistance with small molecules. We demonstrated previously that ePK (eukaryotic protein kinase) inhibitors could inhibit APH enzymes, owing to the structural similarity between these two enzyme families. However, limited structural information of enzyme-inhibitor complexes hindered interpretation of the results. In addition, cross-reactivity of compounds between APHs and ePKs represents an obstacle to their use as aminoglycoside adjuvants to rescue aminoglycoside antibiotic activity. In the present study, we structurally and functionally characterize inhibition of APH(3')-Ia by three diverse chemical scaffolds, anthrapyrazolone, 4-anilinoquinazoline and PP (pyrazolopyrimidine), and reveal distinctions in the binding mode of anthrapyrazolone and PP compounds to APH(3')-Ia compared with ePKs. Using this observation, we identify PP derivatives that select against ePKs, attenuate APH(3')-Ia activity and rescue aminoglycoside antibiotic activity against a resistant Escherichia coli strain. The structures described in the present paper and the inhibition studies provide an important opportunity for structure-based design of compounds to target aminoglycoside phosphotransferases for inhibition, potentially overcoming this form of antibiotic resistance.

  17. Hepatitis B X-interacting protein promotes cisplatin resistance and regulates CD147 via Sp1 in ovarian cancer.

    PubMed

    Zou, Wei; Ma, Xiangdong; Yang, Hong; Hua, Wei; Chen, Biliang; Cai, Guoqing

    2017-03-01

    Ovarian cancer is the highest mortality rate of all female reproductive malignancies. Drug resistance is a major cause of treatment failure in malignant tumors. Hepatitis B X-interacting protein acts as an oncoprotein, regulates cell proliferation, and migration in breast cancer. We aimed to investigate the effects and mechanisms of hepatitis B X-interacting protein on resistance to cisplatin in human ovarian cancer cell lines. The mRNA and protein levels of hepatitis B X-interacting protein were detected using RT-PCR and Western blotting in cisplatin-resistant and cisplatin-sensitive tissues, cisplatin-resistant cell lines A2780/CP and SKOV3/CP, and cisplatin-sensitive cell lines A2780 and SKOV3. Cell viability and apoptosis were measured to evaluate cellular sensitivity to cisplatin in A2780/CP cells. Luciferase reporter gene assay was used to determine the relationship between hepatitis B X-interacting protein and CD147. The in vivo function of hepatitis B X-interacting protein on tumor burden was assessed in cisplatin-resistant xenograft models. The results showed that hepatitis B X-interacting protein was highly expressed in ovarian cancer of cisplatin-resistant tissues and cells. Notably, knockdown of hepatitis B X-interacting protein significantly reduced cell viability in A2780/CP compared with cisplatin treatment alone. Hepatitis B X-interacting protein and cisplatin cooperated to induce apoptosis and increase the expression of c-caspase 3 as well as the Bax/Bcl-2 ratio. We confirmed that hepatitis B X-interacting protein up-regulated CD147 at the protein expression and transcriptional levels. Moreover, we found that hepatitis B X-interacting protein was able to activate the CD147 promoter through Sp1. In vivo, depletion of hepatitis B X-interacting protein decreased the tumor volume and weight induced by cisplatin. Taken together, these results indicate that hepatitis B X-interacting protein promotes cisplatin resistance and regulated CD147 via Sp1 in

  18. Identification of detergent-resistant plasma membrane microdomains in dictyostelium: enrichment of signal transduction proteins.

    PubMed Central

    Xiao, Z; Devreotes, P N

    1997-01-01

    Unlike most other cellular proteins, the chemoattractant receptor, cAR1, of Dictyostelium is resistant to extraction by the zwitterionic detergent, CHAPS. We exploited this property to isolate a subcellular fraction highly enriched in cAR1 by flotation of CHAPS lysates of cells in sucrose density gradients. Immunogold electron microscopy studies revealed a homogeneous preparation of membrane bilayer sheets. This preparation, designated CHAPS-insoluble floating fraction (CHIEF), also contained a defined set of 20 other proteins and a single uncharged lipid. Cell surface biotinylation and preembedding immunoelectron microscopy both confirmed the plasma membrane origin of this preparation. The cell surface phosphodiesterase (PDE) and a downstream effector of cAR1, adenylate cyclase (ACA), were specifically localized in these structures, whereas the cell adhesion molecule gp80, most of the major cell surface membrane proteins, cytoskeletal components, the actin-binding integral membrane protein ponticulin, and G-protein alpha- and beta-subunits were absent. Overall, CHIFF represents about 3-5% of cell externally exposed membrane proteins. All of these results indicate that CHIFF is derived from specialized microdomains of the plasma membrane. The method of isolation is analogous to that of caveolae. However, we were unable to detect distinct caveolae-like structures on the cell surface associated with cAR1, which showed a diffuse staining profile. The discovery of CHIFF facilitates the purification of cAR1 and related signaling proteins and the biochemical characterization of receptor-mediated processes such as G-protein activation and desensitization. It also has important implications for the "fluid mosaic" model of the plasma membrane structures. Images PMID:9168471

  19. Suppression among alleles encoding nucleotide-binding-leucine-rich repeat resistance proteins interferes with resistance in F1 hybrid and allele-pyramided wheat plants.

    PubMed

    Stirnweis, Daniel; Milani, Samira D; Brunner, Susanne; Herren, Gerhard; Buchmann, Gabriele; Peditto, David; Jordan, Tina; Keller, Beat

    2014-09-01

    The development of high-yielding varieties with broad-spectrum durable disease resistance is the ultimate goal of crop breeding. In plants, immune receptors of the nucleotide-binding-leucine-rich repeat (NB-LRR) class mediate race-specific resistance against pathogen attack. When employed in agriculture this type of resistance is often rapidly overcome by newly adapted pathogen races. The stacking of different resistance genes or alleles in F1 hybrids or in pyramided lines is a promising strategy for achieving more durable resistance. Here, we identify a molecular mechanism which can negatively interfere with the allele-pyramiding approach. We show that pairwise combinations of different alleles of the powdery mildew resistance gene Pm3 in F1 hybrids and stacked transgenic wheat lines can result in suppression of Pm3-based resistance. This effect is independent of the genetic background and solely dependent on the Pm3 alleles. Suppression occurs at the post-translational level, as levels of RNA and protein in the suppressed alleles are unaffected. Using a transient expression system in Nicotiana benthamiana, the LRR domain was identified as the domain conferring suppression. The results of this study suggest that the expression of closely related NB-LRR resistance genes or alleles in the same genotype can lead to dominant-negative interactions. These findings provide a molecular explanation for the frequently observed ineffectiveness of resistance genes introduced from the secondary gene pool into polyploid crop species and mark an important step in overcoming this limitation.

  20. Up-regulation of lipolysis genes and increased production of AMP-activated protein kinase protein in the skeletal muscle of rats after resistance training

    PubMed Central

    An, Jae-Heung; Yoon, Jin-Hwan; Suk, Min-Hwa; Shin, Yun-A

    2016-01-01

    The purpose of this study was to investigate the expression of lipogenesis- and lipolysis-related genes and proteins in skeletal muscles after 12 weeks of resistance training. Sprague-Dawley rats (n=12) were randomly divided into control (resting) and resistance training groups. A tower-climbing exercise, in which rats climbed to the top of their cage with a weight applied to their tails, used for resistance training. After 12 weeks, rats from the resistance training group had lower body weights (411.66±14.71 g vs. 478.33±24.63 g in the control), there was no significant difference between the two groups in the concentrations of total cholesterol, and high or low density lipoprotein cholesterol. However, the concentration of triglyceride was lower in resistance-trained rats (59.83±14.05 μg/mL vs 93.33±33.89 μg/mL in the control). The mRNA expression levels of the lipogenesis-related genes sterol regulatory element binding protein-1c, acetyl-CoA carboxylase, and fatty acid synthase were not significantly different between the resistance-trained and control rats; however, mRNA expression of the lipolysis-related carnitine palmitoyl transferase 1 and malonyl-CoA decarboxylase increased significantly with resistance training. AMP-activated protein kinase protein levels also significantly increased in resistance training group compared with in the control group. These results suggested that resistance exercise training contributing to reduced weight gain may be in part be due to increase the lipolysis metabolism and energy expenditure in response to resistance training. PMID:27419110

  1. Group A Streptococcal M1 Protein Provides Resistance against the Antimicrobial Activity of Histones

    PubMed Central

    Döhrmann, Simon; LaRock, Christopher N.; Anderson, Ericka L.; Cole, Jason N.; Ryali, Brinda; Stewart, Chelsea; Nonejuie, Poochit; Pogliano, Joe; Corriden, Ross; Ghosh, Partho; Nizet, Victor

    2017-01-01

    Histones are essential elements of chromatin structure and gene regulation in eukaryotes. An unexpected attribute of these nuclear proteins is their antimicrobial activity. A framework for histone release and function in host defense in vivo was revealed with the discovery of neutrophil extracellular traps, a specialized cell death process in which DNA-based structures containing histones are extruded to ensnare and kill bacteria. Investigating the susceptibility of various Gram-positive pathogens to histones, we found high-level resistance by one leading human pathogen, group A Streptococcus (GAS). A screen of isogenic mutants revealed that the highly surface-expressed M1 protein, a classical GAS virulence factor, was required for high-level histone resistance. Biochemical and microscopic analyses revealed that the N-terminal domain of M1 protein binds and inactivates histones before they reach their cell wall target of action. This finding illustrates a new pathogenic function for this classic GAS virulence factor, and highlights a potential innate immune evasion strategy that may be employed by other bacterial pathogens. PMID:28220899

  2. A Novel Peptide from Soybean Protein Isolate Significantly Enhances Resistance of the Organism under Oxidative Stress

    PubMed Central

    Ma, Heran; Liu, Rui; Zhao, Ziyuan; Zhang, Zhixian; Cao, Yue; Ma, Yudan; Guo, Yi; Xu, Li

    2016-01-01

    Recent studies have indicated that protein hydrolysates have broad biological effects. In the current study we describe a novel antioxidative peptide, FDPAL, from soybean protein isolate (SPI). The aim of this study was to purify and characterize an antioxidative peptide from SPI and determine its antioxidative mechanism. LC–MS/MS was used to isolate and identify the peptide from SPI. The sequence of the peptide was determined to be Phe-Asp-Pro-Ala-Leu (FDPAL, 561 Da). FDPAL can cause significant enhancement of resistance to oxidative stress both in cells as well as simple organisms. In Caenorhabditis elegans (C. elegans), FDPAL can up-regulate the expression of certain genes associated with resistance. The antioxidant activity of this peptide can be attributed to the presence of a specific amino acid sequence. Results from our work suggest that FDPAL can facilitate potential applications of proteins carrying this sequence in the nutraceutical, bioactive material and clinical medicine areas, as well as in cosmetics and health care products. PMID:27455060

  3. Type 2C Protein Phosphatase Is a Key Regulator of Antiviral Extreme Resistance Limiting Virus Spread

    PubMed Central

    Seo, Jang-Kyun; Kwon, Sun-Jung; Cho, Won Kyong; Choi, Hong-Soo; Kim, Kook-Hyung

    2014-01-01

    Effector-triggered immunity (ETI) is an active immune response triggered by interactions between host resistance proteins and their cognate effectors. Although ETI is often associated with the hypersensitive response (HR), various R genes mediate an HR-independent process known as extreme resistance (ER). In the soybean-Soybean mosaic virus (SMV) pathosystem, the strain-specific CI protein of SMV functions as an effector of Rsv3-mediated ER. In this study, we used the soybean (Rsv3)-SMV (CI) pathosystem to gain insight into the molecular signaling pathway involved in ER. We used genome-wide transcriptome analysis to identify a subset of the type 2C protein phophatase (PP2C) genes that are specifically up-regulated in Rsv3-mediated ER. Gain-of-function analysis of the most significantly expressed soybean PP2C gene, GmPP2C3a, showed that ABA-induced GmPP2C3a functions as a key regulator of Rsv3-mediated ER. Our results further suggest that the primary mechanism of ER against viruses is the inhibition of viral cell-to-cell movement by callose deposition in an ABA signaling-dependent manner. PMID:25082428

  4. The giant protein Ebh is a determinant of Staphylococcus aureus cell size and complement resistance.

    PubMed

    Cheng, Alice G; Missiakas, Dominique; Schneewind, Olaf

    2014-03-01

    Staphylococcus aureus USA300, the clonal type associated with epidemic community-acquired methicillin-resistant S. aureus (MRSA) infections, displays the giant protein Ebh on its surface. Mutations that disrupt the ebh reading frame increase the volume of staphylococcal cells and alter the cross wall, a membrane-enclosed peptidoglycan synthesis and assembly compartment. S. aureus ebh variants display increased sensitivity to oxacillin (methicillin) as well as susceptibility to complement-mediated killing. Mutations in ebh are associated with reduced survival of mutant staphylococci in blood and diminished virulence in mice. We propose that Ebh, following its secretion into the cross wall, contributes to the characteristic cell growth and envelope assembly pathways of S. aureus, thereby enabling complement resistance and the pathogenesis of staphylococcal infections.

  5. Bcl-2 family proteins in breast development and cancer: could Mcl-1 targeting overcome therapeutic resistance?

    PubMed Central

    Williams, Michelle M.; Cook, Rebecca S.

    2015-01-01

    Apoptosis, cell death executed by caspases, is essential to normal breast development and homeostasis. Pro-apoptotic and anti-apoptotic signals are tightly regulated in normal breast epithelial cells. Dysregulation of this balance is required for breast tumorigenesis and increases acquired resistance to treatments, including molecularly targeted therapies, radiation and chemotherapies. The pro-apoptotic or anti-apoptotic Bcl-2 family members interact with each other to maintain mitochondrial integrity and regulate cellular commitment to apoptosis. Among the anti-apoptotic Bcl-2 family members, Mcl-1 is uniquely regulated by numerous oncogenic signaling pathways. This review will focus on the role of Bcl-2 family proteins in normal breast development, breast tumorigenesis and acquired resistance to breast cancer treatment strategies, while highlighting Mcl-1 as a promising target to improve breast cancer tumor cell killing. PMID:25784482

  6. Molecular modeling of the human multidrug resistance protein 1 (MRP1/ABCC1)

    SciTech Connect

    DeGorter, Marianne K.; Conseil, Gwenaelle; Deeley, Roger G.; Campbell, Robert L.; Cole, Susan P.C.

    2008-01-04

    Multidrug resistance protein 1 (MRP1/ABCC1) is a 190 kDa member of the ATP-binding cassette (ABC) superfamily of transmembrane transporters that is clinically relevant for its ability to confer multidrug resistance by actively effluxing anticancer drugs. Knowledge of the atomic structure of MRP1 is needed to elucidate its transport mechanism, but only low resolution structural data are currently available. Consequently, comparative modeling has been used to generate models of human MRP1 based on the crystal structure of the ABC transporter Sav1866 from Staphylococcus aureus. In these Sav1866-based models, the arrangement of transmembrane helices differs strikingly from earlier models of MRP1 based on the structure of the bacterial lipid transporter MsbA, both with respect to packing of the twelve helices and their interactions with the nucleotide binding domains. The functional importance of Tyr{sup 324} in transmembrane helix 6 predicted to project into the substrate translocation pathway was investigated.

  7. Quantitative Immunofluorescent Blotting of the Multidrug Resistance-associated Protein 2 (MRP2)

    PubMed Central

    Gerk, Phillip M.

    2011-01-01

    Introduction Quantitation of the expression levels of proteins involved in drug transport and disposition is needed to overcome limitations of film-based detection of chemiluminescent immunoblots. Purpose The purpose was to describe and validate a quantitative immunofluorescent blotting method for detection of ATP-Binding Cassette Transporter Isoform C2/Multidrug Resistance-associated Protein 2 (ABCC2/MRP2). Methods Western blotting was performed by electrophoresis of membrane vesicle protein isolated from Sf9 cells overexpressing MRP2 subsequently blotting with infrared labeled secondary antibody. The bound complex was detected using the Odyssey Infrared Imaging System (Li-Cor; Lincoln, NE). The images were analyzed using the Odyssey Application Software to obtain the integrated intensities, followed by linear regression of the intensity data. Results The limits of quantitation for the time-insensitive technique described here were from 0.001μg to 0.5μg of total membrane protein, the coefficient of variation of the slope was 8.9%; r2 values were 0.986 ± 0.012. The utility and sensitivity of this technique was demonstrated in quantitating expression of MRP2 in human placental tissue samples, in which MRP2 was present in low abundance. Discussion The immunofluorescent blotting technique described provides sensitive, reproducible, and quantitative determinations of large, integral membrane proteins such as MRP2, all with potential long-term cost savings. PMID:21277982

  8. Amino acids in Tobamovirus coat protein controlling pepper L(1a) gene-mediated resistance.

    PubMed

    Mizumoto, Hiroyuki; Nakamura, Ikumi; Shimomoto, Yoshifumi; Sawada, Hiromasa; Tomita, Reiko; Sekine, Ken-Taro; Kiba, Akinori; Nishiguchi, Masamichi; Kobayashi, Kappei; Hikichi, Yasufumi

    2012-10-01

    In pepper plants (genus Capsicum), the resistance against Tobamovirus spp. is conferred by L gene alleles. The recently identified L variant L(1a) can recognize coat proteins (CPs) of Tobacco mild green mosaic virus Japanese strain (TMGMV-J) and Paprika mild mottle virus Japanese strain (PaMMV-J), but not of Pepper mild mottle virus (PMMoV), as the elicitor to induce resistance at 24 °C. Interestingly, L(1a) gene-mediated resistance against TMGMV-J, but not PaMMV-J, is retained at 30 °C. This observation led us to speculate that L(1a) can discriminate between CPs of TMGMV-J and PaMMV-J. In this study, we aimed to determine the region(s) in CP by which L(1a) distinguishes TMGMV-J from PaMMV-J. By using chimeric CPs consisting of TMGMV-J and PaMMV-J, we found that the chimeric TMGMV-J CP, whose residues in the β-sheet domain were replaced by those of PaMMV-J, lost its ability to induce L(1a) gene-mediated resistance at 30 °C. In contrast, the chimeric PaMMV-J CP with the β-sheet domain replaced by TMGMV-J CP was able to induce L(1a) gene-mediated resistance at 30 °C. Furthermore, viral particles were not detected in the leaves inoculated with either chimeric virus. These observations indicated that the amino acids within the β-sheet domain were involved in both the induction of L(1a) gene-mediated resistance and virion formation. Further analyses using chimeric CPs of TMGMV-J and PMMoV indicated that amino acids within the β-sheet domain alone were not sufficient for the induction of L(1a) gene-mediated resistance by TMGMV-J CP. These results suggest that multiple regions in Tobamovirus CP are implicated in the induction of L(1a) gene-mediated resistance.

  9. Stimulation of myofibrillar protein synthesis in hindlimb suspended rats by resistance exercise and growth hormone.

    PubMed

    Linderman, J K; Whittall, J B; Gosselink, K L; Wang, T J; Mukku, V R; Booth, F W; Grindeland, R E

    1995-01-01

    The objective of this study was to determine the ability of a single bout of resistance exercise alone or in combination with recombinant human growth hormone (rhGH) to stimulate myofibrillar protein synthesis (Ks) in hindlimb suspended (HLS) adult female rats. Plantar flexor muscles were stimulated with resistance exercise, consisting of 10 repetitions of ladder climbing on a 1 m grid (85 degrees), carrying an additional 50% of their body weight attached to their tails. Saline or rhGH (1 mg/kg) was administered 30' prior to exercise, and Ks was determined with a constant infusion of 3H-Leucine at 15', 60', 180', and 360' following exercise. Three days of HLS depressed Ks approximately 65% and 30-40% in the soleus and gastrocnemius muscles, respectively (p < or = 0.05). Exercise increased soleus Ks in saline-treated rats 149% 60' following exercise (p < or = 0.05), decaying to that of non-exercised animals during the next 5 hours. Relative to suspended, non-exercised rats rhGH+exercise increased soleus Ks 84%, 108%, and 72% at 15', 60' and 360' following exercise (p < or = 0.05). Gastrocnemius Ks was not significantly increased by exercise or the combination of rhGH and exercise up to 360' post-exercise. Results from this study indicate that resistance exercise stimulated Ks 60' post-exercise in the soleus of HLS rats, with no apparent effect of rhGH to enhance or prolong exercise-induced stimulation. Results suggests that exercise frequency may be important to maintenance of the slow-twitch soleus during non-weightbearing, but that the ability of resistance exercise to maintain myofibrillar protein content in the gastrocnemius of hindlimb suspended rats cannot be explained by acute stimulation of synthesis.

  10. Stimulation of Myofibrillar Protein Synthesis in Hindlimb Suspended Rats by Resistance Exercise and Growth Hormone

    NASA Technical Reports Server (NTRS)

    Linderman, Jon K.; Whittall, Justen B.; Gosselink, Kristin L.; Wang, Tommy J.; Mukku, Venkat R.; Booth, Frank W.; Grindeland, Richard E.

    1995-01-01

    The objective of this study was to determine the ability of a single bout of resistance exercise alone or in combination with recombinant human growth hormone (rhGH) to stimulate myofibrillar protein synthesis (Ks) in hindlimb suspended (HLS) adult female rats. Plantar flexor muscles were stimulated with resistance exercise, consisting of 10 repetitions of ladder climbing on a 1 m grid (85 deg.), carrying an additional 50% of their body weight attached to their tails. Saline or rhGH (1 mg/kg) was administered 30' prior to exercise, and Ks was determined with a constant infusion of H-3-Leucine at 15', 60', 180', and 360' following exercise. Three days of HLS depressed Ks is approx. equal to 65% and 30-40% in the soleus and gastrocnemius muscles, respectively (p is less than or equal to 0.05). Exercise increased soleus Ks in saline-treated rats 149% 60' following exercise (p less than or equal to 0.05), decaying to that of non-exercised animals during the next 5 hours. Relative to suspended, non-exercised rats rhGH + exercise increased soleus Ks 84%, 108%, and 72% at 15', 60' and 360' following exercise (p is less than or equal to 0.05). Gastrocnemius Ks was not significantly increased by exercise or the combination of rhGH and exercise up to 360' post-exercise. Results from this study indicate that resistance exercise stimulated Ks 60' post-exercise in the soleus of HLS rats, with no apparent effect of rhGH to enhance or prolong exercise-induced stimulation. Results suggests that exercise frequency may be important to maintenance of the slow-twitch soleus during non-weightbearing, but that the ability of resistance exercise to maintain myofibrillar protein content in the gastrocnemius of hindlimb suspended rats cannot be explained by acute stimulation of synthesis.

  11. Tracking structural features leading to resistance of activated protein C to alpha 1-antitrypsin.

    PubMed

    Shen, L; Dahlbäck, B; Villoutreix, B O

    2000-03-21

    Activated protein C (APC) is a multi-modular anticoagulant serine protease, which degrades factor V/Va and factor VIIIa. Human APC (hAPC) is inhibited by human alpha 1-antitrypsin (AAT), while the bovine enzyme (bAPC) is fully resistant to this serpin. Structural features in the catalytic domains between the two species cause this difference, but detailed knowledge about the causal molecular difference is missing. To gain insight into the APC-AAT interaction and to create a human protein C resistant to AAT inhibition, we have used molecular modeling and site-directed mutagenesis. First, a structural model for bAPC based on the Gla-domainless X-ray structure of hAPC was built. Screening the molecular surface of the human and bovine APC enzymes suggested that a hAPC molecule resistant to AAT inhibition could be constructed by substituting only a few amino acids. We thus produced recombinant hAPC molecules with a single mutation (S173E, the numbering follows the chymotrypsinogen nomenclature), two mutations (E60aS/S61R) or a combination of all these substitutions (E60aS/S61R/S173E). Amidolytic and anticoagulant activities of the three mutant APC molecules were similar to those of wild-type hAPC. Inhibition of wild-type hAPC by AAT was characterized by a second-order rate constant (k2) of 2.71 M-1 s-1. The amino acid substitution at position 173 (S173E mutant) led to partial resistance to AAT (k2 = 0.84 M-1 s-1). The E60aS/S61R mutant displayed mild resistance to AAT inhibition (k2 = 1.70 M-1 s-1), whereas the E60aS/S61R/S173E mutant was inefficiently inactivated by AAT (k2 = 0.40 M-1 s-1). Inhibition of recombinant APC molecules by the serpin protein C inhibitor (PCI) in the presence and absence of heparin was also investigated.

  12. Resistance to β-Lactams in Neisseria ssp Due to Chromosomally Encoded Penicillin-Binding Proteins

    PubMed Central

    Zapun, André; Morlot, Cécile; Taha, Muhamed-Kheir

    2016-01-01

    Neisseria meningitidis and Neisseria gonorrhoeae are human pathogens that cause a variety of life-threatening systemic and local infections, such as meningitis or gonorrhoea. The treatment of such infection is becoming more difficult due to antibiotic resistance. The focus of this review is on the mechanism of reduced susceptibility to penicillin and other β-lactams due to the modification of chromosomally encoded penicillin-binding proteins (PBP), in particular PBP2 encoded by the penA gene. The variety of penA alleles and resulting variant PBP2 enzymes is described and the important amino acid substitutions are presented and discussed in a structural context. PMID:27690121

  13. [Bt transgenic crops for insect-resistance and modification of Bt protein and utilization of stacking strategy].

    PubMed

    Li, Chen; Liu, Bolin

    2015-01-01

    Insecticidal protein genes from Bacillus thuringiensis are currently the most widely used insect-resistant genes. They have been transferred to many crops for breeding and production. Among them, cotton, maize, potato and other insect-resistant crops are commercialized, creating considerable economic benefit. In this review, we summarized advances in identifying functional genes and transgenic crops for insect resistance, compared different strategies for enhancing vigor of insecticidal protein and utilizing gene stacking as well as listing valuable groups of stacked genes. In addition, the methods for multiple gene transformation was discussed.

  14. Cytoplasmic CopZ-Like Protein and Periplasmic Rusticyanin and AcoP Proteins as Possible Copper Resistance Determinants in Acidithiobacillus ferrooxidans ATCC 23270.

    PubMed

    Navarro, Claudio A; von Bernath, Diego; Martínez-Bussenius, Cristóbal; Castillo, Rodrigo A; Jerez, Carlos A

    2015-12-04

    Acidophilic organisms, such as Acidithiobacillus ferrooxidans, possess high-level resistance to copper and other metals. A. ferrooxidans contains canonical copper resistance determinants present in other bacteria, such as CopA ATPases and RND efflux pumps, but these components do not entirely explain its high metal tolerance. The aim of this study was to find other possible copper resistance determinants in this bacterium. Transcriptional expression of A. ferrooxidans genes coding for a cytoplasmic CopZ-like copper-binding chaperone and the periplasmic copper-binding proteins rusticyanin and AcoP, which form part of an iron-oxidizing supercomplex, was found to increase when the microorganism was grown in the presence of copper. All of these proteins conferred more resistance to copper when expressed heterologously in a copper-sensitive Escherichia coli strain. This effect was absent when site-directed-mutation mutants of these proteins with altered copper-binding sites were used in this metal sensitivity assay. These results strongly suggest that the three copper-binding proteins analyzed here are copper resistance determinants in this extremophile and contribute to the high-level metal resistance of this industrially important biomining bacterium.

  15. Interaction of Herbicides and Quinone with the QB-Protein of the Diuron-Resistant Chlamydomonas reinhardtii Mutant Dr2

    PubMed Central

    Haworth, Philip; Steinback, Katherine E.

    1987-01-01

    We have used the diuron-resistant Dr2 mutant of Chlamydomonas reinhardtii which is altered in the 32 kilodalton QB-protein at amino acid 219 (valine to isoleucine), to investigate the interactions of herbicides and plastoquinone with the 32 kilodalton QB-protein. The data contained in this report demonstrate that the effects of this mutation are different from those of the more completely characterized mutant which confers extreme resistance to triazines in higher plants. The mutation in C. reinhardtii Dr2 confers only slight resistance to a number of inhibitors of photosynthetic electron transport. Extreme triazine resistance results from an increase in the binding constant of the herbicide with the 32 kilodalton QB-protein, in contrast the diuron binding constant for chloroplasts isolated from wild-type (sensitive) Chlamydomonas and the resistant Dr2 are indistinguishable. We conclude that the altered structure in the 32 kilodalton QB-protein of Dr2 does not directly affect the diuron binding site. This mutation appears to alter the steric properties of the binding protein in such a way that diuron and plastoquinone do not directly compete for binding. This steric perturbation confers mild resistance to other herbicidal inhibitors of photosynthesis and alters the kinetics of QA to QB electron transfer. PMID:16665318

  16. CRYPTOCHROME 2 and PHOTOTROPIN 2 regulate resistance protein mediated viral 2 defense by negatively regulating a E3 ubiquitin ligase

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Light harvested by plants is essential for the survival of most life forms. This light perception ability requires the activities of proteins termed photoreceptors. We report a function for photoreceptors in mediating resistance (R) protein-derived plant defense. The blue-light photoreceptors, crypt...

  17. Detergent-resistant membrane subfractions containing proteins of plasma membrane, mitochondrial, and internal membrane origins.

    PubMed

    Mellgren, Ronald L

    2008-04-24

    HEK293 cell detergent-resistant membranes (DRMs) isolated by the standard homogenization protocol employing a Teflon pestle homogenizer yielded a prominent opaque band at approximately 16% sucrose upon density gradient ultracentrifugation. In contrast, cell disruption using a ground glass tissue homogenizer generated three distinct DRM populations migrating at approximately 10%, 14%, and 20% sucrose, named DRM subfractions A, B, and C, respectively. Separation of the DRM subfractions by mechanical disruption suggested that they are physically associated within the cellular environment, but can be dissociated by shear forces generated during vigorous homogenization. All three DRM subfractions possessed cholesterol and ganglioside GM1, but differed in protein composition. Subfraction A was enriched in flotillin-1 and contained little caveolin-1. In contrast, subfractions B and C were enriched in caveolin-1. Subfraction C contained several mitochondrial membrane proteins, including mitofilin and porins. Only subfraction B appeared to contain significant amounts of plasma membrane-associated proteins, as revealed by cell surface labeling studies. A similar distribution of DRM subfractions, as assessed by separation of flotillin-1 and caveolin-1 immunoreactivities, was observed in CHO cells, in 3T3-L1 adipocytes, and in HEK293 cells lysed in detergent-free carbonate. Teflon pestle homogenization of HEK293 cells in the presence of the actin-disrupting agent latrunculin B generated DRM subfractions A-C. The microtubule-disrupting agent vinblastine did not facilitate DRM subfraction separation, and DRMs prepared from fibroblasts of vimentin-null mice were present as a single major band on sucrose gradients, unless pre-treated with latrunculin B. These results suggest that the DRM subfractions are interconnected by the actin cytoskeleton, and not by microtubes or vimentin intermediate filaments. The subfractions described may prove useful in studying discrete protein

  18. Vanadate-resistant mutants of Saccharomyces cerevisiae show alterations in protein phosphorylation and growth control.

    PubMed Central

    Kanik-Ennulat, C; Neff, N

    1990-01-01

    This work describes two spontaneous vanadate-resistant mutants of Saccharomyces cerevisiae with constitutive alterations in protein phosphorylation, growth control, and sporulation. Vanadate has been shown by a number of studies to be an efficient competitor of phosphate in biochemical reactions, especially those that involve phosphoproteins as intermediates or substrates. Resistance to toxic concentrations of vanadate can arise in S. cerevisiae by both recessive and dominant spontaneous mutations in a large number of loci. Mutations in two of the recessive loci, van1-18 and van2-93, resulted in alterations in the phosphorylation of a number of proteins. The mutant van1-18 gene also showed an increase in plasma membrane ATPase activity in vitro and a lowered basal phosphatase activity under alkaline conditions. Cells containing the van2-93 mutant allele had normal levels of plasma membrane ATPase activity, but this activity was not inhibited by vanadate. Both of these mutants failed to enter stationary phase, were heat shock sensitive, showed lowered long-term viability, and sporulated on rich medium in the presence of 2% glucose. The wild-type VAN1 gene was isolated and sequenced. The open reading frame predicts a protein of 522 amino acids, with no significant homology to any genes that have been identified. Diploid cells that contained two mutant alleles of this gene demonstrated defects in spore viability. These data suggest that the VAN1 gene product is involved in regulation of the phosphorylation of a number of proteins, some of which appear to be important in cell growth control. Images PMID:2137555

  19. Light-load resistance exercise increases muscle protein synthesis and hypertrophy signaling in elderly men.

    PubMed

    Agergaard, Jakob; Bülow, Jacob; Jensen, Jacob K; Reitelseder, Søren; Drummond, Micah J; Schjerling, Peter; Scheike, Thomas; Serena, Anja; Holm, Lars

    2017-04-01

    The present study investigated whether well-tolerated light-load resistance exercise (LL-RE) affects skeletal muscle fractional synthetic rate (FSR) and anabolic intracellular signaling as a way to counteract age-related loss of muscle mass. Untrained healthy elderly (>65-yr-old) men were subjected to 13 h of supine rest. After 2.5 h of rest, unilateral LL-RE, consisting of leg extensions (10 sets, 36 repetitions) at 16% of 1 repetition maximum (RM), was conducted. Subsequently, the subjects were randomized to oral intake of 4 g of whey protein per hour (PULSE, n = 10), 28 g of whey protein at 0 h and 12 g of whey protein at 7 h postexercise (BOLUS, n = 10), or 4 g of maltodextrin per hour (placebo, n = 10). Quadriceps muscle biopsies were taken at 0, 3, 7, and 10 h postexercise from the resting and the exercised leg of each subject. Myofibrillar FSR and activity of select targets from the mechanistic target of rapamycin complex 1-signaling cascade were analyzed from the biopsies. LL-RE increased myofibrillar FSR compared with the resting leg throughout the 10-h postexercise period. Phosphorylated (T308) AKT expression increased in the exercised leg immediately after exercise. This increase persisted in the placebo group only. Levels of phosphorylated (T37/46) eukaryotic translation initiation factor 4E-binding protein 1 increased throughout the postexercise period in the exercised leg in the placebo and BOLUS groups and peaked at 7 h. In all three groups, phosphorylated (T56) eukaryotic elongation factor 2 decreased in response to LL-RE. We conclude that resistance exercise at only 16% of 1 RM increased myofibrillar FSR, irrespective of nutrient type and feeding pattern, which indicates an anabolic effect of LL-RE in elderly individuals. This finding was supported by increased signaling for translation initiation and translation elongation in response to LL-RE.

  20. The Aspergillus fumigatus Damage Resistance Protein Family Coordinately Regulates Ergosterol Biosynthesis and Azole Susceptibility

    PubMed Central

    Song, Jinxing; Zhai, Pengfei; Zhang, Yuanwei; Zhang, Caiyun; Sang, Hong; Han, Guanzhu; Keller, Nancy P.

    2016-01-01

    ABSTRACT Ergosterol is a major and specific component of the fungal plasma membrane, and thus, the cytochrome P450 enzymes (Erg proteins) that catalyze ergosterol synthesis have been selected as valuable targets of azole antifungals. However, the opportunistic pathogen Aspergillus fumigatus has developed worldwide resistance to azoles largely through mutations in the cytochrome P450 enzyme Cyp51 (Erg11). In this study, we demonstrate that a cytochrome b5-like heme-binding damage resistance protein (Dap) family, comprised of DapA, DapB, and DapC, coordinately regulates the functionality of cytochrome P450 enzymes Erg5 and Erg11 and oppositely affects susceptibility to azoles. The expression of all three genes is induced in an azole concentration-dependent way, and the decreased susceptibility to azoles requires DapA stabilization of cytochrome P450 protein activity. In contrast, overexpression of DapB and DapC causes dysfunction of Erg5 and Erg11, resulting in abnormal accumulation of sterol intermediates and further accentuating the sensitivity of ΔdapA strains to azoles. The results of exogenous-hemin rescue and heme-binding-site mutagenesis experiments demonstrate that the heme binding of DapA contributes the decreased azole susceptibility, while DapB and -C are capable of reducing the activities of Erg5 and Erg11 through depletion of heme. In vivo data demonstrate that inactivated DapA combined with activated DapB yields an A. fumigatus mutant that is easily treatable with azoles in an immunocompromised mouse model of invasive pulmonary aspergillosis. Compared to the single Dap proteins found in Saccharomyces cerevisiae and Schizosaccharomyces pombe, we suggest that this complex Dap family regulatory system emerged during the evolution of fungi as an adaptive means to regulate ergosterol synthesis in response to environmental stimuli. PMID:26908577

  1. Molecular basis for the role of Staphylococcus aureus penicillin binding protein 4 in antimicrobial resistance.

    PubMed

    Navratna, Vikas; Nadig, Savitha; Sood, Varun; Prasad, K; Arakere, Gayathri; Gopal, B

    2010-01-01

    Penicillin binding proteins (PBPs) are membrane-associated proteins that catalyze the final step of murein biosynthesis. These proteins function as either transpeptidases or carboxypeptidases and in a few cases demonstrate transglycosylase activity. Both transpeptidase and carboxypeptidase activities of PBPs occur at the D-Ala-D-Ala terminus of a murein precursor containing a disaccharide pentapeptide comprising N-acetylglucosamine and N-acetyl-muramic acid-L-Ala-D-Glu-L-Lys-D-Ala-D-Ala. Beta-lactam antibiotics inhibit these enzymes by competing with the pentapeptide precursor for binding to the active site of the enzyme. Here we describe the crystal structure, biochemical characteristics, and expression profile of PBP4, a low-molecular-mass PBP from Staphylococcus aureus strain COL. The crystal structures of PBP4-antibiotic complexes reported here were determined by molecular replacement, using the atomic coordinates deposited by the New York Structural Genomics Consortium. While the pbp4 gene is not essential for the viability of S. aureus, the knockout phenotype of this gene is characterized by a marked reduction in cross-linked muropeptide and increased vancomycin resistance. Unlike other PBPs, we note that expression of PBP4 was not substantially altered under different experimental conditions, nor did it change across representative hospital- or community-associated strains of S. aureus that were examined. In vitro data on purified recombinant S. aureus PBP4 suggest that it is a beta-lactamase and is not trapped as an acyl intermediate with beta-lactam antibiotics. Put together, the expression analysis and biochemical features of PBP4 provide a framework for understanding the function of this protein in S. aureus and its role in antimicrobial resistance.

  2. Endogenous salicylic acid levels correlate with accumulation of pathogenesis-related proteins and virus resistance in tobacco

    SciTech Connect

    Yalpani, N.; Shulaev, V.; Raskin, I. )

    1993-07-01

    Salicylic acid (SA) is hypothesized to be an endogenous regulator of local and systemic disease resistance and an inducer of pathogenesis-related (PR) proteins among plants. High levels of PR proteins have been observed in an uninoculated amphidiploid hybrid of Nicotiana glutinosa [times] N. debneyi, which is highly resistant to tobacco mosaic virus (TMV). Fluoresence, UV, and mass spectral analysis established that the levels of SA in healthy N. glutinosa [times] N. debneyi leaves were 30 times greater than in N. tabacum [open quotes]Xanthi-nc[close quotes] tobacco, which does not constitutively express PR proteins and is less resistant to TMV. Upon TMV-inoculation SA levels increased at least 70-fold leaves of Xanthi-nc but role only slightly in the hybrid. Phloem exudates of N. glutinosa [times] N. debneyi contained at least 500 times more SA than those of Xanthi-nc. SA treatment caused the appearance of PR-1 protein in Xanthi-nc but did not affect constitutively high levels of PR-1 protein in N. glutinosa [times] N. debneyi. In contrast to Xanthi-nc tobacco, TMV-inoculated N. glutinosa [times] N. debneyi kept at 32 C accumulated more than 0.5 [mu]g SA/g fresh weight, maintained high levels of PR proteins, and developed a hypersensitive response to TMV. PR proteins have previously been shown to accumulate in the lower leaves of healthy, flowering Xanthi-nc tobacco, which exhibited increased resistance to TMV. These developmentally induced increases in resistance and PR-1 proteins positively correlated with tissue levels of SA. These results affirm the regulatory role of SA in disease resistance and PR protein production. 31 refs., 9 figs., 1 tab.

  3. [Evaluation of penicillin-binding protein genotypes in penicillin susceptible and resistant Streptococcus pneumoniae isolates].

    PubMed

    Aslan, Gönül; Tezcan, Seda; Delialioğlu, Nuran; Aydın, Fatma Esin; Kuyucu, Necdet; Emekdaş, Gürol

    2012-04-01

    Penicillin-binding proteins (PBPs) are the natural targets of beta-lactam antibiotics and mutations in pbp1a, pbp2b, and pbp2x genes, which encode PBPs, are responsible for resistance to beta-lactams in Streptococcus pneumoniae. In the present study, we intended to determine how often the common mutation patterns occurred within the pbp1a, pbp2b, and pbp2x PBP gene regions and evaluate the PBP genotype mutations which were associated with penicillin resistance in several penicillin-susceptible and - resistant S.pneumoniae isolates in Mersin, Turkey. A total of 62 S.pneumoniae strains isolated from different clinical specimens (32 nasopharyngeal swab, 16 sputum, 3 blood, 3 wound, 2 cerebrospinal fluids and one of each urine, abscess, bronchoalveolar lavage, conjunctival swab, tracheal aspirate, middle ear effusion) were included in the study. Penicillin susceptibilities of the isolates were searched by disc diffusion and E-test methods, and 23 of them were identified as susceptible, 31 were intermediate susceptible, and eight were resistant to penicillin. A rapid DNA extraction procedure was performed for the isolation of nucleic acids from the strains. Distribution of PBP gene mutations in pbp1a, pbp2b, and pbp2x gene regions related to penicillin resistance was determined by using a wild-type specific polymerase chain reaction (PCR) based technique. PBP gene alterations of those isolates were also evaluated in relation to penicillin susceptibility and resistance patterns. Twenty two (95.7%) of 23 penicillin-susceptible S.pneumoniae isolates exhibited no mutation in the three PBP genes (pbp1a, pbp2x, and pbp 2b), while 1 (4.3%) of these harbored mutations in all of the three PBP genes. The penicillin-intermediate susceptible S.pneumoniae isolates exhibited various combinations of mutations. One (3.2%) of 31 penicillin-intermediate susceptible isolates exhibited no mutation in the three PBP genes, while 22 (71%) of them yielded mutations in all of the three PBP

  4. Outer membrane proteomics of kanamycin-resistant Escherichia coli identified MipA as a novel antibiotic resistance-related protein.

    PubMed

    Li, Hui; Zhang, Dan-feng; Lin, Xiang-min; Peng, Xuan-xian

    2015-06-01

    Antibiotic-resistant bacteria are a great threat to human health and food safety and there is an urgent need to understand the mechanisms of resistance for combating these bacteria. In the current study, comparative proteomic methodologies were applied to identify Escherichia coli K-12 outer membrane (OM) proteins related to kanamycin resistance. Mass spectrometry and western blotting results revealed that OM proteins TolC, Tsx and OstA were up-regulated, whereas MipA, OmpA, FadL and OmpW were down-regulated in kanamycin-resistant E. coli K-12 strain. Genetic deletion of tolC (ΔtolC-Km) led to a 2-fold decrease in the minimum inhibitory concentration (MIC) of kanamycin and deletion of mipA (ΔmipA-Km) resulted in a 4-fold increase in the MIC of kanamycin. Changes in the MICs for genetically modified strains could be completely recovered by gene complementation. Compared with the wild-type strain, the survival capability of ΔompA-Km was significantly increased and that of Δtsx-Km was significantly decreased. We further evaluated the role and expression of MipA in response to four other antibiotics including nalidixic acid, streptomycin, chloramphenicol and aureomycin, which suggested that MipA was a novel OM protein related to antibiotic resistance.

  5. Increased chitin biosynthesis contributes to the resistance of Penicillium polonicum against the antifungal protein PgAFP.

    PubMed

    Delgado, Josué; Owens, Rebecca A; Doyle, Sean; Asensio, Miguel A; Núñez, Félix

    2016-01-01

    Antifungal proteins from molds have been proposed as a valuable tool against unwanted molds, but the resistance of some fungi limits their use. Resistance to antimicrobial peptides has been suggested to be due to lack of interaction with the mold or to a successful response. The antifungal protein PgAFP produced by Penicillium chrysogenum inhibits the growth of various ascomycetes, but not Penicillium polonicum. To study the basis for resistance to this antifungal protein, localization of PgAFP and metabolic, structural, and morphological changes were investigated in P. polonicum. PgAFP bound the outer layer of P. polonicum but not regenerated chitin, suggesting an interaction with specific molecules. Comparative two-dimensional gel electrophoresis (2D-PAGE) and comparative quantitative proteomics revealed changes in the relative abundance of several proteins from ribosome, spliceosome, metabolic, and biosynthesis of secondary metabolite pathways. The proteome changes and an altered permeability reveal an active reaction of P. polonicum to PgAFP. The successful response of the resistant mold seems to be based on the higher abundance of protein Rho GTPase Rho1 that would lead to the increased chitin deposition via cell wall integrity (CWI) signaling pathway. Thus, combined treatment with chitinases could provide a complementary means to combat resistance to antifungal proteins.

  6. Expression and purification of an active cecropin-like recombinant protein against multidrug resistance Escherichia coli.

    PubMed

    Téllez, Germán Alberto; Castaño-Osorio, Jhon Carlos

    2014-08-01

    Lucilin is a 36 residue cecropin antimicrobial peptide identified as a partial genetic sequence in Lucilia sericata maggots. The antimicrobial spectrum and toxicity profile of Lucilin is unknown. We first report the expression of Lucilin as an active recombinant fusion protein with a cysteine protease domain (CPD) tag. The fusion protein, GWLK-Lucilin-CPD-His8, showed maximum overexpression in Escherichia coli BL21 cells after 12h induction with 0.5mM IPTG (isopropyl beta-d-thiogalactoside) and growth conditions were 37 °C and 150 rpm shaking. The fusion protein was expressed as a soluble form and was purified by Ni-IMAC. The purified protein was active against E. coli ATCC 35218 with a MIC of 0.68 μM, and a clinical isolate of E. coli with extended spectrum beta-lactamase (ESBL) with a MIC of 0.8 μM. The recombinant GWLK-Lucilin-CPD-His8 was not toxic against human erythrocytes or Vero cells with a therapeutic index >63. The results suggest that GWLK-Lucilin-CPD-His8 represents a potential candidate for therapy against multidrug resistant Gram-negative bacteria.

  7. Water-soluble chlorophyll protein is involved in herbivore resistance activation during greening of Arabidopsis thaliana

    PubMed Central

    Boex-Fontvieille, Edouard; Rustgi, Sachin; von Wettstein, Diter; Reinbothe, Steffen; Reinbothe, Christiane

    2015-01-01

    Water-soluble chlorophyll proteins (WSCPs) constitute a small family of unusual chlorophyll (Chl)-binding proteins that possess a Kunitz-type protease inhibitor domain. In Arabidopsis thaliana, a WSCP has been identified, named AtWSCP, that forms complexes with Chl and the Chl precursor chlorophyllide (Chlide) in vitro. AtWSCP exhibits a quite unexpected expression pattern for a Chl binding protein and accumulated to high levels in the apical hook of etiolated plants. AtWSCP expression was negatively light-regulated. Transgenic expression of AtWSCP fused to green fluorescent protein (GFP) revealed that AtWSCP is localized to cell walls/apoplastic spaces. Biochemical assays identified AtWSCP as interacting with RD21 (RESPONSIVE TO DESICCATION 21), a granulin domain-containing cysteine protease implicated in stress responses and defense. Reconstitution experiments showed tight interactions between RD21 and WSCP that were relieved upon Chlide binding. Laboratory feeding experiments with two herbivorous isopod crustaceans, Porcellio scaber (woodlouse) and Armadillidium vulgare (pillbug), identified the apical hook as Achilles’ heel of etiolated plants and that this was protected by RD21 during greening. Because Chlide is formed in the apical hook during seedling emergence from the soil, our data suggest an unprecedented mechanism of herbivore resistance activation that is triggered by light and involves AtWSCP. PMID:26016527

  8. FAM83 proteins: Fostering new interactions to drive oncogenic signaling and therapeutic resistance

    PubMed Central

    Bartel, Courtney A.; Parameswaran, Neetha; Cipriano, Rocky; Jackson, Mark W.

    2016-01-01

    The FAM83 proteins were recently identified as novel transforming oncogenes that function as intermediaries in EGFR/RAS signaling. Using two distinct forward genetics screens, the Bissell and Jackson laboratories uncovered the importance of the FAM83 proteins in promoting resistance to EGFR tyrosine kinase inhibitors and therapies targeting downstream EGFR signaling effectors. The discovery of this novel oncogene family using distinct genetic screens provides compelling evidence that the FAM83 proteins are key oncogenic players in cancer-associated signaling when they are overexpressed or dysregulated. Consistent with a role in oncogenic transformation, the FAM83 genes are frequently overexpressed in diverse human cancer specimens. Importantly, ablation of numerous FAM83 members results in a marked suppression of cancer-associated signaling and loss of tumorigenic potential. Here, we review the current knowledge of the FAM83 proteins’ involvement in cancer signaling and discuss the potential mechanisms by which they contribute to tumorigenesis. Both redundant activities shared by all 8 FAM83 members and non-redundant activities unique to each member are highlighted. We discuss the promise and challenges of the FAM83 proteins as novel points of attack for future cancer therapies. PMID:27221039

  9. Triclosan Resistome from Metagenome Reveals Diverse Enoyl Acyl Carrier Protein Reductases and Selective Enrichment of Triclosan Resistance Genes

    PubMed Central

    Khan, Raees; Kong, Hyun Gi; Jung, Yong-Hoon; Choi, Jinhee; Baek, Kwang-Yeol; Hwang, Eul Chul; Lee, Seon-Woo

    2016-01-01

    Triclosan (TCS) is a widely used antimicrobial agent and TCS resistance is considered to have evolved in diverse organisms with extensive use of TCS, but distribution of TCS resistance has not been well characterized. Functional screening of the soil metagenome in this study has revealed that a variety of target enoyl acyl carrier protein reductases (ENR) homologues are responsible for the majority of TCS resistance. Diverse ENRs similar to 7-α-hydroxysteroid dehydrogenase (7-α-HSDH), FabG, or the unusual YX7K-type ENR conferred extreme tolerance to TCS. The TCS-refractory 7-α HSDH-like ENR and the TCS-resistant YX7K-type ENR seem to be prevalent in human pathogenic bacteria, suggesting that a selective enrichment occurred in pathogenic bacteria in soil. Additionally, resistance to multiple antibiotics was found to be mediated by antibiotic resistance genes that co-localize with TCS resistance determinants. Further comparative analysis of ENRs from 13 different environments has revealed a huge diversity of both prototypic and metagenomic TCS-resistant ENRs, in addition to a selective enrichment of TCS-resistant specific ENRs in presumably TCS-contaminated environments with reduced ENR diversity. Our results suggest that long-term extensive use of TCS can lead to the selective emergence of TCS-resistant bacterial pathogens, possibly with additional resistance to multiple antibiotics, in natural environments. PMID:27577999

  10. A G-protein-coupled receptor regulation pathway in cytochrome P450-mediated permethrin-resistance in mosquitoes, Culex quinquefasciatus

    PubMed Central

    Li, Ting; Cao, Chuanwang; Yang, Ting; Zhang, Lee; He, Lin; Xi, Zhiyong; Bian, Guowu; Liu, Nannan

    2015-01-01

    Rhodopsin-like G protein-coupled receptors (GPCRs) are known to be involved in the GPCR signal transduction system and regulate many essential physiological processes in organisms. This study, for the first time, revealed that knockdown of the rhodopsin-like GPCR gene in resistant mosquitoes resulted in a reduction of mosquitoes’ resistance to permethrin, simultaneously reducing the expression of two cAMP-dependent protein kinase A genes (PKAs) and four resistance related cytochrome P450 genes. The function of rhodopsin-like GPCR was further confirmed using transgenic lines of Drosophila melanogaster, in which the tolerance to permethrin and the expression of Drosophila resistance P450 genes were both increased. The roles of GPCR signaling pathway second messenger cyclic adenosine monophosphate (cAMP) and downstream effectors PKAs in resistance were investigated using cAMP production inhibitor Bupivacaine HCl and the RNAi technique. Inhibition of cAMP production led to significant decreases in both the expression of four resistance P450 genes and two PKA genes and mosquito resistance to permethrin. Knockdown of the PKA genes had shown the similar effects on permethrin resistance and P450 gene expression. Taken together, our studies revealed, for the first time, the role of the GPCR/cAMP/PKA-mediated regulatory pathway governing P450 gene expression and P450-mediated resistance in Culex mosquitoes. PMID:26656663

  11. A G-protein-coupled receptor regulation pathway in cytochrome P450-mediated permethrin-resistance in mosquitoes, Culex quinquefasciatus.

    PubMed

    Li, Ting; Cao, Chuanwang; Yang, Ting; Zhang, Lee; He, Lin; Xi, Zhiyong; Bian, Guowu; Liu, Nannan

    2015-12-10

    Rhodopsin-like G protein-coupled receptors (GPCRs) are known to be involved in the GPCR signal transduction system and regulate many essential physiological processes in organisms. This study, for the first time, revealed that knockdown of the rhodopsin-like GPCR gene in resistant mosquitoes resulted in a reduction of mosquitoes' resistance to permethrin, simultaneously reducing the expression of two cAMP-dependent protein kinase A genes (PKAs) and four resistance related cytochrome P450 genes. The function of rhodopsin-like GPCR was further confirmed using transgenic lines of Drosophila melanogaster, in which the tolerance to permethrin and the expression of Drosophila resistance P450 genes were both increased. The roles of GPCR signaling pathway second messenger cyclic adenosine monophosphate (cAMP) and downstream effectors PKAs in resistance were investigated using cAMP production inhibitor Bupivacaine HCl and the RNAi technique. Inhibition of cAMP production led to significant decreases in both the expression of four resistance P450 genes and two PKA genes and mosquito resistance to permethrin. Knockdown of the PKA genes had shown the similar effects on permethrin resistance and P450 gene expression. Taken together, our studies revealed, for the first time, the role of the GPCR/cAMP/PKA-mediated regulatory pathway governing P450 gene expression and P450-mediated resistance in Culex mosquitoes.

  12. Cytotoxicity of rhein, the active metabolite of sennoside laxatives, is reduced by multidrug resistance-associated protein 1

    PubMed Central

    van Gorkom, B A P; Timmer-Bosscha, H; de Jong, S; van der Kolk, D M; Kleibeuker, J H; de Vries, E G E

    2002-01-01

    Anthranoid laxatives, belonging to the anthraquinones as do anthracyclines, possibly increase colorectal cancer risk. Anthracyclines interfere with topoisomerase II, intercalate DNA and are substrates for P-glycoprotein and multidrug resistance-associated protein 1. P-glycoprotein and multidrug resistance-associated protein 1 protect colonic epithelial cells against xenobiotics. The aim of this study was to analyse the interference of anthranoids with these natural defence mechanisms and the direct cytotoxicity of anthranoids in cancer cell lines expressing these mechanisms in varying combinations. A cytotoxicity profile of rhein, aloe emodin and danthron was established in related cell lines exhibiting different levels of topoisomerases, multidrug resistance-associated protein 1 and P-glycoprotein. Interaction of rhein with multidrug resistance-associated protein 1 was studied by carboxy fluorescein efflux and direct cytotoxicity by apoptosis induction. Rhein was less cytotoxic in the multidrug resistance-associated protein 1 overexpressing GLC4/ADR cell line compared to GLC4. Multidrug resistance-associated protein 1 inhibition with MK571 increased rhein cytotoxicity. Carboxy fluorescein efflux was blocked by rhein. No P-glycoprotein dependent rhein efflux was observed, nor was topoisomerase II responsible for reduced toxicity. Rhein induced apoptosis but did not intercalate DNA. Aloe emodin and danthron were no substrates for MDR mechanisms. Rhein is a substrate for multidrug resistance-associated protein 1 and induces apoptosis. It could therefore render the colonic epithelium sensitive to cytotoxic agents, apart from being toxic in itself. British Journal of Cancer (2002) 86, 1494–1500. DOI: 10.1038/sj/bjc/6600255 www.bjcancer.com © 2002 Cancer Research UK PMID:11986786

  13. Generic amyloidogenicity of mammalian prion proteins from species susceptible and resistant to prions.

    PubMed

    Nyström, Sofie; Hammarström, Per

    2015-05-11

    Prion diseases are lethal, infectious diseases associated with prion protein (PrP) misfolding. A large number of mammals are susceptible to both sporadic and acquired prion diseases. Although PrP is highly conserved and ubiquitously expressed in all mammals, not all species exhibit prion disease. By employing full length recombinant PrP from five known prion susceptible species (human, cattle, cat, mouse and hamster) and two species considered to be prion resistant (pig and dog) the amyloidogenicity of these PrPs has been delineated. All the mammalian PrPs, even from resistant species, were swiftly converted from the native state to amyloid-like structure when subjected to a native condition conversion assay. The PrPs displayed amyloidotypic tinctorial and ultrastructural hallmarks. Self-seeded conversion of the PrPs displayed significantly decreased lag phases demonstrating that nucleation dependent polymerization is a dominating mechanism in the fibrillation process. Fibrils from Aβ1-40, Aβ1-42, Lysozyme, Insulin and Transthyretin did not accelerate conversion of HuPrP whereas fibrils from HuPrP90-231 and HuPrP121-231 as well as full length PrPs of all PrPs efficiently seeded conversion showing specificity of the assay requiring the C-terminal PrP sequence. Our findings have implications for PrP misfolding and could have ramifications in the context of prion resistant species and silent carriers.

  14. Development of Conformation Independent Computational Models for the Early Recognition of Breast Cancer Resistance Protein Substrates

    PubMed Central

    Gantner, Melisa Edith; Di Ianni, Mauricio Emiliano; Ruiz, María Esperanza; Bruno-Blanch, Luis E.

    2013-01-01

    ABC efflux transporters are polyspecific members of the ABC superfamily that, acting as drug and metabolite carriers, provide a biochemical barrier against drug penetration and contribute to detoxification. Their overexpression is linked to multidrug resistance issues in a diversity of diseases. Breast cancer resistance protein (BCRP) is the most expressed ABC efflux transporter throughout the intestine and the blood-brain barrier, limiting oral absorption and brain bioavailability of its substrates. Early recognition of BCRP substrates is thus essential to optimize oral drug absorption, design of novel therapeutics for central nervous system conditions, and overcome BCRP-mediated cross-resistance issues. We present the development of an ensemble of ligand-based machine learning algorithms for the early recognition of BCRP substrates, from a database of 262 substrates and nonsubstrates compiled from the literature. Such dataset was rationally partitioned into training and test sets by application of a 2-step clustering procedure. The models were developed through application of linear discriminant analysis to random subsamples of Dragon molecular descriptors. Simple data fusion and statistical comparison of partial areas under the curve of ROC curves were applied to obtain the best 2-model combination, which presented 82% and 74.5% of overall accuracy in the training and test set, respectively. PMID:23984415

  15. Association of protein kinase A type I with detergent-resistant structures of mammalian sperm cells.

    PubMed

    Moos, J; Pĕknicová, J; Geussova, G; Philimonenko, V; Hozák, P

    1998-05-01

    The finding that flagellar movement in detergent-permeabilized sperm cells is restored when Mg ATP and cAMP are added implicated detergent-resistant protein kinase A (PKA) in the regulation of sperm motility. It is widely believed that only the PKA regulatory subunit RII can associate with the cytoskeleton and/or organelles. In this paper we used monoclonal antibodies against the PKA catalytic subunit and RI subunit and demonstrated that PKA type I is also associated with the sperm cytoskeleton. To our knowledge, this is the first report showing anchored PKA type I. This association was found in sperm of nonrodent mammalian species and, to a lesser extent, also in mouse sperm. The PKA catalytic subunit is bound to the cytoskeleton secondarily via its complex with the regulatory subunit. The detergent-resistant complexes of RI and catalytic subunits localize predominantly to the flagellum. Ultrastructural immunogold labeling revealed the association of detergent-resistant PKA type I with outer dense fibers (ODF) and the fibrous sheath (FS) but not with microtubules. This location is consistent with a proposed role of PKA in regulation of FS sliding on underlying ODF.

  16. Epstein-Barr virus latent membrane protein 2A contributes to anoikis resistance through ERK activation.

    PubMed

    Iwakiri, Dai; Minamitani, Takeharu; Samanta, Mrinal

    2013-07-01

    Epstein-Barr virus (EBV) is associated with various malignancies, including epithelial cancers. In this study, we analyzed the effect of EBV infection on epithelial cells by using EBV-converted epithelial cells. In EBV-positive cells, the extracellular signal-regulated kinase (ERK) pathway is constitutively activated. Inhibition of ERK activity leads to reduced anoikis resistance; therefore, EBV-positive cells are more resistant to anoikis, a type of apoptosis induced by cell detachment, than are EBV-negative cells. Among the viral genes expressed in EBV-positive cells, the latent membrane protein 2A (LMP2A) is responsible for induction of ERK-mediated anoikis resistance, although the expression level of LMP2A is much lower in EBV-positive cells than in EBV-transformed B cells. Further analysis demonstrated that LMP2A downregulation of the proanoikis mediator Bim through proteasomal degradation is dependent on the immunoreceptor tyrosine-based activation motif (ITAM). These findings suggest that LMP2A-mediated ERK activation is involved in the generation of EBV-associated epithelial malignancies.

  17. The Protein Elicitor PevD1 Enhances Resistance to Pathogens and Promotes Growth in Arabidopsis

    PubMed Central

    Liu, Mengjie; Khan, Najeeb Ullah; Wang, Ningbo; Yang, Xiufen; Qiu, Dewen

    2016-01-01

    The protein elicitor PevD1, isolated from Verticillium dahlia, could enhance resistance to TMV in tobacco and Verticillium wilt in cotton. Here, the pevd1 gene was over-expressed in wild type (WT) Arabidopsis, and its biological functions were investigated. Our results showed that the transgenic lines were more resistant to Botrytis cinerea and Pseudomonas syringae pv. tomato DC3000 than the WT line was. In transgenic plants, both the germination time and bolting time required were significantly shorter and fresh weights and plant heights were significantly higher than those in the WT line. A transcriptomics study using digital gene expression profiling (DGE) was performed in transgenic and WT Arabidopsis. One hundred and thirty-six differentially expressed genes were identified. In transgenic Arabidopsis, three critical regulators of JA biosynthesis were up-regulated and JA levels were slightly increased. Three important repressors of the ABA-responsive pathway were up-regulated, indicating that ABA signal transduction may be suppressed. One CML and two WRKY TFs involved in Ca2+-responsive pathways were up-regulated, indicating that this pathway may have been triggered. In conclusion, we show that PevD1 is involved in regulating several plant endogenous signal transduction pathways and regulatory networks to enhance resistance and promote growth and development in Arabidopsis. PMID:27489497

  18. Topological robustness analysis of protein interaction networks reveals key targets for overcoming chemotherapy resistance in glioma.

    PubMed

    Azevedo, Hátylas; Moreira-Filho, Carlos Alberto

    2015-11-19

    Biological networks display high robustness against random failures but are vulnerable to targeted attacks on central nodes. Thus, network topology analysis represents a powerful tool for investigating network susceptibility against targeted node removal. Here, we built protein interaction networks associated with chemoresistance to temozolomide, an alkylating agent used in glioma therapy, and analyzed their modular structure and robustness against intentional attack. These networks showed functional modules related to DNA repair, immunity, apoptosis, cell stress, proliferation and migration. Subsequently, network vulnerability was assessed by means of centrality-based attacks based on the removal of node fractions in descending orders of degree, betweenness, or the product of degree and betweenness. This analysis revealed that removing nodes with high degree and high betweenness was more effective in altering networks' robustness parameters, suggesting that their corresponding proteins may be particularly relevant to target temozolomide resistance. In silico data was used for validation and confirmed that central nodes are more relevant for altering proliferation rates in temozolomide-resistant glioma cell lines and for predicting survival in glioma patients. Altogether, these results demonstrate how the analysis of network vulnerability to topological attack facilitates target prioritization for overcoming cancer chemoresistance.

  19. Multidrug Resistance Protein-4 Influences Aspirin Toxicity in Human Cell Line

    PubMed Central

    Massimi, Isabella; Ciuffetta, Ambra; Temperilli, Flavia; Ferrandino, Francesca; Zicari, Alessandra; Pulcinelli, Fabio M.; Felli, Maria Pia

    2015-01-01

    Overexpression of efflux transporters, in human cells, is a mechanism of resistance to drug and also to chemotherapy. We found that multidrug resistance protein-4 (MRP4) overexpression has a role in reducing aspirin action in patients after bypass surgery and, very recently, we found that aspirin enhances platelet MRP4 levels through peroxisome proliferator activated receptor-α (PPARα). In the present paper, we verified whether exposure of human embryonic kidney-293 cells (Hek-293) to aspirin modifies MRP4 gene expression and its correlation with drug elimination and cell toxicity. We first investigated the effect of high-dose aspirin in Hek-293 and we showed that aspirin is able to increase cell toxicity dose-dependently. Furthermore, aspirin effects, induced at low dose, already enhance MRP4 gene expression. Based on these findings, we compared cell viability in Hek-293, after high-dose aspirin treatment, in MRP4 overexpressing cells, either after aspirin pretreatment or in MRP4 transfected cells; in both cases, a decrease of selective aspirin cell growth inhibition was observed, in comparison with the control cultures. Altogether, these data suggest that exposing cells to low nontoxic aspirin dosages can induce gene expression alterations that may lead to the efflux transporter protein overexpression, thus increasing cellular detoxification of aspirin. PMID:26491233

  20. Short-Chain Chromate Ion Transporter Proteins from Bacillus subtilis Confer Chromate Resistance in Escherichia coli▿

    PubMed Central

    Díaz-Magaña, Amada; Aguilar-Barajas, Esther; Moreno-Sánchez, Rafael; Ramírez-Díaz, Martha I.; Riveros-Rosas, Héctor; Vargas, Eréndira; Cervantes, Carlos

    2009-01-01

    Tandem paired genes encoding putative short-chain monodomain protein members of the chromate ion transporter (CHR) superfamily (ywrB and ywrA) were cloned from genomic DNA of Bacillus subtilis strain 168. The transcription of the paired genes, renamed chr3N and chr3C, respectively, was shown to occur via a bicistronic mRNA generated from a promoter upstream of the chr3N gene. The chr3N and chr3C genes conferred chromate resistance when expressed in Escherichia coli strain W3110. The cloned chr3N gene alone did not confer chromate resistance on E. coli, suggesting that both chr3N and chr3C genes are required for function. E. coli cells expressing paired chr3N and chr3C genes demonstrated diminished uptake of chromate compared to that by a vector-only control strain. These results suggest that short-chain CHR proteins form heterodimer transporters which efflux chromate ions from the cytoplasm. PMID:19581367

  1. Leaf Treatments with a Protein-Based Resistance Inducer Partially Modify Phyllosphere Microbial Communities of Grapevine

    PubMed Central

    Cappelletti, Martina; Perazzolli, Michele; Antonielli, Livio; Nesler, Andrea; Torboli, Esmeralda; Bianchedi, Pier L.; Pindo, Massimo; Puopolo, Gerardo; Pertot, Ilaria

    2016-01-01

    Protein derivatives and carbohydrates can stimulate plant growth, increase stress tolerance, and activate plant defense mechanisms. However, these molecules can also act as a nutritional substrate for microbial communities living on the plant phyllosphere and possibly affect their biocontrol activity against pathogens. We investigated the mechanisms of action of a protein derivative (nutrient broth, NB) against grapevine downy mildew, specifically focusing on the effects of foliar treatments on plant defense stimulation and on the composition and biocontrol features of the phyllosphere microbial populations. NB reduced downy mildew symptoms and induced the expression of defense-related genes in greenhouse- and in vitro-grown plants, indicating the activation of grapevine resistance mechanisms. Furthermore, NB increased the number of culturable phyllosphere bacteria and altered the composition of bacterial and fungal populations on leaves of greenhouse-grown plants. Although, NB-induced changes on microbial populations were affected by the structure of indigenous communities originally residing on grapevine leaves, degrees of disease reduction and defense gene modulation were consistent among the experiments. Thus, modifications in the structure of phyllosphere populations caused by NB application could partially contribute to downy mildew control by competition for space or other biocontrol strategies. Particularly, changes in the abundance of phyllosphere microorganisms may provide a contribution to resistance induction, partially affecting the hormone-mediated signaling pathways involved. Modifying phyllosphere populations by increasing natural biocontrol agents with the application of selected nutritional factors can open new opportunities in terms of sustainable plant protection strategies. PMID:27486468

  2. Topological robustness analysis of protein interaction networks reveals key targets for overcoming chemotherapy resistance in glioma

    NASA Astrophysics Data System (ADS)

    Azevedo, Hátylas; Moreira-Filho, Carlos Alberto

    2015-11-01

    Biological networks display high robustness against random failures but are vulnerable to targeted attacks on central nodes. Thus, network topology analysis represents a powerful tool for investigating network susceptibility against targeted node removal. Here, we built protein interaction networks associated with chemoresistance to temozolomide, an alkylating agent used in glioma therapy, and analyzed their modular structure and robustness against intentional attack. These networks showed functional modules related to DNA repair, immunity, apoptosis, cell stress, proliferation and migration. Subsequently, network vulnerability was assessed by means of centrality-based attacks based on the removal of node fractions in descending orders of degree, betweenness, or the product of degree and betweenness. This analysis revealed that removing nodes with high degree and high betweenness was more effective in altering networks’ robustness parameters, suggesting that their corresponding proteins may be particularly relevant to target temozolomide resistance. In silico data was used for validation and confirmed that central nodes are more relevant for altering proliferation rates in temozolomide-resistant glioma cell lines and for predicting survival in glioma patients. Altogether, these results demonstrate how the analysis of network vulnerability to topological attack facilitates target prioritization for overcoming cancer chemoresistance.

  3. The Streptomyces peucetius drrC gene encodes a UvrA-like protein involved in daunorubicin resistance and production.

    PubMed Central

    Lomovskaya, N; Hong, S K; Kim, S U; Fonstein, L; Furuya, K; Hutchinson, R C

    1996-01-01

    The drrC gene, cloned from the daunorubicin (DNR)- and doxorubicin-producing strain of Streptomyces peucetius ATCC 29050, encodes a 764-amino-acid protein with a strong sequence similarity to the Escherichia coli and Micrococcus luteus UvrA proteins involved in excision repair of DNA. Expression of drrC was correlated with the timing of DNR production in the growth medium tested and was not dependent on the presence of DNR. Since introduction of drrC into Streptomyces lividans imparted a DNR resistance phenotype, this gene is believed to be a DNR resistance gene. The drrC gene could be disrupted in the non-DNR-producing S. peucetius dnrJ mutant but not in the wild-type strain, and the resulting dnrJ drrC double mutant was significantly more sensitive to DNR in efficiency-of-plating experiments. Expression of drrC in an E. coli uvrA strain conferred significant DNR resistance to this highly DNR-sensitive mutant. However, the DrrC protein did not complement the uvrA mutation to protect the mutant from the lethal effects of UV or mitomycin even though it enhanced the UV resistance of a uvrA+ strain. We speculate that the DrrC protein mediates a novel type of DNR resistance, possibly different from the mechanism of DNR resistance governed by the S. peucetius drrAB genes, which are believed to encode a DNR antiporter. PMID:8655504

  4. Downregulation of mitogen-activated protein kinase 1 of Leishmania donovani field isolates is associated with antimony resistance.

    PubMed

    Ashutosh; Garg, Mansi; Sundar, Shyam; Duncan, Robert; Nakhasi, Hira L; Goyal, Neena

    2012-01-01

    Emergence of resistance to pentavalent antimonials has become a severe obstacle in the treatment of visceral leishmaniasis (VL) on the Indian subcontinent. The mechanisms operating in laboratory-generated strains are somewhat known, but the determinants of clinical antimony resistance are not well understood. By utilizing a DNA microarray expression profiling approach, we identified a gene encoding mitogen-activated protein kinase 1 (MAPK1) for the kinetoplast protozoan Leishmania donovani (LdMAPK1) that was consistently downregulated in antimony-resistant field isolates. The expression level of the gene was validated by real-time PCR. Furthermore, decreased expression of LdMAPK1 was also confirmed at the protein level in resistant isolates. Primary structure analysis of LdMAPK1 revealed the presence of all of the characteristic features of MAPK1. When expressed in Escherichia coli, the recombinant enzyme showed kinase activity with myelin basic protein as the substrate and was inhibited by staurosporine. Interestingly, overexpression of this gene in a drug-sensitive laboratory strain and a resistant field isolate resulted in increased the sensitivity of the transfectants to potassium antimony tartrate, suggesting that it has a role in antimony resistance. Our results demonstrate that downregulation of LdMAPK1 may be in part correlated with antimony drug resistance in Indian VL isolates.

  5. Whey protein hydrolysate augments tendon and muscle hypertrophy independent of resistance exercise contraction mode.

    PubMed

    Farup, J; Rahbek, S K; Vendelbo, M H; Matzon, A; Hindhede, J; Bejder, A; Ringgard, S; Vissing, K

    2014-10-01

    In a comparative study, we investigated the effects of maximal eccentric or concentric resistance training combined with whey protein or placebo on muscle and tendon hypertrophy. 22 subjects were allocated into either a high-leucine whey protein hydrolysate + carbohydrate group (WHD) or a carbohydrate group (PLA). Subjects completed 12 weeks maximal knee extensor training with one leg using eccentric contractions and the other using concentric contractions. Before and after training cross-sectional area (CSA) of m. quadriceps and patellar tendon CSA was quantified with magnetic resonance imaging and a isometric strength test was used to assess maximal voluntary contraction (MVC) and rate of force development (RFD). Quadriceps CSA increased by 7.3 ± 1.0% (P < 0.001) in WHD and 3.4 ± 0.8% (P < 0.01) in PLA, with a greater increase in WHD compared to PLA (P < 0.01). Proximal patellar tendon CSA increased by 14.9 ± 3.1% (P < 0.001) and 8.1 ± 3.2% (P = 0.054) for WHD and PLA, respectively, with a greater increase in WHD compared to PLA (P < 0.05), with no effect of contraction mode. MVC and RFD increased by 15.6 ± 3.5% (P < 0.001) and 12-63% (P < 0.05), respectively, with no group or contraction mode effects. In conclusion, high-leucine whey protein hydrolysate augments muscle and tendon hypertrophy following 12 weeks of resistance training - irrespective of contraction mode.

  6. Recombineering reveals a diverse collection of ribosomal proteins L4 and L22 that confer resistance to macrolide antibiotics

    PubMed Central

    Diner, Elie J.; Hayes, Christopher S.

    2009-01-01

    Summary Mutations in ribosomal proteins L4 and L22 confer resistance to erythromycin and other macrolide antibiotics in a variety of bacteria. L4 and L22 have elongated loops whose tips converge in the peptide exit tunnel near the macrolide binding site, and resistance mutations typically affect residues within these loops. Here, we use bacteriophage λ Red-mediated recombination, or “recombineering”, to uncover new L4 and L22 alleles that confer macrolide resistance in Escherichia coli. We randomized residues at the tips of the L4 and L22 loops using recombineered oligonucleotide libraries, and selected the mutagenized cells for erythromycin-resistant mutants. These experiments led to the identification of 341 different resistance mutations encoding 278 unique L4 and L22 proteins – the overwhelming majority of which are novel. Many resistance mutations were complex, involving multiple missense mutations, in-frame deletions, and insertions. Transfer of L4 and L22 mutations into wild-type cells by phage P1-mediated transduction demonstrated that each allele was sufficient to confer macrolide resistance. Although L4 and L22 mutants are typically resistant to most macrolides, selections carried out on different antibiotics revealed macrolide-specific resistance mutations. L22 Lys90Trp is one such allele, which confers resistance to erythromycin, but not tylosin or spiramycin. Purified L22 Lys90Trp ribosomes show reduced erythromycin binding, but have the same affinity for tylosin as wild-type ribosomes. Moreover, DMS methylation protection assays demonstrated that L22 Lys90Trp ribosomes bind tylosin more readily than erythromycin in vivo. This work underscores the exceptional functional plasticity of the L4 and L22 proteins, and highlights the utility of Red-mediated recombination in targeted genetic selections. PMID:19150357

  7. Dominant negative RPW8.2 fusion proteins reveal the importance of haustorium-oriented protein trafficking for resistance against powdery mildew in Arabidopsis.

    PubMed

    Zhang, Qiong; Berkey, Robert; Pan, Zhiyong; Wang, Wenming; Zhang, Yi; Ma, Xianfeng; King, Harlan; Xiao, Shunyuan

    2015-01-01

    Powdery mildew fungi form feeding structures called haustoria inside epidermal cells of host plants to extract photosynthates for their epiphytic growth and reproduction. The haustorium is encased by an interfacial membrane termed the extrahaustorial membrane (EHM). The atypical resistance protein RPW8.2 from Arabidopsis is specifically targeted to the EHM where RPW8.2 activates haustorium-targeted (thus broad-spectrum) resistance against powdery mildew fungi. EHM-specific localization of RPW8.2 suggests the existence of an EHM-oriented protein/membrane trafficking pathway during EHM biogenesis. However, the importance of this specific trafficking pathway for host defense has not been evaluated via a genetic approach without affecting other trafficking pathways. Here, we report that expression of EHM-oriented, nonfunctional RPW8.2 chimeric proteins exerts dominant negative effect over functional RPW8.2 and potentially over other EHM-localized defense proteins, thereby compromising both RPW8.2-mediated and basal resistance to powdery mildew. Thus, our results highlight the importance of the EHM-oriented protein/membrane trafficking pathway for host resistance against haustorium-forming pathogens such as powdery mildew fungi.

  8. The Plasmin-Sensitive Protein Pls in Methicillin-Resistant Staphylococcus aureus (MRSA) Is a Glycoprotein

    PubMed Central

    Pohlentz, Gottfried; Xia, Guoqing; Hussain, Muzaffar; Foster, Simon; Peters, Georg

    2017-01-01

    Most bacterial glycoproteins identified to date are virulence factors of pathogenic bacteria, i.e. adhesins and invasins. However, the impact of protein glycosylation on the major human pathogen Staphylococcus aureus remains incompletely understood. To study protein glycosylation in staphylococci, we analyzed lysostaphin lysates of methicillin-resistant Staphylococcus aureus (MRSA) strains by SDS-PAGE and subsequent periodic acid-Schiff’s staining. We detected four (>300, ∼250, ∼165, and ∼120 kDa) and two (>300 and ∼175 kDa) glycosylated surface proteins with strain COL and strain 1061, respectively. The ∼250, ∼165, and ∼175 kDa proteins were identified as plasmin-sensitive protein (Pls) by mass spectrometry. Previously, Pls has been demonstrated to be a virulence factor in a mouse septic arthritis model. The pls gene is encoded by the staphylococcal cassette chromosome (SCC)mec type I in MRSA that also encodes the methicillin resistance-conferring mecA and further genes. In a search for glycosyltransferases, we identified two open reading frames encoded downstream of pls on the SCCmec element, which we termed gtfC and gtfD. Expression and deletion analysis revealed that both gtfC and gtfD mediate glycosylation of Pls. Additionally, the recently reported glycosyltransferases SdgA and SdgB are involved in Pls glycosylation. Glycosylation occurs at serine residues in the Pls SD-repeat region and modifying carbohydrates are N-acetylhexosaminyl residues. Functional characterization revealed that Pls can confer increased biofilm formation, which seems to involve two distinct mechanisms. The first mechanism depends on glycosylation of the SD-repeat region by GtfC/GtfD and probably also involves eDNA, while the second seems to be independent of glycosylation as well as eDNA and may involve the centrally located G5 domains. Other previously known Pls properties are not related to the sugar modifications. In conclusion, Pls is a glycoprotein and Pls glycosyl

  9. Lily ASR protein-conferred cold and freezing resistance in Arabidopsis.

    PubMed

    Hsu, Yi-Feng; Yu, Shu-Chuan; Yang, Chin-Ying; Wang, Co-Shine

    2011-09-01

    The lily LLA23 protein is a member of the abscisic acid, stress and ripening-induced (ASR) protein family. Constitutive overexpression of LLA23 under the cauliflower mosaic virus 35S promoter confers cold and freezing tolerance in Arabidopsis. The phenotypical growth and survival percentage of the two transgenic 35S::LLA23 plants showed higher resistance to cold and freezing conditions than those of wild-type (WT) plants. The electrolyte leakage in WT leaves increased by approximately fourfold at -2 °C relative to that at 22 °C whereas both transgenic leaves showed little ion leakage under the same conditions. A microarray analysis of LLA23-overexpressing transgenic line, 35S::LLA23E, under normal growing conditions was previously conducted by Yang et al. (Protoplasma, 2008, 233:241-254). Microarray analysis showed that 12 cold-responsive genes are upregulated and 25 cold-responsive genes are downregulated by lily ASR. Many ASR-regulated genes encode proteins involved in the classes of defense/stress-related, transcription, and metabolism. Quantitative polymerase chain reaction analysis confirms the changes in mRNA levels observed in the microarray analysis. Thus, our results provide in vivo evidence implying that LLA23 mediates cold/freezing stress-responsive signaling. To gain further insight into the functions of LLA23 protein, an in vitro enzyme protection assay was used in which lactate dehydrogenase and malate dehydrogenase were subjected to unfavorable conditions. The assay revealed that both enzyme activities were significantly retained with the addition of LLA23, which was superior to either trehalose or BSA, suggesting that the LLA23 protein can protect enzymatic activities against freeze-thaw cycles. The 35S::LLA23 seedlings also exhibited enzyme activity superior to WT at -4 °C. These results suggest that LLA23 may act as an osmoprotectant as well as a transcription factor to confer 35S::LLA23 plants enhanced cold and freezing resistance.

  10. Ursolic Acid Suppresses Hepatitis B Virus X Protein-mediated Autophagy and Chemotherapeutic Drug Resistance.

    PubMed

    Chang, Ching-Dong; Lin, Ping-Yuan; Hsu, Jue-Liang; Shih, Wen-Ling

    2016-10-01

    Hepatitis B virus X (HBx) protein is a multifunctional oncoprotein that affects diverse cell activities via regulation of various host cell signaling pathways. The current investigation demonstrated that ursolic acid (UA), a pentacyclic triterpenoid, protected hepatoma cells and reduced HBx-mediated autophagy through modulation of Ras homolog gene family member A (RhoA). Low-level ectopic HBx expression in Huh7 cells induced more significant autophagosome formation than high-level HBx expression. HBx activated beclin-1 promoter and enhanced the beclin-1 protein expression under low HBx expression. Transcription factor AP-1 played an essential function in HBx-mediated beclin-1 promoter activation. Inhibition of RhoA and its downstream effector Rho-associated coiled-coil-containing protein kinase 1 (ROCK1) alleviated HBx-mediated autophagy significantly. Transiently-expressed HBx elicited an increased RhoA-GTP level, as well as phospho-ROCK1 transient accumulation. Utilization of transactivation-deficient HBx demonstrated that the transactivation activity of HBx is required for autophagy induction. Furthermore, UA suppressed HBx-mediated RhoA activation, beclin-1 promoter activation and subsequent autophagy induction, while, most importantly, reversed HBx-induced anti-cancer drug resistance.

  11. PMT family of Candida albicans: five protein mannosyltransferase isoforms affect growth, morphogenesis and antifungal resistance.

    PubMed

    Prill, Stephan K-H; Klinkert, Birgit; Timpel, Claudia; Gale, Cheryl A; Schröppel, Klaus; Ernst, Joachim F

    2005-01-01

    Protein O-mannosyltransferases (Pmt proteins) initiate O-mannosylation of secretory proteins. The PMT gene family of the human fungal pathogen Candida albicans consists of PMT1 and PMT6, as well as three additional PMT genes encoding Pmt2, Pmt4 and Pmt5 isoforms described here. Both PMT2 alleles could not be deleted and growth of conditional strains, containing PMT2 controlled by the MET3- or tetOScHOP1-promoters, was blocked in non-permissive conditions, indicating that PMT2 is essential for growth. A homozygous pmt4 mutant was viable, but synthetic lethality of pmt4 was observed in combination with pmt1 mutations. Hyphal morphogenesis of a pmt4 mutant was defective under aerobic induction conditions, yet increased in embedded or hypoxic conditions, suggesting a role of Pmt4p-mediated O-glycosylation for environment-specific morphogenetic signalling. Although a PMT5 transcript was detected, a homozygous pmt5 mutant was phenotypically silent. All other pmt mutants showed variable degrees of supersensitivity to antifungals and to cell wall-destabilizing agents. Cell wall composition was markedly affected in pmt1 and pmt4 mutants, showing a significant decrease in wall mannoproteins. In a mouse model of haematogenously disseminated infection, PMT4 was required for full virulence of C. albicans. Functional analysis of the first complete PMT gene family in a fungal pathogen indicates that Pmt isoforms have variable and specific roles for in vitro and in vivo growth, morphogenesis and antifungal resistance.

  12. Enhanced resistance in Theobroma cacao against oomycete and fungal pathogens by secretion of phosphatidylinositol-3-phosphate-binding proteins.

    PubMed

    Helliwell, Emily E; Vega-Arreguín, Julio; Shi, Zi; Bailey, Bryan; Xiao, Shunyuan; Maximova, Siela N; Tyler, Brett M; Guiltinan, Mark J

    2016-03-01

    The internalization of some oomycete and fungal pathogen effectors into host plant cells has been reported to be blocked by proteins that bind to the effectors' cell entry receptor, phosphatidylinositol-3-phosphate (PI3P). This finding suggested a novel strategy for disease control by engineering plants to secrete PI3P-binding proteins. In this study, we tested this strategy using the chocolate tree Theobroma cacao. Transient expression and secretion of four different PI3P-binding proteins in detached leaves of T. cacao greatly reduced infection by two oomycete pathogens, Phytophthora tropicalis and Phytophthora palmivora, which cause black pod disease. Lesion size and pathogen growth were reduced by up to 85%. Resistance was not conferred by proteins lacking a secretory leader, by proteins with mutations in their PI3P-binding site, or by a secreted PI4P-binding protein. Stably transformed, transgenic T. cacao plants expressing two different PI3P-binding proteins showed substantially enhanced resistance to both P. tropicalis and P. palmivora, as well as to the fungal pathogen Colletotrichum theobromicola. These results demonstrate that secretion of PI3P-binding proteins is an effective way to increase disease resistance in T. cacao, and potentially in other plants, against a broad spectrum of pathogens.

  13. Characterization of resistance to Callosobruchus maculatus (Coleoptera: Bruchidae) in mungbean variety VC6089A and its resistance-associated protein VrD1.

    PubMed

    Lin, Chan; Chen, Ching-San; Horng, Shwu-Bin

    2005-08-01

    Characteristics of resistance of VC6089A, a mungbean, Vigna radiata (L.) Wilczek, bred by using a wild Vigna species, V. sublobata (Roxburgh) Verdcourt (accession no. TC1966), and containing a novel protein, VrD1, were investigated against the cowpea weevil, Callosobruchus maculatus (F.). The seeds of VC6089A showed high level of resistance; > 96% of the bruchid eggs failed to develop into adults, whereas 85% of eggs laid on susceptible cultivar VC1973A became adults. Mortality of surviving bruchids raised for five generations on VC6089A remained higher than 96%; however, female adults maintained high fecundity and thus showed a positive population growth through these generations. We therefore cannot exclude the possibility that the beetles could develop resistance to the resistant mungbean VC6089A. The protein VrD1 purified from seeds of VC6089A showed marked toxicity to C. maculatus when beetles were reared on artificial seeds containing varying levels of VrD1. Thorough inhibition of development was observed when artificial seeds containing 0.2% (wt:wt) VrD1 was provided for insect feeding. Our findings demonstrated the insecticidal activity of VC6089A mungbean seeds and VrD1 protein against C. maculatus. These results may facilitate safer control against bruchid infestation.

  14. Resistance Mechanisms and the Future of Bacterial Enoyl-Acyl Carrier Protein Reductase (FabI) Antibiotics

    PubMed Central

    Yao, Jiangwei; Rock, Charles O.

    2016-01-01

    Missense mutations leading to clinical antibiotic resistance are a liability of single-target inhibitors. The enoyl-acyl carrier protein reductase (FabI) inhibitors have one intracellular protein target and drug resistance is increased by the acquisition of single base pair mutations that alter drug binding. The spectrum of resistance mechanisms to FabI inhibitors suggests criteria that should be considered during the development of single-target antibiotics that would minimize the impact of missense mutations on their clinical usefulness. These criteria include high-affinity, fast on/off kinetics, few drug contacts with residue side chains, and no toxicity. These stringent criteria are achievable by structure-guided design, but this approach will only yield pathogen-specific drugs. Single-step acquisition of resistance may limit the clinical application of broad-spectrum, single-target antibiotics, but appropriately designed, pathogen-specific antibiotics have the potential to overcome this liability. PMID:26931811

  15. Palmitoylation of SARS-CoV S protein is necessary for partitioning into detergent-resistant membranes and cell-cell fusion but not interaction with M protein

    SciTech Connect

    McBride, Corrin E.; Machamer, Carolyn E.

    2010-09-15

    Coronaviruses are enveloped RNA viruses that generally cause mild disease in humans. However, the recently emerged coronavirus that caused severe acute respiratory syndrome (SARS-CoV) is the most pathogenic human coronavirus discovered to date. The SARS-CoV spike (S) protein mediates virus entry by binding cellular receptors and inducing fusion between the viral envelope and the host cell membrane. Coronavirus S proteins are palmitoylated, which may affect function. Here, we created a non-palmitoylated SARS-CoV S protein by mutating all nine cytoplasmic cysteine residues. Palmitoylation of SARS-CoV S was required for partitioning into detergent-resistant membranes and for cell-cell fusion. Surprisingly, however, palmitoylation of S was not required for interaction with SARS-CoV M protein. This contrasts with the requirement for palmitoylation of mouse hepatitis virus S protein for interaction with M protein and may point to important differences in assembly and infectivity of these two coronaviruses.

  16. Altered Protein S-Glutathionylation Identifies a Potential Mechanism of Resistance to Acetaminophen-Induced Hepatotoxicity

    PubMed Central

    McGarry, David J.; Chakravarty, Probir; Wolf, C. Roland

    2015-01-01

    Acetaminophen (APAP) is the most commonly used over-the-counter analgesic. However, hepatotoxicity induced by APAP is a major clinical issue, and the factors that define sensitivity to APAP remain unclear. We have previously demonstrated that mice nulled for glutathione S-transferase Pi (GSTP) are resistant to APAP-induced hepatotoxicity. This study aims to exploit this difference to delineate pathways of importance in APAP toxicity. We used mice nulled for GSTP and heme oxygenase-1 oxidative stress reporter mice, together with a novel nanoflow liquid chromatography–tandem mass spectrometry methodology to investigate the role of oxidative stress, cell signaling, and protein S-glutathionylation in APAP hepatotoxicity. We provide evidence that the sensitivity difference between wild-type and Gstp1/2−/− mice is unrelated to the ability of APAP to induce oxidative stress, despite observing significant increases in c-Jun N-terminal kinase and extracellular signal-regulated kinase phosphorylation in wild-type mice. The major difference in response to APAP was in the levels of protein S-glutathionylation: Gstp1/2−/− mice exhibited a significant increase in the number of S-glutathionylated proteins compared with wild-type animals. Remarkably, these S-glutathionylated proteins are involved in oxidative phosphorylation, respiratory complexes, drug metabolism, and mitochondrial apoptosis. Furthermore, we found that S-glutathionylation of the rate-limiting glutathione-synthesizing enzyme, glutamate cysteine ligase, was markedly increased in Gstp1/2−/− mice in response to APAP. The data demonstrate that S-glutathionylation provides an adaptive response to APAP and, as a consequence, suggest that this is an important determinant in APAP hepatotoxicity. This work identifies potential novel avenues associated with cell survival for the treatment of chemical-induced hepatotoxicity. PMID:26311813

  17. Infectious Bronchitis Coronavirus Inhibits STAT1 Signaling and Requires Accessory Proteins for Resistance to Type I Interferon Activity

    PubMed Central

    Kint, Joeri; Dickhout, Annemiek; Kutter, Jasmin; Maier, Helena J.; Britton, Paul; Koumans, Joseph; Pijlman, Gorben P.; Fros, Jelke J.; Wiegertjes, Geert F.

    2015-01-01

    ABSTRACT The innate immune response is the first line of defense against viruses, and type I interferon (IFN) is a critical component of this response. Similar to other viruses, the gammacoronavirus infectious bronchitis virus (IBV) has evolved under evolutionary pressure to evade and counteract the IFN response to enable its survival. Previously, we reported that IBV induces a delayed activation of the IFN response. In the present work, we describe the resistance of IBV to IFN and the potential role of accessory proteins herein. We show that IBV is fairly resistant to the antiviral state induced by IFN and identify that viral accessory protein 3a is involved in resistance to IFN, as its absence renders IBV less resistant to IFN treatment. In addition to this, we found that independently of its accessory proteins, IBV inhibits IFN-mediated phosphorylation and translocation of STAT1. In summary, we show that IBV uses multiple strategies to counteract the IFN response. IMPORTANCE In the present study, we show that infectious bronchitis virus (IBV) is resistant to IFN treatment and identify a role for accessory protein 3a in the resistance against the type I IFN response. We also demonstrate that, in a time-dependent manner, IBV effectively interferes with IFN signaling and that its accessory proteins are dispensable for this activity. This study demonstrates that the gammacoronavirus IBV, similar to its mammalian counterparts, has evolved multiple strategies to efficiently counteract the IFN response of its avian host, and it identifies accessory protein 3a as multifaceted antagonist of the avian IFN system. PMID:26401035

  18. Salivary gland proteome analysis reveals modulation of anopheline unique proteins in insensitive acetylcholinesterase resistant Anopheles gambiae mosquitoes.

    PubMed

    Cornelie, Sylvie; Rossignol, Marie; Seveno, Martial; Demettre, Edith; Mouchet, François; Djègbè, Innocent; Marin, Philippe; Chandre, Fabrice; Corbel, Vincent; Remoué, Franck; Mathieu-Daudé, Françoise

    2014-01-01

    Insensitive acetylcholinesterase resistance due to a mutation in the acetylcholinesterase (ace) encoding ace-1 gene confers cross-resistance to organophosphate and carbamate insecticides in Anopheles gambiae populations from Central and West Africa. This mutation is associated with a strong genetic cost revealed through alterations of some life history traits but little is known about the physiological and behavioural changes in insects bearing the ace-1(R) allele. Comparative analysis of the salivary gland contents between An. gambiae susceptible and ace-1(R) resistant strains was carried out to charaterize factors that could be involved in modifications of blood meal process, trophic behaviour or pathogen interaction in the insecticide-resistant mosquitoes. Differential analysis of the salivary gland protein profiles revealed differences in abundance for several proteins, two of them showing major differences between the two strains. These two proteins identified as saglin and TRIO are salivary gland-1 related proteins, a family unique to anopheline mosquitoes, one of them playing a crucial role in salivary gland invasion by Plasmodium falciparum sporozoites. Differential expression of two other proteins previously identified in the Anopheles sialome was also observed. The differentially regulated proteins are involved in pathogen invasion, blood feeding process, and protection against oxidation, relevant steps in the outcome of malaria infection. Further functional studies and insect behaviour experiments would confirm the impact of the modification of the sialome composition on blood feeding and pathogen transmission abilities of the resistant mosquitoes. The data supports the hypothesis of alterations linked to insecticide resistance in the biology of the primary vector of human malaria in Africa.

  19. Salivary Gland Proteome Analysis Reveals Modulation of Anopheline Unique Proteins in Insensitive Acetylcholinesterase Resistant Anopheles gambiae Mosquitoes

    PubMed Central

    Cornelie, Sylvie; Rossignol, Marie; Seveno, Martial; Demettre, Edith; Mouchet, François; Djègbè, Innocent; Marin, Philippe; Chandre, Fabrice; Corbel, Vincent; Remoué, Franck; Mathieu-Daudé, Françoise

    2014-01-01

    Insensitive acetylcholinesterase resistance due to a mutation in the acetylcholinesterase (ace) encoding ace-1 gene confers cross-resistance to organophosphate and carbamate insecticides in Anopheles gambiae populations from Central and West Africa. This mutation is associated with a strong genetic cost revealed through alterations of some life history traits but little is known about the physiological and behavioural changes in insects bearing the ace-1R allele. Comparative analysis of the salivary gland contents between An. gambiae susceptible and ace-1R resistant strains was carried out to charaterize factors that could be involved in modifications of blood meal process, trophic behaviour or pathogen interaction in the insecticide-resistant mosquitoes. Differential analysis of the salivary gland protein profiles revealed differences in abundance for several proteins, two of them showing major differences between the two strains. These two proteins identified as saglin and TRIO are salivary gland-1 related proteins, a family unique to anopheline mosquitoes, one of them playing a crucial role in salivary gland invasion by Plasmodium falciparum sporozoites. Differential expression of two other proteins previously identified in the Anopheles sialome was also observed. The differentially regulated proteins are involved in pathogen invasion, blood feeding process, and protection against oxidation, relevant steps in the outcome of malaria infection. Further functional studies and insect behaviour experiments would confirm the impact of the modification of the sialome composition on blood feeding and pathogen transmission abilities of the resistant mosquitoes. The data supports the hypothesis of alterations linked to insecticide resistance in the biology of the primary vector of human malaria in Africa. PMID:25102176

  20. Development of robust biocompatible silicone with high resistance to protein adsorption and bacterial adhesion.

    PubMed

    Lin, Weifeng; Zhang, Juan; Wang, Zhen; Chen, Shengfu

    2011-05-01

    A new biocompatible silicone comprising a carboxybetaine (CB) ester analogue, 3-methacryloxypropyltris(trimethylsiloxy)silane (TRIS) and an organic silicone macromer (bis-α,ω-(methacryloxypropyl) polydimethylsiloxane) has been developed using photo-polymerisation. Following interfacial hydrolysis of the CB ester, the resulting zwitterionic material became significantly more hydrophilic and exhibited high resistance to both non-specific protein adsorption and bacterial adhesion. Moreover, the stability of these non-fouling properties was dramatically improved by using a slow and controlled rate of ester hydrolysis of the original protective hydrophobic matrix. The subsequent ability to maintain the original optical and mechanical properties of the bare silicone following surface activation makes this material an ideal candidate for preparing contact lenses and other medical devices.

  1. Overexpression of patA and patB, which encode ABC transporters, is associated with fluoroquinolone resistance in clinical isolates of Streptococcus pneumoniae.

    PubMed

    Garvey, Mark I; Baylay, Alison J; Wong, Ryan L; Piddock, Laura J V

    2011-01-01

    Fifty-seven clinical isolates of Streptococcus pneumoniae were divided into four groups based on their susceptibilities to the fluoroquinolones ciprofloxacin and norfloxacin and the dyes ethidium bromide and acriflavine. Comparative reverse transcription-PCR was used to determine the level of expression of the genes patA and patB, which encode putative ABC transporters. Overexpression was observed in 14 of the 15 isolates that were resistant to both fluoroquinolones and dyes and in only 3 of 24 of those resistant to fluoroquinolones only. Isolates overexpressing patA and patB accumulated significantly less of the fluorescent dye Hoechst 33342 than wild-type isolates, suggesting that PatA and PatB are involved in efflux. Inactivation of patA and patB by in vitro mariner mutagenesis conferred hypersusceptibility to ethidium bromide and acriflavine in all isolates tested and lowered the MICs of ciprofloxacin in the patAB-overproducing and/or fluoroquinolone-resistant isolates. These data represent the first observation of overexpression of patA and patB in clinical isolates and show that PatA and PatB play a clinically relevant role in fluoroquinolone resistance.

  2. A pitfall in diagnosis of human prion diseases using detection of protease-resistant prion protein in urine. Contamination with bacterial outer membrane proteins.

    PubMed

    Furukawa, Hisako; Doh-ura, Katsumi; Okuwaki, Ryo; Shirabe, Susumu; Yamamoto, Kazuo; Udono, Heiichiro; Ito, Takashi; Katamine, Shigeru; Niwa, Masami

    2004-05-28

    Because a definite diagnosis of prion diseases relies on the detection of the abnormal isoform of prion protein (PrPSc), it has been urgently necessary to establish a non-invasive diagnostic test to detect PrPSc in human prion diseases. To evaluate diagnostic usefulness and reliability of the detection of protease-resistant prion protein in urine, we extensively analyzed proteinase K (PK)-resistant proteins in patients affected with prion diseases and control subjects by Western blot, a coupled liquid chromatography and mass spectrometry analysis, and N-terminal sequence analysis. The PK-resistant signal migrating around 32 kDa previously reported by Shaked et al. (Shaked, G. M., Shaked, Y., Kariv-Inbal, Z., Halimi, M., Avraham, I., and Gabizon, R. (2001) J. Biol. Chem. 276, 31479-31482) was not observed in this study. Instead, discrete protein bands with an apparent molecular mass of approximately 37 kDa were detected in the urine of many patients affected with prion diseases and two diseased controls. Although these proteins also gave strong signals in the Western blot using a variety of anti-PrP antibodies as a primary antibody, we found that the signals were still detectable by incubation of secondary antibodies alone, i.e. in the absence of the primary anti-PrP antibodies. Mass spectrometry and N-terminal protein sequencing analysis revealed that the majority of the PK-resistant 37-kDa proteins in the urine of patients were outer membrane proteins (OMPs) of the Enterobacterial species. OMPs isolated from these bacteria were resistant to PK and the PK-resistant OMPs from the Enterobacterial species migrated around 37 kDa on SDS-PAGE. Furthermore, nonspecific binding of OMPs to antibodies could be mistaken for PrPSc. These findings caution that bacterial contamination can affect the immunological detection of prion protein. Therefore, the presence of Enterobacterial species should be excluded in the immunological tests for PrPSc in clinical samples, in

  3. Nrf2 pathway regulates multidrug-resistance-associated protein 1 in small cell lung cancer.

    PubMed

    Ji, Lili; Li, Hui; Gao, Pan; Shang, Guoguo; Zhang, Donna D; Zhang, Nong; Jiang, Tao

    2013-01-01

    Although multidrug-resistance-associated protein-1 (MRP1) is a major contributor to multi-drug resistance (MDR), the regulatory mechanism of Mrp1 still remains unclear. Nrf2 is a transcription factor that regulates cellular defense response through antioxidant response elements (AREs) in normal tissues. Recently, Nrf2 has emerged as an important contributor to chemo-resistance in tumor tissues. In the present study, the role of Nrf2-ARE pathway on regulation of Mrp1 was investigated. Compared with H69 lung cancer cells, H69AR cells with MDR showed significantly higher Nrf2-ARE pathway activity and expression of Mrp1 as well. When Nrf2 was knocked down in H69AR cells, MRP1's expression decreased accordingly. Moreover, those H69AR cells with reduced Nrf2 level restored sensitivity to chemo-drugs. To explore how Nrf2-ARE pathway regulates Mrp1, the promoter of Mrp1 gene was searched, and two putative AREs--ARE1 and ARE2--were found. Using reporter gene and ChIP assay, both ARE1 and ARE2 showed response to and interaction with Nrf2. In 40 cases of cancer tissues, the expression of Nrf2 and MRP1 was measured by immunohistochemistry (IHC). As the quantitive data of IHC indicated, both Nrf2 and MRP1 showed significantly higher expression in tumor tissue than adjacent non-tumor tissue. And more important, the correlation analysis of the two genes proved that their expression was correlative. Taken together, theses data suggested that Nrf2-ARE pathway is required for the regulatory expression of Mrp1 and implicated Nrf2 as a new therapeutic target for MDR.

  4. Multidrug resistance proteins restrain the intestinal absorption of trans-resveratrol in rats.

    PubMed

    Juan, M Emília; González-Pons, Eulalia; Planas, Joana M

    2010-03-01

    trans-Resveratrol, a natural antioxidant, has been described as a nutraceutic compound with important beneficial effects on health, but its low oral bioavailability hinders its therapeutic activity. Here, we studied the mechanisms of apical transport of trans-resveratrol in enterocytes and the role of ATP-binding cassette (ABC) transporters in the secretion of resveratrol glucuronide and sulfate resulting from the rapid intracellular metabolism. An intestinal perfusion method with recirculation in vivo was used in rats. Jejunal loops were perfused with increasing concentrations of trans-resveratrol and results showed that its uptake occurs by simple diffusion without the participation of a mediated transport. The apparent diffusion constant was 8.1 +/- 0.3 microL/(5 min.mg dry weight). The glycoprotein-P (Pgp, ABCB1), multidrug resistance-associated protein 2 (MRP2, ABCC2), and breast cancer resistance protein (BCRP, ABCG2) located in the apical membrane of enterocytes were investigated using specific inhibitors. The Pgp inhibitors verapamil (5 micromol/L) and cyclosporin A (5 micromol/L) did not affect the efflux of trans-resveratrol and its conjugates. The MRP2 inhibitors probenecid (2 mmol/L) and MK571 (10 micromol/L) reduced the efflux of glucuronide by 61 and 55%, respectively, and of sulfate by 43 and 28%, respectively. The BCRP inhibitor Ko143 (0.5 micromol/L) decreased the secretion of glucuronide by 64% and of sulfate by 46%. Our experiments identify MRP2 and BCRP as the 2 apical transporters involved in the efflux of resveratrol conjugates.

  5. Identification and functional characterization of breast cancer resistance protein in human bronchial epithelial cells (Calu-3)

    PubMed Central

    Paturi, Durga Kalyani; Kwatra, Deep; Ananthula, Hari Krishna; Pal, Dhananjay; Mitra, Ashim K.

    2010-01-01

    Breast cancer resistance protein (BCRP), a 72 kDa protein belongs to the subfamily G of the human ATP binding cassette transporter superfamily. Overexpression of BCRP was found to play a major role in the development of resistance against various chemotherapeutic agents. BCRP plays an important role in absorption, distribution and elimination of several therapeutic agents. BCRP expression and functional activity across human bronchial epithelium and its impact on pulmonary drug accumulation has not been established. The objective of this study is to identify and characterize the BCRP efflux transporter across human bronchial epithelium. Calu-3, a human bronchial epithelial cell line was employed as a model for this study. Reverse transcription-polymerase chain reaction (RT-PCR), western blot and immunocytochemical studies were performed to identify and characterize the expression of BCRP. RT-PCR studies detected ABCG2 mRNA levels in Calu-3 cells. A strong band for BCRP with a molecular weight of approximately 72 kDa was observed in Western blot analysis. Immunocytochemical studies confirmed the presence of BCRP on the apical membrane of human bronchial epithelium. Functional activity of BCRP was determined by performing uptake of radioactive substrate [3H]-mitoxantrone in the presence and absence of BCRP inhibitors. Uptake of [3H]-mitoxantrone was elevated significantly in the presence of GF120918 and fumitremorgin C. An increase in the accumulation of Hoechst 33342, a fluorescent dye was also detected in the presence of BCRP inhibitors when compared to control. In summary, this study provides evidence for the presence of an ATP dependent, membrane bound efflux transporter BCRP across human bronchial epithelial cell line, Calu-3. PMID:19782742

  6. PROTAC-induced BET protein degradation as a therapy for castration-resistant prostate cancer

    PubMed Central

    Raina, Kanak; Lu, Jing; Qian, Yimin; Altieri, Martha; Gordon, Deborah; Rossi, Ann Marie K.; Wang, Jing; Chen, Xin; Dong, Hanqing; Siu, Kam; Winkler, James D.; Crew, Andrew P.; Crews, Craig M.; Coleman, Kevin G.

    2016-01-01

    Prostate cancer has the second highest incidence among cancers in men worldwide and is the second leading cause of cancer deaths of men in the United States. Although androgen deprivation can initially lead to remission, the disease often progresses to castration-resistant prostate cancer (CRPC), which is still reliant on androgen receptor (AR) signaling and is associated with a poor prognosis. Some success against CRPC has been achieved by drugs that target AR signaling, but secondary resistance invariably emerges, and new therapies are urgently needed. Recently, inhibitors of bromodomain and extra-terminal (BET) family proteins have shown growth-inhibitory activity in preclinical models of CRPC. Here, we demonstrate that ARV-771, a small-molecule pan-BET degrader based on proteolysis-targeting chimera (PROTAC) technology, demonstrates dramatically improved efficacy in cellular models of CRPC as compared with BET inhibition. Unlike BET inhibitors, ARV-771 results in suppression of both AR signaling and AR levels and leads to tumor regression in a CRPC mouse xenograft model. This study is, to our knowledge, the first to demonstrate efficacy with a small-molecule BET degrader in a solid-tumor malignancy and potentially represents an important therapeutic advance in the treatment of CRPC. PMID:27274052

  7. Sensitization of Cells Overexpressing Multidrug Resistant Proteins by Pluronic P85

    PubMed Central

    Batrakova, Elena V.; Li, Shu; Alakhov, Valery Yu.; Elmquist, William F.; Miller, Donald W.; Kabanov, Alexander V.

    2011-01-01

    Purpose This study evaluated the chemosensitizing effects of Pluronic P85 (P85) on the cells expressing multidrug resistance-associated proteins, MRP1 and MRP2. Methods Cell models included MRP1- and MRP2-transfected MDCKII cells, as well as doxorubicin-selected COR-L23/R cells overexpressing MRP1. Effects of P85 on cellular accumulation and cytotoxicity of vinblastine and doxorubicin were determined. Mechanistic studies characterized the effects of P85 on ATP and reduced glutathione (GSH) intracellular levels as well as MRPs ATPase and glutathione-S-transferase (GST) activities in these cells. Results Considerable increases of vinblastine and doxorubicin accumulation in the cells overexpressing MRP1 and MRP2 in the presence of P85 were observed, while no statistically significant changes in the drug accumulation in the parental cells were found. P85 treatment caused an inhibition of MRPs ATPase activity. Furthermore, P85 induced ATP depletion in these cells similar to that previously reported for Pgp-overexpressing cells. In addition, reduction of GSH intracellular levels and decrease of GST activity following P85 treatment were observed. Finally, significant enhancement of cytotoxicity of vinblastine and doxorubicin by P85 in MRPs -overexpressing cells was demonstrated. Conclusions This study suggests that P85 can sensitize cells overexpressing MRP1 and MRP2, which could be useful for chemotherapy of cancers that display these resistant mechanisms. PMID:14620511

  8. The breast cancer resistance protein protects against a major chlorophyll-derived dietary phototoxin and protoporphyria.

    PubMed

    Jonker, Johan W; Buitelaar, Marije; Wagenaar, Els; Van Der Valk, Martin A; Scheffer, George L; Scheper, Rik J; Plosch, Torsten; Kuipers, Folkert; Elferink, Ronald P J Oude; Rosing, Hilde; Beijnen, Jos H; Schinkel, Alfred H

    2002-11-26

    The breast cancer resistance protein (BCRPABCG2) is a member of the ATP-binding cassette family of drug transporters and confers resistance to various anticancer drugs. We show here that mice lacking Bcrp1Abcg2 become extremely sensitive to the dietary chlorophyll-breakdown product pheophorbide a, resulting in severe, sometimes lethal phototoxic lesions on light-exposed skin. Pheophorbide a occurs in various plant-derived foods and food supplements. Bcrp1 transports pheophorbide a and is highly efficient in limiting its uptake from ingested food. Bcrp1(-/-) mice also displayed a previously unknown type of protoporphyria. Erythrocyte levels of the heme precursor and phototoxin protoporphyrin IX, which is structurally related to pheophorbide a, were increased 10-fold. Transplantation with wild-type bone marrow cured the protoporphyria and reduced the phototoxin sensitivity of Bcrp1(-/-) mice. These results indicate that humans or animals with low or absent BCRP activity may be at increased risk for developing protoporphyria and diet-dependent phototoxicity and provide a striking illustration of the importance of drug transporters in protection from toxicity of normal food constituents.

  9. Protein-resistant polyurethane via surface-initiated atom transfer radical polymerization of oligo(ethylene glycol) methacrylate.

    PubMed

    Jin, Zhilin; Feng, Wei; Zhu, Shiping; Sheardown, Heather; Brash, John L

    2009-12-15

    Protein-resistant polyurethane (PU) surfaces were prepared by surface-initiated simultaneous normal and reverse atom transfer radical polymerization (s-ATRP) of poly(oligo(ethylene glycol) methacrylate) (poly (OEGMA)). Oxygen plasma treatment was employed for initial activation of the PU surface. The grafted polymer chain length was adjusted by varying the molar ratio of monomer to sacrificial initiator in solution from 5:1 to 200:1. The modified PU surfaces were characterized by water contact angle, X-ray photoelectron spectroscopy (XPS), and atomic force microscopy (AFM). Protein adsorption experiments from tris-buffered saline (TBS) and plasma were carried out to evaluate the protein-resistance of the surfaces. Adsorption from single and binary protein solutions as well as from plasma was significantly reduced after modification. Adsorption decreased with increasing poly(OEGMA) chain length. Fibrinogen (Fg) adsorption on the 200:1 monomer/initiator surface was in the range of 3-33 ng/cm(2) representing 96-99% reduction compared with the unmodified PU. Fg adsorption from 0.01-10% plasma was as low as 1-5 ng/cm(2). Moreover, binary protein adsorption experiments using Fg and lysozyme (Lys) showed that protein size is a factor in the protein resistance of these surfaces.

  10. Mouse breast cancer resistance protein (Bcrp1/Abcg2) mediates etoposide resistance and transport, but etoposide oral availability is limited primarily by P-glycoprotein.

    PubMed

    Allen, John D; Van Dort, Sonja C; Buitelaar, Marije; van Tellingen, Olaf; Schinkel, Alfred H

    2003-03-15

    The breast cancer resistance protein [BCRP (BCRP/ABCG2)] has not previously been directly identified as a source of resistance to epipodophyllotoxins.However, when P-glycoprotein (P-gp)- and Mrp1-deficient mouse fibroblast and kidney cell lines were selected for resistance to etoposide, amplification and overexpression of Bcrp1 emerged as the dominant resistance mechanism in five of five cases. Resistance was accompanied by reduced intracellular etoposide accumulation. Bcrp1 sequence in all of the resistant lines was wild-type in the region spanning the R482 mutation hot spot known to alter the substrate specificity of mouse Bcrp1 (mouse cognate of BCRP) and human BCRP. Transduced wild-type Bcrp1 cDNA mediated resistance to etoposide and teniposide in fibroblast lines and trans-epithelial etoposide transport in polarized Madin-Darby canine kidney II cells. Bcrp1-mediated etoposide resistance was reversed by two structurally different BCRP/Bcrp1 inhibitors, GF120918 and Ko143. BCRP/Bcrp1 (inhibition) might thus impact on the antitumor activity and pharmacokinetics of epipodophyllotoxins. However, treatment of P-gp-deficient mice with GF120918 did not improve etoposide oral uptake, suggesting that Bcrp1 activity is not a major limiting factor in this process. In contrast, use of GF120918 to inhibit P-gp in wild-type mice increased the plasma levels of etoposide after oral administration 4-5-fold. It may thus be worthwhile to test inhibition of P-gp in humans to improve the oral availability of etoposide.

  11. Plasmodesmata-located protein overexpression negatively impacts the manifestation of systemic acquired resistance and the long-distance movement of Defective in Induced Resistance1 in Arabidopsis.

    PubMed

    Carella, P; Isaacs, M; Cameron, R K

    2015-03-01

    Systemic acquired resistance (SAR) is a plant defence response that provides immunity to distant uninfected leaves after an initial localised infection. The lipid transfer protein (LTP) Defective in Induced Resistance1 (DIR1) is an essential component of SAR that moves from induced to distant leaves following a SAR-inducing local infection. To understand how DIR1 is transported to distant leaves during SAR, we analysed DIR1 movement in transgenic Arabidopsis lines with reduced cell-to-cell movement caused by the overexpression of Plasmodesmata-Located Proteins PDLP1 and PDLP5. These PDLP-overexpressing lines were defective for SAR, and DIR1 antibody signals were not observed in phloem sap-enriched petiole exudates collected from distant leaves. Our data support the idea that cell-to-cell movement of DIR1 through plasmodesmata is important during long-distance SAR signalling in Arabidopsis.

  12. Coffee induces breast cancer resistance protein expression in Caco-2 cells.

    PubMed

    Isshiki, Marina; Umezawa, Kazuo; Tamura, Hiroomi

    2011-01-01

    Coffee is a beverage that is consumed world-wide on a daily basis and is known to induce a series of metabolic and pharmacological effects, especially in the digestive tract. However, little is known concerning the effects of coffee on transporters in the gastrointestinal tract. To elucidate the effect of coffee on intestinal transporters, we investigated its effect on expression of the breast cancer resistance protein (BCRP/ABCG2) in a human colorectal cancer cell line, Caco-2. Coffee induced BCRP gene expression in Caco-2 cells in a coffee-dose dependent manner. Coffee treatment of Caco-2 cells also increased the level of BCRP protein, which corresponded to induction of gene expression, and also increased cellular efflux activity, as judged by Hoechst33342 accumulation. None of the major constituents of coffee tested could induce BCRP gene expression. The constituent of coffee that mediated this induction was extractable with ethyl acetate and was produced during the roasting process. Dehydromethylepoxyquinomicin (DHMEQ), an inhibitor of nuclear factor (NF)-κB, inhibited coffee-mediated induction of BCRP gene expression, suggesting involvement of NF-κB in this induction. Our data suggest that daily consumption of coffee might induce BCRP expression in the gastrointestinal tract and may affect the bioavailability of BCRP substrates.

  13. A role for multidrug resistance protein 4 (MRP4; ABCC4) in human dendritic cell migration

    PubMed Central

    van de Ven, Rieneke; Scheffer, George L.; Reurs, Anneke W.; Lindenberg, Jelle J.; Oerlemans, Ruud; Jansen, Gerrit; Gillet, Jean-Pierre; Glasgow, Joel N.; Pereboev, Alexander; Curiel, David T.; Scheper, Rik J.

    2008-01-01

    The capacity of dendritic cells (DCs) to migrate from peripheral organs to lymph nodes (LNs) is important in the initiation of a T cell–mediated immune response. The ATP-binding cassette (ABC) transporters P-glycoprotein (P-gp; ABCB1) and the multidrug resistance protein 1 (MRP1; ABCC1) have been shown to play a role in both human and murine DC migration. Here we show that a more recently discovered family member, MRP4 (ABCC4), is expressed on both epidermal and dermal human skin DCs and contributes to the migratory capacity of DCs. Pharmacological inhibition of MRP4 activity or down-regulation through RNAi in DCs resulted in reduced migration of DCs from human skin explants and of in vitro generated Langerhans cells. The responsible MRP4 substrate remains to be identified as exogenous addition of MRP4's known substrates prostaglandin E2, leukotriene B4 and D4, or cyclic nucleotides (all previously implicated in DC migration) could not restore migration. This notwithstanding, our data show that MRP4 is an important protein, significantly contributing to human DC migration toward the draining lymph nodes, and therefore relevant for the initiation of an immune response and a possible target for immunotherapy. PMID:18625884

  14. Protein adsorption resistance and oxygen permeability of chemically crosslinked phospholipid polymer hydrogel for ophthalmologic biomaterials.

    PubMed

    Goda, Tatsuro; Matsuno, Ryosuke; Konno, Tomohiro; Takai, Madoka; Ishihara, Kazuhiko

    2009-04-01

    The biomimetic structure of a polymer hydrogel bearing phosphorylcholine groups was obtained from 2-methacryloyloxyethylphosphorylcholline (MPC) and a novel crosslinker, 2-(methacryloyloxy)ethyl-N-(2-methacryloyloxy)ethyl]phosphorylcholine (MMPC), to prepare biocompatible ocular materials. MMPC is a dimethacrylate with phosphorylcholine-analogous linkage. Previous reports clarified that the affinity of MMPC to MPC enables the water contents and mechanical properties of the poly(MPC) hydrogels to be varied without disturbing the bulk phases. In this study, we examined the protein adsorption resistance, water wettability, oxygen permeability, and electrolyte permeability of the mechanically enhanced poly(MPC) hydrogel crosslinked with MMPC. The amount of protein adsorbed on this hydrogel was 0.9 microg/cm(2), which accounted for 30% of Omafilcon A and 3% of Etafilcon A. Water contact angle experiments revealed the high wettability of the poly(MPC) hydrogels. The oxygen permeability and NaCl diffusion constant of the poly(MPC) hydrogels were 64 barrer and 48 x 10(-6) cm(2)/s, respectively. This high permeability resulted from the high water content, similar to the case of the human cornea. These results suggested that poly(MPC) hydrogels have good potential for use in ophthalmologic biomaterials.

  15. Mussel-inspired modification of dextran for protein-resistant coatings of titanium oxide.

    PubMed

    Park, Jae Yoon; Kim, Jee Seon; Nam, Yoon Sung

    2013-09-12

    Surface modification of inorganic materials to prevent non-specific protein adsorption is critically important for developing a biocompatible materials' platform for medical implantation, diagnostics, and therapeutics. Here we report mussel-inspired chemical modification of dextran for anti-fouling coatings of metal oxide. Catechols are conjugated to dextran via a carbamate ester linkage, producing catechol-grafted dextran with a grafting density of 7.3 mol.%. Titanium dioxide (TiO₂) is coated with the catechol-grafted dextran, and the anti-fouling effect of dextran coatings is examined by using the adsorption of human serum albumin. The mussel-inspired dextran coatings show excellent resistance to non-specific protein adsorption: the adsorption equilibrium constant (K) is 0.69 Lg(-1) for dextran-coated TiO₂ while that for pristine TiO₂ surface is 3.53 Lg(-1). This study suggests that catechol-grafted dextran is a promising material for effective anti-fouling coatings of implantable inorganic materials.

  16. Muscle strength and hypertrophy occur independently of protein supplementation during short-term resistance training in untrained men.

    PubMed

    Boone, Carleigh H; Stout, Jeffrey R; Beyer, Kyle S; Fukuda, David H; Hoffman, Jay R

    2015-08-01

    Short-term resistance training has consistently demonstrated gains in muscular strength, but not hypertrophy. Post-resistance training protein ingestion is posited to augment the acute anabolic stimulus, thus potentially accelerating changes in muscle size and strength. The purpose of this investigation was to examine the effects of 4 weeks of resistance training with protein supplementation on strength and muscle morphology changes in untrained men. Participants (mean ± SD; N = 18; age, 22.0 ± 2.5 years; body mass index, 25.1 ± 5.4 kg · m(-2)) were randomly assigned to a resistance training + protein group (n = 9; whey (17 g) + colostrum (3 g) + leucine (2 g)) or a resistance training + placebo group (n = 9). One-repetition maximum (1RM) strength in the leg press (LP) and leg extension (LE) exercises, maximal isometric knee extensor strength (MVIC), and muscle morphology (thickness (MT), cross-sectional area (CSA), pennation angle) of the dominant rectus femoris (RF) and vastus lateralis (VL) was assessed before and after training. Participants performed LP and LE exercises (3 × 8-10; at 80% 1RM) 3 days/week for 4 weeks. Data were analyzed using 2-way ANOVA with repeated measures. Four weeks of resistance training resulted in significant increases in LP (p < 0.001), LE (p < 0.001), MVIC (p < 0.001), RF MT (p < 0.001), RF CSA (p < 0.001), VL MT (p < 0.001), and VL CSA (p < 0.001). No between-group differences were observed. Although nutrition can significantly affect training adaptations, these results suggest that short-term resistance training augments muscle strength and size in previously untrained men with no additive benefit from postexercise protein supplementation.

  17. Structure and Function of the Escherichia coli Protein YmgB: A Protein Critical for Biofilm Formation and Acid-resistance

    SciTech Connect

    Lee,J.; Page, R.; Garcia-Contreras, R.; Palermino, J.; Zhang, X.; Doshi, O.; Wood, T.; Peti, W.

    2007-01-01

    The Escherichia coli gene cluster ymgABC was identified in transcriptome studies to have a role in biofilm development and stability. In this study, we showed that YmgB represses biofilm formation in rich medium containing glucose, decreases cellular motility, and protects the cell from acid indicating that YmgB has a major role in acid-resistance in E. coli. Our data show that these phenotypes are potentially mediated through interactions with the important cell signal indole. In addition, gel mobility-shift assays suggest that YmgB may be a non-specific DNA-binding protein. Using nickel-enrichment DNA microarrays, we showed that YmgB binds, either directly or indirectly, via a probable ligand, genes important for biofilm formation. To advance our understanding of the function of YmgB, we used X-ray crystallography to solve the structure of the protein to 1.8 A resolution. YmgB is a biological dimer that is structurally homologous to the E. coli gene regulatory protein Hha, despite having only 5% sequence identity. This supports our DNA microarray data showing that YmgB is a gene regulatory protein. Therefore, this protein, which clearly has a critical role in acid-resistance in E. coli, has been renamed as AriR for regulator of acid resistance influenced by indole.

  18. Correlation between Resistance of Pseudomonas aeruginosa to Quaternary Ammonium Compounds and Expression of Outer Membrane Protein OprR

    PubMed Central

    Tabata, Atsushi; Nagamune, Hideaki; Maeda, Takuya; Murakami, Keiji; Miyake, Yoichiro; Kourai, Hiroki

    2003-01-01

    The adaptation mechanism of Pseudomonas aeruginosa ATCC 10145 to quaternary ammonium compounds (QACs) was investigated. A P. aeruginosa strain with adapted resistance to QACs was developed by a standard broth dilution method. It was revealed that P. aeruginosa exhibited remarkable resistance to N-dodecylpyridinium iodide (P-12), whose structure is similar to that of a common disinfectant, cetylpyridinium chloride. Adapted resistance to benzalkonium chloride (BAC), which is commonly used as a disinfectant, was also observed in P. aeruginosa. Moreover, the P-12-resistant strain exhibited cross-resistance to BAC. Analysis of the outer membrane protein of the P-12-resistant strain by two-dimensional polyacrylamide gel electrophoresis showed a significant increase in the level of expression of a protein (named OprR) whose molecular mass was approximately 26 kDa. The actual function of OprR is not yet clear; however, OprR was expected to be an outer membrane-associated protein with homology to lipoproteins of other bacterial species, according to a search of the National Center for Biotechnology Information website with the BLAST program by use of the N-terminal sequence of OprR. A correlation between the level of expression of OprR and the level of resistance of P. aeruginosa to QACs was observed by using a PA2800 gene knockout mutant derived from the P-12-resistant strain. The knockout mutant recovered susceptibility not only to P-12 but also to BAC. These results suggested that OprR significantly participated in the adaptation of P. aeruginosa to QACs, such as P-12 and BAC. PMID:12821452

  19. Protein supplementation with low fat meat after resistance training: effects on body composition and strength.

    PubMed

    Negro, Massimo; Vandoni, Matteo; Ottobrini, Sara; Codrons, Erwan; Correale, Luca; Buonocore, Daniela; Marzatico, Fulvio

    2014-08-04

    Beef is a nutrient-rich, high-quality protein containing all the essential amino acids in proportions similar to those found in human skeletal muscle. In order to investigate the efficacy of a beef supplementation strategy on strength and body composition, we recruited 26 young healthy adults to participate in a resistance-training program of eight weeks, based on the use of isotonic machines and free weights at 75% of one repetition maximum. Subjects were randomly divided into two groups, food group and control group, of 12 and 14 subjects respectively. Food group were supplemented after resistance training with a 135 g serving of lean beef (tinned meat), providing 20 g of protein and 1.7 g of fat. No supplementation was provided to control group. Fat mass, fat free mass, lean mass, assessed by bioelectrical impedance analyzer, and muscle strength, assessed by one repetition maximum test, were evaluated in all subjects both at the beginning (week 0) and at the end (week 8) of the study. Pre- and post-training differences were evaluated with paired t-tests while group differences for each outcome parameter was evaluated with independent t-tests. At the end of the study the food group showed a significantly decrease in fat mass (week 0: 15.0 ± 6.7 kg; week 8: 13.1 ± 7.6 kg; Δ: -1.9 ± 2.9 kg; p < 0.05) and a significantly increase in fat free mass (week 0: 52.8 kg ± 9.4; week 8: 55.1 kg ± 10.9; Δ: 2.3 ± 2.5 kg; p < 0.01). No significant differences in lean mass were found in either food group or control group. No significant differences in one repetition maximum tests were found between food group and control group. Tinned meat can be considered a nutrition strategy in addition to other proteins or amino acid supplements, but as with any other supplementation strategy, a proper nutrition plan must be coupled.

  20. Intestinal Ciprofloxacin Efflux: The Role of Breast Cancer Resistance Protein (ABCG2)

    PubMed Central

    Wright, J. A.; O'Reilly, D. A.; Sherlock, D. J.; Coleman, T.; Simmons, N. L.

    2011-01-01

    Intestinal secretory movement of the fluoroquinolone antibiotic, ciprofloxacin, may limit its oral bioavailability. Active ATP-binding cassette (ABC) transporters such as breast cancer resistance protein (BCRP) have been implicated in ciprofloxacin transport. The aim of this study was to test the hypothesis that BCRP alone mediates intestinal ciprofloxacin secretion. The involvement of ABC transport proteins in ciprofloxacin secretory flux was investigated with the combined use of transfected cell lines [bcrp1/BCRP-Madin-Darby canine kidney II (MDCKII) and multidrug resistance-related protein 4 (MRP4)-human embryonic kidney (HEK) 293] and human intestinal Caco-2 cells, combined with pharmacological inhibition using 3-(6-isobutyl-9-methoxy-1,4-dioxo-1,2,3,4,6, 7,12,12a-octahydropyrazino[1′,2′:1,6]pyrido[3,4-b]indol-3-yl)-propionic acid tert-butyl ester (Ko143), cyclosporine, 3-[[3-[2-(7-chloroquinolin-2-yl)vinyl]phenyl]-(2-dimethylcarbamoylethylsulfanyl)methylsulfanyl] propionic acid (MK571), and verapamil as ABC-selective inhibitors. In addition, the regional variation in secretory capacity was investigated using male Han Wistar rat intestine mounted in Ussing chambers, and the first indicative measurements of ciprofloxacin transport by ex vivo human jejunum were made. Active, Ko143-sensitive ciprofloxacin secretion was observed in bcrp1-MDCKII cell layers, but in low-passage (BCRP-expressing) Caco-2 cell layers only a 54% fraction was Ko143-sensitive. Ciprofloxacin accumulation was lower in MRP4-HEK293 cells than in the parent line, indicating that ciprofloxacin is also a substrate for this transporter. Ciprofloxacin secretion by Caco-2 cell layers was not inhibited by MK571. Secretory flux showed marked regional variability in the rat intestine, increasing from the duodenum to peak in the ileum. Ciprofloxacin secretion was present in human jejunum and was reduced by Ko143 but showed marked interindividual variability. Ciprofloxacin is a substrate for human

  1. Daily Overfeeding from Protein and/or Carbohydrate Supplementation for Eight Weeks in Conjunction with Resistance Training Does not Improve Body Composition and Muscle Strength or Increase Markers Indicative of Muscle Protein Synthesis and Myogenesis in Resistance-Trained Males

    PubMed Central

    Spillane, Mike; Willoughby, Darryn S.

    2016-01-01

    This study determined the effects of heavy resistance training and daily overfeeding with carbohydrate and/or protein on blood and skeletal muscle markers of protein synthesis (MPS), myogenesis, body composition, and muscle performance. Twenty one resistance-trained males were randomly assigned to either a protein + carbohydrate [HPC (n = 11)] or a carbohydrate [HC (n = 10)] supplement group in a double-blind fashion. Body composition and muscle performance were assessed, and venous blood samples and muscle biopsies were obtained before and after eight weeks of resistance training and supplementation. Data were analyzed by two-way ANOVA (p ≤ 0.05). Total body mass, body water, and fat mass were significantly increased in both groups in response to resistance training, but not supplementation (p < 0.05); however, lean mass was not significantly increased in either group (p = 0.068). Upper- (p = 0.024) and lower-body (p = 0.001) muscle strength and myosin heavy chain (MHC) 1 (p = 0.039) and MHC 2A (p = 0.027) were also significantly increased with resistance training. Serum IGF-1, GH, and HGF were not significantly affected (p > 0.05). Muscle total DNA, total protein, and c-Met were not significantly affected (p > 0.05). In conjunction with resistance training, the peri-exercise and daily overfeeding of protein and/or carbohydrate did not preferentially improve body composition, muscle performance, and markers indicative of MPS and myogenic activation. Key points In response to 56 days of heavy resistance training and HC or HPC supplementation, similar increases in muscle mass and strength in both groups occurred; however, the increases were not different between supplement groups. The supplementation of HPC had no preferential effect on augmenting serum IGF-1 GH, or HGF. The supplementation of HPC had no preferential effect on augmenting increases in total muscle protein content or the myogenic markers, total DNA and muscle cMet content. In response to 56 days of

  2. Daily Overfeeding from Protein and/or Carbohydrate Supplementation for Eight Weeks in Conjunction with Resistance Training Does not Improve Body Composition and Muscle Strength or Increase Markers Indicative of Muscle Protein Synthesis and Myogenesis in Resistance-Trained Males.

    PubMed

    Spillane, Mike; Willoughby, Darryn S

    2016-03-01

    This study determined the effects of heavy resistance training and daily overfeeding with carbohydrate and/or protein on blood and skeletal muscle markers of protein synthesis (MPS), myogenesis, body composition, and muscle performance. Twenty one resistance-trained males were randomly assigned to either a protein + carbohydrate [HPC (n = 11)] or a carbohydrate [HC (n = 10)] supplement group in a double-blind fashion. Body composition and muscle performance were assessed, and venous blood samples and muscle biopsies were obtained before and after eight weeks of resistance training and supplementation. Data were analyzed by two-way ANOVA (p ≤ 0.05). Total body mass, body water, and fat mass were significantly increased in both groups in response to resistance training, but not supplementation (p < 0.05); however, lean mass was not significantly increased in either group (p = 0.068). Upper- (p = 0.024) and lower-body (p = 0.001) muscle strength and myosin heavy chain (MHC) 1 (p = 0.039) and MHC 2A (p = 0.027) were also significantly increased with resistance training. Serum IGF-1, GH, and HGF were not significantly affected (p > 0.05). Muscle total DNA, total protein, and c-Met were not significantly affected (p > 0.05). In conjunction with resistance training, the peri-exercise and daily overfeeding of protein and/or carbohydrate did not preferentially improve body composition, muscle performance, and markers indicative of MPS and myogenic activation. Key pointsIn response to 56 days of heavy resistance training and HC or HPC supplementation, similar increases in muscle mass and strength in both groups occurred; however, the increases were not different between supplement groups.The supplementation of HPC had no preferential effect on augmenting serum IGF-1 GH, or HGF.The supplementation of HPC had no preferential effect on augmenting increases in total muscle protein content or the myogenic markers, total DNA and muscle cMet content.In response to 56 days of a

  3. The natural resistance-associated macrophage protein from the protozoan parasite Perkinsus marinus mediates iron uptake.

    PubMed

    Lin, Zhuoer; Fernández-Robledo, José-Antonio; Cellier, Mathieu F M; Vasta, Gerardo R

    2011-07-26

    Microbial pathogens succeed in acquiring essential metals such as iron and manganese despite their limited availability because of the host's immune response. The eukaryotic natural resistance-associated macrophage proteins mediate uptake of divalent metals and, during infection, may compete directly for metal acquisition with the pathogens' transporters. In this study, we characterize the Nramp gene family of Perkinsus marinus, an intracellular parasite of the eastern oyster, and through yeast complementation, we demonstrate for the first time for a protozoan parasite that Nramp imports environmental Fe. Three PmNramp isogenes differ in their exon-intron structures and encode transcripts that display a trans splicing leader at the 5' end. The protein sequences share conserved properties predicted for the Nramp/Solute carrier 11 (Slc11) family, such as 12-transmembrane segment (TMS) topology (N- and C-termini cytoplasmic) and preferential conservation of four TMS predicted to form a pseudosymmetric proton/metal symport pathway. Yeast fet3fet4 mutant complementation assays showed iron transport activity for PmNramp1 and a fusion chimera of the PmNramp3 hydrophobic core and PmNramp1 N- and C-termini. PmNramp1 site-directed mutagenesis demonstrated that Slc11 invariant and predicted pseudosymmetric motifs (TMS1 Asp-Pro-Gly and TMS6 Met-Pro-His) are key for transport function. PmNramp1 TMS1 mutants D76E, G78A, and D76E/G78A prevented membrane protein expression, while TMS6 M250A, H252Y, and M250A/H252Y specifically abrogated Fe uptake; the TMS6 H252Y mutation also correlates with divergence from Nramp specificity for divalent metals.

  4. Opposite regulation of hepatic breast cancer resistance protein in type 1 and 2 diabetes mellitus.

    PubMed

    He, Lei; Yang, Yuan; Guo, Cen; Yao, Dan; Liu, Hui-hui; Sheng, Jing-jing; Zhou, Wei-ping; Ren, Jin; Liu, Xiao-dong; Pan, Guo-yu

    2014-02-05

    Previous studies on diabetes have reported controversial results with regard to transporters in liver. The present study aimed to explore changes in hepatic breast cancer resistance protein (BCRP) expression and functions, as well as the possible underlying mechanisms, in type 2 diabetic patients, type 1 (streptozotocin-induced), and type 2 (Goto Kakizaki) diabetic rats. Protein and mRNA levels of human (h) and rat (r) BCRP were investigated using Western blot and quantitative polymerase chain reaction analyses. Functions of liver rBCRP were evaluated using rosuvastatin. Sandwich cultured rat hepatocytes (SCRH) were cultured with d-glucose, insulin, or oleic acid for 72 h, and rBCRP mRNA was detected. The effect of oleic acid on rBCRP function in SCRH was also investigated using rosuvastatin. Results showed that liver rBCRP mRNA levels decreased to 20% in type 1 diabetic rats, whereas that in diabetic patients and GK rats significantly increased threefold and twentyfold, respectively. No changes were observed in h/rBCRP protein levels of type 2 diabetic patients and GK rats. The functions of rBCRP significantly declined in type 1 diabetic rats but showed no significant changes in GK rats. The data from SCRH indicated that d-glucose decreased rBCRP mRNA level to 60%. Oleic acid increased rBCRP mRNA in SCRH by approximately eightfold but decreased rBCRP function to 50%. Therefore, h/rBCRP expression and functions were oppositely regulated in type 1 and type 2 diabetes mellitus subjects. Alternations in d-glucose, insulin, and free fatty acid levels in plasma might contribute to the changes in h/rBCRP expression and functions.

  5. Post-Streptococcal Auto-Antibodies Inhibit Protein Disulfide Isomerase and Are Associated with Insulin Resistance

    PubMed Central

    Aran, Adi; Weiner, Karin; Lin, Ling; Finn, Laurel Ann; Greco, Mary Ann; Peppard, Paul; Young, Terry; Ofran, Yanay; Mignot, Emmanuel

    2010-01-01

    Post-streptococcal autoimmunity affects millions worldwide, targeting multiple organs including the heart, brain, and kidneys. To explore the post-streptococcal autoimmunity spectrum, we used western blot analyses, to screen 310 sera from healthy subjects with (33%) and without (67%) markers of recent streptococcal infections [anti-Streptolysin O (ASLO) or anti-DNAse B (ADB)]. A 58 KDa protein, reacting strongly with post-streptococcal sera, was identified as Protein Disulfide Isomerase (PDI), an abundant protein with pleiotropic metabolic, immunologic, and thrombotic effects. Anti-PDI autoantibodies, purified from human sera, targeted similar epitopes in Streptolysin O (SLO, P51-61) and PDI (P328-338). The correlation between post-streptococcal status and anti-human PDI auto-immunity was further confirmed in a total of 2987 samples (13.6% in 530 ASLO positive versus 5.6% in 2457 ASLO negative samples, p<0.0001). Finally, anti-PDI auto-antibodies inhibited PDI-mediated insulin degradation in vitro (n = 90, p<0.001), and correlated with higher serum insulin (14.1 iu/ml vs. 12.2 iu/ml, n = 1215, p = 0.039) and insulin resistance (Homeostatic Model Assessment (HOMA) 4.1 vs. 3.1, n = 1215, p = 0.004), in a population-based cohort. These results identify PDI as a major target of post-streptococcal autoimmunity, and establish a new link between infection, autoimmunity, and metabolic disturbances. PMID:20886095

  6. Breast cancer resistance protein regulates apical ectoplasmic specialization dynamics stage specifically in the rat testis.

    PubMed

    Qian, Xiaojing; Mruk, Dolores D; Wong, Elissa W P; Cheng, C Yan

    2013-04-01

    Drug transporters determine the bioavailability of drugs in the testis behind the blood-testis barrier (BTB). Thus, they are crucial for male contraceptive development if these drugs (e.g., adjudin) exert their effects behind the BTB. Herein breast cancer resistance protein (Bcrp), an efflux drug transporter, was found to be expressed by both Sertoli and germ cells. Interestingly, Bcrp was not a component of the Sertoli cell BTB. Instead, it was highly expressed by peritubular myoid cells at the tunica propria and also endothelial cells of the microvessels in the interstitium at all stages of the epithelial cycle. Unexpectedly, Bcrp was found to be expressed at the Sertoli-step 18-19 spermatid interface but limited to stage VI-early VIII tubules, and an integrated component of the apical ectoplasmic specialization (apical ES). Apparently, Bcrp is being used by late-stage spermatids to safeguard their completion of spermiogenesis by preventing harmful drugs to enter these cells while they transform to spermatozoa. Also, the association of Bcrp with actin, Eps8 (epidermal growth factor receptor pathway substrate 8, an actin barbed end capping and bundling protein), and Arp3 (actin-related protein 3, a component of the Arp2/3 complex known to induce branched actin polymerization) at the apical ES suggest that Bcrp may be involved in regulating the organization of actin filament bundles at the site. Indeed, a knockdown of Bcrp by RNAi in the testis perturbed the apical ES function, disrupting spermatid polarity and adhesion. In summary, Bcrp is a regulator of the F-actin-rich apical ES in the testis.

  7. Differential expression of cardiac muscle mitochondrial matrix proteins in broilers from ascites-resistant and susceptible lines.

    PubMed

    Cisar, C R; Balog, J M; Anthony, N B; Donoghue, A M

    2005-05-01

    Ascites is a metabolic disorder of modern broilers that is distinguished by cardiopulmonary insufficiency in the face of intense oxygen demands of rapidly growing tissues. Broilers with ascites exhibit sustained elevation of pulmonary arterial pressure and right ventricular hypertrophy, the end result of which is heart failure. It has been shown that mitochondrial function is impaired in broilers with ascites. In the current study, mitochondrial matrix protein levels were compared between ascites-resistant line broilers and ascites-susceptible line broilers with and without ascites using two-dimensional (2-D) gel electrophoresis. One hundred seventy-two protein spots were detected on the gels, and 9 of the spots were present at different levels in the 4 groups of broilers. These 9 protein spots were selected for identification by mass spectrometry. Two of the spots were found to contain single mitochondrial matrix proteins. Both mitochondrial matrix proteins, the dihydrolipoamide succinyltransferase component of the 2-oxoglutarate dehydrogenase complex and the alpha-subunit of mitochondrial trifunctional enzyme, were present at higher levels in ascites-resistant line broilers with ascites in the present study. The elevated levels of 2 key proteins in aerobic metabolism in ascites-resistant line broilers with ascites observed in the present study suggests that the mitochondria of broilers with this disease may respond inappropriately to hypoxia.

  8. Mitogen-activated protein kinase phosphatase 1 is involved in tamoxifen resistance in MCF7 cells.

    PubMed

    Ma, Gang; Pan, Yixia; Zhou, Can; Sun, Ruifang; Bai, Jingjing; Liu, Peijun; Ren, Yu; He, Jianjun

    2015-11-01

    Tamoxifen resistance is a major clinical problem for ER-positive breast cancer, but the underlying mechanism is not completely elucidated. In the present study, we reported that mitogen-activated protein kinase (MAPK) phosphatase 1 (MKP-1), a member of the family of MKPs, is involved in tamoxifen resistance. We found that MKP1 expression increased in tamoxifen resistant MCF7 cells. To explore the possible role of MKP1 in tamoxifen resistance, siRNA targeting MKP1 was transfected into tamoxifen resistant MCF7 cells. To our surprise, knockdown of MKP-1 promoted cell death induced by tamoxifen. On the other hand, the MKP1 overexpressed MCF7 cell clone was established and MKP1 overexpression effectively attenuated MCF7 cell death induced by tamoxifen. In addition, we revealed that MKP1 inhibited tamoxifen‑mediated JNK activation in tamoxifen resistant MCF7 and MCF7 cells, and by this mechanism MKP1 was able to inhibit tamoxifen-induced cell death. We also showed that combined appliaction of MKP1 inhibitor triptolide and tamoxifen can effectively increase tamoxifen sensitivity in tamoxifen resistant MCF7 cells. Collectively, our results indicated that MKP-1 can attenuate tamoxifen-induced cell death through inhibiting the JNK signal pathway, which represents a novel mechanism of tamoxifen resistance in MCF7 cells.

  9. A potato pathogenesis-related protein gene, StPRp27, contributes to race-nonspecific resistance against Phytophthora infestans.

    PubMed

    Shi, Xiaolei; Tian, Zhendong; Liu, Jun; van der Vossen, Edwin A G; Xie, Conghua

    2012-02-01

    Late blight caused by Phytophthora infestans is the most important disease of potato. Many efforts have been made to understand molecular mechanism of the durable resistance to address the challenge raised by rapid evolution of the pathogen. A pathogenesis related protein (PR) gene StPRp27 was previously isolated from the potato leaves challenged by P. infestans. The sequence analysis and expression pattern reveal that StPRp27 may be associated with resistance to P. infestans. In present research, transient expression of StPRp27 in Nicotiana benthamiana enhanced resistance to P. infestans isolates 99189 and PY23 indicating its potential contribution to the disease resistance. These findings were also confirmed by over-expression of StPRp27 in potato cv. E-potato 3, which significantly slowed down the development of the disease after inoculation with a mixture of P. infestans races. Further, silencing of StPRp27 homologous genes in N. benthamiana harboring dominant Phytophthora resistance gene Rpi-blb1 or Rpi-blb2 showed no effects on the resistance triggered by these R genes. Our results suggest that StPRp27 contributes to a race-nonspecific resistance against P. infestans by inhibiting the disease development and has a potential use in selection and breeding for durable resistance to late blight.

  10. Multidrug resistance proteins: role of P-glycoprotein, MRP1, MRP2, and BCRP (ABCG2) in tissue defense

    SciTech Connect

    Leslie, Elaine M.; Deeley, Roger G.; Cole, Susan P.C. . E-mail: coles@post.queensu.ca

    2005-05-01

    In tumor cell lines, multidrug resistance is often associated with an ATP-dependent decrease in cellular drug accumulation which is attributed to the overexpression of certain ATP-binding cassette (ABC) transporter proteins. ABC proteins that confer drug resistance include (but are not limited to) P-glycoprotein (gene symbol ABCB1), the multidrug resistance protein 1 (MRP1, gene symbol ABCC1), MRP2 (gene symbol ABCC2), and the breast cancer resistance protein (BCRP, gene symbol ABCG2). In addition to their role in drug resistance, there is substantial evidence that these efflux pumps have overlapping functions in tissue defense. Collectively, these proteins are capable of transporting a vast and chemically diverse array of toxicants including bulky lipophilic cationic, anionic, and neutrally charged drugs and toxins as well as conjugated organic anions that encompass dietary and environmental carcinogens, pesticides, metals, metalloids, and lipid peroxidation products. P-glycoprotein, MRP1, MRP2, and BCRP/ABCG2 are expressed in tissues important for absorption (e.g., lung and gut) and metabolism and elimination (liver and kidney). In addition, these transporters have an important role in maintaining the barrier function of sanctuary site tissues (e.g., blood-brain barrier, blood-cerebral spinal fluid barrier, blood-testis barrier and the maternal-fetal barrier or placenta). Thus, these ABC transporters are increasingly recognized for their ability to modulate the absorption, distribution, metabolism, excretion, and toxicity of xenobiotics. In this review, the role of these four ABC transporter proteins in protecting tissues from a variety of toxicants is discussed. Species variations in substrate specificity and tissue distribution of these transporters are also addressed since these properties have implications for in vivo models of toxicity used for drug discovery and development.

  11. Nonsense Mediated Decay Resistant Mutations Are a Source of Expressed Mutant Proteins in Colon Cancer Cell Lines with Microsatellite Instability

    PubMed Central

    Williams, David S.; Bird, Matthew J.; Jorissen, Robert N.; Yu, Yen Lin; Walker, Franscesa; Zhang, Hui Hua; Nice, Edouard C.; Burgess, Antony W.

    2010-01-01

    Background Frameshift mutations in microsatellite instability high (MSI-High) colorectal cancers are a potential source of targetable neo-antigens. Many nonsense transcripts are subject to rapid degradation due to nonsense-mediated decay (NMD), but nonsense transcripts with a cMS in the last exon or near the last exon-exon junction have intrinsic resistance to nonsense-mediated decay (NMD). NMD-resistant transcripts are therefore a likely source of expressed mutant proteins in MSI-High tumours. Methods Using antibodies to the conserved N-termini of predicted mutant proteins, we analysed MSI-High colorectal cancer cell lines for examples of naturally expressed mutant proteins arising from frameshift mutations in coding microsatellites (cMS) by immunoprecipitation and Western Blot experiments. Detected mutant protein bands from NMD-resistant transcripts were further validated by gene-specific short-interfering RNA (siRNA) knockdown. A genome-wide search was performed to identify cMS-containing genes likely to generate NMD-resistant transcripts that could encode for antigenic expressed mutant proteins in MSI-High colon cancers. These genes were screened for cMS mutations in the MSI-High colon cancer cell lines. Results Mutant protein bands of expected molecular weight were detected in mutated MSI-High cell lines for NMD-resistant transcripts (CREBBP, EP300, TTK), but not NMD-sensitive transcripts (BAX, CASP5, MSH3). Expression of the mutant CREBBP and EP300 proteins was confirmed by siRNA knockdown. Five cMS-bearing genes identified from the genome-wide search and without existing mutation data (SFRS12IP1, MED8, ASXL1, FBXL3 and RGS12) were found to be mutated in at least 5 of 11 (45%) of the MSI-High cell lines tested. Conclusion NMD-resistant transcripts can give rise to expressed mutant proteins in MSI-High colon cancer cells. If commonly expressed in primary MSI-High colon cancers, MSI-derived mutant proteins could be useful as cancer specific immunological

  12. TaCPK2-A, a calcium-dependent protein kinase gene that is required for wheat powdery mildew resistance enhances bacterial blight resistance in transgenic rice.

    PubMed

    Geng, Shuaifeng; Li, Aili; Tang, Lichuan; Yin, Lingjie; Wu, Liang; Lei, Cailin; Guo, Xiuping; Zhang, Xin; Jiang, Guanghuai; Zhai, Wenxue; Wei, Yuming; Zheng, Youliang; Lan, Xiujin; Mao, Long

    2013-08-01

    Calcium-dependent protein kinases (CPKs) are important Ca2+ signalling components involved in complex immune and stress signalling networks; but the knowledge of CPK gene functions in the hexaploid wheat is limited. Previously, TaCPK2 was shown to be inducible by powdery mildew (Blumeria graminis tritici, Bgt) infection in wheat. Here, its functions in disease resistance are characterized further. This study shows the presence of defence-response and cold-response cis-elements on the promoters of the A subgenome homoeologue (TaCPK2-A) and D subgenome homoeologue (TaCPK2-D), respectively. Their expression patterns were then confirmed by quantitative real-time PCR (qRT-PCR) using genome-specific primers, where TaCPK2-A was induced by Bgt treatment while TaCPK2-D mainly responded to cold treatment. Downregulation of TaCPK2-A by virus-induced gene silencing (VIGS) causes loss of resistance to Bgt in resistant wheat lines, indicating that TaCPK2-A is required for powdery mildew resistance. Furthermore, overexpression of TaCPK2-A in rice enhanced bacterial blight (Xanthomonas oryzae pv. oryzae, Xoo) resistance. qRT-PCR analysis showed that overexpression of TaCPK2-A in rice promoted the expression of OsWRKY45-1, a transcription factor involved in both fungal and bacterial resistance by regulating jasmonic acid and salicylic acid signalling genes. The opposite effect was found in wheat TaCPK2-A VIGS plants, where the homologue of OsWRKY45-1 was significantly repressed. These data suggest that modulation of WRKY45-1 and associated defence-response genes by CPK2 genes may be the common mechanism for multiple disease resistance in grass species, which may have undergone subfunctionalization in promoters before the formation of hexaploid wheat.

  13. TaCPK2-A, a calcium-dependent protein kinase gene that is required for wheat powdery mildew resistance enhances bacterial blight resistance in transgenic rice

    PubMed Central

    Geng, Shuaifeng; Li, Aili; Tang, Lichuan; Lan, Xiujin; Mao, Long

    2013-01-01

    Calcium-dependent protein kinases (CPKs) are important Ca2+ signalling components involved in complex immune and stress signalling networks; but the knowledge of CPK gene functions in the hexaploid wheat is limited. Previously, TaCPK2 was shown to be inducible by powdery mildew (Blumeria graminis tritici, Bgt) infection in wheat. Here, its functions in disease resistance are characterized further. This study shows the presence of defence-response and cold-response cis-elements on the promoters of the A subgenome homoeologue (TaCPK2-A) and D subgenome homoeologue (TaCPK2-D), respectively. Their expression patterns were then confirmed by quantitative real-time PCR (qRT-PCR) using genome-specific primers, where TaCPK2-A was induced by Bgt treatment while TaCPK2-D mainly responded to cold treatment. Downregulation of TaCPK2-A by virus-induced gene silencing (VIGS) causes loss of resistance to Bgt in resistant wheat lines, indicating that TaCPK2-A is required for powdery mildew resistance. Furthermore, overexpression of TaCPK2-A in rice enhanced bacterial blight (Xanthomonas oryzae pv. oryzae, Xoo) resistance. qRT-PCR analysis showed that overexpression of TaCPK2-A in rice promoted the expression of OsWRKY45-1, a transcription factor involved in both fungal and bacterial resistance by regulating jasmonic acid and salicylic acid signalling genes. The opposite effect was found in wheat TaCPK2-A VIGS plants, where the homologue of OsWRKY45-1 was significantly repressed. These data suggest that modulation of WRKY45-1 and associated defence-response genes by CPK2 genes may be the common mechanism for multiple disease resistance in grass species, which may have undergone subfunctionalization in promoters before the formation of hexaploid wheat. PMID:23918959

  14. Monocytic differentiation and synthesis of proteins associated with apoptosis in human leukemia U-937 cells acquiring resistance to vincristine.

    PubMed

    Pantazis, P; Chatterjee, D; Han, Z; Wyche, J; DeJesus, A; Giovanella, B

    1996-07-01

    Human leukemia U-937/WT cells were exposed to stepwise increased concentrations of Vincristine so that Vincristine-resistant cell sublines (termed U-937/RV) were developed. Established U-937/RV cell sublines have continuously propagated over a year, both in absence and presence of VCR, and have demonstrated similar features. In contrast to U-937/WT cells, U-937/RV cells have longer doubling time, and are more differentiated as determined by appearance of distinct morphological features and synthesis of mRNA that codes for the monocyte colony-stimulating factor-1 receptor (c-fms). Both apoptosis-suppressing Bcl-2 and Bcl-XL proteins were undectable in U-937/WT cells, whereas Bcl-2 was nearly detectable and Bcl-XL readily detectable in U-937/RV cells. The apoptosis-promoting Bax protein was also absent in U-937/WT cells and readily detected in U-937/RV cells. Vincristine-resistant cells with different levels of resistance synthesize similar levels of c-fms mRNA and Bax protein. Finally, unlike U-937/WT cells, U-937/RV cells have no ability to induce tumors when xenografted in immunodeficient mice. The findings collectively suggest that development of resistance to Vincristine in U-937/WT cells may correlate with cell differentiation and synthesis of proteins that regulate apoptosis.

  15. Physiological quality and gene expression related to heat-resistant proteins at different stages of development of maize seeds.

    PubMed

    Andrade, T; Von Pinho, E V R; Von Pinho, R G; Oliveira, G E; Andrade, V; Fernandes, J S

    2013-09-13

    We quantified and characterized the expression of heat-resistant proteins during seed development of maize lines with distinct levels of tolerance to high drying temperature. A corn field was planted for multiplication of seeds of different lines, two tolerant and two non-tolerant to high drying temperatures. Harvest of the seeds was carried out at various stages of development and they were then subjected to tests of moisture content, germination, first count of germination, accelerated aging, and cold test. The seeds were stored in a freezer for later analysis of expression of heat-resistant proteins by means of real-time PCR, electrophoresis, and spectrophotometry. We observed that heat-resistant proteins are expressed in a differential manner in seeds from different lines and at different stages of development. The expression of heat-resistant proteins was earlier in lines tolerant to high drying temperatures. Greater germination and vigor values was found for seeds collected at the last stage of development.

  16. PROTEOMIC ANALYSIS OF UBIQUITINATED PROTEINS FROM DELTAMETHRIN-RESISTANT AND SUSCEPTIBLE STRAINS OF THE DIAMONDBACK MOTH, Plutella Xylostella L.

    PubMed

    Cheng, Luogen; Du, Yaqiong; Hu, Junli; Jiao, Dongxu; Li, Jin; Zhou, Zhou; Xu, Qin; Li, Fengliang

    2015-10-01

    Ubiquitin, a small protein consisting of 76 amino acids, acts in protein degradation, DNA repair, signal transduction, transcriptional regulation, and receptor control through endocytosis. Using proteomics, we compared the differentially ubiquitinated proteins between a deltamethrin-resistant (DR) strain and a deltamethrin-sensitive (DS) strain in third-instar larvae of the diamondback moth. We used polyubiquitin affinity beads to enrich ubiquitinated proteins and then performed one-dimensional SDS-PAGE separation and mass spectrometric identification. In the DR strain, We found 17 proteins that were upregulated (relative to the DS strain), including carbonic anhydrase family members, ADP ribosylation factor 102F CG11027-PA, protein kinase 61C, phospholipase A2 , dihydrolipoamide dehydrogenase, tyrosine hydroxylase, and heat shock proteins, and five proteins that were downregulated in the DS strain, including carboxylesterase and DNA cytosine-5 methyltransferase. These results were also verified by qPCR. The differentially ubiquitinated proteins/enzymes were mainly responsible for protein binding, catalytic activity, and molecular transducer activity. These results improve our understanding of the relationship between protein ubiquitination and the deltamethrin stress response.

  17. Characterization of site-specific glycosylation of secreted proteins associated with multi-drug resistance of gastric cancer

    PubMed Central

    Li, Ting; Cheng, Kai; Dong, Jiaqiang; Tian, Miaomiao; Chai, Na; Guo, Hao; Li, Jinjing; You, Xin; Dong, Mingming; Ye, Mingliang; Nie, Yongzhan; Zou, Hanfa; Fan, Daiming

    2016-01-01

    Multi-drug resistance (MDR) remains a great obstacle to effective chemotherapy for gastric cancer. A number of secreted glycoproteins have been reported to be involved in the development of MDR in gastric cancer. However, whether glycosylation of secreted glycoproteins changes during MDR of gastric cancer is unclear. Our present work manifested that N-glycosites and site-specific glycoforms of secreted proteins in drug-resistant cell lines were distinctly different from those in the parental cell line for the first time. Further characterization highlighted the significance of some aberrantly glycosylated secretory proteins in MDR, suggesting that manipulating the glycosylation of specific glycoproteins could be a potential target for overcoming multi-drug resistance in gastric cancer. PMID:27015365

  18. Fabrication of protein-resistant blend based on PVDF-HFP and amphiphilic brush copolymer made from PMMA and PEGMA

    NASA Astrophysics Data System (ADS)

    Hwangbo, Kyung-Hee; Kim, Yu-Jeong; Cho, Kuk Young

    2012-12-01

    Polymeric blends provide a facile route to obtaining materials with various synergistic properties arising from the individual components. Poly(vinylidene fluoride-co-hexafluoropropylene) (PVDF-HFP), a hydrophobic polymer, is finding new applications in polymer electrolytes, membranes, and heat-resistant structural materials owing to its high thermal stability, mechanical strength, and weatherability. In this report, blends of PVDF-HFP and polymer brush were prepared with enhanced water uptake and protein resistance, which are important requirements for membranes used in food and biological applications. Polymer brush is composed of poly(methyl methacrylate) main chains, which are miscible with PVDF-HFP, and hydrophilic poly(ethylene glycol) (PEG) brush chains. Incorporation of PEG chains through the polymer brush structure not only enhanced water uptake and protein adsorption resistance but also produced a well-distributed morphology of the blending components through the matrix as evidenced by observation of the morphology after selective extraction of polymer brush from the matrix.

  19. Expression of a truncated form of ribosomal protein L3 confers resistance to pokeweed antiviral protein and the Fusarium mycotoxin deoxynivalenol.

    PubMed

    Di, Rong; Tumer, Nilgun E

    2005-08-01

    The contamination of important agricultural products such as wheat, barley, or maize with the trichothecene mycotoxin deoxynivalenol (DON) due to infection with Fusarium species is a worldwide problem. Trichothecenes inhibit protein synthesis by targeting ribosomal protein L3. Pokeweed antiviral protein (PAP), a ribosome-inactivating protein binds to L3 to depurinate the alpha-sarcin/loop of the large rRNA. Plants transformed with the wild-type PAP show lesions and express very low levels of PAP because PAP autoregulates its expression by destabilizing its own mRNA. We show here that transgenic tobacco plants expressing both the wild-type PAP and a truncated form of yeast L3 (L3delta) are phenotypically normal. PAP mRNA and protein levels are very high in these plants, indicating that L3delta suppresses the autoregulation of PAP mRNA expression. Ribosomes are not depurinated in the transgenic plants expressing PAP and L3delta, even though PAP is associated with ribosomes. The expression of the endogenous tobacco ribosomal protein L3 is up-regulated in these plants and they are resistant to the Fusarium mycotoxin DON. These results demonstrate that expression of an N-terminal fragment of yeast L3 leads to trans-dominant resistance to PAP and the trichothecene mycotoxin DON, providing evidence that both toxins target L3 by a common mechanism.

  20. The CC domain structure from the wheat stem rust resistance protein Sr33 challenges paradigms for dimerization in plant NLR proteins.

    PubMed

    Casey, Lachlan W; Lavrencic, Peter; Bentham, Adam R; Cesari, Stella; Ericsson, Daniel J; Croll, Tristan; Turk, Dušan; Anderson, Peter A; Mark, Alan E; Dodds, Peter N; Mobli, Mehdi; Kobe, Bostjan; Williams, Simon J

    2016-10-17

    Plants use intracellular immunity receptors, known as nucleotide-binding oligomerization domain-like receptors (NLRs), to recognize specific pathogen effector proteins and induce immune responses. These proteins provide resistance to many of the world's most destructive plant pathogens, yet we have a limited understanding of the molecular mechanisms that lead to defense signaling. We examined the wheat NLR protein, Sr33, which is responsible for strain-specific resistance to the wheat stem rust pathogen, Puccinia graminis f. sp. tritici We present the solution structure of a coiled-coil (CC) fragment from Sr33, which adopts a four-helix bundle conformation. Unexpectedly, this structure differs from the published dimeric crystal structure of the equivalent region from the orthologous barley powdery mildew resistance protein, MLA10, but is similar to the structure of the distantly related potato NLR protein, Rx. We demonstrate that these regions are, in fact, largely monomeric and adopt similar folds in solution in all three proteins, suggesting that the CC domains from plant NLRs adopt a conserved fold. However, larger C-terminal fragments of Sr33 and MLA10 can self-associate both in vitro and in planta, and this self-association correlates with their cell death signaling activity. The minimal region of the CC domain required for both cell death signaling and self-association extends to amino acid 142, thus including 22 residues absent from previous biochemical and structural protein studies. These data suggest that self-association of the minimal CC domain is necessary for signaling but is likely to involve a different structural basis than previously suggested by the MLA10 crystallographic dimer.

  1. Protein arginine methyltransferase 1 may be involved in pregnane x receptor-activated overexpression of multidrug resistance 1 gene during acquired multidrug resistant

    PubMed Central

    Li, Tingting; Kong, Ah-Ng Tony; Ma, Zhiqiang; Liu, Haiyan; Liu, Pinghua; Xiao, Yu; Jiang, Xuehua; Wang, Ling

    2016-01-01

    Purpose Pregnane x receptor (PXR) - activated overexpression of the multidrug resistance 1 (MDR1) gene is an important way for tumor cells to acquire drug resistance. However, the detailed mechanism still remains unclear. In the present study, we aimed to investigate whether protein arginine methyl transferase 1(PRMT1) is involved in PXR - activated overexpression of MDR1 during acquired multidrug resistant. Experimental Design Arginine methyltransferase inhibitor 1 (AMI-1) was used to pharmacologically block PRMT1 in resistant breast cancer cells (MCF7/adr). The mRNA and protein levels of MDR1 were detected by real-time PCR and western blotting analysis. Immunofluorescence microscopy and co-immunoprecipitation were used to investigate the physical interaction between PXR and PRMT1. Then, 136 candidate compounds were screened for PRMT1 inhibitors. Lastly, luciferase reporter gene and nude mice bearing resistant breast cancer xenografts were adopted to investigate the anti-tumor effect of PRMT1 inhibitors when combined with adriamycin. Results AMI-1 significantly suppressed the expression of MDR1 in MCF7/adr cells and increased cells sensitivity of MCF7/adr to adriamycin. Physical interaction between PRMT1 and PXR exists in MCF7/adr cells, which could be disrupted by AMI-1. Those results suggest that PRMT1 may be involved in PXR-activated overexpression of MDR1 in resistant breast cancer cells, and AMI-1 may suppress MDR1 by disrupting the interaction between PRMT1 and PXR. Then, five compounds including rutin, isoquercitrin, salvianolic acid A, naproxen, and felodipline were identified to be PRMT1 inhibitors. Finally, those PRMT1 inhibitors were observed to significantly decrease MDR1 promoter activity in vitro and enhance the antitumor effect of adriamycin in nude mice that bearing resistant breast cancer xenografts. Conclusions PRMT1 may be an important co-activator of PXR in activating MDR1 gene during acquired resistance, and PRMT1 inhibitor combined with

  2. Protein Kinase A Activation Promotes Cancer Cell Resistance to Glucose Starvation and Anoikis

    PubMed Central

    Pirola, Yuri; De Vitto, Humberto; De Palma, Sara; Airoldi, Cristina; Vasso, Michele; Ricciardiello, Francesca; Lombardi, Pietro Paolo; Cirulli, Claudia; Rizzi, Raffaella; Nicotra, Francesco; Hiller, Karsten; Gelfi, Cecilia; Alberghina, Lilia; Chiaradonna, Ferdinando

    2016-01-01

    Cancer cells often rely on glycolysis to obtain energy and support anabolic growth. Several studies showed that glycolytic cells are susceptible to cell death when subjected to low glucose availability or to lack of glucose. However, some cancer cells, including glycolytic ones, can efficiently acquire higher tolerance to glucose depletion, leading to their survival and aggressiveness. Although increased resistance to glucose starvation has been shown to be a consequence of signaling pathways and compensatory metabolic routes activation, the full repertoire of the underlying molecular alterations remain elusive. Using omics and computational analyses, we found that cyclic adenosine monophosphate-Protein Kinase A (cAMP-PKA) axis activation is fundamental for cancer cell resistance to glucose starvation and anoikis. Notably, here we show that such a PKA-dependent survival is mediated by parallel activation of autophagy and glutamine utilization that in concert concur to attenuate the endoplasmic reticulum (ER) stress and to sustain cell anabolism. Indeed, the inhibition of PKA-mediated autophagy or glutamine metabolism increased the level of cell death, suggesting that the induction of autophagy and metabolic rewiring by PKA is important for cancer cellular survival under glucose starvation. Importantly, both processes actively participate to cancer cell survival mediated by suspension-activated PKA as well. In addition we identify also a PKA/Src mechanism capable to protect cancer cells from anoikis. Our results reveal for the first time the role of the versatile PKA in cancer cells survival under chronic glucose starvation and anoikis and may be a novel potential target for cancer treatment. PMID:26978032

  3. Molecular evidence and functional expression of multidrug resistance associated protein (MRP) in rabbit corneal epithelial cells.

    PubMed

    Karla, Pradeep K; Pal, Dananjay; Mitra, Ashim K

    2007-01-01

    Multidrug resistance associated protein (MRP) is a major family of efflux transporters involved in drug efflux leading to drug resistance. The objective of this study was to explore physical barriers for ocular drug absorption and to verify if the role of efflux transporters. MRP-2 is a major homologue of MRP family and found to express on the apical side of cell membrane. Cultured Rabbit Corneal Epithelial Cells (rCEC) were selected as an in vitro model for corneal epithelium. [14C]-erythromycin which is a proven substrate for MRP-2 was selected as a model drug for functional expression studies. MK-571, a known specific and potent inhibitor for MRP-2 was added to inhibit MRP mediated efflux. Membrane fraction of rCEC was used for western blot analysis. Polarized transport of [14C]-erythromycin was observed in rCEC and transport from B-->A was significantly high than from A-->B. Permeability's increased significantly from A-->B in the presence of MK-571 and ketoconozole. Uptake of [14C]-erythromycin in the presence of MK-571 was significantly higher than control in rCEC. RT-PCR analysis indicated a unique and distinct band at approximately 498 bp corresponding to MRP-2 in rCEC and MDCK11-MRP-2 cells. Immunoprecipitation followed by Western Blot analysis indicated a specific band at approximately 190 kDa in membrane fraction of rCEC and MDCK11-MRP-2 cells. For the first time we have demonstrated high expression of MRP-2 in rabbit corneal epithelium and its functional activity causing drug efflux. RT-PCR, immunoprecipitation followed by Western blot analysis further confirms the result.

  4. Fetoprotective activity of breast cancer resistance protein (BCRP, ABCG2): expression and function throughout pregnancy.

    PubMed

    Hahnova-Cygalova, Lenka; Ceckova, Martina; Staud, Frantisek

    2011-02-01

    The medical treatment of pregnant women, as well as their fetuses, has become a common clinical practice in developed countries. Therefore, detailed knowledge of maternofetal pharmacokinetics, including the role of drug-efflux transporters in the fetoplacental unit, is crucial to optimize drug choice and dosage schemes and to avoid or exploit possible drug-drug interactions on placental transporters in order to assure appropriate drug levels in the mother and/or fetus. Breast cancer resistance protein (BCRP, ABCG2) is the most recent member of ATP-binding cassette drug-efflux transporters that has been associated with resistance in cancer chemotherapy. Importantly, ABCG2 has also been localized in various normal tissues, affecting the pharmacokinetics of several xenobiotics as well as a number of physiological substances. Extensive expression of ABCG2 in tissue barriers, such as the blood-brain barrier, intestine, testis, or placenta, suggests that ABCG2 plays an important role in the protection of sensitive tissues against toxins. In the placenta, ABCG2 has been experimentally evidenced to actively pump its substrates in the fetal-to-maternal direction and to play an important role in transplacental pharmacokinetics, fetal protection, and detoxication. Further, ABCG2 expression in embryonic and fetal membranes over the course of pregnancy helps ensure proper function of the fetoplacental unit. In this review, we summarize the current knowledge regarding expression and function of ABCG2 in the fetoplacental unit during the development of the fetus and overview the aspects of transplacental pharmacokinetics, ABCG2 regulation, and clinical significance of the transporter for pharmacotherapy in pregnancy.

  5. Cytosolic activation of cell death and stem rust resistance by cereal MLA-family CC–NLR proteins

    PubMed Central

    Cesari, Stella; Moore, John; Chen, Chunhong; Webb, Daryl; Periyannan, Sambasivam; Mago, Rohit; Bernoux, Maud; Lagudah, Evans S.; Dodds, Peter N.

    2016-01-01

    Plants possess intracellular immune receptors designated “nucleotide-binding domain and leucine-rich repeat” (NLR) proteins that translate pathogen-specific recognition into disease-resistance signaling. The wheat immune receptors Sr33 and Sr50 belong to the class of coiled-coil (CC) NLRs. They confer resistance against a broad spectrum of field isolates of Puccinia graminis f. sp. tritici, including the Ug99 lineage, and are homologs of the barley powdery mildew-resistance protein MLA10. Here, we show that, similarly to MLA10, the Sr33 and Sr50 CC domains are sufficient to induce cell death in Nicotiana benthamiana. Autoactive CC domains and full-length Sr33 and Sr50 proteins self-associate in planta. In contrast, truncated CC domains equivalent in size to an MLA10 fragment for which a crystal structure was previously determined fail to induce cell death and do not self-associate. Mutations in the truncated region also abolish self-association and cell-death signaling. Analysis of Sr33 and Sr50 CC domains fused to YFP and either nuclear localization or nuclear export signals in N. benthamiana showed that cell-death induction occurs in the cytosol. In stable transgenic wheat plants, full-length Sr33 proteins targeted to the cytosol provided rust resistance, whereas nuclear-targeted Sr33 was not functional. These data are consistent with CC-mediated induction of both cell-death signaling and stem rust resistance in the cytosolic compartment, whereas previous research had suggested that MLA10-mediated cell-death and disease resistance signaling occur independently, in the cytosol and nucleus, respectively. PMID:27555587

  6. Cytosolic activation of cell death and stem rust resistance by cereal MLA-family CC-NLR proteins.

    PubMed

    Cesari, Stella; Moore, John; Chen, Chunhong; Webb, Daryl; Periyannan, Sambasivam; Mago, Rohit; Bernoux, Maud; Lagudah, Evans S; Dodds, Peter N

    2016-09-06

    Plants possess intracellular immune receptors designated "nucleotide-binding domain and leucine-rich repeat" (NLR) proteins that translate pathogen-specific recognition into disease-resistance signaling. The wheat immune receptors Sr33 and Sr50 belong to the class of coiled-coil (CC) NLRs. They confer resistance against a broad spectrum of field isolates of Puccinia graminis f. sp. tritici, including the Ug99 lineage, and are homologs of the barley powdery mildew-resistance protein MLA10. Here, we show that, similarly to MLA10, the Sr33 and Sr50 CC domains are sufficient to induce cell death in Nicotiana benthamiana Autoactive CC domains and full-length Sr33 and Sr50 proteins self-associate in planta In contrast, truncated CC domains equivalent in size to an MLA10 fragment for which a crystal structure was previously determined fail to induce cell death and do not self-associate. Mutations in the truncated region also abolish self-association and cell-death signaling. Analysis of Sr33 and Sr50 CC domains fused to YFP and either nuclear localization or nuclear export signals in N benthamiana showed that cell-death induction occurs in the cytosol. In stable transgenic wheat plants, full-length Sr33 proteins targeted to the cytosol provided rust resistance, whereas nuclear-targeted Sr33 was not functional. These data are consistent with CC-mediated induction of both cell-death signaling and stem rust resistance in the cytosolic compartment, whereas previous research had suggested that MLA10-mediated cell-death and disease resistance signaling occur independently, in the cytosol and nucleus, respectively.

  7. The involvement of Bcl-2 family proteins in AKT-regulated cell survival in cisplatin resistant epithelial ovarian cancer

    PubMed Central

    Dai, Yan; Jin, Shiguang; Li, Xueping; Wang, Daxin

    2017-01-01

    Many studies involving patients with cisplatin-resistant ovarian cancer have shown that AKT activation leads to inhibition of apoptosis. The aim of this study was to examine the potential involvement of the Bcl-2 family proteins in AKT-regulated cell survival in response to cisplatin treatment. Cisplatin-sensitive (PEO1) and cisplatin-resistant (PEO4) cells were taken from ascites of patients with ovarian cancer before cisplatin treatment and after development of chemoresistance. It was found that cisplatin treatment activated the AKT signaling pathway and promoted cell proliferation in cisplatin-resistant EOC cells. When AKT was transfected into nucleus of cisplatin-resistant ovarian cancer cells, DNA-PK was phosphorylated at S473. The activated AKT (pAKT-S473) in these cells inhibited the death signal induced by cisplatin thereby inhibiting cisplatin-mediated apoptosis. Results from this study showed that the combination of cisplatin, DNA-PK inhibitor NU7441, and AKT inhibitor TCN can overcome drug resistance, increase apoptosis, and re-sensitize PEO4 cells to cisplatin treatment. A decrease in apoptotic activity was seen in PEO4 cells when Bad was downregulated by siRNA, which indicated that Bad promotes apoptosis in PEO4 cells. Use of the Bcl-2 inhibitor ABT-737 showed that ABT-737 binds to Bcl-2 but not Mcl-1 and releases Bax/Bak which leads to cell apoptosis. The combination of ABT-737 and cisplatin leads to a significant increase in the death of PEO1 and PEO4 cells. All together, these results indicate that Bcl-2 family proteins are regulators of drug resistance. The combination of cisplatin and Bcl-2 family protein inhibitor could be a strategy for the treatment of cisplatin-resistant ovarian cancer. PMID:27935869

  8. Nanoscale Drug Delivery Platforms Overcome Platinum-Based Resistance in Cancer Cells Due to Abnormal Membrane Protein Trafficking

    PubMed Central

    Xue, Xue; Hall, Matthew D.; Zhang, Qiang; Wang, Paul C.; Gottesman, Michael M.; Liang, Xing-Jie

    2014-01-01

    The development of cellular resistance to platinum-based chemotherapies is often associated with reduced intracellular platinum concentrations. In some models, this reduction is due to abnormal membrane protein trafficking, resulting in reduced uptake by transporters at the cell surface. Given the central role of platinum drugs in the clinic, it is critical to overcome cisplatin resistance by bypassing the plasma membrane barrier to significantly increase the intracellular cisplatin concentration enough to inhibit the proliferation of cisplatin-resistant cells. Therefore, rational design of appropriate nanoscale drug delivery platforms (nDDPs) loaded with cisplatin or other platinum analogs as payloads is a possible strategy to solve this problem. This review will focus on the known mechanism of membrane trafficking in cisplatin-resistant cells, and the development and employment of nDDPs to improve cell uptake of cisplatin. PMID:24219825

  9. Nanoscale drug delivery platforms overcome platinum-based resistance in cancer cells due to abnormal membrane protein trafficking.

    PubMed

    Xue, Xue; Hall, Matthew D; Zhang, Qiang; Wang, Paul C; Gottesman, Michael M; Liang, Xing-Jie

    2013-12-23

    The development of cellular resistance to platinum-based chemotherapies is often associated with reduced intracellular platinum concentrations. In some models, this reduction is due to abnormal membrane protein trafficking, resulting in reduced uptake by transporters at the cell surface. Given the central role of platinum drugs in the clinic, it is critical to overcome cisplatin resistance by bypassing the plasma membrane barrier to significantly increase the intracellular cisplatin concentration enough to inhibit the proliferation of cisplatin-resistant cells. Therefore, rational design of appropriate nanoscale drug delivery platforms (nDDPs) loaded with cisplatin or other platinum analogues as payloads is a possible strategy to solve this problem. This review will focus on the known mechanism of membrane trafficking in cisplatin-resistant cells and the development and employment of nDDPs to improve cell uptake of cisplatin.

  10. In Silico Prediction of Inhibition of Promiscuous Breast Cancer Resistance Protein (BCRP/ABCG2)

    PubMed Central

    Ding, Yi-Lung; Shih, Yu-Hsuan; Tsai, Fu-Yuan; Leong, Max K.

    2014-01-01

    Background Breast cancer resistant protein has an essential role in active transport of endogenous substances and xenobiotics across extracellular and intracellular membranes along with P-glycoprotein. It also plays a major role in multiple drug resistance and permeation of blood-brain barrier. Therefore, it is of great importance to derive theoretical models to predict the inhibition of both transporters in the process of drug discovery and development. Hitherto, very limited BCRP inhibition predictive models have been proposed as compared with its P-gp counterpart. Methodology/Principal Findings An in silico BCRP inhibition model was developed in this study using the pharmacophore ensemble/support vector machine scheme to take into account the promiscuous nature of BCRP. The predictions by the PhE/SVM model were found to be in good agreement with the observed values for those molecules in the training set (n = 22, r2 = 0.82,  = 0.73, RMSE  =  0.40, s = 0.24), test set (n = 97, q2 = 0.75–0.89, RMSE  = 0.31, s = 0.21), and outlier set (n = 16, q2 = 0.72–0.91, RMSE  =  0.29, s = 0.17). When subjected to a variety of statistical validations, the developed PhE/SVM model consistently met the most stringent criteria. A mock test by HIV protease inhibitors also asserted its predictivity. Conclusions/Significance It was found that this accurate, fast, and robust PhE/SVM model can be employed to predict the BCRP inhibition of structurally diverse molecules that otherwise cannot be carried out by any other methods in a high-throughput fashion to design therapeutic agents with insignificant drug toxicity and unfavorable drug–drug interactions mediated by BCRP to enhance clinical efficacy and/or circumvent drug resistance. PMID:24614353

  11. Identification of Functional Regulatory Residues of the β -Lactam Inducible Penicillin Binding Protein in Methicillin-Resistant Staphylococcus aureus.

    PubMed

    Mbah, Andreas N; Isokpehi, Raphael D

    2013-01-01

    Resistance to methicillin by Staphylococcus aureus is a persistent clinical problem worldwide. A mechanism for resistance has been proposed in which methicillin resistant Staphylococcus aureus (MRSA) isolates acquired a new protein called β -lactam inducible penicillin binding protein (PBP-2'). The PBP-2' functions by substituting other penicillin binding proteins which have been inhibited by β -lactam antibiotics. Presently, there is no structural and regulatory information on PBP-2' protein. We conducted a complete structural and functional regulatory analysis of PBP-2' protein. Our analysis revealed that the PBP-2' is very stable with more hydrophilic amino acids expressing antigenic sites. PBP-2' has three striking regulatory points constituted by first penicillin binding site at Ser25, second penicillin binding site at Ser405, and finally a single metallic ligand binding site at Glu657 which binds to Zn(2+) ions. This report highlights structural features of PBP-2' that can serve as targets for developing new chemotherapeutic agents and conducting site direct mutagenesis experiments.

  12. Design of heat shock-resistant surfaces to prevent protein aggregation: Enhanced chaperone activity of immobilized α-Crystallin.

    PubMed

    Ray, Namrata; Roy, Sarita; Singha, Santiswarup; Chandra, Bappaditya; Dasgupta, Anjan Kr; Sarkar, Amitabha

    2014-05-21

    α-Crystallin is a multimeric protein belonging to the family of small heat shock proteins, which function as molecular chaperones by resisting heat and oxidative stress induced aggregation of other proteins. We immobilized α-Crystallin on a self-assembled monolayer on glass surface and studied its activity in terms of the prevention of aggregation of aldolase. We discovered that playing with grafted protein density led to interesting variations in the chaperone activity of immobilized α-Crystallin. This result is in accordance with the hypothesis that dynamicity of subunits plays a vital role in the functioning of α-Crystallin and might be able to throw light on the structure-activity relationship. We showed that the chaperone activity of a certain number of immobilized α-Crystallins was superior compared to a solution containing an equivalent number of the protein and 10 times the number of the protein at temperatures >60 °C. The α-Crystallin grafted surfaces retained activity on reuse. This could also lead to the design of potent heat-shock resistant surfaces that can find wide applications in storage and shipping of protein based biopharmaceuticals.

  13. Two Genes Encoding Structurally Different CC-NB-LRR Proteins are Required for Lr10-Mediated Leaf Rust Resistance in Wheat of Two Ploidy Levels

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The gene pools of crop plant relatives have been proposed as a source of new functional resistance genes to broaden the basis of genetic resistance. Here, we have studied the allelic diversity of the Lr10 leaf rust resistance gene, encoding a CC-NBS-LRR protein originally identified in hexaploid bre...

  14. Protein tyrosine phosphatase PTPN3 promotes drug resistance and stem cell-like characteristics in ovarian cancer

    PubMed Central

    Li, Shuqin; Cao, Jian; Zhang, Wei; Zhang, Fan; Ni, Guantai; Luo, Qian; Wang, Man; Tao, Xiang; Xia, Hongping

    2016-01-01

    The current standard treatment for ovarian cancer is aggressive surgery followed by platinum-based combination chemotherapy. Recurrence and chemotherapeutic drug resistance are the two main factors that account for the high mortality of most ovarian cancers. Liposomal doxorubicin is primarily used for the treatment of ovarian cancer when the disease has progressed after platinum-based chemotherapy. However, relatively little is known about the genomic changes that contribute to both cisplatin and doxorubicin resistance in high-grade serous ovarian cancer (HGSC) under the selective pressure of chemotherapy. Here, we found that protein tyrosine phosphatase PTPN3 gene expression was substantially increased in both cisplatin and doxorubicin-resistant ovarian cancer cells. Silencing of PTPN3 restored sensitivity to cisplatin and doxorubicin in resistant ovarian cancer cells. Down-regulation of PTPN3 also inhibited cell cycle progression, migration, stemness in vitro and the tumorigenicity of resistant ovarian cancer cells in vivo. Meanwhile, the expression of PTPN3 was found to be regulated by miR-199 in resistant ovarian cancer cells. These findings suggest that PTPN3 promotes tumorigenicity, stemness and drug resistance in ovarian cancer, and thus is a potential therapeutic target for the treatment of ovarian cancer. PMID:27833130

  15. Isolation and characterisation of two multidrug resistance associated protein genes from maize.

    PubMed

    Swarbreck, David; Ripoll, Pierre-Jean; Brown, Donna A; Edwards, Keith J; Theodoulou, Frederica

    2003-10-02

    Two genes encoding ATP-binding cassette (ABC) transporters were isolated from the crop plant Zea mays (maize). The clones, designated ZmMRP1 and ZmMRP2, were highly homologous to members of the multidrug resistance associated protein (MRP) subfamily. Genomic Southern analysis and characterisation of bacterial artificial chromosome (BAC) clones demonstrated that both genes are present in two copies in maize, which are located in proximity to each other, suggesting the occurrence of duplication events. The full-length genomic and cDNA sequences of ZmMRP1 and 2 were obtained, permitting analysis of the intron/exon structures and protein domains. Intron positions and phasing were conserved between ZmMRP1 and 2 and their closest Arabidopsis homologues. Both clones contained two copies each of the membrane spanning domains and nucleotide-binding folds diagnostic of the ABC superfamily, and ZmMRP1 contained an additional N-terminal membrane-spanning domain (MSD0) that is typical of MRP transporters but which is lacking in the most closely related Arabidopsis and rice MRPs. In contrast, ZmMRP2 and its closest rice but not Arabidopsis homologues lacked MSD0, suggesting the repeated loss of this domain in MRP family evolution. ZmMRP1 and 2 were expressed in all tissues examined but displayed distinct expression profiles in response to herbicide safeners and pro-oxidants. ZmMRP1 was induced by aminotriazole and to a lesser extent by menadione, whereas ZmMRP2 was expressed at a lower constitutive level and did not exhibit strong induction by any of the compounds tested. The characterisation of these clones represents an important step in the experimental analysis of the MRP subfamily in a monocotyledonous crop plant.

  16. Multidrug resistance proteins (MRPs, ABCCs): importance for pathophysiology and drug therapy.

    PubMed

    Keppler, Dietrich

    2011-01-01

    The nine multidrug resistance proteins (MRPs) represent the major part of the 12 members of the MRP/CFTR subfamily belonging to the 48 human ATP-binding cassette (ABC) transporters. Cloning, functional characterization, and cellular localization of most MRP subfamily members have identified them as ATP-dependent efflux pumps with a broad substrate specificity for the transport of endogenous and xenobiotic anionic substances localized in cellular plasma membranes. Prototypic substrates include glutathione conjugates such as leukotriene C(4) for MRP1, MRP2, and MRP4, bilirubin glucuronosides for MRP2 and MRP3, and cyclic AMP and cyclic GMP for MRP4, MRP5, and MRP8. Reduced glutathione (GSH), present in living cells at millimolar concentrations, modifies the substrate specificities of several MRPs, as exemplified by the cotransport of vincristine with GSH by MRP1, or by the cotransport of GSH with bile acids or of GSH with leukotriene B(4) by MRP4.The role of MRP subfamily members in pathophysiology may be illustrated by the MRP-mediated release of proinflammatory and immunomodulatory mediators such as leukotrienes and prostanoids. Pathophysiological consequences of many genetic variants leading to a lack of functional MRP protein in the plasma membrane are observed in the hereditary MRP2 deficiency associated with conjugated hyperbilirubinemia in Dubin-Johnson syndrome, in pseudoxanthoma elasticum due to mutations in the MRP6 (ABCC6) gene, or in the type of human earwax and osmidrosis determined by single nucleotide polymorphisms in the MRP8 (ABCC8) gene. The hepatobiliary and renal elimination of many drugs and their metabolites is mediated by MRP2 in the hepatocyte canalicular membrane and by MRP4 as well as MRP2 in the luminal membrane of kidney proximal tubules. Therefore, inhibition of these efflux pumps affects pharmacokinetics, unless compensated by other ATP-dependent efflux pumps with overlapping substrate specificities.

  17. Intestinal multidrug resistance-associated protein 2 is down-regulated in fructose-fed rats.

    PubMed

    Londero, Ana Sofía; Arana, Maite Rocío; Perdomo, Virginia Gabriela; Tocchetti, Guillermo Nicolás; Zecchinati, Felipe; Ghanem, Carolina Inés; Ruiz, María Laura; Rigalli, Juan Pablo; Mottino, Aldo Domingo; García, Fabiana; Villanueva, Silvina Stella Maris

    2017-02-01

    Expression and activity of jejunal multidrug resistance-associated protein 2 (Mrp2) and glutathione-S-transferase (GST) were examined in fructose fed Wistar rats, an experimental model of metabolic syndrome. Animals were fed on (a) control diet or (b) control diet plus 10% w/vol fructose in the drinking water. Mrp2 and the α class of GST proteins as well as their corresponding mRNAs were decreased, suggesting a transcriptional regulation by fructose. Confocal microscopy studies reaffirmed down-regulation of Mrp2. Everted intestinal sacs were incubated with 1-chloro-2,4-dinitrobenzene in the mucosal compartment, and the glutathione-conjugated derivative, dinitrophenyl- S-glutathione (DNP-SG; model Mrp2 substrate), was measured in the same compartment to estimate Mrp2 activity. Excretion of DNP-SG was substantially decreased by fructose treatment, consistent with simultaneous down-regulation of Mrp2 and GST. In addition, the effect of fructose on intestinal barrier function exerted by Mrp2 was evaluated in vivo using valsartan, a recognized Mrp2 substrate of therapeutic use. After intraduodenal administration as a bolus, intestinal absorption of valsartan was increased in fructose-drinking animals. Fructose administration also induced oxidative stress in intestinal tissue as demonstrated by significant increases of intestinal lipid peroxidation end products and activity of the antioxidant enzyme superoxide dismutase, by a decreased GSH/GSSG ratio. Moreover, fructose treatment conduced to increased intestinal levels of the proinflammatory cytokines IL-β1 and IL-6. Collectively, our results demonstrate that metabolic syndrome-like conditions, induced by a fructose-rich diet, result in down-regulation of intestinal Mrp2 expression and activity and consequently in an impairment of its barrier function.

  18. Role of the Porphyromonas gingivalis iron-binding protein PG1777 in oxidative stress resistance

    PubMed Central

    McKenzie, Rachelle M. E.; Henry, Leroy G.; Boutrin, Marie-Claire; Ximinies, Alexia

    2016-01-01

    Whole genome sequencing of the response of Porphyromonas gingivalis W83 to hydrogen peroxide revealed an upregulation of several uncharacterized, novel genes. Under conditions of prolonged oxidative stress in P. gingivalis, increased expression of a unique transcriptional unit carrying the grpE, dnaJ and three other hypothetical genes (PG1777, PG1778 and PG1779) was observed. The transcriptional start site of this operon appears to be located 91 bp upstream of the translational start, with a potential − 10 region at − 3 nt and a − 35 region at − 39 nt. Isogenic P. gingivalis mutants FLL273 (PG1777 : : ermF-ermAM) and FLL293 (PG1779 : : ermF-ermAM) showed increased sensitivity to and decreased survival after treatment with hydrogen peroxide. P. gingivalis FLL273 showed a fivefold increase in the formation of spontaneous mutants when compared with the parent strain after exposure to hydrogen peroxide. The recombinant PG1777 protein displayed iron-binding properties when incubated with FeSO4 and Fe(NH4)2(SO4).6H2O. The rPG1777 protein protected DNA from degradation when exposed to hydrogen peroxide in the presence of iron. Taken together, the data suggest that the grpE-dnaJ-PG1777-PG1778-PG1779 transcriptional unit may play an important role in oxidative stress resistance in P. gingivalis via its ability to protect against DNA damage. PMID:26581883

  19. Soy-dairy protein blend and whey protein ingestion after resistance exercise increases amino acid transport and transporter expression in human skeletal muscle.

    PubMed

    Reidy, P T; Walker, D K; Dickinson, J M; Gundermann, D M; Drummond, M J; Timmerman, K L; Cope, M B; Mukherjea, R; Jennings, K; Volpi, E; Rasmussen, B B

    2014-06-01

    Increasing amino acid availability (via infusion or ingestion) at rest or postexercise enhances amino acid transport into human skeletal muscle. It is unknown whether alterations in amino acid availability, from ingesting different dietary proteins, can enhance amino acid transport rates and amino acid transporter (AAT) mRNA expression. We hypothesized that the prolonged hyperaminoacidemia from ingesting a blend of proteins with different digestion rates postexercise would enhance amino acid transport into muscle and AAT expression compared with the ingestion of a rapidly digested protein. In a double-blind, randomized clinical trial, we studied 16 young adults at rest and after acute resistance exercise coupled with postexercise (1 h) ingestion of either a (soy-dairy) protein blend or whey protein. Phenylalanine net balance and transport rate into skeletal muscle were measured using stable isotopic methods in combination with femoral arteriovenous blood sampling and muscle biopsies obtained at rest and 3 and 5 h postexercise. Phenylalanine transport into muscle and mRNA expression of select AATs [system L amino acid transporter 1/solute-linked carrier (SLC) 7A5, CD98/SLC3A2, system A amino acid transporter 2/SLC38A2, proton-assisted amino acid transporter 1/SLC36A1, cationic amino acid transporter 1/SLC7A1] increased to a similar extent in both groups (P < 0.05). However, the ingestion of the protein blend resulted in a prolonged and positive net phenylalanine balance during postexercise recovery compared with whey protein (P < 0.05). Postexercise myofibrillar protein synthesis increased similarly between groups. We conclude that, while both protein sources enhanced postexercise AAT expression, transport into muscle, and myofibrillar protein synthesis, postexercise ingestion of a protein blend results in a slightly prolonged net amino acid balance across the leg compared with whey protein.

  20. Protein-resistant polymer coatings based on surface-adsorbed poly(aminoethyl methacrylate)/poly(ethylene glycol) copolymers.

    PubMed

    Ionov, Leonid; Synytska, Alla; Kaul, Elisabeth; Diez, Stefan

    2010-01-11

    We report on the protein-resistant properties of glass substrates coated with novel copolymers of 2-aminoethyl methacrylate hydrochloride and poly(ethylene glycol) methyl ether methacrylate (AEM-PEG). In comparison to currently available protein-blocking polymer systems, such as poly-l-lysine-poly(ethylene glycol), silane-based poly(ethylene glycol), and poly(ethylene glycol) brushes prepared by surface-initiated polymerization, the proposed AEM-PEG offers the combined advantages of low cost, simplicity of use, and applicability in aqueous solutions. We demonstrate the capability of AEM-PEG to block the surface binding of globular proteins (tubulin), their assemblies (microtubules), and functional motor proteins (kinesin-1). Moreover, we demonstrate the applicability of AEM-PEG for surface patterning of proteins in microfluidic devices.

  1. DELETION OF PROTEIN TYROSINE PHOSPHATASE 1B IMPROVES PERIPHERAL INSULIN RESISTANCE AND VASCULAR FUNCTION IN OBESE, LEPTIN RESISTANT MICE VIA REDUCED OXIDANT TONE

    PubMed Central

    Ali, M. Irfan; Ketsawatsomkron, Pimonrat; Belin de Chantelemele, Eric J.; Mintz, James D.; Muta, Kenjiro; Salet, Christina; Black, Stephen M.; Tremblay, Michel L.; Fulton, David J.; Marrero, Mario B.; Stepp, David W.

    2009-01-01

    Rationale Obesity is a risk factor for cardiovascular dysfunction, yet the underlying factors driving this impaired function remain poorly understood. Insulin resistance is a common pathology in obese patients and has been shown to impair vascular function. Whether insulin resistance or obesity, itself, is causal remains unclear. Objective The current study tested the hypothesis that insulin resistance is the underlying mediator for impaired nitric oxide mediated dilation in obesity by genetic deletion of the insulin-desensitizing enzyme protein tyrosine phosphatase 1B (PTP1B) in db/db mice. Methods and Results The db/db mouse is morbidly obese, insulin resistant and has tissue-specific elevation in PTP1B expression compared to lean controls. In db/db mice, PTP1B deletion improved glucose clearance, dyslipidemia, and insulin receptor signaling in muscle and fat. Hepatic insulin signaling in db/db mice was not improved by deletion of PTP1B, indicating specific amelioration of peripheral insulin resistance. Additionally, obese mice demonstrate an impaired endothelium dependent and independent vasodilation to acetylcholine and sodium nitroprusside, respectively. This impairment, which correlated with increased superoxide in the db/db mice, was corrected by superoxide scavenging. Increased superoxide production was associated with increased expression of NAD(P)H Oxidase 1 and its molecular regulators, Noxo1 and Noxa1. Conclusion Deletion of PTP1B improved both endothelium dependent and independent nitric oxide mediated dilation and reduced superoxide generation in db/db mice. PTP1B deletion did not affect any vascular function in lean mice. Taken together, these data reveal a role for peripheral insulin resistance as the mediator of vascular dysfunction in obesity. PMID:19797171

  2. Involvement of the Putative ATP-Dependent Efflux Proteins PatA and PatB in Fluoroquinolone Resistance of a Multidrug-Resistant Mutant of Streptococcus pneumoniae

    PubMed Central

    Marrer, Estelle; Schad, Karen; Satoh, Andreas T.; Page, Malcolm G. P.; Johnson, Maggie M.; Piddock, Laura J. V.

    2006-01-01

    The multidrug-resistant mutant Streptococcus pneumoniae M22 constitutively overexpresses two genes (patA and patB) that encode proteins homologous to known efflux proteins belonging to the ABC transporter family. It is shown here that PatA and PatB were strongly induced by quinolone antibiotics and distamycin in fluoroquinolone-sensitive strains. PatA was very important for growth of S. pneumoniae, and it could not be disrupted in strain M22. PatB appeared to control metabolic activity, particularly in amino acid biosynthesis, and it may have a pivotal role in coordination of the response to quinolone antibiotics. The induction of PatA and PatB by antibiotics showed a pattern similar to that exhibited by SP1861, a homologue of ABC-type transporters of choline and other osmoprotectants. A second group of quinolone-induced transporter genes comprising SP1587 and SP0287, which are homologues of, respectively, oxalate/formate antiporters and xanthine or uracil permeases belonging to the major facilitator family, showed a different pattern of induction by other antibiotics. There was no evidence for the involvement of PmrA, the putative proton-dependent multidrug transporter that has been implicated in norfloxacin resistance, in the response to quinolone antibiotics in either the resistant mutant or the fluoroquinolone-sensitive strains. PMID:16436727

  3. Purification and characterization of elicitor protein from Phytophthora colocasiae and basic resistance in Colocasia esculenta.

    PubMed

    Mishra, Ajay Kumar; Sharma, Kamal; Misra, Raj Shekhar

    2009-01-01

    An elicitor was identified in the fungus Phytophthora colocasiae. The molecular weight of the purified elicitor was estimated by means of gel filtration chromatography and SDS-PAGE and was estimated as 15kDa. Protease treatment severely reduced its activity, allowing the conclusion that the elicitor is proteinaceous. Infiltration of a few nanograms of this proteinaceous elicitor into taro leaves caused the formation of lesions that closely resemble hypersensitive response lesions. The elicitation of the cells was effective in the induction of the activity of lipoxygenase. Cellular damage, restricted to the infiltrated zone, occurred only several hours later, after the infiltration of the elicitor protein. After few days, systemic acquired resistance was also induced. Thus, taro plant cells that perceived the glycoprotein generated a cascade of signals acting at local, short, and long distances, and causing the coordinate expression of specific defence. The obtained results give important information regarding the plant-pathogen interactions, mainly as subsidy for taro improvement against Phytophthora leaf blight.

  4. A novel approach for synthesis of zwitterionic polyurethane coating with protein resistance.

    PubMed

    Wang, Chunhua; Ma, Chunfeng; Mu, Changdao; Lin, Wei

    2014-11-04

    We have developed a novel approach to introduce zwitterions into polyurethane for the preparation of antibiofouling coating. First, the thiol-ene click reaction between 2-(dimethylamino)ethyl methacrylate (DMAEMA) and 3-mercapto-1,2-propanediol (TPG) is used to synthesize dihydroxy-terminated DMAEMA (DMA(OH)2) under UV catalysis. The product has been proved by gel permeation chromatography (GPC), Fourier transform infrared spectrum (FT-IR), proton nuclear magnetic resonance ((1)H NMR), and high resolution mass spectrometry (HRMS). DMA(OH)2 is then incorporated into polyurethane as side groups by polyaddition with diisocyanate and further reacts with 1,3-propane sultone to obtain the zwitterionic polyurethanes. The presence of sulfobetaine zwitterions side groups has been demonstrated by FT-IR and X-ray photoelectron spectroscopy (XPS). Thermal analysis indicates that the thermal stability is decreased with the increasing content of zwitterionions. The antibiofouling property of polyurethanes has been investigated by the measurement of adsorption of fibrinogen, bovine serum albumin (BSA), and lysozyme on the polyurethanes surface using quartz crystal microbalance with dissipation (QCM-D). The results show that the polyurethane coatings exhibit effective nonspecific protein resistance at higher content of zwitterionic side groups.

  5. Multidrug transporter ABCG2/breast cancer resistance protein secretes riboflavin (vitamin B2) into milk.

    PubMed

    van Herwaarden, Antonius E; Wagenaar, Els; Merino, Gracia; Jonker, Johan W; Rosing, Hilde; Beijnen, Jos H; Schinkel, Alfred H

    2007-02-01

    The multidrug transporter breast cancer resistance protein (BCRP/ABCG2) is strongly induced in the mammary gland during pregnancy and lactation. We here demonstrate that BCRP is responsible for pumping riboflavin (vitamin B(2)) into milk, thus supplying the young with this important nutrient. In Bcrp1(-/-) mice, milk secretion of riboflavin was reduced >60-fold compared to that in wild-type mice. Yet, under laboratory conditions, Bcrp1(-/-) pups showed no riboflavin deficiency due to concomitant milk secretion of its cofactor flavin adenine dinucleotide, which was not affected. Thus, two independent secretion mechanisms supply vitamin B(2) equivalents to milk. BCRP is the first active riboflavin efflux transporter identified in mammals and the first transporter shown to concentrate a vitamin into milk. BCRP activity elsewhere in the body protects against xenotoxins by reducing their absorption and mediating their excretion. Indeed, Bcrp1 activity increased excretion of riboflavin into the intestine and decreased its systemic availability in adult mice. Surprisingly, the paradoxical dual utilization of BCRP as a xenotoxin and a riboflavin pump is evolutionarily conserved among mammals as diverse as mice and humans. This study establishes the principle that an ABC transporter can transport a vitamin into milk and raises the possibility that other vitamins and nutrients are likewise secreted into milk by ABC transporters.

  6. A Substrate Radical Intermediate in Catalysis by the Antibiotic Resistance Protein Cfr

    PubMed Central

    Grove, Tyler L.; Livada, Jovan; Schwalm, Erica L.; Green, Michael T.; Booker, Squire J.; Silakov, Alexey

    2013-01-01

    Cfr-dependent methylation of C8 of adenosine 2503 (A2503) in 23S rRNA confers bacterial resistance to an array of clinically important antibiotics that target the large subunit of the ribosome, including the synthetic oxazolidinone antibiotic linezolid. The key element of the proposed mechanism for Cfr, a radical S-adenosylmethionine (SAM) enzyme, is the addition of a methylene radical — generated by hydrogen-atom abstraction from the methyl group of an S-methylated cysteine residue (mCys) — onto C8 of A2503 to form a protein – nucleic acid cross-linked species containing an unpaired electron. Herein we use continuous-wave and pulsed electron paramagnetic resonance (EPR) techniques to provide direct spectroscopic evidence for this intermediate, showing a spin-delocalized radical with maximum spin density at N7 of the adenine ring. In addition, we use rapid-freeze quench EPR to show that the radical forms and decays with rate constants that are consistent with the rate of formation of the methylated product. PMID:23644479

  7. Haemophilus influenzae P4 Interacts With Extracellular Matrix Proteins Promoting Adhesion and Serum Resistance.

    PubMed

    Su, Yu-Ching; Mukherjee, Oindrilla; Singh, Birendra; Hallgren, Oskar; Westergren-Thorsson, Gunilla; Hood, Derek; Riesbeck, Kristian

    2016-01-15

    Interaction with the extracellular matrix (ECM) is one of the successful colonization strategies employed by nontypeable Haemophilus influenzae (NTHi). Here we identified Haemophilus lipoprotein e (P4) as a receptor for ECM proteins. Purified recombinant P4 displayed a high binding affinity for laminin (Kd = 9.26 nM) and fibronectin (Kd = 10.19 nM), but slightly less to vitronectin (Kd = 16.51 nM). A P4-deficient NTHi mutant showed a significantly decreased binding to these ECM components. Vitronectin acquisition conferred serum resistance to both P4-expressing NTHi and Escherichia coli transformants. P4-mediated bacterial adherence to pharynx, type II alveolar, and bronchial epithelial cells was mainly attributed to fibronectin. Importantly, a significantly reduced bacterial infection was observed in the middle ear of the Junbo mouse model when NTHi was devoid of P4. In conclusion, our data provide new insight into the role of P4 as an important factor for Haemophilus colonization and subsequent respiratory tract infection.

  8. The bacterial DNA repair protein Mfd confers resistance to the host nitrogen immune response

    PubMed Central

    Guillemet, Elisabeth; Leréec, Alain; Tran, Seav-Ly; Royer, Corinne; Barbosa, Isabelle; Sansonetti, Philippe; Lereclus, Didier; Ramarao, Nalini

    2016-01-01

    Production of reactive nitrogen species (NO) is a key step in the immune response following infections. NO induces lesions to bacterial DNA, thus limiting bacterial growth within hosts. Using two pathogenic bacteria, Bacillus cereus and Shigella flexneri, we show that the DNA-repair protein Mfd (Mutation-Frequency-Decline) is required for bacterial resistance to the host-NO-response. In both species, a mutant deficient for mfd does not survive to NO, produced in vitro or by phagocytic cells. In vivo, the ∆mfd mutant is avirulent and unable to survive the NO-stress. Moreover, NO induces DNA-double-strand-breaks and point mutations in the Δmfd mutant. In overall, these observations demonstrate that NO damages bacterial DNA and that Mfd is required to maintain bacterial genomic integrity. This unexpected discovery reveals that Mfd, a typical housekeeping gene, turns out to be a true virulence factor allowing survival and growth of the pathogen in its host, due to its capacity to protect the bacterium against NO, a key molecule of the innate immune defense. As Mfd is widely conserved in the bacterial kingdom, these data highlight a mechanism that may be used by a large spectrum of bacteria to overcome the host immune response and especially the mutagenic properties of NO. PMID:27435260

  9. Tandem repeat sequence analysis of staphylococcal protein A (spa) gene in methicillin-resistant Staphylococcus pseudintermedius.

    PubMed

    Moodley, Arshnee; Stegger, Marc; Ben Zakour, Nouri L; Fitzgerald, J Ross; Guardabassi, Luca

    2009-03-30

    A putative staphylococcal protein A (spa) gene was discovered in the genome of Staphylococcus pseudintermedius and used for developing a species-specific spa typing protocol. Thirty-one clinical methicillin-resistant S. pseudintermedius (MRSP) isolates from dogs and cats in four countries were characterized by spa typing, pulsed-field gel electrophoresis (PFGE) and staphylococcal cassette chromosome (SCCmec) typing. The results indicated the occurrence of two MRSP clones that acquired distinct SCCmec elements in Europe (t02, PFGE type A, SCCmec type III,) and California (t06, PFGE type B, SCCmec type V). Sequence analysis of mecA revealed the occurrence of four alleles (mecA1 to mecA4), which correlated with the geographical origin of the isolates and enabled discrimination of two distinct subtypes within the European clone. The newly developed spa typing method appeared to be a promising tool for easy and rapid typing of MRSP, either alone or in combination with SCCmec and mecA typing for fine-structure epidemiological analysis.

  10. Characterization of the Soluble NSF Attachment Protein gene family identifies two members involved in additive resistance to a plant pathogen

    PubMed Central

    Lakhssassi, Naoufal; Liu, Shiming; Bekal, Sadia; Zhou, Zhou; Colantonio, Vincent; Lambert, Kris; Barakat, Abdelali; Meksem, Khalid

    2017-01-01

    Proteins with Tetratricopeptide-repeat (TPR) domains are encoded by large gene families and distributed in all plant lineages. In this study, the Soluble NSF-Attachment Protein (SNAP) subfamily of TPR containing proteins is characterized. In soybean, five members constitute the SNAP gene family: GmSNAP18, GmSNAP11, GmSNAP14, GmSNAP02, and GmSNAP09. Recently, GmSNAP18 has been reported to mediate resistance to soybean cyst nematode (SCN). Using a population of recombinant inbred lines from resistant and susceptible parents, the divergence of the SNAP gene family is analysed over time. Phylogenetic analysis of SNAP genes from 22 diverse plant species showed that SNAPs were distributed in six monophyletic clades corresponding to the major plant lineages. Conservation of the four TPR motifs in all species, including ancestral lineages, supports the hypothesis that SNAPs were duplicated and derived from a common ancestor and unique gene still present in chlorophytic algae. Syntenic analysis of regions harbouring GmSNAP genes in soybean reveals that this family expanded from segmental and tandem duplications following a tetraploidization event. qRT-PCR analysis of GmSNAPs indicates a co-regulation following SCN infection. Finally, genetic analysis demonstrates that GmSNAP11 contributes to an additive resistance to SCN. Thus, GmSNAP11 is identified as a novel minor gene conferring resistance to SCN. PMID:28338077

  11. Mutation of G234 amino acid residue in Candida albicans drug-resistance-related protein Rta2p is associated with fluconazole resistance and dihydrosphingosine transport

    PubMed Central

    Zhang, Shi-Qun; Miao, Qi; Li, Li-Ping; Zhang, Lu-lu; Yan, Lan; Jia, Yu; Cao, Yong-Bing; Jiang, Yuan-Ying

    2015-01-01

    Widespread and repeated use of azoles has led to the rapid development of drug resistance in Candida albicans. Our previous study found Rta2p, a membrane protein with 7 transmembrane domains, was involved in calcineurin-mediated azole resistance and sphingoid long-chain base release in C. albicans. Conserved amino acids in the transmembrane domain of Rta2p were subjected to site-directed mutagenesis. The sensitivity of C. albicans to fluconazole in vitro was examined by minimum inhibitory concentration and killing assay, and the therapeutic efficacy of fluconazole in vivo was performed by systemic mice candidiasis model. Furthermore, dihydrosphingosine transport activity was detected by NBD labeled D-erythro-dihydrosphingosine uptake and release assay, and the sensitivity to sphingolipid biosynthesis inhibitors. We successfully constructed 14 mutant strains of Rta2p, screened them by minimum inhibitory concentration and found Ca2+ did not completely induce fluconazole resistance with G158E and G234S mutations. Furthermore, we confirmed that G234S mutant enhanced the therapeutic efficacy of fluconazole against systemic candidiasis and significantly increased the accumulation of dihydrosphingosine by decreasing its release. However, G158E mutant didn't affect drug therapeutic efficacy in vivo and dihydrosphingosine transport in C. albicans. G234 of Rta2p in C. albicans is crucial in calcineurin-mediated fluconazole resistance and dihydrosphingosine transport. PMID:26220356

  12. Mutation of G234 amino acid residue in candida albicans drug-resistance-related protein Rta2p is associated with fluconazole resistance and dihydrosphingosine transport.

    PubMed

    Zhang, Shi-Qun; Miao, Qi; Li, Li-Ping; Zhang, Lu-Lu; Yan, Lan; Jia, Yu; Cao, Yong-Bing; Jiang, Yuan-Ying

    2015-01-01

    Widespread and repeated use of azoles has led to the rapid development of drug resistance in Candida albicans. Our previous study found Rta2p, a membrane protein with 7 transmembrane domains, was involved in calcineurin-mediated azole resistance and sphingoid long-chain base release in C. albicans. Conserved amino acids in the transmembrane domain of Rta2p were subjected to site-directed mutagenesis. The sensitivity of C. albicans to fluconazole in vitro was examined by minimum inhibitory concentration and killing assay, and the therapeutic efficacy of fluconazole in vivo was performed by systemic mice candidiasis model. Furthermore, dihydrosphingosine transport activity was detected by NBD labeled D-erythro-dihydrosphingosine uptake and release assay, and the sensitivity to sphingolipid biosynthesis inhibitors. We successfully constructed 14 mutant strains of Rta2p, screened them by minimum inhibitory concentration and found Ca(2+) did not completely induce fluconazole resistance with G158E and G234S mutations. Furthermore, we confirmed that G234S mutant enhanced the therapeutic efficacy of fluconazole against systemic candidiasis and significantly increased the accumulation of dihydrosphingosine by decreasing its release. However, G158E mutant didn't affect drug therapeutic efficacy in vivo and dihydrosphingosine transport in C. albicans. G234 of Rta2p in C. albicans is crucial in calcineurin-mediated fluconazole resistance and dihydrosphingosine transport.

  13. RTP1 encodes a novel endoplasmic reticulum (ER)-localized protein in Arabidopsis and negatively regulates resistance against biotrophic pathogens.

    PubMed

    Pan, Qiaona; Cui, Beimi; Deng, Fengyan; Quan, Junli; Loake, Gary J; Shan, Weixing

    2016-03-01

    Oomycete pathogens cause serious damage to a wide spectrum of plants. Although host pathogen recognition via pathogen effectors and cognate plant resistance proteins is well established, the genetic basis of host factors that mediate plant susceptibility to oomycete pathogens is relatively unexplored. Here, we report on RTP1, a nodulin-related MtN21 family gene in Arabidopsis that mediates susceptibility to Phytophthora parasitica. RTP1 was identified by screening a T-DNA insertion mutant population and encoded an endoplasmic reticulum (ER)-localized protein. Overexpression of RTP1 rendered Arabidopsis more susceptible, whereas RNA silencing of RTP1 led to enhanced resistance to P. parasitica. Moreover, an RTP1 mutant, rtp1-1, displayed localized cell death, increased reactive oxygen species (ROS) production and accelerated PR1 expression, compared to the wild-type Col-0, in response to P. parasitica infection. rtp1-1 showed a similar disease response to the bacterial pathogen Pseudomonas syringae pv. tomato (Pst) DC3000, including increased disease resistance, cell death and ROS production. Furthermore, rpt1-1 exhibited resistance to the fungal pathogen Golovinomyces cichoracearum, but not to the necrotrophic pathogen Botrytis cinerea. Taken together, these results suggest that RTP1 negatively regulates plant resistance to biotrophic pathogens, possibly by regulating ROS production, cell death progression and PR1 expression.

  14. Ribosomal protein L3 mutations are associated with cfr-mediated linezolid resistance in clinical isolates of Staphylococcus cohnii.

    PubMed

    Xu, Hongtao; Tian, Rui; Li, Yanming; Chen, Dongke; Liu, Yalin; Hu, Yunjian; Xiao, Fei

    2015-06-01

    From June, 2012 to November, 2013 five linezolid-resistant Staphylococcus cohnii isolates were identified in our hospital in Beijing, China. The investigation of the resistance mechanisms confirmed that the cfr-carrying plasmids were the main cause of linezolid resistance in those clinical isolates. Moreover, all the five isolates had ribosomal protein L3 mutations, which had different coordinate effect on cfr-mediated linezolid resistance directly through the substitution of serine 158 by phenylalanine or tyrosine in L3 protein. In this study, two types of plasmids (p432, p438) (Accession No. KM114207) were found, which share high sequence identity with previously reported cfr-carrying pRM01 and pMHZ plasmids originated from northern and southern China, showing wide regional dissemination in China. The stability of linezolid resistance was studied by passaging single colonies serially on antibiotic-free blood medium, which showed that the susceptible derivatives emerged until the passages 39-42 with the elimination of cfr-carrying plasmid. Thus the high stability of this plasmid may pose a risk for the transmission among patients or even cause an outbreak in clinical settings.

  15. Identification of Soybean Proteins and Genes Differentially Regulated in Near Isogenic Lines Differing in Resistance to Aphid Infestation.

    PubMed

    Brechenmacher, Laurent; Nguyen, Tran Hong Nha; Zhang, Ning; Jun, Tae-Hwan; Xu, Dong; Mian, M A Rouf; Stacey, Gary

    2015-10-02

    Soybean aphid is an important pest causing significant yield losses. The Rag2 locus confers resistance to soybean aphid biotypes 1 and 2. Transcriptomic and proteomic analyses were done over a 48 h period after aphid infestation using near isogenic lines (NILs) differing at the Rag2 locus. Comparing the Rag2 and/or rag2 lines identified 3445 proteins, of which 396 were differentially regulated between the two lines, including proteins involved in cell wall metabolism, carbohydrate metabolism, and stress response. RNA-seq transcriptomic analysis identified 2361 genes significantly regulated between the resistant and susceptible lines. Genes upregulated in the Rag2 line were annotated as being involved in cell wall, secondary, and hormone metabolism as well as in stress, signaling, and transcriptional responses. Genes downregulated in the Rag2 line were annotated as being involved in photosynthesis and carbon metabolism. Interestingly, two genes (unknown and mitochondrial protease) located within the defined Rag2 locus were expressed significantly higher in the resistant genotype. The expression of a putative NBS-LRR resistant gene within the Rag2 locus was not different between the two soybean lines, but a second NBL-LRR gene located just at the border of the defined Rag2 locus was. Therefore, this gene may be a candidate R gene controlling aphid resistance.

  16. Hypoenergetic diet-induced reductions in myofibrillar protein synthesis are restored with resistance training and balanced daily protein ingestion in older men

    PubMed Central

    Murphy, Caoileann H.; Churchward-Venne, Tyler A.; Mitchell, Cameron J.; Kolar, Nathan M.; Kassis, Amira; Karagounis, Leonidas G.; Burke, Louise M.; Hawley, John A.

    2015-01-01

    Strategies to enhance weight loss with a high fat-to-lean ratio in overweight/obese older adults are important since lean loss could exacerbate sarcopenia. We examined how dietary protein distribution affected muscle protein synthesis during energy balance (EB), energy restriction (ER), and energy restriction plus resistance training (ER + RT). A 4-wk ER diet was provided to overweight/obese older men (66 ± 4 yr, 31 ± 5 kg/m2) who were randomized to either a balanced (BAL: 25% daily protein/meal × 4) or skewed (SKEW: 7:17:72:4% daily protein/meal; n = 10/group) pattern. Myofibrillar and sarcoplasmic protein fractional synthetic rates (FSR) were measured during a 13-h primed continuous infusion of l-[ring-13C6]phenylalanine with BAL and SKEW pattern of protein intake in EB, after 2 wk ER, and after 2 wk ER + RT. Fed-state myofibrillar FSR was lower in ER than EB in both groups (P < 0.001), but was greater in BAL than SKEW (P = 0.014). In ER + RT, fed-state myofibrillar FSR increased above ER in both groups and in BAL was not different from EB (P = 0.903). In SKEW myofibrillar FSR remained lower than EB (P = 0.002) and lower than BAL (P = 0.006). Fed-state sarcoplasmic protein FSR was reduced similarly in ER and ER + RT compared with EB (P < 0.01) in both groups. During ER in overweight/obese older men a BAL consumption of protein stimulated the synthesis of muscle contractile proteins more effectively than traditional, SKEW distribution. Combining RT with a BAL protein distribution “rescued” the lower rates of myofibrillar protein synthesis during moderate ER. PMID:25738784

  17. Therapeutic silencing of fat-specific protein 27 improves glycemic control in mouse models of obesity and insulin resistance[S

    PubMed Central

    Langhi, Cédric; Arias, Noemí; Rajamoorthi, Ananthi; Basta, Jeannine; Lee, Richard G.; Baldán, Ángel

    2017-01-01

    Obesity is a component of the metabolic syndrome, mechanistically linked to diabetes, fatty liver disease, and cardiovascular disease. Proteins that regulate the metabolic fate of intracellular lipid droplets are potential therapeutic candidates to treat obesity and its related consequences. CIDEC (cell death-inducing DFFA-like effector C), also known in mice as Fsp27 (fat-specific protein 27), is a lipid droplet-associated protein that prevents lipid mobilization and promotes intracellular lipid storage. The consequences of complete loss of FSP27 on hepatic metabolism and on insulin resistance are controversial, as both healthy and deleterious lipodystrophic phenotypes have been reported in Fsp27−/− mice. To test whether therapeutic silencing of Fsp27 might be useful to improve obesity, fatty liver, and glycemic control, we used antisense oligonucleotides (ASOs) in both nutritional (high-fat diet) and genetic (leptin-deficient ob/ob) mouse models of obesity, hyperglycemia, and hepatosteatosis. We show that partial silencing Fsp27 in either model results in the robust decrease in visceral fat, improved insulin sensitivity and whole-body glycemic control, and tissue-specific changes in transcripts controlling lipid oxidation and synthesis. These data suggest that partial reduction of FSP27 activity (e.g., using ASOs) might be exploited therapeutically in insulin-resistant obese or overweight patients. PMID:27884961

  18. Insect Resistance to Bacillus thuringiensis Toxin Cry2Ab Is Conferred by Mutations in an ABC Transporter Subfamily A Protein

    PubMed Central

    Tay, Wee Tek; Mahon, Rod J.; Heckel, David G.; Walsh, Thomas K.; Downes, Sharon; James, William J.; Lee, Sui-Fai; Reineke, Annette; Williams, Adam K.; Gordon, Karl H. J.

    2015-01-01

    The use of conventional chemical insecticides and bacterial toxins to control lepidopteran pests of global agriculture has imposed significant selection pressure leading to the rapid evolution of insecticide resistance. Transgenic crops (e.g., cotton) expressing the Bt Cry toxins are now used world wide to control these pests, including the highly polyphagous and invasive cotton bollworm Helicoverpa armigera. Since 2004, the Cry2Ab toxin has become widely used for controlling H. armigera, often used in combination with Cry1Ac to delay resistance evolution. Isolation of H. armigera and H. punctigera individuals heterozygous for Cry2Ab resistance in 2002 and 2004, respectively, allowed aspects of Cry2Ab resistance (level, fitness costs, genetic dominance, complementation tests) to be characterised in both species. However, the gene identity and genetic changes conferring this resistance were unknown, as was the detailed Cry2Ab mode of action. No cross-resistance to Cry1Ac was observed in mutant lines. Biphasic linkage analysis of a Cry2Ab-resistant H. armigera family followed by exon-primed intron-crossing (EPIC) marker mapping and candidate gene sequencing identified three independent resistance-associated INDEL mutations in an ATP-Binding Cassette (ABC) transporter gene we named HaABCA2. A deletion mutation was also identified in the H. punctigera homolog from the resistant line. All mutations truncate the ABCA2 protein. Isolation of further Cry2Ab resistance alleles in the same gene from field H. armigera populations indicates unequal resistance allele frequencies and the potential for Bt resistance evolution. Identification of the gene involved in resistance as an ABC transporter of the A subfamily adds to the body of evidence on the crucial role this gene family plays in the mode of action of the Bt Cry toxins. The structural differences between the ABCA2, and that of the C subfamily required for Cry1Ac toxicity, indicate differences in the detailed mode

  19. Role of ribosomal protein S12 in peptide chain elongation: analysis of pleiotropic, streptomycin-resistant mutants of Escherichia coli.

    PubMed Central

    Zengel, J M; Young, R; Dennis, P P; Nomura, M

    1977-01-01

    Some of the spontaneous streptomycin-resistant mutants of Escherichia coli strain C600 exhibit pleiotropic effects in addition to the antibiotic resistance. These effects include decreased growth rates, reduced levels of certain enzymes, and poor support of bacteriophage growth. One of these mutants, strain SM3, was studied further. We have examined the question of whether the reduced growth rate of the mutant SM3 is related to the reduction in relative amounts of ribosomes or to the reduction in the efficiency of ribosomes in protein synthesis. Measurements of alpha, the differential synthesis rate of ribosomal protein, revealed that the protein synthesis effeciency of ribosomes from the mutant strain SM3 was reduced about twofold relative to that of the parent strain C600. Measurements of the induction lag for beta-galactosidase and of the synthesis time of several different molecular-weight classes of proteins indicated that the mutation resulted in a marked reduction in the peptide chain growth rate. This reduction in the chain growth rate probably accounted for most of the observed reduction in the growth rate of the mutant strain. These experimental results show that the strA gene product, the S12 protein of the 30S subunit, is involved in some aspect of protein chain elongation. Presumably this involvement occurs during the messenger ribonucleic acid-directed binding of transfer ribonucleic acid to the ribosome. PMID:321423

  20. Role of ribosomal protein S12 in peptide chain elongation: analysis of pleiotropic, streptomycin-resistant mutants of Escherichia coli.

    PubMed

    Zengel, J M; Young, R; Dennis, P P; Nomura, M

    1977-03-01

    Some of the spontaneous streptomycin-resistant mutants of Escherichia coli strain C600 exhibit pleiotropic effects in addition to the antibiotic resistance. These effects include decreased growth rates, reduced levels of certain enzymes, and poor support of bacteriophage growth. One of these mutants, strain SM3, was studied further. We have examined the question of whether the reduced growth rate of the mutant SM3 is related to the reduction in relative amounts of ribosomes or to the reduction in the efficiency of ribosomes in protein synthesis. Measurements of alpha, the differential synthesis rate of ribosomal protein, revealed that the protein synthesis effeciency of ribosomes from the mutant strain SM3 was reduced about twofold relative to that of the parent strain C600. Measurements of the induction lag for beta-galactosidase and of the synthesis time of several different molecular-weight classes of proteins indicated that the mutation resulted in a marked reduction in the peptide chain growth rate. This reduction in the chain growth rate probably accounted for most of the observed reduction in the growth rate of the mutant strain. These experimental results show that the strA gene product, the S12 protein of the 30S subunit, is involved in some aspect of protein chain elongation. Presumably this involvement occurs during the messenger ribonucleic acid-directed binding of transfer ribonucleic acid to the ribosome.

  1. Characterization of a mitogen-activated protein kinase gene from cucumber required for trichoderma-conferred plant resistance.

    PubMed

    Shoresh, Michal; Gal-On, Amit; Leibman, Diana; Chet, Ilan

    2006-11-01

    The fungal biocontrol agent Trichoderma asperellum has been recently shown to induce systemic resistance in plants through a mechanism that employs jasmonic acid and ethylene signal transduction pathways. Mitogen-activated protein kinase (MAPK) proteins have been implicated in the signal transduction of a wide variety of plant stress responses. Here we report the identification and characterization of a Trichoderma-induced MAPK (TIPK) gene function in cucumber (Cucumis sativus). Similar to its homologs, wound-induced protein kinase, MPK3, and MPK3a, TIPK is also induced by wounding. Normally, preinoculation of roots with Trichoderma activates plant defense mechanisms, which result in resistance to the leaf pathogen Pseudomonas syringae pv lachrymans. We used a unique attenuated virus vector, Zucchini yellow mosaic virus (ZYMV-AGII), to overexpress TIPK protein and antisense (AS) RNA. Plants overexpressing TIPK were more resistant to pathogenic bacterial attack than control plants, even in the absence of Trichoderma preinoculation. On the other hand, plants expressing TIPK-AS revealed increased sensitivity to pathogen attack. Moreover, Trichoderma preinoculation could not protect these AS plants against subsequent pathogen attack. We therefore demonstrate that Trichoderma exerts its protective effect on plants through activation of the TIPK gene, a MAPK that is involved in signal transduction pathways of defense responses.

  2. Emergence of clonally related multidrug resistant Haemophilus influenzae with penicillin-binding protein 3-mediated resistance to extended-spectrum cephalosporins, Norway, 2006 to 2013.

    PubMed

    Skaare, D; Anthonisen, I L; Kahlmeter, G; Matuschek, E; Natås, O B; Steinbakk, M; Sundsfjord, A; Kristiansen, B E

    2014-12-11

    Resistance to cephalosporins in Haemophilus influenzae is usually caused by characteristic alterations in penicillin-binding protein 3 (PBP3), encoded by the ftsI gene. Resistance to extended-spectrum cephalosporins is associated with high-level PBP3-mediated resistance (high-rPBP3), defined by the second stage S385T substitution in addition to a first stage substitution (R517H or N526K). The third stage L389F substitution is present in some high-rPBP3 strains. High-rPBP3 H. influenzae are considered rare outside Japan and Korea. In this study, 30 high-rPBP3 isolates from Norway, collected between 2006 and 2013, were examined by serotyping, multilocus sequence typing (MLST), ftsI sequencing, detection of beta-lactamase genes and minimum inhibitory concentration (MIC) determination. MICs were interpreted according to clinical breakpoints from the European Committee on Antimicrobial Susceptibility Testing (EUCAST). Respiratory isolates predominated (proportion: 24/30). The 30 isolates included one serotype f isolate, while the remaining 29 lacked polysaccharide capsule genes. Resistance to extended-spectrum cephalosporins (cefixime, 29 isolates/30 isolates; cefepime, 28/30; cefotaxime, 26 /30; ceftaroline, 26/30; ceftriaxone, 14/30), beta-lactamase production (11/30) and co-resistance to non-beta-lactams (trimethoprim-sulfamethoxazole, 13/30; tetracycline, 4/30; chloramphenicol, 4/30; ciprofloxacin, 3/30) was frequent. The N526K substitution in PBP3 was present in 23 of 30 isolates; these included a blood isolate which represents the first invasive S385T + N526K isolate reported from Europe. The L389F substitution, present in 16 of 30 isolates, coincided with higher beta-lactam MICs. Non-susceptibility to meropenem was frequent in S385T + L389F + N526K isolates (8/12). All 11 beta-lactamase positive isolates were TEM-1. Five clonal groups of two to 10 isolates with identical MLST-ftsI allelic profiles were observed, including the first reported high-rPBP3

  3. Crystal Structures of Penicillin-Binding Protein 2 From Penicillin-Susceptible And -Resistant Strains of Neisseria Gonorrhoeae Reveal An Unexpectedly Subtle Mechanism for Antibiotic Resistance

    SciTech Connect

    Powell, A.J.; Tomberg, J.; Deacon, A.M.; Nicholas, R.A.; Davies, C.

    2009-05-21

    Penicillin-binding protein 2 (PBP2) from N. gonorrhoeae is the major molecular target for {beta}-lactam antibiotics used to treat gonococcal infections. PBP2 from penicillin-resistant strains of N. gonorrhoeae harbors an aspartate insertion after position 345 (Asp-345a) and 4-8 additional mutations, but how these alter the architecture of the protein is unknown. We have determined the crystal structure of PBP2 derived from the penicillin-susceptible strain FA19, which shows that the likely effect of Asp-345a is to alter a hydrogen-bonding network involving Asp-346 and the SXN triad at the active site. We have also solved the crystal structure of PBP2 derived from the penicillin-resistant strain FA6140 that contains four mutations near the C terminus of the protein. Although these mutations lower the second order rate of acylation for penicillin by 5-fold relative to wild type, comparison of the two structures shows only minor structural differences, with the positions of the conserved residues in the active site essentially the same in both. Kinetic analyses indicate that two mutations, P551S and F504L, are mainly responsible for the decrease in acylation rate. Melting curves show that the four mutations lower the thermal stability of the enzyme. Overall, these data suggest that the molecular mechanism underlying antibiotic resistance contributed by the four mutations is subtle and involves a small but measurable disordering of residues in the active site region that either restricts the binding of antibiotic or impedes conformational changes that are required for acylation by {beta}-lactam antibiotics.

  4. Fludarabine resistance mediated by aminoglycoside-3'-phosphotransferase-IIa and the structurally related eukaryotic cAMP-dependent protein kinase.

    PubMed

    Sánchez-Carrera, Dámaso; Bravo-Navas, Sara; Cabezón, Elena; Arechaga, Ignacio; Cabezas, Matilde; Yáñez, Lucrecia; Pipaón, Carlos

    2017-04-03

    While working with G418-resistant stably transfected cells, we realized the neomycin resistance gene (NeoR), which encodes the aminoglycoside-3'-phosphotransferase-IIa [APH(3')-IIa], also confers resistance to the nucleoside analog fludarabine. Fludarabine is a cytostatic drug widely used in the treatment of hematologic and solid tumors as well as in the conditioning of patients before transplantation of hematopoietic progenitors. We present evidence that NeoR-transfected cells do not incorporate fludarabine, thus avoiding DNA damage caused by the drug, evidenced by a lack of FANCD2 monoubiquitination and impaired apoptosis. A screening of other nucleoside analogs revealed that APH(3')-IIa only protects against ATP purine analogs. Moreover, APH(3')-IIa ATPase activity is inhibited by fludarabine monophosphate, suggesting that APH(3')-IIa blocks fludarabine incorporation into DNA by dephosphorylating its active fludarabine triphosphate form. Furthermore, overexpression of the catalytic subunit of the eukaryotic kinase PKA, which is structurally related to APHs, also provides resistance to fludarabine, anticipating its putative utility as a response marker to the drug. Our results preclude the use of Neo marker plasmids in the study of purine analogs and unveils a new resistance mechanism against these chemotherapeuticals.-Sánchez-Carrera, D., Bravo-Navas, S., Cabezón, E., Arechaga, I., Cabezas, M., Yáñez, L., Pipaón, C. Fludarabine resistance mediated by aminoglycoside-3'-phosphotransferase-IIa and the structurally related eukaryotic cAMP-dependent protein kinase.

  5. Riboflavin-Induced Disease Resistance Requires the Mitogen-Activated Protein Kinases 3 and 6 in Arabidopsis thaliana

    PubMed Central

    Nie, Shengjun; Xu, Huilian

    2016-01-01

    As a resistance elicitor, riboflavin (vitamin B2) protects plants against a wide range of pathogens. At molecular biological levels, it is important to elucidate the signaling pathways underlying the disease resistance induced by riboflavin. Here, riboflavin was tested to induce resistance against virulent Pseudomonas syringae pv. Tomato DC3000 (Pst DC3000) in Arabidopsis. Results showed that riboflavin induced disease resistance based on MAPK-dependent priming for the expression of PR1 gene. Riboflavin induced transient expression of PR1 gene. However, following Pst DC3000 inoculation, riboflavin potentiated stronger PR1 gene transcription. Further was suggested that the transcript levels of mitogen-activated protein kinases, MPK3 and MPK6, were primed under riboflavin. Upon infection by Pst DC3000, these two enzymes were more strongly activated. The elevated activation of both MPK3 and MPK6 was responsible for enhanced defense gene expression and resistance after riboflavin treatment. Moreover, riboflavin significantly reduced the transcript levels of MPK3 and MPK6 by application of AsA and BAPTA, an H2O2 scavenger and a calcium (Ca2+) scavenger, respectively. In conclusion, MPK3 and MPK6 were responsible for riboflavin-induced resistance, and played an important role in H2O2- and Ca2+-related signaling pathways, and this study could provide a new insight into the mechanistic study of riboflavin-induced defense responses. PMID:27054585

  6. Optimal dietary protein level improved growth, disease resistance, intestinal immune and physical barrier function of young grass carp (Ctenopharyngodon idella).

    PubMed

    Xu, Jing; Wu, Pei; Jiang, Wei-Dan; Liu, Yang; Jiang, Jun; Kuang, Sheng-Yao; Tang, Ling; Tang, Wu-Neng; Zhang, Yong-An; Zhou, Xiao-Qiu; Feng, Lin

    2016-08-01

    This study investigated the effects of dietary proteins on the growth, disease resistance, intestinal immune and physical barrier functions of young grass carp (Ctenopharyngodon idella). A total of 540 young grass carp (264.11 ± 0.76 g) were fed six diets containing graded levels of protein (143.1, 176.7, 217.2, 257.5, 292.2 and 322.8 g digestible protein kg(-1) diet) for 8 weeks. After the growth trial, fish were challenged with Aeromonas hydrophila and mortalities were recorded for 14 days. The results indicated that optimal dietary protein levels: increased the production of antibacterial components, up-regulated anti-inflammatory cytokines, inhibitor of κBα, target of rapamycin and ribosomal protein S6 kinases 1 mRNA levels, whereas down-regulated pro-inflammatory cytokines, nuclear factor kappa B (NF-κB) P65, NF-κB P52, c-Rel, IκB kinase β, IκB kinase γ and eIF4E-binding proteins 2 mRNA levels in three intestinal segments of young grass carp (P < 0.05), suggesting that optimal dietary protein level could enhance fish intestinal immune barrier function; up-regulated the mRNA levels of tight junction complexes, B-cell lymphoma protein-2, inhibitor of apoptosis proteins, myeloid cell leukemia-1 and NF-E2-related factor 2, and increased the activities and mRNA levels of antioxidant enzymes, whereas down-regulated myosin light chain kinase, cysteinyl aspartic acid-protease 2, 3, 7, 8, 9, fatty acid synthetase ligand, apoptotic protease activating factor-1, Bcl-2 associated X protein, p38 mitogen-activated protein kinase, c-Jun N-terminal protein kinase and Kelch-like-ECH-associated protein 1b mRNA levels, and decreased reactive oxygen species, malondialdehyde and protein carbonyl contents in three intestinal segments of young grass carp (P < 0.05), indicating that optimal dietary protein level could improve fish intestinal physical barrier function. Finally, the optimal dietary protein levels for the growth performance (PWG) and against enteritis

  7. The Tomato mosaic virus 30 kDa movement protein interacts differentially with the resistance genes Tm-2 and Tm-2(2).

    PubMed

    Weber, H; Ohnesorge, S; Silber, M V; Pfitzner, A J P

    2004-08-01

    In tomato plants ( Lycopersicon esculentum Mill.), the genes Tm-2 and Tm-2(2) confer resistance to Tomato mosaic virus (ToMV). Sequence analysis of ToMV strains able to break the Tm-2 or Tm-2(2) resistance revealed distinct amino acid exchanges in the viral 30 kDa protein, suggesting that the movement protein is recognized by both resistance genes to induce the plant defense reaction. To analyze the interactions between the ToMV movement protein and the Tm-2 and Tm-2(2) genes in detail, we generated transgenic tomato lines expressing various movement protein gene constructs. Crosses of the transgenic tomato lines with cultivars containing either the Tm-2 or the Tm-2(2) gene demonstrated that both genes are able to elicit a hypersensitive reaction in response to movement proteins from resistance inducing ToMV strains. However, the domains and the structural requirements for induction of the necrotic response by the ToMV movement protein are completely different for either resistance gene. In the context of the Tm-2 gene, the resistance determinant lies within the N-terminal 188 amino acids of the ToMV movement protein. Interaction of the 30 kDa protein with the Tm-2(2) gene requires two distinct domains localized at the C-terminus and in a different region of the protein, respectively.

  8. Altered chlorplast ribosomal proteins associated with erythromycin-resistant mutants in two genetic systems of Chlamydomonas reinhardi.

    PubMed

    Mets, L; Bogorad, L

    1972-12-01

    The phenotype of several erythromycin-resistant mutants of Chlamydomonas reinhardi was further characterized in terms of the electrophoretic properties of their chloroplast ribosomal proteins. In mutant ery-M2d a single protein of the large (52 S) subunit has altered properties, which probably result from a change in its primary sequence. This mutation is inherited in a Meudelian manner. In mutant ery-U1a, which is inherited in a uniparental manner, a different single protein of the 52 S subunit is altered. This change might result from a change in either the primary sequence of the protein or in some form of secondary modification. These results indicate that these two distinct genetic systems must cooperate in the production of chloroplast ribosomes.

  9. Effects of divergent resistance exercise contraction mode and dietary supplementation type on anabolic signalling, muscle protein synthesis and muscle hypertrophy.

    PubMed

    Rahbek, Stine Klejs; Farup, Jean; Møller, Andreas Buch; Vendelbo, Mikkel Holm; Holm, Lars; Jessen, Niels; Vissing, Kristian

    2014-10-01

    Greater force produced with eccentric (ECC) compared to concentric (CONC) contractions, may comprise a stronger driver of muscle growth, which may be further augmented by protein supplementation. We investigated the effect of differentiated contraction mode with either whey protein hydrolysate and carbohydrate (WPH + CHO) or isocaloric carbohydrate (CHO) supplementation on regulation of anabolic signalling, muscle protein synthesis (MPS) and muscle hypertrophy. Twenty-four human participants performed unilateral isolated maximal ECC versus CONC contractions during exercise habituation, single-bout exercise and 12 weeks of training combined with WPH + CHO or CHO supplements. In the exercise-habituated state, p-mTOR, p-p70S6K, p-rpS6 increased by approximately 42, 206 and 213 %, respectively, at 1 h post-exercise, with resistance exercise per se; whereas, the phosphorylation was exclusively maintained with ECC at 3 and 5 h post-exercise. This acute anabolic signalling response did not differ between the isocaloric supplement types, neither did protein fractional synthesis rate differ between interventions. Twelve weeks of ECC as well as CONC resistance training augmented hypertrophy with WPH + CHO group compared to the CHO group (7.3 ± 1.0 versus 3.4 ± 0.8 %), independently of exercise contraction type. Training did not produce major changes in basal levels of Akt-mTOR pathway components. In conclusion, maximal ECC contraction mode may constitute a superior driver of acute anabolic signalling that may not be mirrored in the muscle protein synthesis rate. Furthermore, with prolonged high-volume resistance training, contraction mode seems less influential on the magnitude of muscle hypertrophy, whereas protein and carbohydrate supplementation augments muscle hypertrophy as compared to isocaloric carbohydrate supplementation .

  10. Involvement of the Pepper Antimicrobial Protein CaAMP1 Gene in Broad Spectrum Disease Resistance1[C][OA

    PubMed Central

    Lee, Sung Chul; Hwang, In Sun; Choi, Hyong Woo; Hwang, Byung Kook

    2008-01-01

    Pathogen-inducible antimicrobial defense-related proteins have emerged as key antibiotic peptides and enzymes involved in disease resistance in plants. A novel antimicrobial protein gene, CaAMP1 (for Capsicum annuum ANTIMICROBIAL PROTEIN1), was isolated from pepper (C. annuum) leaves infected with Xanthomonas campestris pv vesicatoria. Expression of the CaAMP1 gene was strongly induced in pepper leaves not only during pathogen infection but also after exposure to abiotic elicitors. The purified recombinant CaAMP1 protein possessed broad-spectrum antimicrobial activity against phytopathogenic bacteria and fungi. CaAMP1:smGFP fusion protein was localized mainly in the external and intercellular regions of onion (Allium cepa) epidermal cells. The virus-induced gene silencing technique and gain-of-function transgenic plants were used to determine the CaAMP1 gene function in plant defense. Silencing of CaAMP1 led to enhanced susceptibility to X. campestris pv vesicatoria and Colletotrichum coccodes infection, accompanied by reduced PATHOGENESIS-RELATED (PR) gene expression. In contrast, overexpression of CaAMP1 in Arabidopsis (Arabidopsis thaliana) conferred broad-spectrum resistance to the hemibiotrophic bacterial pathogen Pseudomonas syringae pv tomato, the biotrophic oomycete Hyaloperonospora parasitica, and the fungal necrotrophic pathogens Fusarium oxysporum f. sp. matthiolae and Alternaria brassicicola. CaAMP1 overexpression induced the salicylic acid pathway-dependent genes PR1 and PR5 but not the jasmonic acid-dependent defense gene PDF1.2 during P. syringae pv tomato infection. Together, these results suggest that the antimicrobial CaAMP1 protein is involved in broad-spectrum resistance to bacterial and fungal pathogen infection. PMID:18676663

  11. Elicitin-Induced Distal Systemic Resistance in Plants is Mediated Through the Protein-Protein Interactions Influenced by Selected Lysine Residues.

    PubMed

    Uhlíková, Hana; Obořil, Michal; Klempová, Jitka; Šedo, Ondrej; Zdráhal, Zbyněk; Kašparovský, Tomáš; Skládal, Petr; Lochman, Jan

    2016-01-01

    Elicitins are a family of small proteins with sterol-binding activity that are secreted by Phytophthora and Pythium sp. classified as oomycete PAMPs. Although α- and β-elicitins bind with the same affinity to one high affinity binding site on the plasma membrane, β-elicitins (possessing 6-7 lysine residues) are generally 50- to 100-fold more active at inducing distal HR and systemic resistance than the α-isoforms (with only 1-3 lysine residues). To examine the role of lysine residues in elicitin biological activity, we employed site-directed mutagenesis to prepare a series of β-elicitin cryptogein variants with mutations on specific lysine residues. In contrast to direct infiltration of protein into leaves, application to the stem revealed a rough correlation between protein's charge and biological activity, resulting in protection against Phytophthora parasitica. A detailed analysis of proteins' movement in plants showed no substantial differences in distribution through phloem indicating differences in consequent apoplastic or symplastic transport. In this process, an important role of homodimer formation together with the ability to form a heterodimer with potential partner represented by endogenous plants LTPs is suggested. Our work demonstrates a key role of selected lysine residues in these interactions and stresses the importance of processes preceding elicitin recognition responsible for induction of distal systemic resistance.

  12. Electroacupuncture Improves Insulin Resistance by Reducing Neuroprotein Y/Agouti-Related Protein Levels and Inhibiting Expression of Protein Tyrosine Phosphatase 1B in Diet-induced Obese Rats.

    PubMed

    Liu, Xia; He, Jun-Feng; Qu, Ya-Ting; Liu, Zhi-Jun; Pu, Qing-Yang; Guo, Sheng-Tong; Du, Jia; Jiang, Peng-Fei

    2016-04-01

    Electroacupuncture (EA) has been shown to exert beneficial effects on obesity, but the mechanism is unclear. This study investigated the effects of EA on diet-induced obese (DIO) rats. Fifty male Sprague-Dawley rats were randomly divided into low-fat diet (LFD, 10 rats) and high-fat diet (HFD, 40 rats) groups. After the DIO models had been established, successful model rats were randomly divided into HFD, EA, and orlistat (OLST) groups. The EA group received EA at Zusanli (ST36) and Quchi (LI11) for 20 minutes once per day for 28 days. The OLST group was treated with orlistat by gavage. The body weight, homeostasis model assessment-insulin resistance index, adipocyte diameters, and neuroprotein Y/agouti-related protein and protein tyrosine phosphatase 1B levels were significantly lower in the EA group than in the HFD group. The rats of the OLST group showed watery stools and yellow hairs whereas those of the EA group had regular stools and sleek coats. The effect of EA on weight loss may be related to improved insulin resistance caused by changes in the adipocyte size and by reductions in the expressions of neuroprotein Y/agouti-related protein and protein tyrosine phosphatase 1B. This study indicates that EA may be a better method of alternative therapy for treating obesity and other metabolic diseases.

  13. A novel small acid soluble protein variant is important for spore resistance of most Clostridium perfringens food poisoning isolates.

    PubMed

    Li, Jihong; McClane, Bruce A

    2008-05-02

    Clostridium perfringens is a major cause of food poisoning (FP) in developed countries. C. perfringens isolates usually induce the gastrointestinal symptoms of this FP by producing an enterotoxin that is encoded by a chromosomal (cpe) gene. Those typical FP strains also produce spores that are extremely resistant to food preservation approaches such as heating and chemical preservatives. This resistance favors their survival and subsequent germination in improperly cooked, prepared, or stored foods. The current study identified a novel alpha/beta-type small acid soluble protein, now named Ssp4, and showed that sporulating cultures of FP isolates producing resistant spores consistently express a variant Ssp4 with an Asp substitution at residue 36. In contrast, Gly was detected at Ssp4 residue 36 in C. perfringens strains producing sensitive spores. Studies with isogenic mutants and complementing strains demonstrated the importance of the Asp 36 Ssp4 variant for the exceptional heat and sodium nitrite resistance of spores made by most FP strains carrying a chromosomal cpe gene. Electrophoretic mobility shift assays and DNA binding studies showed that Ssp4 variants with an Asp at residue 36 bind more efficiently and tightly to DNA than do Ssp4 variants with Gly at residue 36. Besides suggesting one possible mechanistic explanation for the highly resistant spore phenotype of most FP strains carrying a chromosomal cpe gene, these findings may facilitate eventual development of targeted strategies to increase killing of the resistant spores in foods. They also provide the first indication that SASP variants can be important contributors to intra-species (and perhaps inter-species) variations in bacterial spore resistance phenotypes. Finally, Ssp4 may contribute to spore resistance properties throughout the genus Clostridium since ssp4 genes also exist in the genomes of other clostridial species.

  14. Resistance to Bt maize in Mythimna unipuncta (Lepidoptera: Noctuidae) is mediated by alteration in Cry1Ab protein activation.

    PubMed

    González-Cabrera, Joel; García, Matías; Hernández-Crespo, Pedro; Farinós, Gema P; Ortego, Félix; Castañera, Pedro

    2013-08-01

    Bt maize cultivars based on the event MON810 (expressing Cry1Ab) have shown high efficacy for controlling corn borers. However, their efficiency for controlling some secondary lepidopteran pests such as Mythimna unipuncta has been questioned, raising concerns about potential outbreaks and its economic consequences. We have selected a resistant strain (MR) of M. unipuncta, which is capable of completing its life cycle on Bt maize and displays a similar performance when feeding on both Bt and non-Bt maize. The proteolytic activation of the protoxin and the binding of active toxin to brush border membrane vesicles were investigated in the resistant and a control strain. A reduction in the activity of proteolytic enzymes, which correlates with impaired capacity of midgut extracts to activate the Cry1Ab protoxin has been observed in the resistant strain. Moreover, resistance in larvae of the MR strain was reverted when treated with Cry1Ab toxin activated with midgut juice from the control strain. All these data indicate that resistance in the MR strain is mediated by alteration of toxin activation rather than to an increase in the proteolytic degradation of the protein. By contrast, binding assays performed with biotin labelled Cry1Ab suggest that binding to midgut receptors does not play a major role in the resistance to Bt maize. Our results emphasize the risk of development of resistance in field populations of M. unipuncta and the need to consider this secondary pest in ongoing resistance management programs to avoid the likely negative agronomic and environmental consequences.

  15. Molecular Epidemiology and Distribution of Serotypes, Surface Proteins, and Antibiotic Resistance among Group B Streptococci in Italy▿

    PubMed Central

    Gherardi, Giovanni; Imperi, Monica; Baldassarri, Lucilla; Pataracchia, Marco; Alfarone, Giovanna; Recchia, Simona; Orefici, Graziella; Dicuonzo, Giordano; Creti, Roberta

    2007-01-01

    Group B streptococci (GBS) comprising three different sets of isolates (31 invasive, 36 noninvasive, and 24 colonizing isolates) were collected in Italy during the years 2002 to 2005. Clonal groups were established by pulsed-field gel electrophoresis (PFGE), and selected isolates were studied by multilocus sequence typing (MLST). GBS isolates were also characterized by classical and molecular techniques for serotyping and protein gene and antibiotic resistance profiling. Some serotypes were significantly associated with a particular isolate population: serotype Ia more frequently corresponded to invasive strains than other strains, serotype V was more frequently encountered among noninvasive strains, and nontypeable strains were more common among isolates from carriers. Four major clonal groups accounted for 52.7% of all isolates: PFGE type 1/clonal complex 1 (CC1) comprised mainly serotype V isolates carrying the alp3 gene, PFGE type 2/CC23 encompassed serotype Ia isolates with the alp1 or alpha gene, PFGE type 3/CC17 comprised serotype III isolates carrying the rib gene, and PFGE type 4/CC19 consisted mainly of serotype II isolates possessing the rib gene. The same serotypes were shared by isolates of different clonal groups, and conversely, isolates belonging to the same clonal groups were found to be of different serotypes, presumably due to capsular switching by the horizontal transfer of capsular genes. Erythromycin resistance (prevalence, 16.5%; 15 resistant isolates of 91) was restricted to strains isolated from patients with noninvasive infections and carriers, while tetracycline resistance was evenly distributed (prevalence, 68.1%; 62 resistant isolates of 91). Most erythromycin-resistant GBS strains were of serotype V, were erm(B) positive, and belonged to the PFGE type 1/CC1 group, suggesting that macrolide resistance may have arisen both by clonal dissemination and by the horizontal transfer of resistance genes. PMID:17634303

  16. X-linked inhibitor of apoptosis protein mediates tumor cell resistance to antibody-dependent cellular cytotoxicity

    PubMed Central

    Evans, M K; Sauer, S J; Nath, S; Robinson, T J; Morse, M A; Devi, G R

    2016-01-01

    Inflammatory breast cancer (IBC) is the deadliest, distinct subtype of breast cancer. High expression of epidermal growth factor receptors [EGFR or human epidermal growth factor receptor 2 (HER2)] in IBC tumors has prompted trials of anti-EGFR/HER2 monoclonal antibodies to inhibit oncogenic signaling; however, de novo and acquired therapeutic resistance is common. Another critical function of these antibodies is to mediate antibody-dependent cellular cytotoxicity (ADCC), which enables immune effector cells to engage tumors and deliver granzymes, activating executioner caspases. We hypothesized that high expression of anti-apoptotic molecules in tumors would render them resistant to ADCC. Herein, we demonstrate that the most potent caspase inhibitor, X-linked inhibitor of apoptosis protein (XIAP), overexpressed in IBC, drives resistance to ADCC mediated by cetuximab (anti-EGFR) and trastuzumab (anti-HER2). Overexpression of XIAP in parental IBC cell lines enhances resistance to ADCC; conversely, targeted downregulation of XIAP in ADCC-resistant IBC cells renders them sensitive. As hypothesized, this ADCC resistance is in part a result of the ability of XIAP to inhibit caspase activity; however, we also unexpectedly found that resistance was dependent on XIAP-mediated, caspase-independent suppression of reactive oxygen species (ROS) accumulation, which otherwise occurs during ADCC. Transcriptome analysis supported these observations by revealing modulation of genes involved in immunosuppression and oxidative stress response in XIAP-overexpressing, ADCC-resistant cells. We conclude that XIAP is a critical modulator of ADCC responsiveness, operating through both caspase-dependent and -independent mechanisms. These results suggest that strategies targeting the effects of XIAP on caspase activation and ROS suppression have the potential to enhance the activity of monoclonal antibody-based immunotherapy. PMID:26821068

  17. Natural-resistance-associated macrophage protein 1 is an H+/bivalent cation antiporter.

    PubMed Central

    Goswami, T; Bhattacharjee, A; Babal, P; Searle, S; Moore, E; Li, M; Blackwell, J M

    2001-01-01

    In mammals, natural-resistance-associated macrophage protein 1 (Nramp1) regulates macrophage activation and is associated with infectious and autoimmune diseases. Nramp2 is associated with anaemia. Both belong to a highly conserved eukaryote/prokaryote protein family. We used Xenopus oocytes to demonstrate that, like Nramp2, Nramp1 is a bivalent cation (Fe2+, Zn2+ and Mn2+) transporter. Strikingly, however, where Nramp2 is a symporter of H+ and metal ions, Nramp1 is a highly pH-dependent antiporter that fluxes metal ions in either direction against a proton gradient. At pH 9.0, oocytes injected with cRNA from wild-type murine Nramp1 with a glycine residue at position 169 (Nramp1(G169); P=3.22x10(-6)) and human NRAMP1 (P=3.87x10(-5)) showed significantly enhanced uptake of radiolabelled Zn2+ compared with water-injected controls. At pH 5.5, Nramp1(G169) (P=1.34x10(-13)) and NRAMP1 (P=1.09x10(-6)) oocytes showed significant efflux of Zn2+. Zn2+ transport was abolished when the proton gradient was dissipated using carbonyl cyanide p-trifluoromethoxyphenylhydrazone. Using pre-acidified oocytes, currents of 130+/-57 nA were evoked by 100 microM Zn2+ at pH 7.5, and 139+/-47 nA by 100 microM Fe2+ at pH 7.0, in Nramp1(G169) oocytes; currents of 254+/-49 nA and 242+/-26 nA were evoked, respectively, in NRAMP1 oocytes. Steady-state currents evoked by increasing concentrations of Zn2+ were saturable, with apparent affinity constants of approx. 614 nM for Nramp1(G169) and approx. 562 nM for NRAMP1 oocytes, and a curvilinear voltage dependence of transporter activity (i.e. the data points approximate to a curve that approaches a linear asymptote). In the present study we propose a new model for metal ion homoeostasis in macrophages. Under normal physiological conditions, Nramp2, localized to early endosomal membranes, delivers extracellularly acquired bivalent cations into the cytosol. Nramp1, localized to late endosomal/lysosomal membranes, delivers bivalent cations from the

  18. A High Protein Diet Has No Harmful Effects: A One-Year Crossover Study in Resistance-Trained Males

    PubMed Central

    Ellerbroek, Anya; Silver, Tobin; Vargas, Leonel; Tamayo, Armando; Buehn, Richard

    2016-01-01

    The purpose of this investigation was to determine the effects of a high protein diet over a one-year period. Fourteen healthy resistance-trained men completed the study (mean ± SD; age 26.3 ± 3.9 yr; height 178.5 ± 8.4 cm; and average years of training 8.9 ± 3.4 yr). In a randomized crossover design, subjects consumed their habitual or normal diet for 2 months and 4 months and alternated that with a higher protein diet (>3 g/kg/d) for 2 months and 4 months. Thus, on average, each subject was on their normal diet for 6 months and a higher protein diet for 6 months. Body composition was assessed via the Bod Pod®. Each subject provided approximately 100–168 daily dietary self-reports. During the subjects' normal eating phase, they consumed (mean ± SD) 29.94 ± 5.65 kcals/kg/day and 2.51 ± 0.69 g/kg/day of protein. This significantly increased (p < 0.05) during the high protein phase to 34.37 ± 5.88 kcals/kg/day and 3.32 ± 0.87 g/kg/day of protein. Our investigation discovered that, in resistance-trained men that consumed a high protein diet (~2.51–3.32 g/kg/d) for one year, there were no harmful effects on measures of blood lipids as well as liver and kidney function. In addition, despite the total increase in energy intake during the high protein phase, subjects did not experience an increase in fat mass. PMID:27807480

  19. Resistance of human glioblastoma multiforme cells to growth factor inhibitors is overcome by blockade of inhibitor of apoptosis proteins

    PubMed Central

    Ziegler, David S.; Wright, Renee D.; Kesari, Santosh; Lemieux, Madeleine E.; Tran, Mary A.; Jain, Monish; Zawel, Leigh; Kung, Andrew L.

    2008-01-01

    Multiple receptor tyrosine kinases (RTKs), including PDGFR, have been validated as therapeutic targets in glioblastoma multiforme (GBM), yet inhibitors of RTKs have had limited clinical success. As various antiapoptotic mechanisms render GBM cells resistant to chemo- and radiotherapy, we hypothesized that these antiapoptotic mechanisms also confer resistance to RTK inhibition. We found that in vitro inhibition of PDGFR in human GBM cells initiated the intrinsic pathway of apoptosis, as evidenced by mitochondrial outer membrane permeabilization, but downstream caspase activation was blocked by inhibitor of apoptosis proteins (IAPs). Consistent with this, inhibition of PDGFR combined with small molecule inactivation of IAPs induced apoptosis in human GBM cells in vitro and had synergistic antitumor effects in orthotopic mouse models of GBM and in primary human GBM neurospheres. These results demonstrate that concomitant inhibition of IAPs can overcome resistance to RTK inhibitors in human malignant GBM cells, and suggest that blockade of IAPs has the potential to improve treatment outcomes in patients with GBM. PMID:18677408

  20. Design of protease-resistant myelin basic protein-derived peptides by cleavage site directed amino acid substitutions.

    PubMed

    Burster, Timo; Marin-Esteban, Viviana; Boehm, Bernhard O; Dunn, Shannon; Rotzschke, Olaf; Falk, Kirsten; Weber, Ekkehard; Verhelst, Steven H L; Kalbacher, Hubert; Driessen, Christoph

    2007-11-15

    Multiple Sclerosis (MS) is considered to be a T cell-mediated autoimmune disease. An attractive strategy to prevent activation of autoaggressive T cells in MS, is the use of altered peptide ligands (APL), which bind to major histocompatibility complex class II (MHC II) molecules. To be of clinical use, APL must be capable of resisting hostile environments including the proteolytic machinery of antigen presenting cells (APC). The current design of APL relies on cost- and labour-intensive strategies. To overcome these major drawbacks, we used a deductive approach which involved modifying proteolytic cleavage sites in APL. Cleavage site-directed amino acid substitution of the autoantigen myelin basic protein (MBP) resulted in lysosomal protease-resistant, high-affinity binding peptides. In addition, these peptides mitigated T cell activation in a similar fashion as conventional APL. The strategy outlined allows the development of protease-resistant APL and provides a universal design strategy to improve peptide-based immunotherapeutics.

  1. Effect of glucose transport inhibitors on vincristine efflux in multidrug-resistant murine erythroleukaemia cells overexpressing the multidrug resistance-associated protein (MRP) and two glucose transport proteins, GLUT1 and GLUT3.

    PubMed Central

    Martell, R. L.; Slapak, C. A.; Levy, S. B.

    1997-01-01

    The relationship between mammalian facilitative glucose transport proteins (GLUT) and multidrug resistance was examined in two vincristine (VCR)-selected murine erythroleukaemia (MEL) PC4 cell lines. GLUT proteins, GLUT1 and GLUT3, were constitutively coexpressed in the parental cell line and also in the VCR-selected cell lines. Increased expression of the GLUT1 isoform was noted both in the PC-V40 (a non-P-glycoprotein, mrp-overexpressing subline) and in the more resistant PC-V160 (overexpressing mrp and mdr3) cell lines. Overexpression of GLUT3 was detected only in the PC-V160 subline. An increased rate of facilitative glucose transport (Vmax) and level of plasma membrane GLUT protein expression paralleled increased VCR resistance, active VCR efflux and decreased VCR steady-state accumulation in these cell lines. Glucose transport inhibitors (GTIs), cytochalasin B (CB) and phloretin blocked the active efflux and decreased steady-state accumulation of VCR in the PC-V40 subline. GTIs did not significantly affect VCR accumulation in the parental or PC-V160 cells. A comparison of protein sequences among GLUT1, GLUT3 and MRP revealed a putative cytochalasin B binding site in MRP, which displayed 44% sequence similarity/12% identity with that previously identified in GLUT1 and GLUT3; these regions also exhibited a similar hydropathy plot pattern. The findings suggested that CB bound to MRP and directly or indirectly lowered VCR efflux and/or CB bound to one or both GLUT proteins, which acted to lower the VCR efflux mediated by MRP. This is the first report of a non-neuronal murine cell line that expressed GLUT3. Images Figure 3 PMID:9010020

  2. A review of resistance training-induced changes in skeletal muscle protein synthesis and their contribution to hypertrophy.

    PubMed

    Damas, Felipe; Phillips, Stuart; Vechin, Felipe Cassaro; Ugrinowitsch, Carlos

    2015-06-01

    Muscle protein synthesis (MPS) is stimulated by resistance exercise (RE) and is further stimulated by protein ingestion. The summation of periods of RE-induced increases in MPS can induce hypertrophy chronically. As such, studying the response of MPS with resistance training (RT) is informative, as adaptations in this process can modulate muscle mass gain. Previous studies have shown that the amplitude and duration of increases in MPS after an acute bout of RE are modulated by an individual's training status. Nevertheless, it has been shown that the initial responses of MPS to RE and nutrition are not correlated with subsequent hypertrophy. Thus, early acute responses of MPS in the hours after RE, in an untrained state, do not capture how MPS can affect RE-induced muscle hypertrophy. The purpose of this review is provide an in-depth understanding of the dynamic process of muscle hypertrophy throughout RT by examining all of the available data on MPS after RE and in different phases of an RT programme. Analysis of the time course and the overall response of MPS is critical to determine the potential protein accretion after an RE bout. Exercise-induced increases in MPS are shorter lived and peak earlier in the trained state than in the untrained state, resulting in a smaller overall muscle protein synthetic response in the trained state. Thus, RT induces a dampening of the MPS response, potentially limiting protein accretion, but when this occurs remains unknown.

  3. Multidrug Resistance Protein 1 Protects the Oropharyngeal Mucosal Layer and the Testicular Tubules against Drug-induced Damage

    PubMed Central

    Wijnholds, Jan; Scheffer, George L.; van der  Valk, Martin; van der  Valk, Paul; Beijnen, Jos H.; Scheper, Rik J.; Borst, Piet

    1998-01-01

    The multidrug resistance protein 1 (MRP1) gene encodes a transporter protein that helps to protect cells against xenobiotics. Elevated levels of MRP1 in tumor cells can result in active extrusion of a wide range of (anticancer) drugs with different cellular targets, a phenomenon called multidrug resistance (MDR). To explore the protective function of the mouse mrp1 protein during drug treatment, we investigated the toxicity caused by the anticancer drug etoposide-phosphate (ETOPOPHOS) in mice lacking the mrp1 gene (mrp1−/− mice). We show here that the lack of mrp1 protein results in increased etoposide-induced damage to the mucosa of the oropharyngeal cavity and to the seminiferous tubules of the testis. The high concentrations of mrp1 that we find in the basal layers of the oropharyngeal mucosa and in the basal membrane of the Sertoli cells in the testis apparently protect wild-type mice against this tissue damage. We also find drug-induced polyuria in mrp1−/− mice, which correlates with the presence of mrp1 protein in the urinary collecting tubules, the major site of kidney water reabsorption. Our results indicate that specific inhibitors of MRP1 used to reverse MDR, in combination with carcinostatic drugs transported by MRP1, might lead to drug-induced mucositis, (temporary) infertility, and diabetes insipidus. PMID:9730882

  4. Myxovirus Resistance Protein A mRNA Expression Kinetics in Multiple Sclerosis Patients Treated with IFNβ

    PubMed Central

    Libertinova, Jana; Meluzinova, Eva; Tomek, Ales; Horakova, Dana; Kovarova, Ivana; Matoska, Vaclav; Kumstyrova, Simona; Zajac, Miroslav; Hyncicova, Eva; Liskova, Petra; Houzvickova, Eva; Martinkovic, Lukas; Bojar, Martin; Havrdova, Eva; Marusic, Petr

    2017-01-01

    Introduction Interferon-β (IFNß) is the first-line treatment for relapsing-remitting multiple sclerosis. Myxovirus resistance protein A (MxA) is a marker of IFNß bioactivity, which may be reduced by neutralizing antibodies (NAbs) against IFNß. The aim of the study was to analyze the kinetics of MxA mRNA expression during long-term IFNβ treatment and assess its predictive value. Methods A prospective, observational, open-label, non-randomized study was designed in multiple sclerosis patients starting IFNß treatment. MxA mRNA was assessed prior to initiation of IFNß therapy and every three months subsequently. NAbs were assessed every six months. Assessment of relapses was scheduled every three months during 24 months of follow up. The disease activity was correlated to the pretreatment baseline MxA mRNA value. In NAb negative patients, clinical status was correlated to MxA mRNA values. Results 119 patients were consecutively enrolled and 107 were included in the final analysis. There was no correlation of MxA mRNA expression levels between baseline and month three. Using survival analysis, none of the selected baseline MxA mRNA cut off points allowed prediction of time to first relapse on the treatment. In NAb negative patients, mean MxA mRNA levels did not significantly differ in patients irrespective of relapse status. Conclusion Baseline MxA mRNA does not predict the response to IFNß treatment or the clinical status of the disease and the level of MxA mRNA does not correlate with disease activity in NAb negative patients. PMID:28081207

  5. Involvement of multidrug resistance-associated protein 2 in intestinal secretion of grepafloxacin in rats.

    PubMed

    Naruhashi, Kazumasa; Tamai, Ikumi; Inoue, Natsuko; Muraoka, Hiromi; Sai, Yoshimichi; Suzuki, Nagao; Tsuji, Akira

    2002-02-01

    We investigated the contribution of multidrug resistance-associated protein 2 (MRP2) to the secretory transport of grepafloxacin and compared its functional role with that of P-glycoprotein (P-gp) by using Sprague-Dawley rats (SDRs) and Eisai hyperbilirubinemic rats (EHBRs), in which MRP2 is hereditarily defective. In intestinal tissue from SDRs mounted in Ussing chambers, the level of transport in the direction from the serosal layer to the mucosal layer was twofold greater than that in the direction from the mucosal layer to the serosal layer. This secretory transport of grepafloxacin was diminished by both probenecid, an MRP2 inhibitor, and cyclosporine, a P-gp inhibitor. In intestinal tissue from EHBRs, the secretory transport of grepafloxacin was lower than that in intestinal tissue from SDRs and was inhibited by cyclosporine but not by probenecid. The absorption of grepafloxacin from intestinal loops in SDRs was in the order of duodenum > jejunum > ileum and was increased by cyclosporine but not by probenecid. The absorption in EHBRs was not higher than that in SDRs. The intestinal secretory clearance in SDRs after intravenous administration of grepafloxacin was shown to be greater for the ileum than for the duodenum, which is in good agreement with the previously reported regional expression profile of MRP2 mRNA. The intestinal secretory clearance was lower in EHBRs than in SDRs. Accordingly, in addition to P-gp, MRP2 might play a role in the secretory transport of grepafloxacin. The function of MRP2 in facilitating grepafloxacin transport in the secretory direction is more pronounced both in vitro and in vivo, while the restriction of entry from the lumen into the cell by MRP2 seems to be negligible, compared with that by P-gp, in the case of grepafloxacin.

  6. Effects of hypergravity on the expression of multidrug resistance proteins in human melanocytic cells

    NASA Astrophysics Data System (ADS)

    Lambers, B.; Stieber, C.; Grigorieva, O.; Block, I.; Bromeis, B.; Buravkova, L.; Gerzer, R.; Ivanova, K.

    In humans the skin serves as a barrier against potentially harmful effects of the environment Human melanocytes constitute the principal cells for skin pigmentation by synthesizing the pigment melanin Melanin acts as a scavenger for free radicals that may arise during metabolic stress The melanocytes are also able to secrete a wide range of signal molecules In previous studies we found that normal human melanocytes NHMs and non-metastatic melanoma cells respond to long-time exposure to hypergravity up to 5 g for 24 h with elevated efflux of guanosine 3 5 -cyclic monophosphate cGMP in the presence of phosphodiesterase PDE inhibitors e g 3-isobutyl-1-methylxanthine Cyclic GMP is known to play a signaling role in human melanocyte physiology It controls the signaling activities of nitric oxide NO in relation to melanogenesis as well as in melanocyte-extracellular matrix interactions that may be important for some pathological processes including metastasis The present study investigated the effects of hypergravity on the expression of the multidrug resistance proteins MRP 4 and 5 as highly selective cGMP exporters in non-stimulated and NO-stimulated NHMs and melanoma cells MCs on mRNA levels using semi-quantitative RT-PCR analysis Hypergravity up to 5 g for 24 h was produced by horizontal centrifugal acceleration The NONOate DETA-NO 0 1 mM was used as a direct NO donor for cell stimulation For 5-g experiments the mRNA levels for the highly specific cGMP transporter MRP5 appeared to be

  7. Eel green fluorescent protein is associated with resistance to oxidative stress.

    PubMed

    Funahashi, Aki; Komatsu, Masaharu; Furukawa, Tatsuhiko; Yoshizono, Yuki; Yoshizono, Hikari; Orikawa, Yasuhiro; Takumi, Shota; Shiozaki, Kazuhiro; Hayashi, Seiichi; Kaminishi, Yoshio; Itakura, Takao

    2016-01-01

    Green fluorescent protein (GFP) from eel (Anguilla japonica) muscle (eelGFP) is unique in the vertebrates and requires bilirubin as a ligand to emit fluorescence. This study was performed to clarify the physiological function of the unique GFP. Investigation of susceptibility to oxidative stress was carried out using three types of cell lines including jellyfish (Aequorea coerulescens) GFP (jfGFP)-, or eel GFP (eelGFP)-expressing HEK293 cells, and control vector-transfected HEK293 cells. Binding of eelGFP to bilirubin was confirmed by the observation of green fluorescence in HEK293-eelGFP cells. The growth rate was compared with the three types of cells in the presence or absence of phenol red which possessed antioxidant activity. The growth rates of HEK293-CV and HEK293-jfGFP under phenol red-free conditions were reduced to 52 and 31% of those under phenol red. Under the phenol red-free condition, HEK293-eelGFP had a growth rate of approximately 70% of the phenol red-containing condition. The eelGFP-expressing cells were approximately 2-fold resistant to oxidative stress such as H2O2 exposure. The fluorescence intensity partially decreased or disappeared after exposure to H2O2, and heterogeneous intensity of fluorescence was also observed in isolated eel skeletal muscle cells. These results suggested eelGFP, but not jfGFP, coupled with bilirubin provided the antioxidant activity to the cells as compared to non-bound free bilirubin.

  8. Protease resistance of infectious prions is suppressed by removal of a single atom in the cellular prion protein.

    PubMed

    Leske, Henning; Hornemann, Simone; Herrmann, Uli Simon; Zhu, Caihong; Dametto, Paolo; Li, Bei; Laferriere, Florent; Polymenidou, Magdalini; Pelczar, Pawel; Reimann, Regina Rose; Schwarz, Petra; Rushing, Elisabeth Jane; Wüthrich, Kurt; Aguzzi, Adriano

    2017-01-01

    Resistance to proteolytic digestion has long been considered a defining trait of prions in tissues of organisms suffering from transmissible spongiform encephalopathies. Detection of proteinase K-resistant prion protein (PrPSc) still represents the diagnostic gold standard for prion diseases in humans, sheep and cattle. However, it has become increasingly apparent that the accumulation of PrPSc does not always accompany prion infections: high titers of prion infectivity can be reached also in the absence of protease resistant PrPSc. Here, we describe a structural basis for the phenomenon of protease-sensitive prion infectivity. We studied the effect on proteinase K (PK) resistance of the amino acid substitution Y169F, which removes a single oxygen atom from the β2-α2 loop of the cellular prion protein (PrPC). When infected with RML or the 263K strain of prions, transgenic mice lacking wild-type (wt) PrPC but expressing MoPrP169F generated prion infectivity at levels comparable to wt mice. The newly generated MoPrP169F prions were biologically indistinguishable from those recovered from prion-infected wt mice, and elicited similar pathologies in vivo. Surprisingly, MoPrP169F prions showed greatly reduced PK resistance and density gradient analyses showed a significant reduction in high-density aggregates. Passage of MoPrP169F prions into mice expressing wt MoPrP led to full recovery of protease resistance, indicating that no strain shift had taken place. We conclude that a subtle structural variation in the β2-α2 loop of PrPC affects the sensitivity of PrPSc to protease but does not impact prion replication and infectivity. With these findings a specific structural feature of PrPC can be linked to a physicochemical property of the corresponding PrPSc.

  9. Protease resistance of infectious prions is suppressed by removal of a single atom in the cellular prion protein

    PubMed Central

    Hornemann, Simone; Herrmann, Uli Simon; Zhu, Caihong; Dametto, Paolo; Li, Bei; Laferriere, Florent; Polymenidou, Magdalini; Pelczar, Pawel; Schwarz, Petra; Rushing, Elisabeth Jane; Wüthrich, Kurt; Aguzzi, Adriano

    2017-01-01

    Resistance to proteolytic digestion has long been considered a defining trait of prions in tissues of organisms suffering from transmissible spongiform encephalopathies. Detection of proteinase K-resistant prion protein (PrPSc) still represents the diagnostic gold standard for prion diseases in humans, sheep and cattle. However, it has become increasingly apparent that the accumulation of PrPSc does not always accompany prion infections: high titers of prion infectivity can be reached also in the absence of protease resistant PrPSc. Here, we describe a structural basis for the phenomenon of protease-sensitive prion infectivity. We studied the effect on proteinase K (PK) resistance of the amino acid substitution Y169F, which removes a single oxygen atom from the β2–α2 loop of the cellular prion protein (PrPC). When infected with RML or the 263K strain of prions, transgenic mice lacking wild-type (wt) PrPC but expressing MoPrP169F generated prion infectivity at levels comparable to wt mice. The newly generated MoPrP169F prions were biologically indistinguishable from those recovered from prion-infected wt mice, and elicited similar pathologies in vivo. Surprisingly, MoPrP169F prions showed greatly reduced PK resistance and density gradient analyses showed a significant reduction in high-density aggregates. Passage of MoPrP169F prions into mice expressing wt MoPrP led to full recovery of protease resistance, indicating that no strain shift had taken place. We conclude that a subtle structural variation in the β2–α2 loop of PrPC affects the sensitivity of PrPSc to protease but does not impact prion replication and infectivity. With these findings a specific structural feature of PrPC can be linked to a physicochemical property of the corresponding PrPSc. PMID:28207746

  10. Protein, cell and bacterial fouling resistance of polypeptoid-modified surfaces: effect of side-chain chemistry†

    PubMed Central

    Statz, Andrea R.; Barron, Annelise E.; Messersmith, Phillip B.

    2011-01-01

    Peptidomimetic polymers consisting of poly-N-substituted glycine oligomers (polypeptoids) conjugated to biomimetic adhesive polypeptides were investigated as antifouling surface coatings. The polymers were immobilized onto TiO2 surfaces via an anchoring peptide consisting of alternating residues of 3,4-dihydroxyphenylalanine (DOPA) and lysine. Three polypeptoid side-chain compositions were investigated for antifouling performance and stability toward enzymatic degradation. Ellipsometry and XPS analysis confirmed that purified polymers adsorbed strongly to TiO2 surfaces, and the immobilized polymers were resistant to enzymatic degradation as demonstrated by mass spectrometry. All polypeptoid-modified surfaces exhibited significant reductions in adsorption of lysozyme, fibrinogen and serum proteins, and were resistant to 3T3 fibroblast cell attachment for up to seven days. Long-term in vitro cell attachment studies conducted for six weeks revealed the importance of polypeptoid side-chain composition, with a methoxyethyl side chain providing superior long-term fouling resistance compared to hydroxyethyl and hydroxypropyl side chains. Finally, attachment of both gram-positive and gram-negative bacteria for up to four days under continuous-flow conditions was significantly reduced on the polypeptoid-modified surfaces compared to unmodified TiO2 surfaces. The results reveal the influence of polypeptoid side-chain chemistry on short-term and long-term protein, cell and bacterial fouling resistance. PMID:21472038

  11. Virulent Diuraphis noxia Aphids Over-Express Calcium Signaling Proteins to Overcome Defenses of Aphid-Resistant Wheat Plants

    PubMed Central

    Sinha, Deepak K.; Chandran, Predeesh; Timm, Alicia E.; Aguirre-Rojas, Lina; Smith, C. Michael

    2016-01-01

    The Russian wheat aphid, Diuraphis noxia, an invasive phytotoxic pest of wheat, Triticum aestivum, and barley, Hordeum vulgare, causes huge economic losses in Africa, South America, and North America. Most acceptable and ecologically beneficial aphid management strategies include selection and breeding of D. noxia-resistant varieties, and numerous D. noxia resistance genes have been identified in T. aestivum and H. vulgare. North American D. noxia biotype 1 is avirulent to T. aestivum varieties possessing Dn4 or Dn7 genes, while biotype 2 is virulent to Dn4 and avirulent to Dn7. The current investigation utilized next-generation RNAseq technology to reveal that biotype 2 over expresses proteins involved in calcium signaling, which activates phosphoinositide (PI) metabolism. Calcium signaling proteins comprised 36% of all transcripts identified in the two D. noxia biotypes. Depending on plant resistance gene-aphid biotype interaction, additional transcript groups included those involved in tissue growth; defense and stress response; zinc ion and related cofactor binding; and apoptosis. Activation of enzymes involved in PI metabolism by D. noxia biotype 2 aphids allows depletion of plant calcium that normally blocks aphid feeding sites in phloem sieve elements and enables successful, continuous feeding on plants resistant to avirulent biotype 1. Inhibition of the key enzyme phospholipase C significantly reduced biotype 2 salivation into phloem and phloem sap ingestion. PMID:26815857

  12. CopM is a novel copper-binding protein involved in copper resistance in Synechocystis sp. PCC 6803.

    PubMed

    Giner-Lamia, Joaquín; López-Maury, Luis; Florencio, Francisco J

    2015-02-01

    Copper resistance system in the cyanobacterium Synechocystis sp. PCC 6803 comprises two operons, copMRS and copBAC, which are expressed in response to copper in the media. copBAC codes for a heavy-metal efflux-resistance nodulation and division (HME-RND) system, while copMRS codes for a protein of unknown function, CopM, and a two-component system CopRS, which controls the expression of these two operons. Here, we report that CopM is a periplasmic protein able to bind Cu(I) with high affinity (KD ~3 × 10(-16) ). Mutants lacking copM showed a sensitive copper phenotype similar to mutants affected in copB, but lower than mutants of the two-component system CopRS, suggesting that CopBAC and CopM constitute two independent resistance mechanisms. Moreover, constitutive expression of copM is able to partially suppress the copper sensitivity of the copR mutant strain, pointing out that CopM per se is able to confer copper resistance. Furthermore, constitutive expression of copM was able to reduce total cellular copper content of the copR mutant to the levels determined in the wild-type (WT) strain. Finally, CopM was localized not only in the periplasm but also in the extracellular space, suggesting that CopM can also prevent copper accumulation probably by direct copper binding outside the cell.

  13. Enhancing the protein resistance of silicone via surface-restructuring PEO-silane amphiphiles with variable PEO length

    PubMed Central

    Rufin, M. A.; Gruetzner, J. A.; Hurley, M. J.; Hawkins, M. L.; Raymond, E. S.; Raymond, J. E.

    2015-01-01

    Silicones with superior protein resistance were produced by bulk-modification with poly(ethylene oxide) (PEO)-silane amphiphiles that demonstrated a higher capacity to restructure to the surface-water interface versus conventional non-amphiphilic PEO-silanes. The PEO-silane amphiphiles were prepared with a single siloxane tether length but variable PEO segment lengths: α-(EtO)3Si(CH2)2-oligodimethylsiloxane13-block-poly(ethylene oxide)n-OCH3 (n = 3, 8, and 16). Conventional PEO-silane analogues (n = 3, 8 and 16) as well as a siloxane tether-silane (i.e. no PEO segment) were prepared as controls. When surface-grafted onto silicon wafer, PEO-silane amphiphiles produced surfaces that were more hydrophobic and thus more adherent towards fibrinogen versus the corresponding PEO-silane. However, when blended into a silicone, PEO-silane amphiphiles exhibited rapid restructuring to the surface-water interface and excellent protein resistance whereas the PEO-silanes did not. Silicones modified with PEO-silane amphiphiles of PEO segment lengths n = 8 and 16 achieved the highest protein resistance. PMID:26339488

  14. Protein-resistant properties of a chemical vapor deposited alkyl-functional carboxysilane coating characterized using quartz crystal microbalance

    NASA Astrophysics Data System (ADS)

    Vaidya, Shyam V.; Yuan, Min; Narváez, Alfredo R.; Daghfal, David; Mattzela, James; Smith, David

    2016-02-01

    The protein-resistant properties of a chemical vapor deposited alkyl-functional carboxysilane coating (Dursan®) were compared to that of an amorphous fluoropolymer (AF1600) coating and bare 316L grade stainless steel by studying non-specific adsorption of various proteins onto these surfaces using quartz crystal microbalance with dissipation monitoring (QCM-D). A wash solution with nonionic surfactant, polyoxyethyleneglycol dodecyl ether (or Brij 35), facilitated 100% removal of the adsorbed bovine serum albumin (BSA), mouse immunoglobulin G (IgG), and normal human plasma proteins from the Dursan surface and of the adsorbed normal human plasma proteins from the AF1600 surface, whereas these proteins remained adsorbed on the bare stainless steel surface. Mechanical stress in the form of sonication demonstrated durability of the Dursan coating to mechanical wear and showed no negative impact on the coating's ability to prevent adsorption of plasma proteins. Surface delamination was observed in case of the sonicated AF1600 coating, which further led to adsorption of normal human plasma proteins.

  15. Role of extracellular matrix protein CabA in resistance of Vibrio vulnificus biofilms to decontamination strategies.

    PubMed

    Park, Jin Hwan; Lee, Byungho; Jo, Youmi; Choi, Sang Ho

    2016-11-07

    Biofilms are recalcitrant and raise safety problems in the food industry. In this study, the role of CabA, an extracellular matrix protein, in the resistance of the biofilms of Vibrio vulnificus, a foodborne pathogen, to decontamination strategies was investigated. Biofilms of the cabA mutant revealed reduced resistance to detachment by vibration and disinfection by sodium hypochlorite compared to the biofilms of the parental wild type in vitro. The reduced resistance of the cabA mutant biofilms was complemented by introducing a recombinant cabA, indicating that the reduced resistance of the cabA mutant biofilms is caused by the inactivation of cabA. The expression of cabA was induced in cells bound to oyster, the primary vehicle of the pathogen. The cabA mutant biofilms on oyster are defective in biomass and resistance to detachment and disinfection. The bacterial cells in the wild-type biofilms are clustered by filaments which are not apparent in the cabA mutant biofilms. The combined results indicated that CabA contributes to the structural integrity of V. vulnificus biofilms possibly by forming filaments in the matrix and thus rendering the biofilms robust, suggesting that CabA could be a target to control V. vulnificus biofilms on oyster.

  16. TRIB2 confers resistance to anti-cancer therapy by activating the serine/threonine protein kinase AKT

    PubMed Central

    Hill, Richard; Madureira, Patricia A.; Ferreira, Bibiana; Baptista, Inês; Machado, Susana; Colaço, Laura; dos Santos, Marta; Liu, Ningshu; Dopazo, Ana; Ugurel, Selma; Adrienn, Angyal; Kiss-Toth, Endre; Isbilen, Murat; Gure, Ali O.; Link, Wolfgang

    2017-01-01

    Intrinsic and acquired resistance to chemotherapy is the fundamental reason for treatment failure for many cancer patients. The identification of molecular mechanisms involved in drug resistance or sensitization is imperative. Here we report that tribbles homologue 2 (TRIB2) ablates forkhead box O activation and disrupts the p53/MDM2 regulatory axis, conferring resistance to various chemotherapeutics. TRIB2 suppression is exerted via direct interaction with AKT a key signalling protein in cell proliferation, survival and metabolism pathways. Ectopic or intrinsic high expression of TRIB2 induces drug resistance by promoting phospho-AKT (at Ser473) via its COP1 domain. TRIB2 expression is significantly increased in tumour tissues from patients correlating with an increased phosphorylation of AKT, FOXO3a, MDM2 and an impaired therapeutic response. This culminates in an extremely poor clinical outcome. Our study reveals a novel regulatory mechanism underlying drug resistance and suggests that TRIB2 functions as a regulatory component of the PI3K network, activating AKT in cancer cells. PMID:28276427

  17. Gastrodia anti-fungal protein from the orchid Gastrodia elata confers disease resistance to root pathogens in transgenic tobacco.

    PubMed

    Cox, K D; Layne, D R; Scorza, R; Schnabel, G

    2006-11-01

    Diseases of agricultural crops are caused by pathogens from several higher-order phylogenetic lineages including fungi, straminipila, eubacteria, and metazoa. These pathogens are commonly managed with pesticides due to the lack of broad-spectrum host resistance. Gastrodia anti-fungal protein (GAFP; gastrodianin) may provide a level of broad-spectrum resistance due to its documented anti-fungal activity in vitro and structural similarity to insecticidal lectins. We transformed tobacco (Nicotiana tabacum cv. Wisconsin 38) with GAFP-1 and challenged transformants with agriculturally important plant pathogens from several higher-order lineages including Rhizoctonia solani (fungus), Phytophthora nicotianae (straminipile), Ralstonia solanacearum (eubacterium), and Meloidogyne incognita (metazoan). Quantitative real-time PCR and western blotting analysis indicated that GAFP-1 was transcribed and translated in transgenic lines. When challenged by R. solani and P. nicotianae, GAFP-1 expressing lines had reduced symptom development and improved plant vigor compared to non-transformed and empty vector control lines. These lines also exhibited reduced root galling when challenged by M. incognita. Against R. solanacearum expression of GAFP-1 neither conferred resistance, nor exacerbated disease development. These results indicate that heterologous expression of GAFP-1 can confer enhanced resistance to a diverse set of plant pathogens and may be a good candidate gene for the development of transgenic, root-disease-resistant crops.

  18. Recognition of the Protein Kinase AVRPPHB SUSCEPTIBLE1 by the Disease Resistance Protein RESISTANCE TO PSEUDOMONAS SYRINGAE5 Is Dependent on S-Acylation and an Exposed Loop in AVRPPHB SUSCEPTIBLE11[W][OPEN

    PubMed Central

    Qi, Dong; Dubiella, Ullrich; Kim, Sang Hee; Sloss, D. Isaiah; Dowen, Robert H.; Dixon, Jack E.; Innes, Roger W.

    2014-01-01

    The recognition of pathogen effector proteins by plants is typically mediated by intracellular receptors belonging to the nucleotide-binding leucine-rich repeat (NLR) family. NLR proteins often detect pathogen effector proteins indirectly by detecting modification of their targets. How NLR proteins detect such modifications is poorly understood. To address these questions, we have been investigating the Arabidopsis (Arabidopsis thaliana) NLR protein RESISTANCE TO PSEUDOMONAS SYRINGAE5 (RPS5), which detects the Pseudomonas syringae effector protein Avirulence protein Pseudomonas phaseolicolaB (AvrPphB). AvrPphB is a cysteine protease that specifically targets a subfamily of receptor-like cytoplasmic kinases, including the Arabidopsis protein kinase AVRPPHB Susceptible1 (PBS1). RPS5 is activated by the cleavage of PBS1 at the apex of its activation loop. Here, we show that RPS5 activation requires that PBS1 be localized to the plasma membrane and that plasma membrane localization of PBS1 is mediated by amino-terminal S-acylation. We also describe the development of a high-throughput screen for mutations in PBS1 that block RPS5 activation, which uncovered four new pbs1 alleles, two of which blocked cleavage by AvrPphB. Lastly, we show that RPS5 distinguishes among closely related kinases by the amino acid sequence (SEMPH) within an exposed loop in the C-terminal one-third of PBS1. The SEMPH loop is located on the opposite side of PBS1 from the AvrPphB cleavage site, suggesting that RPS5 associates with the SEMPH loop while leaving the AvrPphB cleavage site exposed. These findings provide support for a model of NLR activation in which NLR proteins form a preactivation complex with effector targets and then sense a conformational change in the target induced by effector modification. PMID:24225654

  19. The Role of a Host Protein (TIP) in the Resistance Response of Arabidopsis to Turnip Crinkle Virus Infection.

    SciTech Connect

    T. Jack Morris, School of Biological Sciences, University of Nebraska, Lincoln, NE 68588-0118

    2008-10-20

    Our research on Turnip crinkle virus (TCV) has shown that the viral capsid protein (CP) is both a virulence factor as well as the elicitor of a hypersensitive resistance response (HR) to the virus in Arabidopsis. Initially, we identified a protein from Arabidopsis that specifically interacted with the viral CP using a yeast two-hybrid screen. This protein, designated TIP for TCV-Interacting Protein, is a member of the NAC family of plant transcription factors implicated in the regulation of development and senescence. When TCV CP was mutated to eliminate its ability to interact with TIP, the corresponding virus mutants broke the HR-mediated resistance conferred by the HRT resistance (R) gene in Arabidopsis ecotype Dijon (Di)-17. This result suggested that TIP is a component of the signal transduction pathway that leads to the genetically specified TCV resistance. We next confirmed that TIP and the viral CP interact in plant cells and that this interaction prevents nuclear localization of this transcription factor. We demonstrated that TCV CP suppresses post-transcriptional gene silencing (PTGS), a newly discovered RNA-mediated defense system in plants. Together these results suggest that the CP is a virulence factor that could well be functioning through its interaction with TIP. We have proposed a model involving the role of TIP and CP in triggering HR mediated plant defense that fits with the current thinking about how gene-for-gene resistance may function. A unique component of our system is the opportunity to link R-gene function with the newly discovered RNA silencing pathway that is not only a potent defense against viral pathogens, but also regulates early development in plants. In the current funding period we made several significant findings: First, we completed an array analysis comparing gene expression in Arabidopsis infected with TCV and a mutant virus unable to bind TIP. Second, we produced transgenic lines that over-express and inducibly under

  20. Expression and activity of multidrug resistance proteins in mature endothelial cells and their precursors: A challenging correlation

    PubMed Central

    Bielawska-Pohl, Aleksandra; Wojtowicz, Karolina; Jura, Roksana; Paprocka, Maria; Wojdat, Elżbieta; Kozłowska, Urszula; Klimczak, Aleksandra; Grillon, Catherine; Kieda, Claudine; Duś, Danuta

    2017-01-01

    Active cellular transporters of harmful agents—multidrug resistance (mdr) proteins—are present in tumor, stem and endothelial cells, among others. While mdr proteins are broadly studied in tumor cells, their role in non-tumor cells and the significance of their action not connected with removal of harmful xenobiotics is less extensively documented. Proper assessment of mdr proteins expression is difficult. Mdr mRNA presence is most often evaluated but that does not necessarily correlate with the protein level. The protein expression itself is difficult to determine; usually cells with mdr overexpression are studied, not cells under physiological conditions, in which a low expression level of mdr protein is often insufficient for detection in vitro. Various methods are used to identify mdr mRNA and protein expression, together with functional tests demonstrating their biological drug transporting activities. Data comparing different methods of investigating expression of mdr mRNAs and their corresponding proteins are still scarce. In this article we present the results of a study concerning mdr mRNA and protein expression. Our goal was to search for the best method to investigate the expression level and functional activity of five selected mdr proteins—MDR1, BCRP, MRP1, MRP4 and MRP5—in established in vitro cell lines of human endothelial cells (ECs) and their progenitors. Endothelial cells demonstrated mdr presence at the mRNA level, which was not always confirmed at the protein level or in functional tests. Therefore, several different assays had to be applied for evaluation of mdr proteins expression and functions in endothelial cells. Among them functional tests seemed to be the most conclusive, although not very specific. PMID:28212450

  1. Transgenic plants expressing ω-ACTX-Hv1a and snowdrop lectin (GNA) fusion protein show enhanced resistance to aphids

    PubMed Central

    Nakasu, Erich Y. T.; Edwards, Martin G.; Fitches, Elaine; Gatehouse, John A.; Gatehouse, Angharad M. R.

    2014-01-01

    Recombinant fusion proteins containing arthropod toxins have been developed as a new class of biopesticides. The recombinant fusion protein Hv1a/GNA, containing the spider venom toxin ω-ACTX-Hv1a linked to snowdrop lectin (GNA) was shown to reduce survival of the peach-potato aphid Myzus persicae when delivered in artificial diet, with survival <10% after 8 days exposure to fusion protein at 1 mg/ml. Although the fusion protein was rapidly degraded by proteases in the insect, Hv1a/GNA oral toxicity to M. persicae was significantly greater than GNA alone. A construct encoding the fusion protein, including the GNA leader sequence, under control of the constitutive CaMV 35S promoter was transformed into Arabidopsis; the resulting plants contained intact fusion protein in leaf tissues at an estimated level of 25.6 ± 4.1 ng/mg FW. Transgenic Arabidopsis expressing Hv1a/GNA induced up to 40% mortality of M. persicae after 7 days exposure in detached leaf bioassays, demonstrating that transgenic plants can deliver fusion proteins to aphids. Grain aphids (Sitobion avenae) were more susceptible than M. persicae to the Hv1a/GNA fusion protein in artificial diet bioassays (LC50 = 0.73 mg/ml after 2 days against LC50 = 1.81 mg/ml for M. persicae), as they were not able to hydrolyze the fusion protein as readily as M. persicae. Expression of this fusion protein in suitable host plants for the grain aphid is likely to confer higher levels of resistance than that shown with the M. persicae/Arabidopsis model system. PMID:25506351

  2. Ageing has no effect on the regulation of the ubiquitin proteasome-related genes and proteins following resistance exercise

    PubMed Central

    Stefanetti, Renae J.; Zacharewicz, Evelyn; Della Gatta, Paul; Garnham, Andrew; Russell, Aaron P.; Lamon, Séverine

    2014-01-01

    Skeletal muscle atrophy is a critical component of the ageing process. Age-related muscle wasting is due to disrupted muscle protein turnover, a process mediated in part by the ubiquitin proteasome pathway (UPP). Additionally, older subjects have been observed to have an attenuated anabolic response, at both the molecular and physiological levels, following a single-bout of resistance exercise (RE). We investigated the expression levels of the UPP-related genes and proteins involved in muscle protein degradation in 10 older (60–75 years) vs. 10 younger (18–30 years) healthy male subjects at basal as well as 2 h after a single-bout of RE. MURF1, atrogin-1 and FBXO40, their substrate targets PKM2, myogenin, MYOD, MHC and EIF3F as well as MURF1 and atrogin-1 transcriptional regulators FOXO1 and FOXO3 gene and/or protein expression levels were measured via real time PCR and western blotting, respectively. At basal, no age-related difference was observed in the gene/protein levels of atrogin-1, MURF1, myogenin, MYOD and FOXO1/3. However, a decrease in FBXO40 mRNA and protein levels was observed in older subjects, while PKM2 protein was increased. In response to RE, MURF1, atrogin-1 and FBXO40 mRNA were upregulated in both the younger and older subjects, with changes observed in protein levels. In conclusion, UPP-related gene/protein expression is comparably regulated in healthy young and old male subjects at basal and following RE. These findings suggest that UPP signaling plays a limited role in the process of age-related muscle wasting. Future studies are required to investigate additional proteolytic mechanisms in conjunction with skeletal muscle protein breakdown (MPB) measurements following RE in older vs. younger subjects. PMID:24550841

  3. Mecillinam resistance in Escherichia coli is conferred by loss of a second activity of the AroK protein.

    PubMed Central

    Vinella, D; Gagny, B; Joseleau-Petit, D; D'Ari, R; Cashel, M

    1996-01-01

    Mecillinam, a beta-lactam antibiotic specific to penicillin-binding protein 2 (PBP 2) in Escherichia coli, blocks cell wall elongation and, indirectly, cell division, but its lethality can be overcome by increased levels of ppGpp, the nucleotide effector of the stringent response. We have subjected an E. coli K-12 strain to random insertional mutagenesis with a mini-Tn10 element. One insertion, which was found to confer resistance to mecillinam in relA+ and relA strains, was mapped at 75.5 min on the E. coli map and was located between the promoters and the coding sequence of the aroK gene, which codes for shikimate kinase 1, one of two E. coli shikimate kinases, both of which are involved in aromatic amino acid biosynthesis. The mecillinam resistance conferred by the insertion was abolished in a delta relA delta spoT strain completely lacking ppGpp, and it thus depends on the presence of ppGpp. Furthermore, the insertion increased the ppGpp pool approximately twofold in a relA+ strain. However, this increase was not observed in relA strains, although the insertion still conferred mecillinam resistance in these backgrounds, showing that mecillinam resistance is not due to an increased ppGpp pool. The resistance was also abolished in an ftsZ84(Ts) strain under semipermissive conditions, and the aroK::mini-Tn10 allele partially suppressed ftsZ84(Ts); however, it did not increase the concentration of the FtsZ cell division protein. The insertion greatly decreased or abolished the shikimate kinase activity of AroK in vivo and in vitro. The two shikimate kinases of E. coli are not equivalent; the loss of AroK confers mecillinam resistance, whereas the loss of Arol, does not. Furthermore, the ability of the aroK mutation to confer mecillinam resistance is shown to be independent of polar effects on operon expression and of effects on the availability of aromatic amino acids or shikimic acid. Instead, we conclude that the AroK protein has a second activity, possibly

  4. Overexpression of a stress-responsive U-box protein gene VaPUB affects the accumulation of resistance related proteins in Vitis vinifera 'Thompson Seedless'.

    PubMed

    Jiao, Li; Zhang, Yali; Lu, Jiang

    2017-03-01

    Many U-box proteins have been identified and characterized as important factors against environmental stresses such as chilling, heat, salinity and pathogen attack in plant. Our previous research reported the cloning of a novel U-box protein gene VaPUB from Vitis amurensis 'Zuoshanyi' grape and suggested a function of it in related to cold stress in the model plant Arabidopsis system. In this study, the role of VaPUB in response to biotic and abiotic stress was further analyzed in the homologous grapevine system by studying the transcript regulation and the protein accumulation in VaPUB transgenic vines. The expression analysis assay shown that VaPUB was significantly up-regulated 6 h after cold treatment and as early as 2 h post inoculation with Plasmopara viticola, a pathogen causing downy mildew disease in grapevine. Over-expressing VaPUB in V. Vinifera 'Thompson Seedless' affected the microstructure of leaves. The proteome assay shown that the accumulation of pathogenesis-related protein PR10 and many proteins involved in carbon and energy metabolism, oxidation reaction and protein metabolism were significantly altered in transgenic vines. In comparison with wild type plants, the expression level of PR10 family genes was significantly decreased in VaPUB transgenic vines under P. viticola treatment or cold stress. Results from this study showed that the U-box protein gene PUB quickly responded to both biotic stress and abiotic stress and significantly influenced the accumulation of resistance related proteins in grapevine.

  5. Analyzing pepsin degradation assay conditions used for allergenicity assessments to ensure that pepsin susceptible and pepsin resistant dietary proteins are distinguishable

    PubMed Central

    Edrington, Thomas C.; Storrs, S. Bradley; Crowley, Kathleen S.; Ward, Jason M.; Lee, Thomas C.; Liu, Zi L.; Li, Bin; Glenn, Kevin C.

    2017-01-01

    The susceptibility of a dietary protein to proteolytic degradation by digestive enzymes, such as gastric pepsin, provides information on the likelihood of systemic exposure to a structurally intact and biologically active macromolecule, thus informing on the safety of proteins for human and animal consumption. Therefore, the purpose of standardized in vitro degradation studies that are performed during protein safety assessments is to distinguish whether proteins of interest are susceptible or resistant to pepsin degradation via a study design that enables study-to-study comparison. Attempting to assess pepsin degradation under a wide-range of possible physiological conditions poses a problem because of the lack of robust and consistent data collected under a large-range of sub-optimal conditions, which undermines the needs to harmonize in vitro degradation conditions. This report systematically compares the effects of pH, incubation time, and pepsin-to-substrate protein ratio on the relative degradation of five dietary proteins: three pepsin susceptible proteins [ribulose 1,5-bisphosphate carboxylase-oxygenase (Rubisco), horseradish peroxidase (HRP), hemoglobin (Hb)], and two pepsin resistant proteins [lipid transfer protein (LTP) and soybean trypsin inhibitor (STI)]. The results indicate that proteins susceptible to pepsin degradation are readily distinguishable from pepsin-resistant proteins when the reaction conditions are within the well-characterized optima for pepsin. The current standardized in vitro pepsin resistant assay with low pH and high pepsin-to-substrate ratio fits this purpose. Using non-optimal pH and/or pepsin-to-substrate protein ratios resulted in susceptible proteins no longer being reliably degraded by this stomach enzyme, which compromises the ability of this in vitro assay to distinguish between resistant and susceptible proteins and, therefore, no longer providing useful data to an overall weight-of-evidence approach to assessing safety

  6. Analyzing pepsin degradation assay conditions used for allergenicity assessments to ensure that pepsin susceptible and pepsin resistant dietary proteins are distinguishable.

    PubMed

    Wang, Rong; Edrington, Thomas C; Storrs, S Bradley; Crowley, Kathleen S; Ward, Jason M; Lee, Thomas C; Liu, Zi L; Li, Bin; Glenn, Kevin C

    2017-01-01

    The susceptibility of a dietary protein to proteolytic degradation by digestive enzymes, such as gastric pepsin, provides information on the likelihood of systemic exposure to a structurally intact and biologically active macromolecule, thus informing on the safety of proteins for human and animal consumption. Therefore, the purpose of standardized in vitro degradation studies that are performed during protein safety assessments is to distinguish whether proteins of interest are susceptible or resistant to pepsin degradation via a study design that enables study-to-study comparison. Attempting to assess pepsin degradation under a wide-range of possible physiological conditions poses a problem because of the lack of robust and consistent data collected under a large-range of sub-optimal conditions, which undermines the needs to harmonize in vitro degradation conditions. This report systematically compares the effects of pH, incubation time, and pepsin-to-substrate protein ratio on the relative degradation of five dietary proteins: three pepsin susceptible proteins [ribulose 1,5-bisphosphate carboxylase-oxygenase (Rubisco), horseradish peroxidase (HRP), hemoglobin (Hb)], and two pepsin resistant proteins [lipid transfer protein (LTP) and soybean trypsin inhibitor (STI)]. The results indicate that proteins susceptible to pepsin degradation are readily distinguishable from pepsin-resistant proteins when the reaction conditions are within the well-characterized optima for pepsin. The current standardized in vitro pepsin resistant assay with low pH and high pepsin-to-substrate ratio fits this purpose. Using non-optimal pH and/or pepsin-to-substrate protein ratios resulted in susceptible proteins no longer being reliably degraded by this stomach enzyme, which compromises the ability of this in vitro assay to distinguish between resistant and susceptible proteins and, therefore, no longer providing useful data to an overall weight-of-evidence approach to assessing safety

  7. Involvement of the Eukaryote-Like Kinase-Phosphatase System and a Protein That Interacts with Penicillin-Binding Protein 5 in Emergence of Cephalosporin Resistance in Cephalosporin-Sensitive Class A Penicillin-Binding Protein Mutants in Enterococcus faecium

    PubMed Central

    Desbonnet, Charlene; Tait-Kamradt, Amelia; Garcia-Solache, Monica; Dunman, Paul; Coleman, Jeffrey; Arthur, Michel

    2016-01-01

    ABSTRACT The intrinsic resistance of Enterococcus faecium to ceftriaxone and cefepime (here referred to as “cephalosporins”) is reliant on the presence of class A penicillin-binding proteins (Pbps) PbpF and PonA. Mutants lacking these Pbps exhibit cephalosporin susceptibility that is reversible by exposure to penicillin and by selection on cephalosporin-containing medium. We selected two cephalosporin-resistant mutants (Cro1 and Cro2) of class A Pbp-deficient E. faecium CV598. Genome analysis revealed changes in the serine-threonine kinase Stk in Cro1 and a truncation in the associated phosphatase StpA in Cro2 whose respective involvements in resistance were confirmed in separate complementation experiments. In an additional effort to identify proteins linked to cephalosporin resistance, we performed tandem affinity purification using Pbp5 as bait in penicillin-exposed E. faecium; these experiments yielded a protein designated Pbp5-associated protein (P5AP). Transcription of the P5AP gene was increased after exposure to penicillin in wild-type strains and in Cro2 and suppressed in Cro2 complemented with the wild-type stpA. Transformation of class A Pbp-deficient strains with the plasmid-carried P5AP gene conferred cephalosporin resistance. These data suggest that Pbp5-associated cephalosporin resistance in E. faecium devoid of typical class A Pbps is related to the presence of P5AP, whose expression is influenced by the activity of the serine-threonine phosphatase/kinase system. PMID:27048803

  8. Improving the resistance of a eukaryotic β-barrel protein to thermal and chemical perturbations.

    PubMed

    Gessmann, Dennis; Mager, Frauke; Naveed, Hammad; Arnold, Thomas; Weirich, Sara; Linke, Dirk; Liang, Jie; Nussberger, Stephan

    2011-10-14

    β-Barrel membrane proteins have regular structures with extensive hydrogen-bond networks between their transmembrane (TM) β-strands, which stabilize their protein fold. Nevertheless, weakly stable TM regions, which are important for the protein function and interaction with other proteins, exist. Here, we report on the apparent stability of human Tom40A, a member of the "mitochondrial porin family" and main constituent of the mitochondrial protein-conducting channel TOM (translocase of the outer membrane). Using a physical interaction model, TmSIP, for β-barrel membrane proteins, we have identified three unfavorable β-strands in the TM domain of the protein. Substitution of key residues inside these strands with hydrophobic amino acids results in a decreased sensitivity of the protein to chemical and/or thermal denaturation. The apparent melting temperature observed when denatured at a rate of 1 °C per minute is shifted from 73 to 84 °C. Moreover, the sensitivity of the protein to denaturant agents is significantly lowered. Further, we find a reduced tendency for the mutated protein to form dimers. We propose that the identified weakly stable β-strands 1, 2 and 9 of human Tom40A play an important role in quaternary protein-protein interactions within the mammalian TOM machinery. Our results show that the use of empirical energy functions to model the apparent stability of β-barrel membrane proteins may be a useful tool in the field of nanopore bioengineering.

  9. Improving the Resistance of a Eukaryotic β-Barrel Protein to Thermal and Chemical Perturbation

    PubMed Central

    Gessmann, Dennis; Mager, Frauke; Naveed, Hammad; Arnold, Thomas; Weirich, Sara; Linke, Dirk; Liang, Jie; Nussberger, Stephan

    2013-01-01

    Beta-barrel membrane proteins have regular structures with extensive hydrogen bonding networks between their transmembrane (TM) β-strands, which stabilize their protein fold. Nevertheless, weakly stable TM regions exist, which are important for the protein function and interaction with other proteins. Here, we report on the apparent stability of human Tom40A, a member of the ‘mitochondrial porin family’ and main constituent of the mitochondrial protein-conducting channel TOM. Using a physical interaction model TmSIP for β-barrel membrane proteins, we have identified three β-strands unfavorable in the TM domain of the protein. Substitution of key residues inside these strands with hydrophobic amino acids results in a decreased sensitivity of the protein to chemical and/or thermal denaturation. The apparent melting temperature observed when denatured at a rate of one degree per minute, is shifted from 73 to 84 °C. Moreover, the sensitivity of the protein to denaturant agents is significantly lowered. Further, we find a reduced tendency for the mutated protein to form dimers. We propose that the identified weakly stable β-strands 1, 2 and 9 of human Tom40A play an important role in quaternary protein-protein interactions within the mammalian TOM machinery. Our results show that the use of empirical energy functions to model the apparent stability of β-barrel membrane proteins may be a useful tool in the field of nanopore bioengineering. PMID:21835183

  10. Effects of standard chemotherapy on tumor growth and regulation of multidrug resistance genes and proteins in childhood rhabdomyosarcoma.

    PubMed

    Seitz, Guido; Warmann, Steven W; Vokuhl, Christian O; Heitmann, Heike; Treuner, Claudia; Leuschner, Ivo; Fuchs, Jörg

    2007-05-01

    The prognosis of rhabdomyosarcoma (RMS) in advanced stages is still sobering. Therapy is limited due to local tumor recurrence, development of metastases and multidrug resistance. The aim of this study was to investigate the development of multidrug resistance in cell lines and in xenografts of alveolar and embryonal RMS treated according to the German Soft Tissue Sarcoma Study (CWS). Alveolar and embryonal RMS cell lines were treated with Vincristine, Topotecan, Carboplatin, Actinomycin D, or Ifosfamide. Expression levels of resistance-associated genes were assessed using Real time-PCR. Nude mice (NMRI nu/nu, n = 10 per group) underwent xenotransplantation of human embryonal or alveolar RMS. Animals were treated with standard chemotherapeutic drugs Vincristine, Topotecan, Carboplatin, Actinomycin D, or Ifosfamide according to treatment schedules of the CWS-study. Tumor sizes were measured and relative tumor volumes were calculated. Animals were sacrificed after 20 days and standard histology, Real-time-PCR for MDR1-, MRP-, LRP- and MDM2-gene as well as immunohistochemistry for MDR1-, LRP-, and MRP-protein were performed. In the cell lines, an up-regulation of MDR-1 gene was found in alveolar rhabdomyosarcoma. In embryonal rhabdomyosarcoma, an up-regulation of LRP and MRP was found. Standard chemotherapy of alveolar rhabdomyosarcoma resulted in a significant reduction of tumor growth (P < 0.05) in all groups. In embryonal rhabdomyosarcoma strongest effects were found after treatment with Ifosfamide, Vincristine and Carboplatin (P < 0.05). RT-PCR revealed a MDR1-dependent mechanism in alveolar rhabdomyosarcoma. In embryonal rhabdomyosarcoma, MDR1 occurred to a lower degree. Immunohistochemistry revealed correlating expression levels of multidrug resistance-associated proteins. The use of established chemotherapy on human RMS in vivo had strong effects on xenografts compared to their controls. In all cases, there was only a reduction of tumor growth, but not a

  11. Protein resistant surfaces: comparison of acrylate graft polymers bearing oligo-ethylene oxide and phosphorylcholine side chains.

    PubMed

    Feng, Wei; Zhu, Shiping; Ishihara, Kazuhiko; Brash, John L

    2006-03-01

    The objective of this work was to compare poly(ethylene glycol) (PEG) and phosphorylcholine (PC) moieties as surface modifiers with respect to their ability to inhibit protein adsorption. Surfaces were prepared by graft polymerization of the methacrylate monomers oligo(ethylene glycol) methyl ether methacrylate (OEGMA, MW 300, PEG side chains of length n=4.5) and 2-methacryloyloxyethyl phosphorylcholine (MPC, MW 295). The grafted polymers thus contained short PEG chains and PC, respectively, as side groups. Grafting on silicon was carried out using surface-initiated atom transfer radical polymerization (ATRP). Graft density was controlled via the surface density of the ATRP initiator, and chain length of the grafts was controlled via the ratio of monomer to sacrificial initiator. The grafted surfaces were characterized by water contact angle, x-ray photoelectron spectroscopy, and atomic force microscopy. The effect of graft density and chain length on fibrinogen adsorption from buffer was investigated using radio labeling methods. Adsorption to both MPC- and OEGMA-grafted surfaces was found to decrease with increasing graft density and chain length. Adsorption on the MPC and OEGMA surfaces for a given chain length and density was essentially the same. Very low adsorption levels of the order of 7 ngcm(2) were seen on the most resistant surfaces. The effect of protein size on resistance to adsorption was studied using binary solutions of lysozyme (MW 14 600) and fibrinogen (MW 340 000). Adsorption levels in these experiments were also greatly reduced on the grafted surfaces compared to the control surfaces. It was concluded that at the lowest graft density, both proteins had unrestricted access to the substrate, and the relative affinities of the proteins for the substrate (higher affinity of fibrinogen) determined the composition of the layer. At the highest graft density also, where the adsorption of both proteins was very low, no preference for one or the other

  12. F-Box Protein FBXO22 Mediates Polyubiquitination and Degradation of CD147 to Reverse Cisplatin Resistance of Tumor Cells

    PubMed Central

    Wu, Bo; Liu, Zhen-Yu; Cui, Jian; Yang, Xiang-Min; Jing, Lin; Zhou, Yang; Chen, Zhi-Nan; Jiang, Jian-Li

    2017-01-01

    Drug resistance remains a major clinical obstacle to successful treatment of cancer. As posttranslational modification is becoming widely recognized to affect the function of oncoproteins, targeting specific posttranslational protein modification provides an attractive strategy for anticancer drug development. CD147 is a transmembrane glycoprotein contributing to chemo-resistance of cancer cells in a variety of human malignancies. Ubiquitination is an important posttranslational modification mediating protein degradation. Degradation of oncoproteins, CD147 included, emerges as an attractive alternative for tumor inhibition. However, the ubiquitination of CD147 remains elusive. Here in this study, we found that deletion of the CD147 intracellular domain (CD147-ICD) prolonged the half-life of CD147 in HEK293T cells, and we identified that CD147-ICD interacts with FBXO22 using mass spectrometry and Western blot. Then, we demonstrated that FBXO22 mediates the polyubiquitination and degradation of CD147 by recognizing CD147-ICD. While knocking down of FBXO22 prolonged the half-life of CD147 in HEK293T cells, we found that FBXO22 regulates CD147 protein turnover in SMMC-7721, Huh-7 and A549 cells. Moreover, we found that the low level of FBXO22 contributes to the accumulation of CD147 and thereafter the cisplatin resistance of A549/DDP cells. To conclude, our study demonstrated that FBXO22 mediated the polyubiquitination and degradation of CD147 by interacting with CD147-ICD, and CD147 polyubiquitination by FBXO22 reversed cisplatin resistance of tumor cells. PMID:28117675

  13. Solute Transport Proteins and the Outer Membrane Protein NmpC Contribute to Heat Resistance of Escherichia coli AW1.7▿

    PubMed Central

    Ruan, Lifang; Pleitner, Aaron; Gänzle, Michael G.; McMullen, Lynn M.

    2011-01-01

    This study aimed to elucidate determinants of heat resistance in Escherichia coli by comparing the composition of membrane lipids, as well as gene expression, in heat-resistant E. coli AW1.7 and heat-sensitive E. coli GGG10 with or without heat shock. The survival of E. coli AW1.7 at late exponential phase was 100-fold higher than that of E. coli GGG10 after incubation at 60°C for 15 min. The cytoplasmic membrane of E. coli AW1.7 contained a higher proportion of saturated and cyclopropane fatty acids than that of E. coli GGG10. Microarray hybridization of cDNA libraries obtained from exponentially growing or heat-shocked cultures was performed to compare gene expression in these two strains. Expression of selected genes from different functional groups was quantified by quantitative PCR. DnaK and 30S and 50S ribosomal subunits were overexpressed in E. coli GGG10 relative to E. coli AW1.7 upon heat shock at 50°C, indicating improved ribosome stability. The outer membrane porin NmpC and several transport proteins were overexpressed in exponentially growing E. coli AW1.7. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of membrane properties confirmed that NmpC is present in the outer membrane of E. coli AW1.7 but not in that of E. coli GGG10. Expression of NmpC in E. coli GGG10 increased survival at 60°C 50- to 1,000-fold. In conclusion, the outer membrane porin NmpC contributes to heat resistance in E. coli AW1.7, but the heat resistance of this strain is dependent on additional factors, which likely include the composition of membrane lipids, as well as solute transport proteins. PMID:21398480

  14. Chloroplast-targeted expression of recombinant crystal-protein gene in cotton: an unconventional combat with resistant pests.

    PubMed

    Kiani, Sarfraz; Mohamed, Bahaeldeen Babiker; Shehzad, Kamran; Jamal, Adil; Shahid, Muhammad Naveed; Shahid, Ahmad Ali; Husnain, Tayyab

    2013-07-10

    Plants transformed with single Bt gene are liable to develop insect resistance and this has already been reported in a number of studies carried out around the world where Bt cotton was cultivated on commercial scale. Later, it was envisaged to transform plants with more than one Bt genes in order to combat with resistant larvae. This approach seems valid as various Bt genes possess different binding domains which could delay the likely hazards of insect resistance against a particular Bt toxin. But it is difficult under field conditions to develop homozygous plants expressing all Bt genes equally after many generations without undergoing recombination effects. A number of researches claiming to transform plants from three to seven transgenes in a single plant were reported during the last decade but none has yet applied for patent of homozygous transgenic lines. A better strategy might be to use hybrid-Bt gene(s) modified for improved lectin-binding domains to boost Bt receptor sites in insect midgut. These recombinant-Bt gene(s) would express different lectin domains in a single polypeptide and it is relatively easy to develop homozygous transgenic lines under field conditions. Enhanced chloroplast-localized expression of hybrid-Bt gene would leave no room for insects to develop resistance. We devised and successfully applied this strategy in cotton (Gossypium hirsutum) and data up to T3 generation showed that our transgenic cotton plants were displaying enhanced chloroplast-targeted Cry1Ac-RB expression. Laboratory and field bioassays gave promising results against American bollworm (Heliothis armigera), pink bollworm (Pictinophora scutigera) and fall armyworm (Spodoptera frugiperda) that otherwise, were reported to have evolved resistance against Cry1Ac toxin. Elevated levels of hybrid-Bt toxin were confirmed by ELISA of chloroplast-enriched protein samples extracted from leaves of transgenic cotton lines. While, localization of recombinant Cry1Ac-RB protein in

  15. Modulatory effects of plant polyphenols on human multidrug resistance proteins 1, 4, and 5 (ABCC1, 4, and 5)

    PubMed Central

    Wu, Chung-Pu; Calcagno, Anna Maria; Hladky, Stephen B.; Ambudkar, Suresh V.; Barrand, Margery A.

    2005-01-01

    SUMMARY Plant flavonoids are polyphenolic compounds commonly found in vegetables, fruits and many food sources that form a significant portion of our diet. These compounds have been shown to interact with several ATP-Binding Cassette transporters that are linked with anticancer and antiviral drug resistance and as such, may be beneficial in modulating drug resistance. The present study investigates the interactions of six common polyphenols; quercetin, silymarin, resveratrol, naringenin, daidzein and hesperetin with the multidrug resistance associated proteins, MRP1, MRP4 and MRP5. At non-toxic concentrations, several of the polyphenols were able to modulate MRP1-, MRP4- and MRP5- mediated drug resistance though to varying extents. The polyphenols also reversed resistance to NSC251820, a compound that appears to be a good substrate for MRP4 as had been predicted by data mining studies. Furthermore, most of the polyphenols showed direct inhibition of MRP1-mediated [3H]-dinitrophenyl S-glutathione and MRP4-mediated [3H]-cGMP transport in inside-out vesicles prepared from human erythrocytes. Additionally, both quercetin and silymarin were found to inhibit MRP1-, MRP4-, and MRP5-mediated transport from intact cells with high affinity. They also had significant effects on ATPase activity of MRP1 and MRP4 without having any effect on [α-32P]8-azidoATP binding to these proteins. This suggests that these flavonoids most likely interact at the transporter’s substrate-binding sites. Collectively, these results suggest that dietary flavonoids such as quercetin and silymarin can modulate transport activities of MRP1, 4 and 5. Such interactions could influence bioavailability of anticancer and antiviral drugs in vivo and thus, should be considered for increasing efficacy in drug therapies. PMID:16156793

  16. Selection and characterization of human respiratory syncytial virus escape mutants resistant to a polyclonal antiserum raised against the F protein.

    PubMed

    Tomé, Lorena; Frabasile, Sandra; Candia, Claudia; Pittini, Alvaro; Farina, Natalia; Melero, José Antonio; Arbiza, Juan

    2012-06-01

    A human respiratory syncytial virus (HRSV) neutralization escape mutant was obtained after 56 serial passages in the presence of a polyclonal antiserum raised against the F protein. Nucleotide sequence analysis of this escape mutant virus revealed two amino acid substitutions: Asn268Ile and Val533Met. When this virus was allowed to grow in the absence of the anti-F polyclonal serum, only the mutation Asn268Ile was stably maintained. Both the double and single escape mutant viruses lost reactivity with mAbs belonging to antigenic site II of the fusion protein of RSV. Mutation Asn268Ile has already been reported in RS viruses that are resistant to mAbs 47F and 11 and palivizumab (PZ). We have thus identified a novel mutation (Val533Met) in the transmembrane domain of the F protein that was selected under immune pressure.

  17. Identification and characterization of gsp65, an organic hydroperoxide resistance (ohr) gene encoding a general stress protein in Enterococcus faecalis.

    PubMed

    Rincé, A; Giard, J C; Pichereau, V; Flahaut, S; Auffray, Y

    2001-02-01

    The Enterococcus faecalis general stress protein Gsp65 has been purified from two-dimensional gel electrophoresis. Determination of its N-terminal sequence and characterization of the corresponding gene revealed that the gsp65 product is a 133-amino-acid protein sharing homologies with organic hydroperoxide resistance (Ohr) proteins. Transcriptional analysis of gsp65 gave evidence for a monocistronic mRNA initiated 52 nucleotides upstream of the ATG start codon and for an induction in response to hydrogen peroxide, heat shock, acid pH, detergents, ethanol, sodium chloride, and tert-butylhydroperoxide (tBOOH). A gsp65 mutant showed increased sensitivity to the organic hydroperoxide tBOOH and to ethanol.

  18. Protein adsorption resistance of PVP-modified polyurethane film prepared by surface-initiated atom transfer radical polymerization

    NASA Astrophysics Data System (ADS)

    Yuan, Huihui; Qian, Bin; Zhang, Wei; Lan, Minbo

    2016-02-01

    An anti-fouling surface of polyurethane (PU) film grafted with Poly(N-vinylpyrrolidone) (PVP) was prepared through surface-initiated atom transfer radical polymerization (SI-ATRP). And the polymerization time was investigated to obtain PU films with PVP brushes of different lengths. The surface properties and protein adsorption of modified PU films were evaluated. The results showed that the hydrophilicity of PU-PVP films were improved with the increase of polymerization time, which was not positive correlation with the surface roughness due to the brush structure. Additionally, the protein resistance performance was promoted when prolonging the polymerization time. The best antifouling PU-PVP (6.0 h) film reduced the adsoption level of bovine serum albumin (BSA), lysozyme (LYS), and brovin serum fibrinogen (BFG) by 93.4%, 68.3%, 85.6%, respectively, compared to the unmodified PU film. The competitive adsorption of three proteins indicated that LYS preferentially adsorbed on the modified PU film, while BFG had the lowest adsorption selectivity. And the amount of BFG on PU-PVP (6.0 h) film reduced greatly to 0.08 μg/cm2, which was almost one-tenth of its adsorption from the single-protein system. Presented results suggested that both hydrophilicity and surface roughness might be the important factors in all cases of protein adsorption, and the competitive or selective adsorption might be related to the size of the proteins, especially on the non-charged films.

  19. Assay of urinary protein-bound sialic acid can differentiate steroidsensitive nephrotic syndrome from steroid-resistant cases.

    PubMed

    Gopal, Niranjan; Koner, Bidhan Chandra; Bhattacharjee, Atanu; Bhat, Vishnu

    2016-01-01

    The protein selectivity index as measured from the ratio of urinary immunoglobulin to albumin failed to differentiate between steroid-sensitive (SS) and steroid-resistant (SR) cases of nephrotic syndrome (NS). Sialic acid contributes negative charges to many plasma proteins. The negative charge is a determinant of protein excretion rate. The prognostic significance of assay of urinary excretion of protein-bound sialic acid in NS has not been evaluated. Hence, the present study was designed to evaluate whether measurement of urinary protein bound sialic acid (UPBSA) can be used as a marker to differentiate SS from SR cases of NS. The urine samples of 70 (47 SS and 23 SR) pediatric NS children were assayed for UPBSA by Aminoff's method. The levels were compared and the receiver-operator curve was drawn to determine the optimum cutoff point to differentiate among the groups before starting the therapy. The excretion of UPBSA in SR cases of NS was significantly higher than that of SS cases (P<0.05). The optimum cutoff limit for UPBSA was 2.71 μg/mg of proteins with 75% sensitivity and 75.5% specificity for differentiating SS cases from SR cases (area under the plasma- concentration time curve=0.814, P=0.009). We conclude that UPBSA can differentiate SR cases from SS cases of NS in pediatric patients and may help in predicting the response to steroid therapy.

  20. The Proline Rich Homeodomain Protein PRH/Hhex Forms Stable Oligomers That Are Highly Resistant to Denaturation

    PubMed Central

    Shukla, Anshuman; Burton, Nicholas M.; Jayaraman, Padma-Sheela; Gaston, Kevin

    2012-01-01

    Background Many transcription factors control gene expression by binding to specific DNA sequences at or near the genes that they regulate. However, some transcription factors play more global roles in the control of gene expression by altering the architecture of sections of chromatin or even the whole genome. The ability to form oligomeric protein assemblies allows many of these proteins to manipulate extensive segments of DNA or chromatin via the formation of structures such as DNA loops or protein-DNA fibres. Principal Findings Here we show that the proline rich homeodomain protein PRH/Hhex forms predominantly octameric and/or hexadecameric species in solution as well as larger assemblies. We show that these assemblies are highly stable resisting denaturation by temperature and chemical denaturants. Conclusion These data indicate that PRH is functionally and structurally related to the Lrp/AsnC family of proteins, a group of proteins that are known to act globally to control gene expression in bacteria and archaea. PMID:22540015

  1. Identification of a chitinase modifying protein from Fusarium verticillioides: truncation of a host resistance protein by a fungalysin metalloprotease

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Chitinase modifying proteins (cmps) are proteases, secreted by fungal pathogens, which truncate the plant class IV chitinases ChitA and ChitB during maize ear rot. Cmp activity has been characterized for Bipolaris zeicola and Stenocarpella maydis, but the identities of the proteases are not known. H...

  2. The effects of protein and amino acid supplementation on performance and training adaptations during ten weeks of resistance training.

    PubMed

    Kerksick, Chad M; Rasmussen, Christopher J; Lancaster, Stacy L; Magu, Bharat; Smith, Penney; Melton, Charles; Greenwood, Michael; Almada, Anthony L; Earnest, Conrad P; Kreider, Richard B

    2006-08-01

    The purpose of this study was to examine the effects of whey protein supplementation on body composition, muscular strength, muscular endurance, and anaerobic capacity during 10 weeks of resistance training. Thirty-six resistance-trained males (31.0 +/- 8.0 years, 179.1 +/- 8.0 cm, 84.0 +/- 12.9 kg, 17.8 +/- 6.6%) followed a 4 days-per-week split body part resistance training program for 10 weeks. Three groups of supplements were randomly assigned, prior to the beginning of the exercise program, in a double-blind manner to all subjects: 48 g per day (g.d(-1)) carbohydrate placebo (P), 40 g.d(-1) of whey protein + 8 g.d(-1) of casein (WC), or 40 g.d(-1) of whey protein + 3 g.d(-1) branched-chain amino acids + 5 g.d(-1) L-glutamine (WBG). At 0, 5, and 10 weeks, subjects were tested for fasting blood samples, body mass, body composition using dual-energy x-ray absorptiometry (DEXA), 1 repetition maximum (1RM) bench and leg press, 80% 1RM maximal repetitions to fatigue for bench press and leg press, and 30-second Wingate anaerobic capacity tests. No changes (p > 0.05) were noted in all groups for energy intake, training volume, blood parameters, and anaerobic capacity. WC experienced the greatest increases in DEXA lean mass (P = 0.0 +/- 0.9; WC = 1.9 +/- 0.6; WBG = -0.1 +/- 0.3 kg, p < 0.05) and DEXA fat-free mass (P = 0.1 +/- 1.0; WC = 1.8 +/- 0.6; WBG = -0.1 +/- 0.2 kg, p < 0.05). Significant increases in 1RM bench press and leg press were observed in all groups after 10 weeks. In this study, the combination of whey and casein protein promoted the greatest increases in fat-free mass after 10 weeks of heavy resistance training. Athletes, coaches, and nutritionists can use these findings to increase fat-free mass and to improve body composition during resistance training.

  3. Antibiotic Resistance, Core-Genome and Protein Expression in IncHI1 Plasmids in Salmonella Typhimurium.

    PubMed

    Kubasova, Tereza; Cejkova, Darina; Matiasovicova, Jitka; Sekelova, Zuzana; Polansky, Ondrej; Medvecky, Matej; Rychlik, Ivan; Juricova, Helena

    2016-06-13

    Conjugative plasmids from the IncHI1 incompatibility group play an important role in transferring antibiotic resistance in Salmonella Typhimurium. However, knowledge of their genome structure or gene expression is limited. In this study, we determined the complete nucleotide sequences of four IncHI1 plasmids transferring resistance to antibiotics by two different next generation sequencing protocols and protein expression by mass spectrometry. Sequence data including additional 11 IncHI1 plasmids from GenBank were used for the definition of the IncHI1 plasmid core-genome and pan-genome. The core-genome consisted of approximately 123 kbp and 122 genes while the total pan-genome represented approximately 600 kbp. When the core-genome sequences were used for multiple alignments, the 15 tested IncHI1 plasmids were separated into two main lineages. GC content in core-genome genes was around 46% and 50% in accessory genome genes. A multidrug resistance region present in all 4 sequenced plasmids extended over 20 kbp and, except for tet(B), the genes responsible for antibiotic resistance were those with the highest GC content. IncHI1 plasmids therefore represent replicons that evolved in low GC content bacteria. From their original host, they spread to Salmonella and during this spread these plasmids acquired multiple accessory genes including those coding for antibiotic resistance. Antibiotic-resistance genes belonged to genes with the highest level of expression and were constitutively expressed even in the absence of antibiotics. This is the likely mechanism that facilitates host cell survival when antibiotics suddenly emerge in the environment.

  4. Functional Characterization of Triclosan-Resistant Enoyl-acyl-carrier Protein Reductase (FabV) in Pseudomonas aeruginosa

    PubMed Central

    Huang, Yong-Heng; Lin, Jin-Shui; Ma, Jin-Cheng; Wang, Hai-Hong

    2016-01-01

    Pseudomonas aeruginosa is extremely resistant to triclosan. Previous studies have shown that P. aeruginosa encodes a triclosan-resistant enoyl-acyl-carrier protein reductase (ENR), FabV, and that deletion of fabV causes P. aeruginosa to be extremely sensitive to triclosan. In this report, we complemented a P. aeruginosa fabV deletion strain with several triclosan-resistant ENR encoding genes, including Vibrio cholerae fabV, Bacillus subtilis fabL and Enterococcus faecalis fabK. All complemented strains restored triclosan resistance to the level of the wild-type strain, which confirmed that triclosan-resistant ENR allows P. aeruginosa to be extremely resistant to triclosan. Moreover, fabV exhibits pleiotropic effects. Deletion of fabV led P. aeruginosa to show attenuated swarming motility, decreased rhamnolipid, pyoverdine and acyl-homoserine lactones (AHLs) production. Complementation of the fabV mutant with any one ENR encoding gene could restore these features to some extent, in comparison with the wild-type strain. Furthermore, we found that addition of exogenous AHLs could restore the fabV mutant strain to swarm on semisolid plates and to produce more virulence factors than the fabV mutant strain. These findings indicate that deletion of fabV reduced the activity of ENR in P. aeruginosa, decreased fatty acid synthesis, and subsequently depressed the production of AHLs and other virulence factors, which finally may led to a reduction in the pathogenicity of P. aeruginosa. Therefore, fabV should be an ideal target for the control of P. aeruginosa infectivity. PMID:27965638

  5. Effect of membrane length, membrane resistance, and filtration conditions on the fractionation of milk proteins by microfiltration.

    PubMed

    Piry, A; Heino, A; Kühnl, W; Grein, T; Ripperger, S; Kulozik, U

    2012-04-01

    We investigated the fractionation of casein micelles and the whey protein β-lactoglobulin (β-LG) of skim milk by crossflow microfiltration (0.1 μm) for the first time by a novel approach as a function of membrane length and membrane resistance. A special module was constructed with 4 sections and used to assess the effects of membrane length by measuring flux and β-LG permeation (or transmission) as a function of transmembrane pressure and membrane length. Depending on the position, the membranes were partly controlled by a deposit layer. A maximum for β-LG mass flow through the various membrane sections was found, depending on the position along the membrane. To study the effect of convective flow toward the membrane, membranes with 4 different intrinsic permeation resistances were assessed in terms of the permeation and fouling effects along the flow channel. From these findings, we derived a ratio between transmembrane pressure and membrane resistance, which was useful in reducing the effect of deposit formation and, thus, to optimize the protein permeation. In addition, the fouling effect was investigated in terms of reversible and irreversible fouling and, in addition, by differentiation between pressure-induced fouling and adsorption-induced (pressure-independent) fouling, again as a function of membrane length.

  6. Implication of protein tyrosine phosphatase 1B in MCF-7 cell proliferation and resistance to 4-OH tamoxifen

    SciTech Connect

    Blanquart, Christophe; Karouri, Salah-Eddine; Issad, Tarik

    2009-10-02

    The protein tyrosine phosphatase 1B (PTP1B) and the T-cell protein tyrosine phosphatase (TC-PTP) were initially thought to be mainly anti-oncogenic. However, overexpression of PTP1B and TC-PTP has been observed in human tumors, and recent studies have demonstrated that PTP1B contributes to the appearance of breast tumors by modulating ERK pathway. In the present work, we observed that decreasing the expression of TC-PTP or PTP1B in MCF-7 cells using siRNA reduced cell proliferation without affecting cell death. This reduction in proliferation was associated with decreased ERK phosphorylation. Moreover, selection of tamoxifen-resistant MCF-7 cells, by long-term culture in presence of 4-OH tamoxifen, resulted in cells that display overexpression of PTP1B and TC-PTP, and concomitant increase in ERK and STAT3 phosphorylation. siRNA experiments showed that PTP1B, but not TC-PTP, is necessary for resistance to 4-OH tamoxifen. Therefore, our work indicates that PTP1B could be a relevant therapeutic target for treatment of tamoxifen-resistant breast cancers.

  7. Bovine spongiform encephalopathy induces misfolding of alleged prion-resistant species cellular prion protein without altering its pathobiological features.

    PubMed

    Vidal, Enric; Fernández-Borges, Natalia; Pintado, Belén; Ordóñez, Montserrat; Márquez, Mercedes; Fondevila, Dolors; Torres, Juan María; Pumarola, Martí; Castilla, Joaquín

    2013-05-01

    Bovine spongiform encephalopathy (BSE) prions were responsible for an unforeseen epizootic in cattle which had a vast social, economic, and public health impact. This was primarily because BSE prions were found to be transmissible to humans. Other species were also susceptible to BSE either by natural infection (e.g., felids, caprids) or in experimental settings (e.g., sheep, mice). However, certain species closely related to humans, such as canids and leporids, were apparently resistant to BSE. In vitro prion amplification techniques (saPMCA) were used to successfully misfold the cellular prion protein (PrP(c)) of these allegedly resistant species into a BSE-type prion protein. The biochemical and biological properties of the new prions generated in vitro after seeding rabbit and dog brain homogenates with classical BSE were studied. Pathobiological features of the resultant prion strains were determined after their inoculation into transgenic mice expressing bovine and human PrP(C). Strain characteristics of the in vitro-adapted rabbit and dog BSE agent remained invariable with respect to the original cattle BSE prion, suggesting that the naturally low susceptibility of rabbits and dogs to prion infections should not alter their zoonotic potential if these animals became infected with BSE. This study provides a sound basis for risk assessment regarding prion diseases in purportedly resistant species.

  8. TaTypA, a Ribosome-Binding GTPase Protein, Positively Regulates Wheat Resistance to the Stripe Rust Fungus

    PubMed Central

    Liu, Peng; Myo, Thwin; Ma, Wei; Lan, Dingyun; Qi, Tuo; Guo, Jia; Song, Ping; Guo, Jun; Kang, Zhensheng

    2016-01-01

    Tyrosine phosphorylation protein A (TypA/BipA) belongs to the ribosome-binding GTPase superfamily. In many bacterial species, TypA acts as a global stress and virulence regulator and also mediates resistance to the antimicrobial peptide bactericidal permeability-increasing protein. However, the function of TypA in plants under biotic stresses is not known. In this study, we isolated and functionally characterized a stress-responsive TypA gene (TaTypA) from wheat, with three copies located on chromosomes 6A, 6B, and 6D, respectively. Transient expression assays indicated chloroplast localization of TaTypA. The transcript levels of TaTypA were up-regulated in response to treatment with methyl viologen, which induces reactive oxygen species (ROS) in chloroplasts through photoreaction, cold stress, and infection by an avirulent strain of the stripe rust pathogen. Knock down of the expression of TaTypA through virus-induced gene silencing decreased the resistance of wheat to stripe rust accompanied by weakened ROS accumulation and hypersensitive response, an increase in TaCAT and TaSOD expression, and an increase in pathogen hyphal growth and branching. Our findings suggest that TaTypA contributes to resistance in an ROS-dependent manner. PMID:27446108

  9. Characterisation of the protein corona using tunable resistive pulse sensing: determining the change and distribution of a particle's surface charge.

    PubMed

    Blundell, Emma L C J; Healey, Matthew J; Holton, Elizabeth; Sivakumaran, Muttuswamy; Manstana, Sarabjit; Platt, Mark

    2016-08-01

    The zeta potential of the protein corona around carboxyl particles has been measured using tunable resistive pulse sensing (TRPS). A simple and rapid assay for characterising zeta potentials within buffer, serum and plasma is presented monitoring the change, magnitude and distribution of proteins on the particle surface. First, we measure the change in zeta potential of carboxyl-functionalised nanoparticles in solutions that contain biologically relevant concentrations of individual proteins, typically constituted in plasma and serum, and observe a significant difference in distributions and zeta values between room temperature and 37 °C assays. The effect is protein dependent, and the largest difference between the two temperatures is recorded for the γ-globulin protein where the mean zeta potential changes from -16.7 to -9.0 mV for 25 and 37 °C, respectively. This method is further applied to monitor particles placed into serum and/or plasma. A temperature-dependent change is again observed with serum showing a 4.9 mV difference in zeta potential between samples incubated at 25 and 37 °C; this shift was larger than that observed for samples in plasma (0.4 mV). Finally, we monitor the kinetics of the corona reorientation for particles initially placed into serum and then adding 5 % (V/V) plasma. The technology presented offers an interesting insight into protein corona structure and kinetics of formation measured in biologically relevant solutions, i.e. high protein, high salt levels, and its particle-by-particle analysis gives a measure of the distribution of particle zeta potential that may offer a better understanding of the behaviour of nanoparticles in solution. Graphical Abstract The relative velocity of a nanoparticle as it traverses a nanopore can be used to determine its zeta potential. Monitoring the changes in translocation speeds can therefore be used to follow changes to the surface chemistry/composition of 210 nm particles that were placed

  10. Crystal Structure of the Zorbamycin-Binding Protein ZbmA, the Primary Self-Resistance Element in Streptomyces flavoviridis ATCC21892

    SciTech Connect

    Rudolf, Jeffrey D.; Bigelow, Lance; Chang, Changsoo; Cuff, Marianne E.; Lohman, Jeremy R.; Chang, Chin-Yuan; Ma, Ming; Yang, Dong; Clancy, Shonda; Babnigg, Gyorgy; Joachimiak, Andrzej; Phillips, George N.; Shen, Ben

    2015-11-17

    The bleomycins (BLMs), tallysomycins (TLMs), phleomycin, and zorbamycin (ZBM) are members of the BLM family of glycopeptide-derived antitumor antibiotics. The BLM-producing Streptomyces verticillus ATCC15003 and the TLM-producing Streptoalloteichus hindustanus E465-94 ATCC31158 both possess at least two self-resistance elements, an N-acetyltransferase and a binding protein. The N-acetyltransferase provides resistance by disrupting the metal-binding domain of the antibiotic that is required for activity, while the binding protein confers resistance by sequestering the metal-bound antibiotic and preventing drug activation via molecular oxygen. We recently established that the ZBM producer, Streptomyces flavoviridis ATCC21892, lacks the N-acetyltransferase resistance gene and that the ZBM-binding protein, ZbmA, is sufficient to confer resistance in the producing strain. To investigate the resistance mechanism attributed to ZbmA, we determined the crystal structures of apo and Cu(II)-ZBM-bound ZbmA at high resolutions of 1.90 and 1.65 angstrom, respectively. A comparison and contrast with other structurally characterized members of the BLM-binding protein family revealed key differences in the protein ligand binding environment that fine-tunes the ability of ZbmA to sequester metal-bound ZBM and supports drug sequestration as the primary resistance mechanism in the producing organisms of the BLM family of antitumor antibiotics.

  11. Role of protein and amino acids in promoting lean mass accretion with resistance exercise and attenuating lean mass loss during energy deficit in humans.

    PubMed

    Churchward-Venne, Tyler A; Murphy, Caoileann H; Longland, Thomas M; Phillips, Stuart M

    2013-08-01

    Amino acids are major nutrient regulators of muscle protein turnover. After protein ingestion, hyperaminoacidemia stimulates increased rates of skeletal muscle protein synthesis, suppresses muscle protein breakdown, and promotes net muscle protein accretion for several hours. These acute observations form the basis for strategized protein intake to promote lean mass accretion, or prevent lean mass loss over the long term. However, factors such as protein dose, protein source, and timing of intake are important in mediating the anabolic effects of amino acids on skeletal muscle and must be considered within the context of evaluating the reported efficacy of long-term studies investigating protein supplementation as part of a dietary strategy to promote lean mass accretion and/or prevent lean mass loss. Current research suggests that dietary protein supplementation can augment resistance exercise-mediated gains in skeletal muscle mass and strength and can preserve skeletal muscle mass during periods of diet-induced energy restriction. Perhaps less appreciated, protein supplementation can augment resistance training-mediated gains in skeletal muscle mass even in individuals habitually consuming 'adequate' (i.e., >0.8 g kg⁻¹ day⁻¹) protein. Additionally, overfeeding energy with moderate to high-protein intake (15-25 % protein or 1.8-3.0 g kg⁻¹ day⁻¹) is associated with lean, but not fat mass accretion, when compared to overfeeding energy with low protein intake (5 % protein or ~0.68 g kg⁻¹ day⁻¹). Amino acids represent primary nutrient regulators of skeletal muscle anabolism, capable of enhancing lean mass accretion with resistance exercise and attenuating the loss of lean mass during periods of energy deficit, although factors such as protein dose, protein source, and timing of intake are likely important in mediating these effects.

  12. Overexpression of the multidrug resistance-associated protein (MRP1) in human heavy metal-selected tumor cells.

    PubMed

    Vernhet, L; Courtois, A; Allain, N; Payen, L; Anger, J P; Guillouzo, A; Fardel, O

    1999-01-29

    Cellular and molecular mechanisms involved in the resistance to cytotoxic heavy metals remain largely to be characterized in mammalian cells. To this end, we have analyzed a metal-resistant variant of the human lung cancer GLC4 cell line that we have selected by a step-wise procedure in potassium antimony tartrate. Antimony-selected cells, termed GLC4/Sb30 cells, poorly accumulated antimony through an enhanced cellular efflux of metal, thus suggesting up-regulation of a membrane export system in these cells. Indeed, GLC4/Sb30 cells were found to display a functional overexpression of the multidrug resistance-associated protein MRP1, a drug export pump, as demonstrated by Western blotting, reverse transcriptase-polymerase chain reaction and calcein accumulation assays. Moreover, MK571, a potent inhibitor of MRP1 activity, was found to markedly down-modulate resistance of GLC4/Sb30 cells to antimony and to decrease cellular export of the metal. Taken together, our data support the conclusion that overexpression of functional MRP1 likely represents one major mechanism by which human cells can escape the cytotoxic effects of heavy metals.

  13. miR-145 sensitizes breast cancer to doxorubicin by targeting multidrug resistance-associated protein-1

    PubMed Central

    Gao, Man; Miao, Lingling; Liu, Mingxia; Li, Chenggang; Yu, Cunzhi; Yan, Hong; Yin, Yongxiang; Wang, Yizheng; Qi, Xinming; Ren, Jin

    2016-01-01

    Multidrug resistance-associated protein 1 (MRP1) is an important efflux transporter and overexpression of MRP1 usually leads to chemoresistance in breast cancer. Here, we found MRP1 overexpressed in human breast cancer tissues and breast cancer cell lines (compared with normal breast tissues and cell line, respectively). And MRP1 level increased in doxorubicin resistant MCF-7 cells compared with parental MCF-7 cells. Increasing evidences suggest microRNAs (miRNAs) influence chemotherapy response. We found miR-145 level decreased in human breast cancer tissues, breast cancer cell lines and doxorubicin resistant MCF-7 cells, and inversely correlated with MRP1 expression level. In the process of constructing MCF-7 doxorubicin resistant cell line, escalating doxorubicin markedly decreased miR-145 level, following by increased MRP1 level. Further study showed, miR-145 suppressed MRP1 expression by directly targeting MRP1 3′-untranslated regions. Overexpression of miR-145 sensitized breast cancer cells to doxorubicin in vitro and enhanced to doxorubicin chemotherapy in vivo through inducing intracellular doxorubicin accumulation via inhibiting MRP1. Taken together, our study revealed miR-145 sensitizes breast cancer to doxorubicin by targeting MRP1 and indicated the potential application in developing MRP1 inhibitor. PMID:27487127

  14. Prunus domestica pathogenesis-related protein-5 activates the defense response pathway and enhances the resistance to fungal infection.

    PubMed

    El-kereamy, Ashraf; El-sharkawy, Islam; Ramamoorthy, Rengasamy; Taheri, Ali; Errampalli, Deena; Kumar, Prakash; Jayasankar, Subramanian

    2011-03-23

    Pathogenesis-related protein-5 (PR-5) has been implicated in plant disease resistance and its antifungal activity has been demonstrated in some fruit species. However, their roles, especially their interactions with the other defense responses in plant cells, are still not fully understood. In this study, we have cloned and characterized a new PR-5 cDNA named PdPR5-1 from the European plum (Prunus domestica). Expression of PdPR5-1 was studied in different cultivars varying in resistance to the brown rot disease caused by the necrotrophic fungus Monilinia fructicola. In addition transgenic Arabidopsis, ectopically expressing PdPR5-1 was used to study its role in other plant defense responses after fungal infection. We show that the resistant cultivars exhibited much higher levels of transcripts than the susceptible cultivars during fruit ripening. However, significant rise in the transcript levels after infection with M. fructicola was observed in the susceptible cultivars too. Transgenic Arabidopsis plants exhibited more resistance to Alternaria brassicicola. Further, there was a significant increase in the transcripts of genes involved in the phenylpropanoid biosynthesis pathway such as phenylalanine ammonia-lyase (PAL) and phytoalexin (camalexin) pathway leading to an increase in camalexin content after fungal infection. Our results show that PdPR5-1 gene, in addition to its anti-fungal properties, has a possible role in activating other defense pathways, including phytoalexin production.

  15. SET protein overexpression contributes to paclitaxel resistance in MCF-7/S cells through PI3K/Akt pathway.

    PubMed

    Zhang, Weipeng; Zheng, Xiaowei; Meng, Ti; You, Haisheng; Dong, Yalin; Xing, Jianfeng; Chen, Siying

    2017-03-01

    Patient SE translation (SET) is a carcinogen in facilitating cellular growth and proliferation, and promoting tumorigenesis and metastasis. The present study was to investigate the resistance mechanisms associated with SET in paclitaxel-induced human breast cancer cells. The different expressions of SET, ATP-binding cassette (ABC) transporters and PI3K/Akt pathway between paclitaxel sensitive MCF-7/S and paclitaxel resistant MCF-7/PTX cells were identified using western blotting. We adopted plasmid transfection to upregulate SET in MCF-7/S cells and a novel SET antagonist COG112 to decrease SET in MCF-7/PTX cells. Subsequently, cell viability to paclitaxel was assessed by MTT assay and cell apoptosis was analyzed by flow cytometry. We found that levels of SET, ABC transporters and PI3K/Akt pathway were elevated in MCF-7/PTX. Upregulation of SET in MCF-7/S cells expressed resistant to paclitaxel and decreased cell apoptosis. Moreover, overexpression of SET promoted the mRNA and protein level of ABC transporters and PI3K/Akt signal pathway in MCF-7/S cells. Conversely, decreased level of SET by COG112 not only significantly sensitized MCF-7/PTX cells to paclitaxel, but also enhanced paclitaxel-induced cell apoptosis. Additionally, the levels of the ABC transporters and PI3K/Akt signal pathway were also reduced in the COG112-treated MCF-7/PTX cells. The above results demonstrated that SET was associated with paclitaxel resistance in MCF-7/PTX cells.

  16. Uptake and Degradation of Protease-Sensitive and -Resistant Forms of Abnormal Human Prion Protein Aggregates by Human Astrocytes

    PubMed Central

    Choi, Young Pyo; Head, Mark W.; Ironside, James W.; Priola, Suzette A.

    2015-01-01

    Sporadic Creutzfeldt-Jakob disease is the most common of the human prion diseases, a group of rare, transmissible, and fatal neurologic diseases associated with the accumulation of an abnormal form (PrPSc) of the host prion protein. In sporadic Creutzfeldt-Jakob disease, disease-associated PrPSc is present not only as an aggregated, protease-resistant form but also as an aggregated protease-sensitive form (sPrPSc). Although evidence suggests that sPrPSc may play a role in prion pathogenesis, little is known about how it interacts with cells during prion infection. Here, we show that protease-sensitive abnormal PrP aggregates derived from patients with sporadic Creutzfeldt-Jakob disease are taken up and degraded by immortalized human astrocytes similarly to abnormal PrP aggregates that are resistant to proteases. Our data suggest that relative proteinase K resistance does not significantly influence the astrocyte's ability to degrade PrPSc. Furthermore, the cell does not appear to distinguish between sPrPSc and protease-resistant PrPSc, suggesting that sPrPSc could contribute to prion infection. PMID:25280631

  17. A TolC-Like Protein of Actinobacillus pleuropneumoniae Is Involved in Antibiotic Resistance and Biofilm Formation

    PubMed Central

    Li, Ying; Cao, Sanjie; Zhang, Luhua; Lau, Gee W.; Wen, Yiping; Wu, Rui; Zhao, Qin; Huang, Xiaobo; Yan, Qigui; Huang, Yong; Wen, Xintian

    2016-01-01

    Actinobacillus pleuropneumoniae is the etiologic agent of porcine contagious pleuropneumonia, a significant disease that causes serious economic losses to the swine industry worldwide. Persistent infections caused by bacterial biofilms are recalcitrant to treat because of the particular drug resistance of biofilm-dwelling cells. TolC, a key component of multidrug efflux pumps, are responsible for multidrug resistance (MDR) in many Gram-negative bacteria. In this study, we identified two TolC-like proteins, TolC1 and TolC2, in A. pleuropneumoniae. Deletion of tolC1, but not tolC2, caused a significant reduction in biofilm formation, as well as increased drug sensitivity of both planktonic and biofilm cells. The genetic-complementation of the tolC1 mutation restored the competent biofilm and drug resistance. Besides, biofilm formation was inhibited and drug sensitivity was increased by the addition of phenylalanine-arginine beta-naphthylamide (PAβN), a well-known efflux pump inhibitor (EPI), suggesting a role for EPI in antibacterial strategies toward drug tolerance of A. pleuropneumoniae. Taken together, TolC1 is required for biofilm formation and is a part of the MDR machinery of both planktonic and biofilm cells, which could supplement therapeutic strategies for resistant bacteria and biofilm-related infections of A. pleuropneumoniae clinical isolate SC1516. PMID:27822201

  18. Association of the emerging carbapenemase NDM-1 with a bleomycin resistance protein in Enterobacteriaceae and Acinetobacter baumannii.

    PubMed

    Dortet, Laurent; Nordmann, Patrice; Poirel, Laurent

    2012-04-01

    The carbapenemase NDM-1 has been identified recently in Enterobacteriaceae and Acinetobacter baumannii as a source of multidrug resistance, including resistance to carbapenems. By analyzing the immediate genetic environment of the bla(NDM-1) carbapenemase gene among a series of NDM-1-producing enterobacterial isolates, a novel gene (ble(MBL), for ble gene associated with the metallo-β-lactamase NDM-1) was identified. The ble(MBL) gene encodes a novel bleomycin resistance protein (BRP), named BRP(MBL), that shares weak similarities with known BRPs (less than 60% amino acid identity). The expression of BRP(MBL) conferred resistance to bleomycin and to bleomycin-like molecules in Enterobacteriaceae and A. baumannii. The bla(NDM-1) and ble(MBL) genes were coexpressed under the control of the same promoter, located upstream of the bla(NDM-1) gene and at the extremity of the insertion sequence ISAba125. Most of the NDM producers possessed the ble(MBL) gene. Although BRP(MBL) did not modify the growth or death rates of Escherichia coli under experimental conditions, it suppressed the mutation rate of hypermutable E. coli and therefore may stabilize the plasmid-borne bla(NDM-1) gene. This study suggests that the emerging carbapenemase NDM-1 is selected by bleomycin-like molecules, and that BRP(MBL) producers (and consequently NDM producers) are better suited to various environments.

  19. Breast Cancer Resistance Protein Abundance, but Not mRNA Expression, Correlates With Estrone-3-Sulfate Transport in Caco-2.

    PubMed

    Harwood, Matthew D; Neuhoff, Sibylle; Rostami-Hodjegan, Amin; Warhurst, Geoffrey

    2016-04-01

    Transporter mRNA and protein expression data are used to extrapolate in vitro transporter kinetics to in vivo drug disposition predictions. Breast cancer resistance protein (BCRP) possesses broad substrate specificity; therefore, understanding BCRP expression-activity relationships are necessary for the translation to in vivo. Bidirectional transport of estrone-3-sulfate (E-3-S), a BCRP probe, was evaluated with respect to relative BCRP mRNA expression and absolute protein abundance in 10- and 29-day cultured Caco-2 cells. BCRP mRNA expression was quantified by real-time PCR against a housekeeper gene, Cyclophilin A. The BCRP protein abundance in total membrane fractions was quantified by targeted proteomics, and [(3)H]-E-3-S bidirectional transport was determined in the presence or absence of Ko143, a potent BCRP inhibitor. BCRP mRNA expression was 1.5-fold higher in 29- versus 10-day cultured cells (n = 3), whereas a 2.4-fold lower (p < 0.001) BCRP protein abundance was observed in 29- versus 10-day cultured cells (1.28 ± 0.33 and 3.06 ± 0.22 fmol/μg protein, n = 6, respectively). This correlated to a 2.45-fold lower (p < 0.01) efflux ratio for E-3-S in 29- versus 10-day cultured cells (8.97 ± 2.51 and 3.32 ± 0.66, n = 6, respectively). Caco-2 cell BCRP protein abundance, but not mRNA levels, correlates with BCRP activity, suggesting that extrapolation strategies incorporating BCRP protein abundance-activity relationships may be more successful.

  20. Thermal stability and flame resistance of cotton fabrics treated with whey proteins.

    PubMed

    Bosco, Francesca; Carletto, Riccardo Andrea; Alongi, Jenny; Marmo, Luca; Di Blasio, Alessandro; Malucelli, Giulio

    2013-04-15

    It is well described in the literature that whey proteins are able to form coatings, which exhibit high mechanical and oxygen barrier properties, notwithstanding a great water vapour adsorption. These peculiarities have been exploited for applying a novel protein-based finishing treatment to cotton and for assessing the protein effect on the thermal and thermo-oxidative stability and on the flame retardant properties of the cellulosic fabric. Indeed, the deposited whey protein coatings have turned out to significantly affect the thermal degradation of cotton in inert and oxidative atmosphere, and to somehow modify its combustion when a flame has been applied. Furthermore, the influence of the secondary and tertiary structure of these proteins on the morphology of the deposited coating, and thus on the thermal and flame retardant properties of the treated fabrics, has been evaluated by performing a denaturation thermal treatment before the protein application.

  1. Fludarabine nucleoside modulates nuclear "survival and death" proteins in resistant chronic lymphocytic leukemia cells.

    PubMed

    Henrich, Silke; Mactier, Swetlana; Best, Giles; Mulligan, Stephen P; Crossett, Ben; Christopherson, Richard Ian

    2011-12-01

    The nuclear mechanisms by which fludarabine nucleoside (F-ara-A) induces apoptosis have been investigated in human MEC1 cells derived from B-cell chronic lymphocytic leukemia. Upon treatment of cells with F-ara-A (100 μM, 72 hours), 15 nuclear proteins changed in abundance by more than 2-fold. Nuclear proteins up-regulated included calm