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Sample records for acriflavine resistance protein

  1. 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.

  2. 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. PMID:23052789

  3. 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

  4. Resonance energy transfer from dibucaine to acriflavine in polystyrene latex dispersions.

    PubMed

    Li, Yuan; Kuwabara, Hiroe; Gong, Yong-Kuan; Takaki, Yuki; Nakashima, Kenichi

    2003-07-01

    Resonance energy transfer from dibucaine (DC) to acriflavine (AF) has been investigated both in homogeneous aqueous solutions and in polystyrene latex dispersions. The energy transfer reaction is observed by monitoring fluorescence quenching of DC as well as sensitized emission of AF. It is found that the energy transfer from DC to AF is remarkably enhanced on going from the aqueous homogeneous solution to the latex dispersion. This is mainly attributed to the fact that both the donor and acceptor are effectively adsorbed onto the latex particles, as evidenced by the measurement of adsorption isotherms. From the adsorption experiments, it is also elucidated that electrostatic interaction is significant at low DC concentration, while hydrophobic interaction dominates at the higher concentration especially at higher pH.

  5. 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.

  6. Molecular basis of polyspecificity of the Small Multidrug Resistance Efflux Pump AbeS from Acinetobacter baumannii.

    PubMed

    Lytvynenko, Iryna; Brill, Shlomo; Oswald, Christine; Pos, Klaas M

    2016-02-13

    Secondary multidrug efflux transporters play a key role in the bacterial resistance phenotype. One of the major questions concerns the polyspecific recognition of substrates by these efflux pumps. To understand the molecular basis of this promiscuous recognition, we compared the substrate specificity of the well-studied Escherichia coli small multidrug resistance protein EmrE with that of the poorly studied Acinetobacter baumannii homologue AbeS. The latter drug/H(+) antiporter is a 109-amino-acid membrane protein with predicted four transmembrane helices. It effectively confers resistance toward ethidium, acriflavine and benzalkonium in an E. coli ΔemrEΔmdfA background. Purified AbeS and the substrate-specific hyperactive variant A16G bind tetraphenylphosphonium with nanomolar affinity and exhibit electrogenic transport of 1-methyl-4-phenylpyridinium after reconstitution into liposomes. A16G hyperactivity was apparent toward acriflavine and ethidium, resulting in 7- to 10-fold higher normalized IC50 values, respectively, but not toward substrates 1-methyl-4-phenylpyridinium and benzalkonium. Substitution of Y3 and A42 with Ala or Ser, respectively, also displayed a substrate-dependent phenotype, as these variants were strongly affected in their properties to confer resistance against acriflavine and ethidium, but not against benzalkonium. The size and planarity of the conjugated aromatic moieties appear to be a critical and subtle criterion for substrate recognition by these transporters. Rather moderate changes in the property of side chains postulated to be part of the substrate binding site result in a large phenotypical difference. These observations provide indications for the molecular basis of specificity within the binding pocket of polyspecific transporters. PMID:26707198

  7. 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.

  8. Multidrug Resistance Proteins (MRPs) and Cancer Therapy.

    PubMed

    Zhang, Yun-Kai; Wang, Yi-Jun; Gupta, Pranav; Chen, Zhe-Sheng

    2015-07-01

    The ATP-binding cassette (ABC) transporters are members of a protein superfamily that are known to translocate various substrates across membranes, including metabolic products, lipids and sterols, and xenobiotic drugs. Multidrug resistance proteins (MRPs) belong to the subfamily C in the ABC transporter superfamily. MRPs have been implicated in mediating multidrug resistance by actively extruding chemotherapeutic substrates. Moreover, some MRPs are known to be essential in physiological excretory or regulatory pathways. The importance of MRPs in cancer therapy is also implied by their clinical insights. Modulating the function of MRPs to re-sensitize chemotherapeutic agents in cancer therapy shows great promise in cancer therapy; thus, multiple MRP inhibitors have been developed recently. This review article summarizes the structure, distribution, and physiological as well as pharmacological function of MRP1-MRP9 in cancer chemotherapy. Several novel modulators targeting MRPs in cancer therapy are also discussed. PMID:25840885

  9. Multidrug Resistance Proteins (MRPs) and Cancer Therapy.

    PubMed

    Zhang, Yun-Kai; Wang, Yi-Jun; Gupta, Pranav; Chen, Zhe-Sheng

    2015-07-01

    The ATP-binding cassette (ABC) transporters are members of a protein superfamily that are known to translocate various substrates across membranes, including metabolic products, lipids and sterols, and xenobiotic drugs. Multidrug resistance proteins (MRPs) belong to the subfamily C in the ABC transporter superfamily. MRPs have been implicated in mediating multidrug resistance by actively extruding chemotherapeutic substrates. Moreover, some MRPs are known to be essential in physiological excretory or regulatory pathways. The importance of MRPs in cancer therapy is also implied by their clinical insights. Modulating the function of MRPs to re-sensitize chemotherapeutic agents in cancer therapy shows great promise in cancer therapy; thus, multiple MRP inhibitors have been developed recently. This review article summarizes the structure, distribution, and physiological as well as pharmacological function of MRP1-MRP9 in cancer chemotherapy. Several novel modulators targeting MRPs in cancer therapy are also discussed.

  10. Dietary protein to maximize resistance training: a review and examination of protein spread and change theories.

    PubMed

    Bosse, John D; Dixon, Brian M

    2012-01-01

    An appreciable volume of human clinical data supports increased dietary protein for greater gains from resistance training, but not all findings are in agreement. We recently proposed "protein spread theory" and "protein change theory" in an effort to explain discrepancies in the response to increased dietary protein in weight management interventions. The present review aimed to extend "protein spread theory" and "protein change theory" to studies examining the effects of protein on resistance training induced muscle and strength gains. Protein spread theory proposed that there must have been a sufficient spread or % difference in g/kg/day protein intake between groups during a protein intervention to see muscle and strength differences. Protein change theory postulated that for the higher protein group, there must be a sufficient change from baseline g/kg/day protein intake to during study g/kg/day protein intake to see muscle and strength benefits. Seventeen studies met inclusion criteria. In studies where a higher protein intervention was deemed successful there was, on average, a 66.1% g/kg/day between group intake spread versus a 10.2% g/kg/day spread in studies where a higher protein diet was no more effective than control. The average change in habitual protein intake in studies showing higher protein to be more effective than control was +59.5% compared to +6.5% when additional protein was no more effective than control. The magnitudes of difference between the mean spreads and changes of the present review are similar to our previous review on these theories in a weight management context. Providing sufficient deviation from habitual intake appears to be an important factor in determining the success of additional protein in enhancing muscle and strength gains from resistance training. An increase in dietary protein favorably effects muscle and strength during resistance training. PMID:22958314

  11. Dietary protein to maximize resistance training: a review and examination of protein spread and change theories

    PubMed Central

    2012-01-01

    An appreciable volume of human clinical data supports increased dietary protein for greater gains from resistance training, but not all findings are in agreement. We recently proposed “protein spread theory” and “protein change theory” in an effort to explain discrepancies in the response to increased dietary protein in weight management interventions. The present review aimed to extend “protein spread theory” and “protein change theory” to studies examining the effects of protein on resistance training induced muscle and strength gains. Protein spread theory proposed that there must have been a sufficient spread or % difference in g/kg/day protein intake between groups during a protein intervention to see muscle and strength differences. Protein change theory postulated that for the higher protein group, there must be a sufficient change from baseline g/kg/day protein intake to during study g/kg/day protein intake to see muscle and strength benefits. Seventeen studies met inclusion criteria. In studies where a higher protein intervention was deemed successful there was, on average, a 66.1% g/kg/day between group intake spread versus a 10.2% g/kg/day spread in studies where a higher protein diet was no more effective than control. The average change in habitual protein intake in studies showing higher protein to be more effective than control was +59.5% compared to +6.5% when additional protein was no more effective than control. The magnitudes of difference between the mean spreads and changes of the present review are similar to our previous review on these theories in a weight management context. Providing sufficient deviation from habitual intake appears to be an important factor in determining the success of additional protein in enhancing muscle and strength gains from resistance training. An increase in dietary protein favorably effects muscle and strength during resistance training. PMID:22958314

  12. 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

  13. Bacterial histone-like proteins: roles in stress resistance.

    PubMed

    Wang, Ge; Maier, Robert J

    2015-11-01

    Histone-like proteins (HLPs) are small and basic bacterial proteins that are associated with a nucleoid and play roles in maintaining DNA architecture and regulating DNA transactions such as replication, recombination/repair and transcription. The studies on HLPs from a variety of bacterial species in recent years are summarized in this mini-review. A recent study reported a novel DNA-binding protein (HP119) in Helicobacter pylori that shows some HLP features. It plays a large role in aiding bacterial stress resistance. We provide herein additional evidence that HP119 is a nucleoid-associated protein, and present some perspectives for future study.

  14. 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.

  15. The pharmacogenomics of drug resistance to protein kinase inhibitors.

    PubMed

    Gillis, Nancy K; McLeod, Howard L

    2016-09-01

    Dysregulation of growth factor cell signaling is a major driver of most human cancers. This has led to development of numerous drugs targeting protein kinases, with demonstrated efficacy in the treatment of a wide spectrum of cancers. Despite their high initial response rates and survival benefits, the majority of patients eventually develop resistance to these targeted therapies. This review article discusses examples of established mechanisms of drug resistance to anticancer therapies, including drug target mutations or gene amplifications, emergence of alternate signaling pathways, and pharmacokinetic variation. This reveals a role for pharmacogenomic analysis to identify and monitor for resistance, with possible therapeutic strategies to combat chemoresistance. PMID:27620953

  16. The pharmacogenomics of drug resistance to protein kinase inhibitors.

    PubMed

    Gillis, Nancy K; McLeod, Howard L

    2016-09-01

    Dysregulation of growth factor cell signaling is a major driver of most human cancers. This has led to development of numerous drugs targeting protein kinases, with demonstrated efficacy in the treatment of a wide spectrum of cancers. Despite their high initial response rates and survival benefits, the majority of patients eventually develop resistance to these targeted therapies. This review article discusses examples of established mechanisms of drug resistance to anticancer therapies, including drug target mutations or gene amplifications, emergence of alternate signaling pathways, and pharmacokinetic variation. This reveals a role for pharmacogenomic analysis to identify and monitor for resistance, with possible therapeutic strategies to combat chemoresistance.

  17. 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

  18. 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.

  19. Cycloheximide resistance in yeast: the gene and its protein.

    PubMed Central

    Käufer, N F; Fried, H M; Schwindinger, W F; Jasin, M; Warner, J R

    1983-01-01

    Mutations in the yeast gene CYH2 can lead to resistance to cycloheximide, an inhibitor of eukaryotic protein synthesis. The gene product of CYH2 is ribosomal protein L29, a component of the 60S ribosomal subunit. We have cloned the wild-type and resistance alleles of CYH2 and determined their nucleotide sequence. Transcription of CYH2 appears to initiate and terminate at multiple sites, as judged by S1 nuclease analysis. The gene is transcribed into an RNA molecule of about 1082 nucleotides, containing an intervening sequence of 510 nucleotides. The splice junction of the intron resides within a codon near the 5' end of the gene. In confirmation of peptide analysis by Stocklein et al. (1) we find that resistance to cycloheximide is due to a transversion mutation resulting in the replacement of a glutamine by glutamic acid in position 37 of L29. Images PMID:6304624

  20. 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.

  1. An etoposide-resistant lung cancer subline overexpresses the multidrug resistance-associated protein.

    PubMed Central

    Doyle, L. A.; Ross, D. D.; Ordonez, J. V.; Yang, W.; Gao, Y.; Tong, Y.; Belani, C. P.; Gutheil, J. C.

    1995-01-01

    We have characterised an etoposide-resistant subline of the small-cell lung cancer cell line, UMCC-1, derived at our centre. Subline UMCC-1/VP was developed by culturing the parent line in increasing concentrations of etoposide over 16 months. UMCC-1/VP is 20-fold resistant to etoposide by MTT assays, relative to the parent line, and is cross-resistant to doxorubicin, vincristine and actinomycin D, but not to taxol, cisplatin, melphalan, thiotepa or idarubicin. Topoisomerase II immunoblotting demonstrates a 50% reduction of the protein in the resistant subline. The UMCC-1/VP subline demonstrates a marked decrease in the accumulation of [3H]etoposide relative to the parent line, as well as a modest reduction in the accumulation of daunorubicin. Reverse transcription-polymerase chain reaction assays demonstrate no detectable mdr1 expression but marked expression of the multidrug resistance-associated protein (MRP) gene in the resistant subline. Northern blotting with an MRP cDNA probe confirms marked overexpression of the MRP gene only in the UMCC-1/VP subline. Western blotting with antisera against MRP peptide confirms a 195 kDa protein band in the UMCC-1/VP subline. Southern blotting experiments demonstrate a 10-fold amplification of the MRP gene in the resistant subline. Depletion of glutathione with buthionine sulphoximine sensitised UMCC-1/VP cells to daunorubicin and etoposide. Our studies indicate that MRP gene expression may be induced by etoposide and may lead to reduced accumulation of the drug. Images Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 PMID:7669558

  2. 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.

  3. Diversity and evolution of the small multidrug resistance protein family

    PubMed Central

    Bay, Denice C; Turner, Raymond J

    2009-01-01

    Background Members of the small multidrug resistance (SMR) protein family are integral membrane proteins characterized by four α-helical transmembrane strands that confer resistance to a broad range of antiseptics and lipophilic quaternary ammonium compounds (QAC) in bacteria. Due to their short length and broad substrate profile, SMR proteins are suggested to be the progenitors for larger α-helical transporters such as the major facilitator superfamily (MFS) and drug/metabolite transporter (DMT) superfamily. To explore their evolutionary association with larger multidrug transporters, an extensive bioinformatics analysis of SMR sequences (> 300 Bacteria taxa) was performed to expand upon previous evolutionary studies of the SMR protein family and its origins. Results A thorough annotation of unidentified/putative SMR sequences was performed placing sequences into each of the three SMR protein subclass designations, namely small multidrug proteins (SMP), suppressor of groEL mutations (SUG), and paired small multidrug resistance (PSMR) using protein alignments and phylogenetic analysis. Examination of SMR subclass distribution within Bacteria and Archaea taxa identified specific Bacterial classes that uniquely encode for particular SMR subclass members. The extent of selective pressure acting upon each SMR subclass was determined by calculating the rate of synonymous to non-synonymous nucleotide substitutions using Syn-SCAN analysis. SUG and SMP subclasses are maintained under moderate selection pressure in comparison to integron and plasmid encoded SMR homologues. Conversely, PSMR sequences are maintained under lower levels of selection pressure, where one of the two PSMR pairs diverges in sequence more rapidly than the other. SMR genomic loci surveys identified potential SMR efflux substrates based on its gene association to putative operons that encode for genes regulating amino acid biogenesis and QAC-like metabolites. SMR subclass protein transmembrane domain

  4. The effect of body protein supply on resistance to cadmium.

    PubMed Central

    Gontzea, I; Popescu, F

    1978-01-01

    Six groups of 15 rats were fed on three diets, each differing in the quantity and quality of protein (17.87 as opposed to 8.85 g%, with or without the addition of 0.5 g methionine), one group of each pair of animals being injected subcutaneously with 0.3 mg Cd/kg body weight/day, for 13 weeks. The low protein diet increased the effects of cadmium, rendering them significantly more harmful than in animals which were given the normal protein diet. The incorporation of 0.5 g% DL-methionine in the low protein diet, without increasing the total nitrogen content, diminished the most marked effects induced by the same amounts of cadmium, so that their mean values were not significantly different from those found in the normal protein group treated with the same dose of the metal. The results show that a quantitatively and qualitatively adequate protein supply increased the resistance of the organism to cadmium, diminishing significantly the severity of symptoms induced by the metal. PMID:656340

  5. Nuclear export of proteins and drug resistance in cancer.

    PubMed

    Turner, Joel G; Dawson, Jana; Sullivan, Daniel M

    2012-04-15

    The intracellular location of a protein is crucial to its normal functioning in a cell. Cancer cells utilize the normal processes of nuclear-cytoplasmic transport through the nuclear pore complex of a cell to effectively evade anti-neoplastic mechanisms. CRM1-mediated export is increased in various cancers. Proteins that are exported in cancer include tumor-suppressive proteins such as retinoblastoma, APC, p53, BRAC1, FOXO proteins, INI1/hSNF5, galectin-3, Bok, nucleophosmin, RASSF2, Merlin, p21(CIP), p27(KIP1), N-WASP/FAK, estradiol receptor and Tob, drug targets topoisomerase I and IIα and BCR-ABL, and the molecular chaperone protein Hsp90. Here, we review in detail the current processes and known structures involved in the export of a protein through the nuclear pore complex. We also discuss the export receptor molecule CRM1 and its binding to the leucine-rich nuclear export signal of the cargo protein and the formation of a nuclear export trimer with RanGTP. The therapeutic potential of various CRM1 inhibitors will be addressed, including leptomycin B, ratjadone, KOS-2464, and specific small molecule inhibitors of CRM1, N-azolylacrylate analogs, FOXO export inhibitors, valtrate, acetoxychavicol acetate, CBS9106, and SINE inhibitors. We will also discuss examples of how drug resistance may be reversed by targeting the exported proteins topoisomerase IIα, BCR-ABL, and galectin-3. As effective and less toxic CRM1 export inhibitors become available, they may be used as both single agents and in combination with current chemotherapeutic drugs. We believe that the future development of low-toxicity, small-molecule CRM1 inhibitors may provide a new approach to treating cancer.

  6. Multidrug resistance-associated protein 4 is a determinant of arsenite resistance.

    PubMed

    Yuan, Bo; Yoshino, Yuta; Fukushima, Hisayo; Markova, Svetlana; Takagi, Norio; Toyoda, Hiroo; Kroetz, Deanna L

    2016-01-01

    Although arsenic trioxide (arsenite, As(III)) has shown a remarkable efficacy in the treatment of acute promyelocytic leukemia patients, multidrug resistance is still a major concern for its clinical use. Multidrug resistance-associated protein 4 (MRP4), which belongs to the ATP-binding cassette (ABC) superfamily of transporters, is localized to the basolateral membrane of hepatocytes and the apical membrane of renal proximal tubule cells. Due to its characteristic localization, MRP4 is proposed as a candidate in the elimination of arsenic and may contribute to resistance to As(III). To test this hypothesis, stable HEK293 cells overexpressing MRP4 or MRP2 were used to establish the role of these two transporters in As(III) resistance. The IC50 values of As(III) in MRP4 cells were approximately 6-fold higher than those in MRP2 cells, supporting an important role for MRP4 in resistance to As(III). The capacity of MRP4 to confer resistance to As(III) was further confirmed by a dramatic decrease in the IC50 values with the addition of MK571, an MRP4 inhibitor, and cyclosporine A, a well-known broad-spectrum inhibitor of ABC transporters. Surprisingly, the sensitivity of the MRP2 cells to As(III) was similar to that of the parent cells, although insufficient formation of glutathione and/or Se conjugated arsenic compounds in the MRP2 cells might limit transport. Given that MRP4 is a major contributor to arsenic resistance in vitro, further investigation into the correlation between MRP4 expression and treatment outcome of leukemia patients treated with arsenic-based regimens is warranted. PMID:26497925

  7. N-terminal motifs in some plant disease resistance proteins function in membrane attachment and contribute to disease resistance.

    PubMed

    Takemoto, Daigo; Rafiqi, Maryam; Hurley, Ursula; Lawrence, Greg J; Bernoux, Maud; Hardham, Adrienne R; Ellis, Jeffrey G; Dodds, Peter N; Jones, David A

    2012-03-01

    To investigate the role of N-terminal domains of plant disease resistance proteins in membrane targeting, the N termini of a number of Arabidopsis and flax disease resistance proteins were fused to green fluorescent protein (GFP) and the fusion proteins localized in planta using confocal microscopy. The N termini of the Arabidopsis RPP1-WsB and RPS5 resistance proteins and the PBS1 protein, which is required for RPS5 resistance, targeted GFP to the plasma membrane, and mutation of predicted myristoylation and potential palmitoylation sites resulted in a shift to nucleocytosolic localization. The N-terminal domain of the membrane-attached Arabidopsis RPS2 resistance protein was targeted incompletely to the plasma membrane. In contrast, the N-terminal domains of the Arabidopsis RPP1-WsA and flax L6 and M resistance proteins, which carry predicted signal anchors, were targeted to the endomembrane system, RPP1-WsA to the endoplasmic reticulum and the Golgi apparatus, L6 to the Golgi apparatus, and M to the tonoplast. Full-length L6 was also targeted to the Golgi apparatus. Site-directed mutagenesis of six nonconserved amino acid residues in the signal anchor domains of L6 and M was used to change the localization of the L6 N-terminal fusion protein to that of M and vice versa, showing that these residues control the targeting specificity of the signal anchor. Replacement of the signal anchor domain of L6 by that of M did not affect L6 protein accumulation or resistance against flax rust expressing AvrL567 but removal of the signal anchor domain reduced L6 protein accumulation and L6 resistance, suggesting that membrane attachment is required to stabilize the L6 protein.

  8. 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

  9. 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

  10. Protease-resistant prions selectively decrease Shadoo protein.

    PubMed

    Watts, Joel C; Stöhr, Jan; Bhardwaj, Sumita; Wille, Holger; Oehler, Abby; Dearmond, Stephen J; Giles, Kurt; Prusiner, Stanley B

    2011-11-01

    The central event in prion diseases is the conformational conversion of the cellular prion protein (PrP(C)) into PrP(Sc), a partially protease-resistant and infectious conformer. However, the mechanism by which PrP(Sc) causes neuronal dysfunction remains poorly understood. Levels of Shadoo (Sho), a protein that resembles the flexibly disordered N-terminal domain of PrP(C), were found to be reduced in the brains of mice infected with the RML strain of prions [1], implying that Sho levels may reflect the presence of PrP(Sc) in the brain. To test this hypothesis, we examined levels of Sho during prion infection using a variety of experimental systems. Sho protein levels were decreased in the brains of mice, hamsters, voles, and sheep infected with different natural and experimental prion strains. Furthermore, Sho levels were decreased in the brains of prion-infected, transgenic mice overexpressing Sho and in infected neuroblastoma cells. Time-course experiments revealed that Sho levels were inversely proportional to levels of protease-resistant PrP(Sc). Membrane anchoring and the N-terminal domain of PrP both influenced the inverse relationship between Sho and PrP(Sc). Although increased Sho levels had no discernible effect on prion replication in mice, we conclude that Sho is the first non-PrP marker specific for prion disease. Additional studies using this paradigm may provide insight into the cellular pathways and systems subverted by PrP(Sc) during prion disease. PMID:22163178

  11. 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

  12. SynProt: A Database for Proteins of Detergent-Resistant Synaptic Protein Preparations

    PubMed Central

    Pielot, Rainer; Smalla, Karl-Heinz; Müller, Anke; Landgraf, Peter; Lehmann, Anne-Christin; Eisenschmidt, Elke; Haus, Utz-Uwe; Weismantel, Robert; Gundelfinger, Eckart D.; Dieterich, Daniela C.

    2012-01-01

    Chemical synapses are highly specialized cell–cell contacts for communication between neurons in the CNS characterized by complex and dynamic protein networks at both synaptic membranes. The cytomatrix at the active zone (CAZ) organizes the apparatus for the regulated release of transmitters from the presynapse. At the postsynaptic side, the postsynaptic density constitutes the machinery for detection, integration, and transduction of the transmitter signal. Both pre- and postsynaptic protein networks represent the molecular substrates for synaptic plasticity. Their function can be altered both by regulating their composition and by post-translational modification of their components. For a comprehensive understanding of synaptic networks the entire ensemble of synaptic proteins has to be considered. To support this, we established a comprehensive database for synaptic junction proteins (SynProt database) primarily based on proteomics data obtained from biochemical preparations of detergent-resistant synaptic junctions. The database currently contains 2,788 non-redundant entries of rat, mouse, and some human proteins, which mainly have been manually extracted from 12 proteomic studies and annotated for synaptic subcellular localization. Each dataset is completed with manually added information including protein classifiers as well as automatically retrieved and updated information from public databases (UniProt and PubMed). We intend that the database will be used to support modeling of synaptic protein networks and rational experimental design. PMID:22737123

  13. Theoretical model of the three-dimensional structure of a disease resistance gene homolog encoding resistance protein in Vigna mungo.

    PubMed

    Basak, Jolly; Bahadur, Ranjit P

    2006-10-01

    Plant disease resistance (R) genes, the key players of innate immunity system in plants encode 'R' proteins. 'R' protein recognizes product of avirulance gene from the pathogen and activate downstream signaling responses leading to disease resistance. No three dimensional (3D) structural information of any 'R' proteins is available as yet. We have reported a 'R' gene homolog, the 'VMYR1', encoding 'R' protein in Vigna mungo. Here, we describe the homology modeling of the 'VMYR1' protein. The model was created by using the 3D structure of an ATP-binding cassette transporter protein from Vibrio cholerae as a template. The strategy for homology modeling was based on the high structural conservation in the superfamily of P-loop containing nucleoside triphosphate hydrolase in which target and template proteins belong. This is the first report of theoretical model structure of any 'R' proteins.

  14. 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.

  15. HIV-1 Tat Protein Enhances Expression and Function of Breast Cancer Resistance Protein.

    PubMed

    Zhou, Yancong; Zhang, Kun; Yin, Xiaojie; Nie, Qichang; Ma, Yonggang

    2016-01-01

    ATP binding cassette (ABC) transporters can transfer a variety of antiviral agents from the cytoplasm to body fluid, which results in a reduced intracellular concentration of the drugs. Proteins of HIV-1, e.g., Tat and gp120, altered some types of ABC transporter expression in brain microvascular endothelial cells and astrocytes. However, the effect of Tat on ABC transporters in T lymphocytes is unclear. In this study the status of breast cancer resistance protein (BCRP) in Tat expressing cell lines was examined with real-time PCR and flow cytometry. It was found that HIV-1 Tat protein upregulated BCRP expression and enhanced efflux mediated by BCRP significantly, which could inhibit antiviral drugs from entering infected cells and interfere with the therapeutic effect of HAART. PMID:26367065

  16. [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

  17. Mutagenesis of SugE, a small multidrug resistance protein.

    PubMed

    Son, Mike S; Del Castilho, Colin; Duncalf, Karen A; Carney, Dominic; Weiner, Joel H; Turner, Raymond J

    2003-12-26

    The small multidrug resistance protein family has two subclasses. In this study we used a mutation approach to see what is necessary to convert a SUG subgroup member into a quaternary ammonium compound (QAC) transporter. We chose four key residues (H24, M39, I43, and A44) conserved within SUGs but conserved differently within the QAC transporters. Altogether, seven mutants were generated in Citrobacter freundii SugE. Surprisingly, the mutated SugE demonstrated an increased sensitivity to representative QACs. Additionally, ethidium uptake is found to be more prominent in the hypersensitive mutants. We conducted orientation studies using topology reporter gene fusions which indicated that SugE and the QAC transporter EmrE both have their N- and C-termini in the cytoplasm as predicted. The results imply that SugE can be converted to a QAC transporter with only a single mutation. However, because hypersensitivity was observed, the SugE mutant proteins are behaving as importers rather than as exporters. PMID:14651958

  18. Retinol Binding Protein: Marker for Insulin Resistance and Inflammation Postburn?

    PubMed Central

    Kraft, Robert; Herndon, David N.; Kulp, Gabriela A.; Mecott, Gabriel A.; Trentzsch, Heiko; Jeschke, Marc G.

    2013-01-01

    Introduction Burn injury leads to vast changes in both metabolic and inflammatory responses and is associated with increased morbidity and mortality. Insulin resistance (IR) and hyperglycemia are major components of the hypermetabolic response found in burn-injured patients and subsequently contribute to adverse outcomes. Studies have shown that increased systemic retinol binding protein (RBP) levels are associated with IR and hyperinflammation in diabetic and obese patients. The aim of this study was to determine RBP profiles and to test the hypothesis that elevated RBP levels are associated with both IR and the inflammatory response in burned patients. Methods RBP was measured in 372 patients during the acute stay postburn. Patients’ demographics, glucose levels, and insulin administration were recorded. Cytokines, hormones, plasma proteins, and organ markers were measured. The average of all measurements of RBP (2.1 mg/dL) was used to divide patients into high and low groups. Statistical analysis was performed by Student t test. Statistical significance was accepted at P < .05. Results Fifty-one patients (high group) had elevated RBP levels during acute hospitalization and demonstrated a significant higher incidence of multiorgan failure, sepsis, and mortality (P < .05). Moreover, in the high group, a significant increase of IR, inflammatory cytokines, and catabolic and organ-specific markers were detected (P < .05). Conclusions Increased RBP levels postburn correlate with increased IR, inflammatory and catabolic responses, incidence of multiorgan failure, and mortality. RBP may be a novel biomarker to monitor these detrimental responses postburn. PMID:22042048

  19. Tumor promotion by caspase-resistant retinoblastoma protein.

    PubMed

    Borges, Helena L; Bird, Jeff; Wasson, Katherine; Cardiff, Robert D; Varki, Nissi; Eckmann, Lars; Wang, Jean Y J

    2005-10-25

    The retinoblastoma (RB) protein regulates cell proliferation and cell death. RB is cleaved by caspase during apoptosis. A mutation of the caspase-cleavage site in the RB C terminus has been made in the mouse Rb-1 locus; the resulting Rb-MI mice are resistant to endotoxin-induced apoptosis in the intestine. The Rb-MI mice do not exhibit increased tumor incidence, because the MI mutation does not disrupt the Rb tumor suppressor function. In this study, we show that Rb-MI can promote the formation of colonic adenomas in the p53-null genetic background. Consistent with this tumor phenotype, Rb-MI reduces colorectal epithelial apoptosis and ulceration caused by dextran sulfate sodium. By contrast, Rb-MI does not affect the lymphoma phenotype of p53-null mice, in keeping with its inability to protect thymocytes and splenocytes from apoptosis. The Rb-MI protein is expressed and phosphorylated in the tumors, thereby inactivating its growth suppression function. These results suggest that RB tumor suppressor function, i.e., inhibition of proliferation, is inactivated by phosphorylation, whereas RB tumor promoting function, i.e., inhibition of apoptosis, is inactivated by caspase cleavage. PMID:16227443

  20. Heterochromatin Protein 1 Binding Protein 3 Expression as a Candidate Marker of Intrinsic 5-Fluorouracil Resistance

    PubMed Central

    HADAC, JAMIE N.; MILLER, DEVON D.; GRIMES, IAN C.; CLIPSON, LINDA; NEWTON, MICHAEL A.; SCHELMAN, WILLIAM R.; HALBERG, RICHARD B.

    2016-01-01

    Background Despite receiving post-operative 5-fluorouracil (5-FU)-based chemotherapy, approximately 50% of patients with stage IIIC colon cancer experience recurrence. Currently, no molecular signature can predict response to 5-FU. Materials and Methods Mouse models of colon cancer have been developed and characterized. Individual tumors in these mice can be longitudinally monitored and assessed to identify differences between those that are responsive and those that are resistant to therapy. Gene expression was analyzed in serial biopsies that were collected before and after treatment with 5-FU. Colon tumors had heterogeneous responses to treatment with 5-FU. Microarray analysis of pretreatment biopsies revealed that Hp1bp3, a gene encoding heterochromatin protein 1 binding protein 3, was differentially expressed between sensitive and resistant tumors. Conclusion Using mouse models of human colorectal cancer, Hp1bp3 was identified as a candidate marker of intrinsic 5-FU resistance and may represent a potential biomarker for patient stratification or a target of clinical importance. PMID:26976970

  1. Modulation of drug resistance in Staphylococcus aureus by a kaempferol glycoside from Herissantia tiubae (Malvaceae).

    PubMed

    Falcão-Silva, Vivyanne S; Silva, Davi A; Souza, Maria de Fátima V; Siqueira-Junior, José P

    2009-10-01

    In an ongoing project to evaluate natural compounds isolated from plants from the Brazilian biodiversity as modulators of antibiotic resistance, kaempferol-3-O-beta-d-(6''-E-p-coumaroyl) glucopyranoside (tiliroside), isolated from Herissantia tiubae (Malvaceae) was investigated using the strain SA-1199B of Staphylococcus aureus, which overexpresses the norA gene encoding the NorA efflux protein which extrudes hydrophilic fluorquinolones and some biocides, such as benzalkonium chloride, cetrimide, acriflavine and ethidium bromide. The minimum inhibitory concentrations (MICs) of the antibiotics and biocides were determined by the microdilution assay in the absence and in the presence of sub-inhibitory concentration of tiliroside. Although tiliroside did not display relevant antibacterial activity (MIC = 256 microg/mL), it modulated the activity of antibiotics, i.e. in combination with antibiotics a reduction in the MIC was observed for norfloxacin (16-fold), ciprofloxacin (16-fold), lomefloxacin (four-fold) and ofloxacin (two-fold), and an impressive reduction in the MICs for the biocides (up to 128-fold). The results presented here represent the first report of a kaempferol glycoside as a putative efflux pump inhibitor in bacteria. The present finding indicates that H. tiubae (and broadly Malvaceae) could serve as a source of plant-derived natural products that modulate bacterial resistance, i.e. a source of potential adjuvants of antibiotics.

  2. 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

  3. Protein Defense Systems against the Lantibiotic Nisin: Function of the Immunity Protein NisI and the Resistance Protein NSR

    PubMed Central

    Khosa, Sakshi; Lagedroste, Marcel; Smits, Sander H. J.

    2016-01-01

    Lantibiotics are potential alternatives to antibiotics because of their broad-range killing spectrum. The producer strain is immune against its own synthesized lantibiotic via the expression of two proteins LanI and LanFEG. Recently, gene operons are found in mainly human pathogenic strains, which confer resistance against lantibiotics. Of all the lantibiotics discovered till date, nisin produced by some Lactococcus lactis strains is the most prominent member. Nisin has multiple mode of actions of which binding to the cell wall precursor lipid II and subsequent insertion into the bacterial membrane to form pores are the most effective. The nisin producing strains express the lipoprotein NisI to prevent a suicidal effect. NisI binds nisin, inducing a reversible cell clustering to prevent nisin from reaching the membrane. Importantly NisI does not modify nisin and releases it as soon as the concentration in the media drops below a certain level. The human pathogen Streptococcus agalactiae is naturally resistant against nisin by expressing a resistance protein called SaNSR, which is a nisin degrading enzyme. By cleaving off the last six amino acids of nisin, its effectiveness is 100-fold reduced. This cleavage reaction appears to be specific for nisin since SaNSR recognizes the C-terminal located lanthionine rings. Recently, the structures of both NisI and SaNSR were determined by NMR and X-ray crystallography, respectively. Furthermore, for both proteins the binding site for nisin was determined. Within this review, the structures of both proteins and their different defense mechanisms are described. PMID:27148193

  4. Novel channel enzyme fusion proteins confer arsenate resistance.

    PubMed

    Wu, Binghua; Song, Jie; Beitz, Eric

    2010-12-17

    Steady exposure to environmental arsenic has led to the evolution of vital cellular detoxification mechanisms. Under aerobic conditions, a two-step process appears most common among microorganisms involving reduction of predominant, oxidized arsenate (H(2)As(V)O(4)(-)/HAs(V)O(4)(2-)) to arsenite (As(III)(OH)(3)) by a cytosolic enzyme (ArsC; Escherichia coli type arsenate reductase) and subsequent extrusion via ArsB (E. coli type arsenite transporter)/ACR3 (yeast type arsenite transporter). Here, we describe novel fusion proteins consisting of an aquaglyceroporin-derived arsenite channel with a C-terminal arsenate reductase domain of phosphotyrosine-phosphatase origin, providing transposable, single gene-encoded arsenate resistance. The fusion occurred in actinobacteria from soil, Frankia alni, and marine environments, Salinispora tropica; Mycobacterium tuberculosis encodes an analogous ACR3-ArsC fusion. Mutations rendered the aquaglyceroporin channel more polar resulting in lower glycerol permeability and enhanced arsenite selectivity. The arsenate reductase domain couples to thioredoxin and can complement arsenate-sensitive yeast strains. A second isoform with a nonfunctional channel may use the mycothiol/mycoredoxin cofactor pool. These channel enzymes constitute prototypes of a novel concept in metabolism in which a substrate is generated and compartmentalized by the same molecule. Immediate diffusion maintains the dynamic equilibrium and prevents toxic accumulation of metabolites in an energy-saving fashion.

  5. Novel channel enzyme fusion proteins confer arsenate resistance.

    PubMed

    Wu, Binghua; Song, Jie; Beitz, Eric

    2010-12-17

    Steady exposure to environmental arsenic has led to the evolution of vital cellular detoxification mechanisms. Under aerobic conditions, a two-step process appears most common among microorganisms involving reduction of predominant, oxidized arsenate (H(2)As(V)O(4)(-)/HAs(V)O(4)(2-)) to arsenite (As(III)(OH)(3)) by a cytosolic enzyme (ArsC; Escherichia coli type arsenate reductase) and subsequent extrusion via ArsB (E. coli type arsenite transporter)/ACR3 (yeast type arsenite transporter). Here, we describe novel fusion proteins consisting of an aquaglyceroporin-derived arsenite channel with a C-terminal arsenate reductase domain of phosphotyrosine-phosphatase origin, providing transposable, single gene-encoded arsenate resistance. The fusion occurred in actinobacteria from soil, Frankia alni, and marine environments, Salinispora tropica; Mycobacterium tuberculosis encodes an analogous ACR3-ArsC fusion. Mutations rendered the aquaglyceroporin channel more polar resulting in lower glycerol permeability and enhanced arsenite selectivity. The arsenate reductase domain couples to thioredoxin and can complement arsenate-sensitive yeast strains. A second isoform with a nonfunctional channel may use the mycothiol/mycoredoxin cofactor pool. These channel enzymes constitute prototypes of a novel concept in metabolism in which a substrate is generated and compartmentalized by the same molecule. Immediate diffusion maintains the dynamic equilibrium and prevents toxic accumulation of metabolites in an energy-saving fashion. PMID:20947511

  6. Novel Channel Enzyme Fusion Proteins Confer Arsenate Resistance*

    PubMed Central

    Wu, Binghua; Song, Jie; Beitz, Eric

    2010-01-01

    Steady exposure to environmental arsenic has led to the evolution of vital cellular detoxification mechanisms. Under aerobic conditions, a two-step process appears most common among microorganisms involving reduction of predominant, oxidized arsenate (H2AsVO4−/HAsVO42−) to arsenite (AsIII(OH)3) by a cytosolic enzyme (ArsC; Escherichia coli type arsenate reductase) and subsequent extrusion via ArsB (E. coli type arsenite transporter)/ACR3 (yeast type arsenite transporter). Here, we describe novel fusion proteins consisting of an aquaglyceroporin-derived arsenite channel with a C-terminal arsenate reductase domain of phosphotyrosine-phosphatase origin, providing transposable, single gene-encoded arsenate resistance. The fusion occurred in actinobacteria from soil, Frankia alni, and marine environments, Salinispora tropica; Mycobacterium tuberculosis encodes an analogous ACR3-ArsC fusion. Mutations rendered the aquaglyceroporin channel more polar resulting in lower glycerol permeability and enhanced arsenite selectivity. The arsenate reductase domain couples to thioredoxin and can complement arsenate-sensitive yeast strains. A second isoform with a nonfunctional channel may use the mycothiol/mycoredoxin cofactor pool. These channel enzymes constitute prototypes of a novel concept in metabolism in which a substrate is generated and compartmentalized by the same molecule. Immediate diffusion maintains the dynamic equilibrium and prevents toxic accumulation of metabolites in an energy-saving fashion. PMID:20947511

  7. Proteomic Analysis of Protease Resistant Proteins in the Diabetic Rat Kidney

    PubMed Central

    Bansode, Sneha B.; Chougale, Ashok D.; Joshi, Rakesh S.; Giri, Ashok P.; Bodhankar, Subhash L.; Harsulkar, Abhay M.; Kulkarni, Mahesh J.

    2013-01-01

    Glycation induced protein aggregation has been implicated in the development of diabetic complications and neurodegenerative diseases. These aggregates are known to be resistant to proteolytic digestion. Here we report the identification of protease resistant proteins from the streptozotocin induced diabetic rat kidney, which included enzymes in glucose metabolism and stress response proteins. These protease resistant proteins were characterized to be advanced glycation end products modified and ubiquitinated by immunological and mass spectrometry analysis. Further, diabetic rat kidney exhibited significantly impaired proteasomal activity. The functional analysis of identified physiologically important enzymes showed that their activity was reduced in diabetic condition. Loss of functional activity of these proteins was compensated by enhanced gene expression. Aggregation prone regions were predicted by in silico analysis and compared with advanced glycation end products modification sites. These findings suggested that the accumulation of protein aggregates is an inevitable consequence of impaired proteasomal activity and protease resistance due to advanced glycation end products modification. PMID:23118466

  8. Characteristics of protein variants in trichlorphon-resistant Bactrocera dorsalis (Diptera; Tephritidae) larvae.

    PubMed

    Jin, T; Zeng, L; Lin, Y-Y; Lu, Y-Y; Liang, G-W

    2012-08-16

    Functional proteins in larvae of Bactrocera dorsalis, a major fruit pest, play a central role in their resistance to organophosphorus insecticides. Changes in proteins in B. dorsalis larvae after trichlorphon treatment may have a role in the resistance response to trichlorphon. We analyzed 14 protein spots of crude proteins from B. dorsalis larvae post-treatment with trichlorphon in two-dimensional gel electrophoresis through mass spectrometry and protein sequencing. We found functional proteins that are responsible for signal transduction (pkaap and dual specificity tyrosine-phosphorylation-regulated kinase), immunity (hemolectin), synthesis and decomposition (twinstar, cathepsin B, RE66325p), oxidative stress metabolism (glutathione S transferase and CG7320), energy metabolism (Act57B), and cytoskeleton formation (actin). These proteins appear to be involved in the resistance response to trichlorphon.

  9. Resistance to β-Lactamase Inhibitor Protein Does Not Parallel Resistance to Clavulanic Acid in TEM β-Lactamase Mutants

    PubMed Central

    Schroeder, William A.; Locke, Troy R.; Jensen, Susan E.

    2002-01-01

    In order to compare patterns of resistance to inhibition by clavulanic acid with patterns of resistance to inhibition by a β-lactamase inhibitor protein (BLIP), R164S, R244S, and R164S/R244S mutant forms of TEM β-lactamase were prepared by site-directed mutagenesis. When kinetic parameters were determined for these mutant and wild-type forms of TEM, the single mutants showed properties that were similar to those in the literature but the double mutant showed properties that were very different. The R164S/R244S double mutant form of TEM retained its resistance to inhibition by clavulanic acid (characteristic of the R244S mutation) but lost all its ability to hydrolyze ceftazidime (characteristic of the R164S mutation). While these characteristics are contrary to those previously reported for an R164S/R244S double mutant, this discrepancy resulted from the use of a defective mutant in the earlier study. Both the single and double mutant forms of TEM remained highly sensitive when tested for inhibition by BLIP, showing only slightly increased resistance compared to that of the wild type; this pattern of resistance is quite different from the pattern of clavulanic acid resistance. The slight increases in resistance to inhibition by BLIP seen in the mutants may have been related to the fact that all of the mutations effected changes in the net charge on the TEM protein that could impede interactions with BLIP. PMID:12384366

  10. 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.

  11. Protein-resistant NTA-functionalized polymer brushes for selective and stable immobilization of histidine-tagged proteins.

    PubMed

    Gautrot, Julien E; Huck, Wilhelm T S; Welch, Martin; Ramstedt, Madeleine

    2010-01-01

    Protein-resistant polymeric coatings that allow highly selective immobilization of specific biomolecules are essential for biomedical applications such as microarrays, biosensing, heterogeneous catalysis, and bioengineering. Polymer brushes are particularly interesting for this purpose because their chemical structure and physical properties can easily be tailored to meet specific needs. This article explores the functionalization of two protein-resistant polymer brushes, poly(oligoethylene glycol methacrylate) (POEGMA) and poly(hydroxyethyl methacrylate) (PHEMA), with nitrilotriacetic acid (NTA) moieties that can complex histidine-tagged (His-tagged) proteins selectively and reversibly. Using fluorescence microscopy, IR spectroscopy, X-ray photoelectron spectroscopy, surface plasmon resonanace, and ellipsometry, we demonstrate that His-tagged green fluorescent protein can be immobilized on NTA brushes with high stability and loading. The loading saturation reached for NTA-POEGMA is higher than that for NTA-PHEMA because of increased swelling of the former brush. Despite this higher loading capacity, NTA-POEGMA remained highly protein-resistant, which shows its potential for "clean" and specific protein immobilization. Finally, we showed that the preserved protein resistance of NTA-POEGMA brushes can be used to generate well-defined binary biofunctional patterns via a simple protocol of incubations and washes. These patterns may find applications in cell arraying and screening. PMID:20356235

  12. Crystal Structure of the Carbapenem Intrinsic Resistance Protein CarG

    PubMed Central

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

    2015-01-01

    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. PMID:24583229

  13. An NB-LRR protein required for HR signalling mediated by both extra- and intracellular resistance proteins.

    PubMed

    Gabriëls, Suzan H E J; Vossen, Jack H; Ekengren, Sophia K; van Ooijen, Gerben; Abd-El-Haliem, Ahmed M; van den Berg, Grardy C M; Rainey, Daphne Y; Martin, Gregory B; Takken, Frank L W; de Wit, Pierre J G M; Joosten, Matthieu H A J

    2007-04-01

    Tomato (Solanum lycopersicum) Cf resistance genes confer hypersensitive response (HR)-associated resistance to strains of the pathogenic fungus Cladosporium fulvum that express the matching avirulence (Avr) gene. Previously, we identified an Avr4-responsive tomato (ART) gene that is required for Cf-4/Avr4-induced HR in Nicotiana benthamiana as demonstrated by virus-induced gene silencing (VIGS). The gene encodes a CC-NB-LRR type resistance (R) protein analogue that we have designated NRC1 (NB-LRR protein required for HR-associated cell death 1). Here we describe that knock-down of NRC1 in tomato not only affects the Cf-4/Avr4-induced HR but also compromises Cf-4-mediated resistance to C. fulvum. In addition, VIGS using NRC1 in N. benthamiana revealed that this protein is also required for the HR induced by the R proteins Cf-9, LeEix, Pto, Rx and Mi. Transient expression of NRC1(D481V), which encodes a constitutively active NRC1 mutant protein, triggers an elicitor-independent HR. Subsequently, we transiently expressed this auto-activating protein in N. benthamiana silenced for genes known to be involved in HR signalling, thereby allowing NRC1 to be positioned in an HR signalling pathway. We found that NRC1 requires RAR1 and SGT1 to be functional, whereas it does not require NDR1 and EDS1. As the Cf-4 protein requires EDS1 for its function, we hypothesize that NRC1 functions downstream of EDS1. We also found that NRC1 acts upstream of a MAP kinase pathway. We conclude that Cf-mediated resistance signalling requires a downstream NB-LRR protein that also functions in cell death signalling pathways triggered by other R proteins.

  14. Stabilization of mutant BRCA1 protein confers PARP inhibitor and platinum resistance

    PubMed Central

    Johnson, Neil; Johnson, Shawn F.; Yao, Wei; Li, Yu-Chen; Choi, Young-Eun; Bernhardy, Andrea J.; Wang, Yifan; Capelletti, Marzia; Sarosiek, Kristopher A.; Moreau, Lisa A.; Chowdhury, Dipanjan; Wickramanayake, Anneka; Harrell, Maria I.; Liu, Joyce F.; D’Andrea, Alan D.; Miron, Alexander; Swisher, Elizabeth M.; Shapiro, Geoffrey I.

    2013-01-01

    Breast Cancer Type 1 Susceptibility Protein (BRCA1)-deficient cells have compromised DNA repair and are sensitive to poly(ADP-ribose) polymerase (PARP) inhibitors. Despite initial responses, the development of resistance limits clinical efficacy. Mutations in the BRCA C-terminal (BRCT) domain of BRCA1 frequently create protein products unable to fold that are subject to protease-mediated degradation. Here, we show HSP90-mediated stabilization of a BRCT domain mutant BRCA1 protein under PARP inhibitor selection pressure. The stabilized mutant BRCA1 protein interacted with PALB2-BRCA2-RAD51, was essential for RAD51 focus formation, and conferred PARP inhibitor as well as cisplatin resistance. Treatment of resistant cells with the HSP90 inhibitor 17-dimethylaminoethylamino-17-demethoxygeldanamycin reduced mutant BRCA1 protein levels and restored their sensitivity to PARP inhibition. Resistant cells also acquired a TP53BP1 mutation that facilitated DNA end resection in the absence of a BRCA1 protein capable of binding CtIP. Finally, concomitant increased mutant BRCA1 and decreased 53BP1 protein expression occur in clinical samples of BRCA1-mutated recurrent ovarian carcinomas that have developed resistance to platinum. These results provide evidence for a two-event mechanism by which BRCA1-mutant tumors acquire anticancer therapy resistance. PMID:24085845

  15. 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.

  16. Effect of whey and soy protein supplementation combined with resistance training in young adults.

    PubMed

    Candow, Darren G; Burke, Natalie C; Smith-Palmer, T; Burke, Darren G

    2006-06-01

    The purpose was to compare changes in lean tissue mass, strength, and myofibrillar protein catabolism resulting from combining whey protein or soy protein with resistance training. Twenty-seven untrained healthy subjects (18 female, 9 male) age 18 to 35 y were randomly assigned (double blind) to supplement with whey protein (W; 1.2 g/kg body mass whey protein + 0.3 g/kg body mass sucrose power, N = 9: 6 female, 3 male), soy protein (S; 1.2 g/kg body mass soy protein + 0.3 g/kg body mass sucrose powder, N= 9: 6 female, 3 male) or placebo (P; 1.2 g/kg body mass maltodextrine + 0.3 g/kg body mass sucrose powder, N = 9: 6 female, 3 male) for 6 wk. Before and after training, measurements were taken for lean tissue mass (dual energy X-ray absorptiometry), strength (1-RM for bench press and hack squat), and an indicator of myofibrillar protein catabolism (urinary 3-methylhistidine). Results showed that protein supplementation during resistance training, independent of source, increased lean tissue mass and strength over isocaloric placebo and resistance training (P < 0.05). We conclude that young adults who supplement with protein during a structured resistance training program experience minimal beneficial effects in lean tissue mass and strength.

  17. 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.

  18. Purification of the M flax-rust resistance protein expressed in Pichia pastoris.

    PubMed

    Schmidt, Simon A; Williams, Simon J; Wang, Ching-I A; Sornaraj, Pradeep; James, Ben; Kobe, Bostjan; Dodds, Peter N; Ellis, Jeffrey G; Anderson, Peter A

    2007-06-01

    The M flax-rust resistance (R) gene is predicted to encode a 150-kDa protein of the Toll-interleukin-like receptor-nucleotide binding site-leucine rich repeat (TIR-NBS-LRR) class of plant disease resistance proteins and provides resistance against the Melampsora lini (flax rust) fungus carrying the AvrM avirulence gene. The extremely low level of this class of R proteins found in plant tissue has precluded their biochemical and structural analysis, and the study of these proteins has been largely restricted to genetic analyses and in vivo investigations. Here we report the production and purification of the M protein in the methalotrophic yeast, Pichia pastoris. Expression trials with five different constructs reveals optimum levels of soluble native M protein can be obtained as an N-terminally 9x His-tagged protein, in which the first 21 amino acids of the predicted wild-type protein are deleted. Expression was achieved using a high cell density fed-batch bioreactor culture at low temperature. M protein was purified to near homogeneity from whole-cell lysates using cation exchange, immobilised metal ion affinity chromatography and gel filtration with a final yield of approximately 3 mg of protein/1000 g wet weight of yeast cells lysed. The successful expression and purification of soluble M protein opens the way for biochemical and structural analysis of this class of important plant proteins.

  19. 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

  20. Resistance of Klebsiella Pneumoniae clinical isolates: linkage of outer membrane proteins (omps) with production esbls

    PubMed Central

    Marques, Lívia Érika Carlos; de Oliveira, Danielle Ferreira; Marques, Márcia Maria Mendes; da Silva, Ana Raquel Araújo; Alves, Carlucio Roberto; Guedes, Maria Izabel Florindo

    2011-01-01

    Three isolates of Klebsiella pneumoniae, collected from the University Hospital in Fortaleza, Brazil, were analyzed to determine their resistance to multiple antibiotics. The results of this study showed that the resistance of the clinically isolated bacteria is associated with the production of extended-spectrum beta-lactamases (ESLBs) and loss of outer membrane proteins. PMID:24031656

  1. 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

  2. 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

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

    NASA Astrophysics Data System (ADS)

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

    2015-03-01

    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 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.

  4. 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.

  5. Expression of the breast cancer resistance protein and 5-fluorouracil resistance in clinical breast cancer tissue specimens

    PubMed Central

    WANG, MIN; WANG, XIANMING; YUAN, JIANHUI; GUO, LIANGFENG

    2013-01-01

    The breast cancer resistance protein (BCRP) is a recently characterized xenobiotic half-transporter protein that acts as an energy-dependent efflux pump and may be associated with the multidrug-resistant phenotype. The aim of this study was to determine the association between BCRP expression and 5-fluorouracil (5-FU) resistance in clinical breast cancer tissue specimens. The BCRP expression was investigated using quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) by use of the Master SYBR-Green I reagent and immunohistochemistry (IHC) by use of the BXP-21 anti-BCRP monoclonal antibody in clinical breast cancer tissue specimens. Chemosensitivity to 5-FU for BCRP-positive clinical breast cancer tissue specimens was colorimetrically assessed with the cytotoxicity assay through methyl thiazolyl tetrazolium (MTT) reduction. A total of 37 BCRP-positive clinical breast cancer tissue specimens were identified with quantitative RT-PCR and IHC. There was a significant correlation in BCRP expression between the results of quantitative RT-PCR and IHC in the specimens. The fold resistance to 5-FU was 7–12 compared to sensitivity to paclitaxel as determined by the colorimetric assay through MTT reduction in the 37 specimens. Our study results indicated that 5-FU resistance may be mediated by BCRP expression in clinical breast cancer tissue specimens, which may help optimize the design of breast cancer clinical chemotherapy schemes in BCRP-positive specimens. PMID:24649260

  6. 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. PMID:22434723

  7. 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.

  8. Dietary protein and resistance training effects on muscle and body composition in older persons.

    PubMed

    Campbell, Wayne W; Leidy, Heather J

    2007-12-01

    The regular performance of resistance exercises and the habitual ingestion of adequate amounts of dietary protein from high-quality sources are two important ways for older persons to slow the progression of and treat sarcopenia, the age-related loss of skeletal muscle mass and function. Resistance training can help older people gain muscle strength, hypertrophy muscle, and increase whole body fat-free mass. It can also help frail elderly people improve balance and physical functioning capabilities. Inadequate protein intake will cause adverse metabolic and physiological accommodation responses that include the loss of fat-free mass and muscle strength and size. Findings from controlled feeding studies show that older persons retain the capacity to metabolically adjust to lower protein intakes by increasing the efficiency of nitrogen retention and amino acid utilization. However, they also suggest that the recommended dietary allowance of 0.8 g protein x kg(-1) x d(-1) might not be sufficient to prevent subtle accommodations and blunt desired changes in body composition and muscle size with resistance training. Most of the limited research suggests that resistance training-induced improvements in body composition, muscle strength and size, and physical functioning are not enhanced when older people who habitually consume adequate protein (modestly above the RDA) increase their protein intake by either increasing the ingestion of higher-protein foods or consuming protein-enriched nutritional supplements. PMID:18187436

  9. Characterization of protein changes associated with sugar beet (Beta vulgaris) resistance and susceptibility to Fusarium oxysporum.

    PubMed

    Larson, Rebecca L; Hill, Amy L; Nuñez, Alberto

    2007-09-19

    Fusarium oxysporum (F-19) is a serious threat to sugar beet. Resistance exists, but the basis for resistance and disease is unknown. Protein extracts from sugar beet genotypes C1200.XH024 (resistant, R) and Fus7 (susceptible, S) were analyzed by multidimensional liquid chromatography at 2 and 5 days postinoculation (dpi) and compared to mock-inoculated controls. One hundred twenty-one (R) and 73 (S) protein peaks were induced/repressed by F-19, approximately 12 (R) and 8% (S) of the total proteome detected. Temporal protein regulation occurred within and between each genotype, indicating that the timing of expression may be important for resistance. Thirty-one (R) and 48 (S) of the differentially expressed peaks were identified using matrix-assisted laser desorption-ionization with tandem time-of-flight mass spectrometry; others were below detection level. Comparison between the two genotypes uncovered R- and S-specific proteins with potential roles in resistance and disease development, respectively. Use of these proteins to select for new sources of resistance and to develop novel disease control strategies is discussed.

  10. [The roles of epigenetics and protein post-translational modifications in bacterial antibiotic resistance].

    PubMed

    Xie, Longxiang; Yu, Zhaoxiao; Guo, Siyao; Li, Ping; Abdalla, Abualgasim Elgaili; Xie, Jianping

    2015-08-01

    The increasing antibiotic resistance is now threatening to take us back to a pre-antibiotic era. Bacteria have evolved diverse resistance mechanisms, on which in-depth research could help the development of new strategies to control antibiotic-resistant infections. Epigenetic alterations and protein post-translational modifications (PTMs) play important roles in multiple cellular processes such as metabolism, signal transduction, protein degradation, DNA replication regulation and stress response. Recent studies demonstrated that epigenetics and PTMs also play vital roles in bacterial antibiotic resistance. In this review, we summarize the regulatory roles of epigenetic factors including DNA methylation and regulatory RNAs as well as PTMs such as phosphorylation and succinylation in bacterial antibiotic resistance, which may provide innovative perspectives on selecting antibacterial targets and developing antibiotics. PMID:26266782

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

    PubMed Central

    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. PMID:25970790

  12. 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.

  13. 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. PMID:25970790

  14. Heat-resistant variants of Chinese hamster fibroblasts altered in expression of heat shock protein.

    PubMed Central

    Laszlo, A; Li, G C

    1985-01-01

    Heat-resistant variants of the Chinese hamster HA-1 line have been isolated after repeated heat treatments. The heat-resistant phenotype has been stable for over 70 passages. One of the members of the 70-kDa heat shock protein family was found to be synthesized at greater levels in the heat-resistant variants under normal growth conditions. Mild heat treatment of the variant lines induced a transient thermotolerance that was accompanied by additional increase in the synthesis of the 70-kDa heat shock proteins. Cell-free translation of total cellular RNA revealed greater amounts of 70-kDa heat shock protein mRNA in both control and heated variant cells. The greater levels of 70-kDa heat shock protein synthesized in the variant cells presumably are a reflection of altered levels of its messenger mRNA. In addition, we found that translational control plays a role in the elevated expression of heat shock proteins in heat-shocked HA-1 cells and their heat-resistant variants. The association of the heat-resistant phenotype with increased levels of a 70-kDa heat shock protein suggests strongly that this gene product plays a role in protecting cells from damage inflicted by elevated temperatures. Images PMID:3865213

  15. 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

    2016-01-01

    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. PMID:27525950

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

    PubMed

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

    2013-05-01

    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.

  17. Discovery and characterization of proteins associated with aflatoxin-resistance: evaluating their potential as breeding markers.

    PubMed

    Brown, Robert L; Chen, Zhi-Yuan; Warburton, Marilyn; Luo, Meng; Menkir, Abebe; Fakhoury, Ahmad; Bhatnagar, Deepak

    2010-04-01

    Host resistance has become a viable approach to eliminating aflatoxin contamination of maize since the discovery of several maize lines with natural resistance. However, to derive commercial benefit from this resistance and develop lines that can aid growers, markers need to be identified to facilitate the transfer of resistance into commercially useful genetic backgrounds without transfer of unwanted traits. To accomplish this, research efforts have focused on the identification of kernel resistance-associated proteins (RAPs) including the employment of comparative proteomics to investigate closely-related maize lines that vary in aflatoxin accumulation. RAPs have been identified and several further characterized through physiological and biochemical investigations to determine their causal role in resistance and, therefore, their suitability as breeding markers. Three RAPs, a 14 kDa trypsin inhibitor, pathogenesis-related protein 10 and glyoxalase I are being investigated using RNAi gene silencing and plant transformation. Several resistant lines have been subjected to QTL mapping to identify loci associated with the aflatoxin-resistance phenotype. Results of proteome and characterization studies are discussed. PMID:22069617

  18. Discovery and Characterization of Proteins Associated with Aflatoxin-Resistance: Evaluating Their Potential as Breeding Markers

    PubMed Central

    Brown, Robert L.; Chen, Zhi-Yuan; Warburton, Marilyn; Luo, Meng; Menkir, Abebe; Fakhoury, Ahmad; Bhatnagar, Deepak

    2010-01-01

    Host resistance has become a viable approach to eliminating aflatoxin contamination of maize since the discovery of several maize lines with natural resistance. However, to derive commercial benefit from this resistance and develop lines that can aid growers, markers need to be identified to facilitate the transfer of resistance into commercially useful genetic backgrounds without transfer of unwanted traits. To accomplish this, research efforts have focused on the identification of kernel resistance-associated proteins (RAPs) including the employment of comparative proteomics to investigate closely-related maize lines that vary in aflatoxin accumulation. RAPs have been identified and several further characterized through physiological and biochemical investigations to determine their causal role in resistance and, therefore, their suitability as breeding markers. Three RAPs, a 14 kDa trypsin inhibitor, pathogenesis-related protein 10 and glyoxalase I are being investigated using RNAi gene silencing and plant transformation. Several resistant lines have been subjected to QTL mapping to identify loci associated with the aflatoxin-resistance phenotype. Results of proteome and characterization studies are discussed. PMID:22069617

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

    PubMed

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

    2015-08-20

    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.

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

    PubMed

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

    2015-08-01

    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

  1. 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

  2. Increased Levels of Antinutritional and/or Defense Proteins Reduced the Protein Quality of a Disease-Resistant Soybean Cultivar.

    PubMed

    Sousa, Daniele O B; Carvalho, Ana F U; Oliveira, José Tadeu A; Farias, Davi F; Castelar, Ivan; Oliveira, Henrique P; Vasconcelos, Ilka M

    2015-07-01

    The biochemical and nutritional attributes of two soybean (Glycine max (L.) Merr.) cultivars, one susceptible (Seridó) and the other resistant (Seridó-RCH) to stem canker, were examined to assess whether the resistance to pathogens was related to levels of antinutritional and/or defense proteins in the plant and subsequently affected the nutritional quality. Lectin, urease, trypsin inhibitor, peroxidase and chitinase activities were higher in the resistant cultivar. Growing rats were fed with isocaloric and isoproteic diets prepared with defatted raw soybean meals. Those on the Seridó-RCH diet showed the worst performance in terms of protein quality indicators. Based on regression analysis, lectin, trypsin inhibitor, peroxidase and chitinase appear to be involved in the resistance trait but also in the poorer nutritional quality of Seridó-RCH. Thus, the development of cultivars for disease resistance may lead to higher concentrations of antinutritional compounds, affecting the quality of soybean seeds. Further research that includes the assessment of more cultivars/genotypes is needed. PMID:26205163

  3. Increased Levels of Antinutritional and/or Defense Proteins Reduced the Protein Quality of a Disease-Resistant Soybean Cultivar

    PubMed Central

    Sousa, Daniele O. B.; Carvalho, Ana F. U.; Oliveira, José Tadeu A.; Farias, Davi F.; Castelar, Ivan; Oliveira, Henrique P.; Vasconcelos, Ilka M.

    2015-01-01

    The biochemical and nutritional attributes of two soybean (Glycine max (L.) Merr.) cultivars, one susceptible (Seridó) and the other resistant (Seridó-RCH) to stem canker, were examined to assess whether the resistance to pathogens was related to levels of antinutritional and/or defense proteins in the plant and subsequently affected the nutritional quality. Lectin, urease, trypsin inhibitor, peroxidase and chitinase activities were higher in the resistant cultivar. Growing rats were fed with isocaloric and isoproteic diets prepared with defatted raw soybean meals. Those on the Seridó-RCH diet showed the worst performance in terms of protein quality indicators. Based on regression analysis, lectin, trypsin inhibitor, peroxidase and chitinase appear to be involved in the resistance trait but also in the poorer nutritional quality of Seridó-RCH. Thus, the development of cultivars for disease resistance may lead to higher concentrations of antinutritional compounds, affecting the quality of soybean seeds. Further research that includes the assessment of more cultivars/genotypes is needed. PMID:26205163

  4. Tobacco serine/threonine protein kinase gene NrSTK enhances black shank resistance.

    PubMed

    Gao, Y-L; Wang, B-W; Xu, Z-L; Li, M-Y; Song, Z-B; Li, W-Z; Li, Y-P

    2015-01-01

    A serine/threonine protein kinase gene (NrSTK) was cloned from Nicotiana repanda based on the sequence of a previously isolated resistance gene analog (RGA). Expression of RGA was induced by challenge with the pathogen black shank. The NrSTK gene was predicted to encode a protein kinase that contained an ATP binding site at residues 41-69 and a serine/threonine protein kinase activation sequence spanning the region 161-173. Overexpression of NrSTK in the susceptible tobacco variety Honghuadajinyuan significantly enhanced resistance to black shank, indicating that NrSTK plays a role in incompatibility reactions between tobacco and the pathogen. Characterization of NrSTK will help elucidate the molecular mechanisms involved in black shank resistance in N. repanda.

  5. Quantitative Proteomic Analysis of Ovarian Cancer Cells Identified Mitochondrial Proteins Associated with Paclitaxel Resistance

    PubMed Central

    Tian, Yuan; Tan, Aik-Choon; Sun, Xiaer; Olson, Matthew T; Xie, Zhi; Jinawath, Natini; Chan, Daniel W.; Shih, Ie-Ming; Zhang, Zhen; Zhang, Hui

    2010-01-01

    Paclitaxel has been widely used as an anti-mitotic agent in chemotherapy for a variety of cancers and adds substantial efficacy as the first-line chemotherapeutic regimen for ovarian cancers. However, the frequent occurrence of paclitaxel resistance limits its function in long-term management. Despite abundant clinical and cellular demonstration of paclitaxel resistant tumors, the molecular mechanisms leading to paclitaxel resistance are poorly understood. Using genomic approaches, we have previously identified an association between a BTB/POZ gene, Nac1, and paclitaxel resistance in ovarian cancer. The experiments presented here have applied multiple quantitative proteomic methods to identify protein changes associated with paclitaxel resistance and Nac1 function. The SKOV-3 ovarian serous carcinoma cell line, which has inducible expression of dominant negative Nac1, was used to determine the paclitaxel treatment associated changes in the presence and absence of functional Nac1. Quantitative proteomic analyses were performed using iTRAQ labeling and mass spectrometry. Two label-free quantitative proteomic methods: LC-MS and spectral count were used to increase confidence of proteomic quantification. A total of 1371 proteins were quantified by at least one of the quantitative proteomic methods. Candidate proteins related to paclitaxel and NAC1 function were identified in this study. Go analysis of the protein changes identified upon paclitaxel resistance revealed that cell component enrichment related to mitochondria. Moreover, tubulin and mitochondrial proteins were the major cellular components with changes associated with paclitaxel treatment. This suggests that mitochondria may play a role in paclitaxel resistance. PMID:21113235

  6. Neisseria gonorrhoeae strain with high-level resistance to spectinomycin due to a novel resistance mechanism (mutated ribosomal protein S5) verified in Norway.

    PubMed

    Unemo, Magnus; Golparian, Daniel; Skogen, Vegard; Olsen, Anne Olaug; Moi, Harald; Syversen, Gaute; Hjelmevoll, Stig Ove

    2013-02-01

    Gonorrhea may become untreatable, and new treatment options are essential. Verified resistance to spectinomycin is exceedingly rare. However, we describe a high-level spectinomycin-resistant (MIC, >1,024 μg/ml) Neisseria gonorrhoeae strain from Norway with a novel resistance mechanism. The resistance determinant was a deletion of codon 27 (valine) and a K28E alteration in the ribosomal protein 5S. The traditional spectinomycin resistance gene (16S rRNA) was wild type. Despite this exceedingly rare finding, spectinomycin available for treatment of ceftriaxone-resistant urogenital gonorrhea would be very valuable. PMID:23183436

  7. 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

  8. 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.

  9. 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

  10. Crystallization of DIR1, a LTP2-like resistance signalling protein from Arabidopsis thaliana

    SciTech Connect

    Lascombe, Marie-Bernard; Buhot, Nathalie; Bakan, Bénédicte; Marion, Didier; Blein, Jean Pierre; Lamb, Chris J.; Prangé, Thierry

    2006-07-01

    DIR1, a putative LTP2 protein from Arabidopsis thaliana implicated in systemic acquired resistance in planta, has been crystallized in space group P2{sub 1}2{sub 1}2{sub 1} with one molecule per asymmetric unit. DIR1, a putative LTP2 protein from Arabidopsis thaliana implicated in systemic acquired resistance in planta, has been crystallized in space group P2{sub 1}2{sub 1}2{sub 1} with one molecule per asymmetric unit. The crystals diffract to a resolution of 1.6 Å.

  11. Penicillin-Binding Protein 2a of Methicillin-Resistant Staphylococcus aureus

    PubMed Central

    Fishovitz, Jennifer; Hermoso, Juan A.; Chang, Mayland

    2014-01-01

    Summary High-level resistance to β-lactam antibiotics in methicillin-resistant Staphylococcus aureus (MRSA) is due to expression of penicillin-binding protein 2a (PBP2a), a transpeptidase that catalyzes cell-wall crosslinking in the face of the challenge by β-lactam antibiotics. The activity of this protein is regulated by allostery at a site 60 Å distant from the active site, where crosslinking of cell wall takes place. This review discusses the state of knowledge on this important enzyme of cell-wall biosynthesis in MRSA. PMID:25044998

  12. A serine (threonine) protein kinase confers fungicide resistance in the phytopathogenic fungus Ustilago maydis.

    PubMed Central

    Orth, A B; Rzhetskaya, M; Pell, E J; Tien, M

    1995-01-01

    A mutant of Ustilago maydis (VR43) with single-gene resistance to the dicarboximide fungicide vinclozolin was previously isolated and characterized. A genomic library was constructed, and an 8.7-kb resistance-conferring fragment was isolated by sib selection. Sequencing this fragment, we identified an 1,218-bp open reading frame, which, if disrupted by deletion, no longer confers resistance. Analyses of the data in GenBank demonstrated a high degree of homology between the product of the 1,218-bp open reading frame, referred to as the adr-1 gene, and Ser (Thr) protein kinases. PMID:7793954

  13. Annotated Differentially Expressed Salivary Proteins of Susceptible and Insecticide-Resistant Mosquitoes of Anopheles stephensi

    PubMed Central

    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

  14. 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

  15. Dietary protein safety and resistance exercise: what do we really know?

    PubMed

    Lowery, Lonnie M; Devia, Lorena

    2009-01-01

    Resistance trainers continue to receive mixed messages about the safety of purposely seeking ample dietary protein in their quest for stimulating protein synthesis, improving performance, or maintaining health. Despite protein's lay popularity and the routinely high intakes exhibited by strength athletes, liberal and purposeful protein consumption is often maligned by "experts". University textbooks, instructors, and various forms of literature from personal training groups and athletic organizations continue to use dissuasive language surrounding dietary protein. Due to the widely known health benefits of dietary protein and a growing body of evidence on its safety profile, this is unfortunate. In response, researchers have critiqued unfounded educational messages. As a recent summarizing example, the International Society of Sports Nutrition (ISSN) Position Stand: Protein and Exercise reviewed general literature on renal and bone health. The concluding remark that "Concerns that protein intake within this range [1.4 - 2.0 g/kg body weight per day] is unhealthy are unfounded in healthy, exercising individuals." was based largely upon data from non-athletes due to "a lack of scientific evidence". Future studies were deemed necessary. This assessment is not unique in the scientific literature. Investigators continue to cite controversy, debate, and the lack of direct evidence that allows it. This review discusses the few existing safety studies done specific to athletes and calls for protein research specific to resistance trainers. Population-specific, long term data will be necessary for effective education in dietetics textbooks and from sports governing bodies. PMID:19138405

  16. 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

  17. 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.

  18. Is carbohydrate needed to further stimulate muscle protein synthesis/hypertrophy following resistance exercise?

    PubMed Central

    2013-01-01

    It is now well established that protein supplementation after resistance exercise promotes increased muscle protein synthesis, which ultimately results in greater net muscle accretion, relative to exercise alone or exercise with supplementary carbohydrate ingestion. However, it is not known whether combining carbohydrate with protein produces a greater anabolic response than protein alone. Recent recommendations have been made that the composition of the ideal supplement post-exercise would be a combination of a protein source with a high glycemic index carbohydrate. This is based on the hypothesis that insulin promotes protein synthesis, thus maximising insulin secretion will maximally potentiate this action. However, it is still controversial as to whether raising insulin level, within the physiological range, has any effect to further stimulate muscle protein synthesis. The present commentary will review the evidence underpinning the recommendation to consume carbohydrates in addition to a protein supplementation after resistance exercise for the specific purpose of increasing muscle mass. The paucity of data will be discussed, thus our conclusions are that further studies are necessary prior to any conclusions that enable evidence-based recommendations to be made. PMID:24066806

  19. Is carbohydrate needed to further stimulate muscle protein synthesis/hypertrophy following resistance exercise?

    PubMed

    Figueiredo, Vandré Casagrande; Cameron-Smith, David

    2013-01-01

    It is now well established that protein supplementation after resistance exercise promotes increased muscle protein synthesis, which ultimately results in greater net muscle accretion, relative to exercise alone or exercise with supplementary carbohydrate ingestion. However, it is not known whether combining carbohydrate with protein produces a greater anabolic response than protein alone. Recent recommendations have been made that the composition of the ideal supplement post-exercise would be a combination of a protein source with a high glycemic index carbohydrate. This is based on the hypothesis that insulin promotes protein synthesis, thus maximising insulin secretion will maximally potentiate this action. However, it is still controversial as to whether raising insulin level, within the physiological range, has any effect to further stimulate muscle protein synthesis. The present commentary will review the evidence underpinning the recommendation to consume carbohydrates in addition to a protein supplementation after resistance exercise for the specific purpose of increasing muscle mass. The paucity of data will be discussed, thus our conclusions are that further studies are necessary prior to any conclusions that enable evidence-based recommendations to be made.

  20. Identification of novel γ-secretase-associated proteins in detergent-resistant membranes from brain.

    PubMed

    Hur, Ji-Yeun; Teranishi, Yasuhiro; Kihara, Takahiro; Yamamoto, Natsuko Goto; Inoue, Mitsuhiro; Hosia, Waltteri; Hashimoto, Masakazu; Winblad, Bengt; Frykman, Susanne; Tjernberg, Lars O

    2012-04-01

    In Alzheimer disease, oligomeric amyloid β-peptide (Aβ) species lead to synapse loss and neuronal death. γ-Secretase, the transmembrane protease complex that mediates the final catalytic step that liberates Aβ from its precursor protein (APP), has a multitude of substrates, and therapeutics aimed at reducing Aβ production should ideally be specific for APP cleavage. It has been shown that APP can be processed in lipid rafts, and γ-secretase-associated proteins can affect Aβ production. Here, we use a biotinylated inhibitor for affinity purification of γ-secretase and associated proteins and mass spectrometry for identification of the purified proteins, and we identify novel γ-secretase-associated proteins in detergent-resistant membranes from brain. Furthermore, we show by small interfering RNA-mediated knockdown of gene expression that a subset of the γ-secretase-associated proteins, in particular voltage-dependent anion channel 1 (VDAC1) and contactin-associated protein 1 (CNTNAP1), reduced Aβ production (Aβ40 and Aβ42) by around 70%, whereas knockdown of presenilin 1, one of the essential γ-secretase complex components, reduced Aβ production by 50%. Importantly, these proteins had a less pronounced effect on Notch processing. We conclude that VDAC1 and CNTNAP1 associate with γ-secretase in detergent-resistant membranes and affect APP processing and suggest that molecules that interfere with this interaction could be of therapeutic use for Alzheimer disease. PMID:22315232

  1. Identification of Novel γ-Secretase-associated Proteins in Detergent-resistant Membranes from Brain*

    PubMed Central

    Hur, Ji-Yeun; Teranishi, Yasuhiro; Kihara, Takahiro; Yamamoto, Natsuko Goto; Inoue, Mitsuhiro; Hosia, Waltteri; Hashimoto, Masakazu; Winblad, Bengt; Frykman, Susanne; Tjernberg, Lars O.

    2012-01-01

    In Alzheimer disease, oligomeric amyloid β-peptide (Aβ) species lead to synapse loss and neuronal death. γ-Secretase, the transmembrane protease complex that mediates the final catalytic step that liberates Aβ from its precursor protein (APP), has a multitude of substrates, and therapeutics aimed at reducing Aβ production should ideally be specific for APP cleavage. It has been shown that APP can be processed in lipid rafts, and γ-secretase-associated proteins can affect Aβ production. Here, we use a biotinylated inhibitor for affinity purification of γ-secretase and associated proteins and mass spectrometry for identification of the purified proteins, and we identify novel γ-secretase-associated proteins in detergent-resistant membranes from brain. Furthermore, we show by small interfering RNA-mediated knockdown of gene expression that a subset of the γ-secretase-associated proteins, in particular voltage-dependent anion channel 1 (VDAC1) and contactin-associated protein 1 (CNTNAP1), reduced Aβ production (Aβ40 and Aβ42) by around 70%, whereas knockdown of presenilin 1, one of the essential γ-secretase complex components, reduced Aβ production by 50%. Importantly, these proteins had a less pronounced effect on Notch processing. We conclude that VDAC1 and CNTNAP1 associate with γ-secretase in detergent-resistant membranes and affect APP processing and suggest that molecules that interfere with this interaction could be of therapeutic use for Alzheimer disease. PMID:22315232

  2. Activated Protein C Resistance Does Not Increase Risk for Recurrent Stroke or Death in Stroke Patients

    PubMed Central

    Thaler, Christoph; Sonntag, Natalie; Schleef, Michael; Rondak, Ina-Christine; Poppert, Holger

    2016-01-01

    Background Activated protein C (APC) resistance is the most common inherited prothrombotic disorder. The role of APC resistance in ischemic stroke is controversially discussed. Objectives The aim of this single center follow up study was to investigate the effect of APC resistance on stroke recurrence and survival in stroke patients. Patients/Methods We retrospectively identified 966 patients who had had an ischemic stroke or transitory ischemic attack (TIA) and in whom laboratory tests for APC resistance had been conducted. These patients were contacted to determine the primary outcomes of recurrent ischemic stroke or death. Results A total of 858 patients with an average follow up time of 8.48 years were included. APC resistance did not influence cumulative incidence functions for stroke free and total survival. In multivariate analyses, crude and adjusted hazard ratios for recurrent stroke as well as for death where not significantly increased in patients with APC resistance. This also applies to the subgroups of young patients, patients with cryptogenic stroke and patients with atrial fibrillation. Conclusion APC-resistance is not a risk factor for subsequent stroke or death in patients with a first ischemic stroke or TIA. Testing for APC-resistance in stroke patients therefore cannot be routinely recommended. PMID:27508300

  3. A Novel Membrane Protein, VanJ, Conferring Resistance to Teicoplanin

    PubMed Central

    Novotna, Gabriela; Hill, Chris; Vincent, Karen; Liu, Chang

    2012-01-01

    Bacterial resistance to the glycopeptide antibiotic teicoplanin shows some important differences from the closely related compound vancomycin. They are currently poorly understood but may reflect significant differences in the mode of action of each antibiotic. Streptomyces coelicolor possesses a vanRSJKHAX gene cluster that when expressed confers resistance to both vancomycin and teicoplanin. The resistance to vancomycin is mediated by the enzymes encoded by vanKHAX, but not by vanJ. vanHAX effect a reprogramming of peptidoglycan biosynthesis, which is considered to be generic, conferring resistance to all glycopeptide antibiotics. Here, we show that vanKHAX are not in fact required for teicoplanin resistance in S. coelicolor, which instead is mediated solely by vanJ. vanJ is shown to encode a membrane protein oriented with its C-terminal active site exposed to the extracytoplasmic space. VanJ also confers resistance to the teicoplanin-like antibiotics ristocetin and A47934 and to a broad range of semisynthetic teicoplanin derivatives, but not generally to antibiotics or semisynthetic derivatives with vancomycin-like structures. vanJ homologues are found ubiquitously in streptomycetes and include staP from the Streptomyces toyocaensis A47934 biosynthetic gene cluster. While overexpression of staP also conferred resistance to teicoplanin, similar expression of other vanJ homologues (SCO2255, SCO7017, and SAV5946) did not. The vanJ and staP orthologues, therefore, appear to represent a subset of a larger protein family whose members have acquired specialist roles in antibiotic resistance. Future characterization of the divergent enzymatic activity within this new family will contribute to defining the molecular mechanisms important for teicoplanin activity and resistance. PMID:22232274

  4. 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

  5. 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.

  6. A chemo-resistant protein expression pattern of glioblastoma cells (A172) to perillyl alcohol

    PubMed Central

    Fischer, Juliana de Saldanha da Gama; Carvalho, Paulo Costa; Fonseca, Clovis Orlando da; Liao, Lujian; Degrave, Wim M; Carvalho, Maria da Gloria da Costa; Yates, John R; Domont, Gilberto B

    2010-01-01

    Glioblastoma multiform (GBM) is by far the most malignant glioma. We have introduced a new treatment for GBMs that comprises the inhalation of a naturally occurring terpene with chemotherapeutic properties known as perillyl alcohol (POH). Clinical trial results on recurrent GBM patients showed that POH extends the average life by more than eight months, temporarily slows tumor growth, and in some cases even decreases tumor size. After approximately seven months the tumor continues to grow and leads to a dismal prognosis. To investigate how these tumors become resistant to POH we generated an A172 human glioblastoma cell culture tolerant to 0.06 mM of POH (A172r). We used Multidimensional Protein Identification Technology (MudPIT) to compare the protein expression profile of A172r cells to the established glioblastoma A172 cell line. Our results include a list of identified proteins unique to either the resistant or the non-resistant cell line. These proteins are related to cellular growth, negative apoptosis regulation, Ras pathway, and other key cellular functions that could be connected to the underlying mechanisms of resistance. PMID:20806975

  7. Generation of PVY coat protein siRNAs in transgenic potatoes resistant to PVY.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Transgenic potatoes expressing the potato virus Y coat protein (PVY-CP) inverted hairpin RNA (ihRNA) construct driven by the Solanum bulbocastanum ubiquitin 409s promoter exhibited resistance to PVY in glass house studies using PVYNTN and PVYO as inocula and in field studies using naturally occurrin...

  8. Transgenic maize plants expressing the Totivirus antifungal protein, KP4, are highly resistant to corn smut.

    PubMed

    Allen, Aron; Islamovic, Emir; Kaur, Jagdeep; Gold, Scott; Shah, Dilip; Smith, Thomas J

    2011-10-01

    The corn smut fungus, Ustilago maydis, is a global pathogen responsible for extensive agricultural losses. Control of corn smut using traditional breeding has met with limited success because natural resistance to U. maydis is organ specific and involves numerous maize genes. Here, we present a transgenic approach by constitutively expressing the Totivirus antifungal protein KP4, in maize. Transgenic maize plants expressed high levels of KP4 with no apparent negative impact on plant development and displayed robust resistance to U. maydis challenges to both the stem and ear tissues in the greenhouse. More broadly, these results demonstrate that a high level of organ independent fungal resistance can be afforded by transgenic expression of this family of antifungal proteins.

  9. Dual mode of action of Bt proteins: protoxin efficacy against resistant insects

    PubMed Central

    Tabashnik, Bruce E.; Zhang, Min; Fabrick, Jeffrey A.; Wu, Yidong; Gao, Meijing; Huang, Fangneng; Wei, Jizhen; Zhang, Jie; Yelich, Alexander; Unnithan, Gopalan C.; Bravo, Alejandra; Soberón, Mario; Carrière, Yves; Li, Xianchun

    2015-01-01

    Transgenic crops that produce Bacillus thuringiensis (Bt) proteins for pest control are grown extensively, but insect adaptation can reduce their effectiveness. Established mode of action models assert that Bt proteins Cry1Ab and Cry1Ac are produced as inactive protoxins that require conversion to a smaller activated form to exert toxicity. However, contrary to this widely accepted paradigm, we report evidence from seven resistant strains of three major crop pests showing that Cry1Ab and Cry1Ac protoxins were generally more potent than the corresponding activated toxins. Moreover, resistance was higher to activated toxins than protoxins in eight of nine cases evaluated in this study. These data and previously reported results support a new model in which protoxins and activated toxins kill insects via different pathways. Recognizing that protoxins can be more potent than activated toxins against resistant insects may help to enhance and sustain the efficacy of transgenic Bt crops. PMID:26455902

  10. Role of Ingested Amino Acids and Protein in the Promotion of Resistance Exercise-Induced Muscle Protein Anabolism.

    PubMed

    Reidy, Paul T; Rasmussen, Blake B

    2016-02-01

    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 on

  11. Global protein synthesis in human trophoblast is resistant to inhibition by hypoxia

    PubMed Central

    Williams, S.F.; Fik, E.; Zamudio, S.; Illsley, N.P.

    2012-01-01

    Placental growth and function depend on syncytial cell processes which require the continuing synthesis of cellular proteins. The substantial energy demands of protein synthesis are met primarily from oxidative metabolism. Although the responses of individual proteins produced by the syncytiotrophoblast to oxygen deprivation have been investigated previously, there is no information available on global protein synthesis in syncytiotrophoblast under conditions of hypoxia. These studies were designed to test the hypothesis that syncytial protein synthesis is decreased in a dose-dependent manner by hypoxia. Experiments were performed to measure amino acid incorporation into proteins in primary syncytiotrophoblast cells exposed to oxygen concentrations ranging from 0 to 10%. Compared to cells exposed to normoxia (10% O2), no changes were observed following exposure to 5% or 3% O2, but after exposure to 1% O2, protein synthesis after 24 and 48 h decreased by 24% and 23% and with exposure to 0% O2, by 65% and 50%. As a consequence of these results, we hypothesized that global protein synthesis in conditions of severe hypoxia was being supported by glucose metabolism. Additional experiments were performed therefore to examine the role of glucose in supporting protein synthesis. These demonstrated that at each oxygen concentration there was a significant, decreasing linear trend in protein synthesis as glucose concentration was reduced. Under conditions of near-anoxia and in the absence of glucose, protein synthesis was reduced by >85%. Even under normoxic conditions (defined as 10% O2) and in the presence of oxidative substrates, reductions in glucose were accompanied by decreases in protein synthesis. These experiments demonstrate that syncytiotrophoblast cells are resistant to reductions in protein synthesis at O2 concentrations greater than 1%. This could be explained by our finding that a significant fraction of protein synthesis in the syncytiotrophoblast is

  12. Identification of glycan structure alterations on cell membrane proteins in desoxyepothilone B resistant leukemia cells.

    PubMed

    Nakano, Miyako; Saldanha, Rohit; Göbel, Anja; Kavallaris, Maria; Packer, Nicolle H

    2011-11-01

    Resistance to tubulin-binding agents used in cancer is often multifactorial and can include changes in drug accumulation and modified expression of tubulin isotypes. Glycans on cell membrane proteins play important roles in many cellular processes such as recognition and apoptosis, and this study investigated whether changes to the glycan structures on cell membrane proteins occur when cells become resistant to drugs. Specifically, we investigated the alteration of glycan structures on the cell membrane proteins of human T-cell acute lymphoblastic leukemia (CEM) cells that were selected for resistance to desoxyepothilone B (CEM/dEpoB). The glycan profile of the cell membrane glycoproteins was obtained by sequential release of N- and O-glycans from cell membrane fraction dotted onto polyvinylidene difluoride membrane with PNGase F and β-elimination respectively. The released glycan alditols were analyzed by liquid chromatography (graphitized carbon)-electrospray ionization tandem MS. The major N-glycan on CEM cell was the core fucosylated α2-6 monosialo-biantennary structure. Resistant CEM/dEpoB cells had a significant decrease of α2-6 linked sialic acid on N-glycans. The lower α2-6 sialylation was caused by a decrease in activity of β-galactoside α2-6 sialyltransferase (ST6Gal), and decreased expression of the mRNA. It is clear that the membrane glycosylation of leukemia cells changes during acquired resistance to dEpoB drugs and that this change occurs globally on all cell membrane glycoproteins. This is the first identification of a specific glycan modification on the surface of drug resistant cells and the mechanism of this downstream effect on microtubule targeting drugs may offer a route to new interventions to overcome drug resistance.

  13. 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. PMID:27495375

  14. The role of organic proteins on the crack growth resistance of human enamel.

    PubMed

    Yahyazadehfar, Mobin; Arola, Dwayne

    2015-06-01

    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.

  15. 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

  16. Kinetic Ductility and Force-Spike Resistance of Proteins from Single-Molecule Force Spectroscopy.

    PubMed

    Cossio, Pilar; Hummer, Gerhard; Szabo, Attila

    2016-08-23

    Ductile materials can absorb spikes in mechanical force, whereas brittle ones fail catastrophically. Here we develop a theory to quantify the kinetic ductility of single molecules from force spectroscopy experiments, relating force-spike resistance to the differential responses of the intact protein and the unfolding transition state to an applied mechanical force. We introduce a class of unistable one-dimensional potential surfaces that encompass previous models as special cases and continuously cover the entire range from ductile to brittle. Compact analytic expressions for force-dependent rates and rupture-force distributions allow us to analyze force-clamp and force-ramp pulling experiments. We find that the force-transmitting protein domains of filamin and titin are kinetically ductile when pulled from their two termini, making them resistant to force spikes. For the mechanostable muscle protein titin, a highly ductile model reconciles data over 10 orders of magnitude in force loading rate from experiment and simulation.

  17. Kinetic Ductility and Force-Spike Resistance of Proteins from Single-Molecule Force Spectroscopy.

    PubMed

    Cossio, Pilar; Hummer, Gerhard; Szabo, Attila

    2016-08-23

    Ductile materials can absorb spikes in mechanical force, whereas brittle ones fail catastrophically. Here we develop a theory to quantify the kinetic ductility of single molecules from force spectroscopy experiments, relating force-spike resistance to the differential responses of the intact protein and the unfolding transition state to an applied mechanical force. We introduce a class of unistable one-dimensional potential surfaces that encompass previous models as special cases and continuously cover the entire range from ductile to brittle. Compact analytic expressions for force-dependent rates and rupture-force distributions allow us to analyze force-clamp and force-ramp pulling experiments. We find that the force-transmitting protein domains of filamin and titin are kinetically ductile when pulled from their two termini, making them resistant to force spikes. For the mechanostable muscle protein titin, a highly ductile model reconciles data over 10 orders of magnitude in force loading rate from experiment and simulation. PMID:27558726

  18. 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.

  19. The small heat shock protein B8 (HSPB8) confers resistance to bortezomib by promoting autophagic removal of misfolded proteins in multiple myeloma cells.

    PubMed

    Hamouda, Mohamed-Amine; Belhacene, Nathalie; Puissant, Alexandre; Colosetti, Pascal; Robert, Guillaume; Jacquel, Arnaud; Mari, Bernard; Auberger, Patrick; Luciano, Frederic

    2014-08-15

    Velcade is one of the inescapable drug to treat patient suffering from multiple myeloma (MM) and resistance to this drug represents a major drawback for patients. However, the mechanisms underlying velcade resistance remain incompletely understood. We derived several U266 MM cell clones that resist to velcade. U266-resistant cells were resistant to velcade-induced cell death but exhibited a similar sensitivity to various proapoptotic stimuli. Careful analysis of proteosomal subunits and proteasome enzymatic activities showed that neither the composition nor the activity of the proteasome was affected in velcade-resistant cells. Elimination of velcade-induced poly-ubiquitinated proteins and protein aggregates was drastically stimulated in the resistant cells and correlated with increased cell survival. Inhibition of the lysosomal activity in velcade-resistant cells resulted in an increase of cell aggregates and decrease survival, indicating that aggregates are eliminated through lysosomal degradation. In addition, pangenomic profiling of velcade-sensitive and resistant cells showed that the small heat shock protein HSPB8 was overexpressed in resistant cells. Finally, gain and loss of function experiment demonstrated that HSPB8 is a key factor for velcade resistance. In conclusion, HSPB8 plays an important role for the elimination of aggregates in velcade-resistant cells that contributes to their enhanced survival.

  20. Identification of seed proteins associated with resistance to pre-harvested aflatoxin contamination in peanut (Arachis hypogaea L)

    PubMed Central

    2010-01-01

    Background Pre-harvest infection of peanuts by Aspergillus flavus and subsequent aflatoxin contamination is one of the food safety factors that most severely impair peanut productivity and human and animal health, especially in arid and semi-arid tropical areas. Some peanut cultivars with natural pre-harvest resistance to aflatoxin contamination have been identified through field screening. However, little is known about the resistance mechanism, which has slowed the incorporation of resistance into cultivars with commercially acceptable genetic background. Therefore, it is necessary to identify resistance-associated proteins, and then to recognize candidate resistance genes potentially underlying the resistance mechanism. Results The objective of this study was to identify resistance-associated proteins in response to A. flavus infection under drought stress using two-dimensional electrophoresis with mass spectrometry. To identify proteins involved in the resistance to pre-harvest aflatoxin contamination, we compared the differential expression profiles of seed proteins between a resistant cultivar (YJ-1) and a susceptible cultivar (Yueyou 7) under well-watered condition, drought stress, and A. flavus infection with drought stress. A total of 29 spots showed differential expression between resistant and susceptible cultivars in response to A. flavus attack under drought stress. Among these spots, 12 protein spots that consistently exhibited an altered expression were screened by Image Master 5.0 software and successfully identified by MALDI-TOF MS. Five protein spots, including Oso7g0179400, PII protein, CDK1, Oxalate oxidase, SAP domain-containing protein, were uniquely expressed in the resistant cultivar. Six protein spots including low molecular weight heat shock protein precursor, RIO kinase, L-ascorbate peroxidase, iso-Ara h3, 50 S ribosomal protein L22 and putative 30 S ribosomal S9 were significantly up-regulated in the resistant cultivar challenged by A

  1. Comparisons of protein profiles of beech bark disease resistant and susceptible American beech (Fagus grandifolia)

    PubMed Central

    2013-01-01

    Background Beech bark disease is an insect-fungus complex that damages and often kills American beech trees and has major ecological and economic impacts on forests of the northeastern United States and southeastern Canadian forests. The disease begins when exotic beech scale insects feed on the bark of trees, and is followed by infection of damaged bark tissues by one of the Neonectria species of fungi. Proteomic analysis was conducted of beech bark proteins from diseased trees and healthy trees in areas heavily infested with beech bark disease. All of the diseased trees had signs of Neonectria infection such as cankers or fruiting bodies. In previous tests reported elsewhere, all of the diseased trees were demonstrated to be susceptible to the scale insect and all of the healthy trees were demonstrated to be resistant to the scale insect. Sixteen trees were sampled from eight geographically isolated stands, the sample consisting of 10 healthy (scale-resistant) and 6 diseased/infested (scale-susceptible) trees. Results Proteins were extracted from each tree and analysed in triplicate by isoelectric focusing followed by denaturing gel electrophoresis. Gels were stained and protein spots identified and intensity quantified, then a statistical model was fit to identify significant differences between trees. A subset of BBD differential proteins were analysed by mass spectrometry and matched to known protein sequences for identification. Identified proteins had homology to stress, insect, and pathogen related proteins in other plant systems. Protein spots significantly different in diseased and healthy trees having no stand or disease-by-stand interaction effects were identified. Conclusions Further study of these proteins should help to understand processes critical to resistance to beech bark disease and to develop biomarkers for use in tree breeding programs and for the selection of resistant trees prior to or in early stages of BBD development in stands. Early

  2. Timing of postexercise protein intake is important for muscle hypertrophy with resistance training in elderly humans

    PubMed Central

    Esmarck, B; Andersen, J L; Olsen, S; Richter, E A; Mizuno, M; Kjær, M

    2001-01-01

    Age-associated loss of skeletal muscle mass and strength can partly be counteracted by resistance training, causing a net synthesis of muscular proteins. Protein synthesis is influenced synergistically by postexercise amino acid supplementation, but the importance of the timing of protein intake remains unresolved. The study investigated the importance of immediate (P0) or delayed (P2) intake of an oral protein supplement upon muscle hypertrophy and strength over a period of resistance training in elderly males. Thirteen men (age, 74 ± 1 years; body mass index (BMI), 25 ± 1 kg m−2 (means ± S.E.M.)) completed a 12 week resistance training programme (3 times per week) receiving oral protein in liquid form (10 g protein, 7 g carbohydrate, 3 g fat) immediately after (P0) or 2 h after (P2) each training session. Muscle hypertrophy was evaluated by magnetic resonance imaging (MRI) and from muscle biopsies and muscle strength was determined using dynamic and isokinetic strength measurements. Body composition was determined from dual-energy X-ray absorptiometry (DEXA) and food records were obtained over 4 days. The plasma insulin response to protein supplementation was also determined. In response to training, the cross-sectional area of m. quadriceps femoris (54.6 ± 0.5 to 58.3 ± 0.5 cm2) and mean fibre area (4047 ± 320 to 5019 ± 615 μm2) increased in the P0 group, whereas no significant increase was observed in P2. For P0 both dynamic and isokinetic strength increased, by 46 and 15 %, respectively (P < 0.05), whereas P2 only improved in dynamic strength, by 36 % (P < 0.05). No differences in glucose or insulin response were observed between protein intake at 0 and 2 h postexercise. We conclude that early intake of an oral protein supplement after resistance training is important for the development of hypertrophy in skeletal muscle of elderly men in response to resistance training. PMID:11507179

  3. 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

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

    PubMed

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

    2013-08-01

    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

  5. 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

  6. Mutations in G protein beta subunits promote transformation and kinase inhibitor resistance

    PubMed Central

    Yoda, Akinori; Adelmant, Guillaume; Tamburini, Jerome; Chapuy, Bjoern; Shindoh, Nobuaki; Yoda, Yuka; Weigert, Oliver; Kopp, Nadja; Wu, Shuo-Chieh; Kim, Sunhee S.; Liu, Huiyun; Tivey, Trevor; Christie, Amanda L.; Elpek, Kutlu G.; Card, Joseph; Gritsman, Kira; Gotlib, Jason; Deininger, Michael W.; Makishima, Hideki; Turley, Shannon J.; Javidi-Sharifi, Nathalie; Maciejewski, Jaroslaw P.; Jaiswal, Siddhartha; Ebert, Benjamin L.; Rodig, Scott J.; Tyner, Jeffrey W.; Marto, Jarrod A.; Weinstock, David M.; Lane, Andrew A.

    2014-01-01

    Activating mutations of G protein alpha subunits (Gα) occur in 4–5% of all human cancers1 but oncogenic alterations in beta subunits (Gβ) have not been defined. Here we demonstrate that recurrent mutations in the Gβ proteins GNB1 and GNB2 confer cytokine-independent growth and activate canonical G protein signaling. Multiple mutations in GNB1 affect the protein interface that binds Gα subunits as well as downstream effectors, and disrupt Gα-Gβγ interactions. Different mutations in Gβ proteins clustered to some extent based on lineage; for example, all eleven GNB1 K57 mutations were in myeloid neoplasms while 7 of 8 GNB1 I80 mutations were in B cell neoplasms. Expression of patient-derived GNB1 alleles in Cdkn2a-deficient bone marrow followed by transplantation resulted in either myeloid or B cell malignancies. In vivo treatment with the dual PI3K/mTOR inhibitor BEZ235 suppressed GNB1-induced signaling and markedly increased survival. In several human tumors, GNB1 mutations co-occurred with oncogenic kinase alterations, including BCR/ABL, JAK2 V617F and BRAF V600K. Co-expression of patient-derived GNB1 alleles with these mutant kinases resulted in inhibitor resistance in each context. Thus, GNB1 and GNB2 mutations confer transformed and resistance phenotypes across a range of human tumors and may be targetable with inhibitors of G protein signaling. PMID:25485910

  7. SRFR1 Negatively Regulates Plant NB-LRR Resistance Protein Accumulation to Prevent Autoimmunity

    PubMed Central

    Li, Yingzhong; Li, Shuxin; Bi, Dongling; Cheng, Yu Ti; Li, Xin; Zhang, Yuelin

    2010-01-01

    Plant defense responses need to be tightly regulated to prevent auto-immunity, which is detrimental to growth and development. To identify negative regulators of Resistance (R) protein-mediated resistance, we screened for mutants with constitutive defense responses in the npr1-1 background. Map-based cloning revealed that one of the mutant genes encodes a conserved TPR domain-containing protein previously known as SRFR1 (SUPPRESSOR OF rps4-RLD). The constitutive defense responses in the srfr1 mutants in Col-0 background are suppressed by mutations in SNC1, which encodes a TIR-NB-LRR (Toll Interleukin1 Receptor-Nucleotide Binding-Leu-Rich Repeat) R protein. Yeast two-hybrid screens identified SGT1a and SGT1b as interacting proteins of SRFR1. The interactions between SGT1 and SRFR1 were further confirmed by co-immunoprecipitation analysis. In srfr1 mutants, levels of multiple NB-LRR R proteins including SNC1, RPS2 and RPS4 are increased. Increased accumulation of SNC1 is also observed in the sgt1b mutant. Our data suggest that SRFR1 functions together with SGT1 to negatively regulate R protein accumulation, which is required for preventing auto-activation of plant immunity. PMID:20862316

  8. Accelerated degradation of caspase-8 protein correlates with TRAIL resistance in a DLD1 human colon cancer cell line.

    PubMed

    Zhang, Lidong; Zhu, Hongbo; Teraishi, Fuminori; Davis, John J; Guo, Wei; Fan, Zhen; Fang, Bingliang

    2005-06-01

    The tumor-selective cytotoxic effect of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) makes TRAIL an attractive candidate as an anticancer agent. However, resistance to TRAIL poses a challenge in anticancer therapy with TRAIL. Therefore, characterizing the mechanisms of resistance and developing strategies to overcome the resistance are important steps toward successful TRAIL-mediated cancer therapy. In this study, we investigated mechanisms of acquired TRAIL resistance in a colon cancer DLD1 cell line. Compared with the TRAIL-susceptible DLD1 cell line, TRAIL-resistant DLD1/TRAIL-R cells have a low level of caspase-8 protein, but not its mRNA. Suppression of caspase-8 expression by siRNA in parental DLD1 cells led to TRAIL resistance. Restoration of caspase-8 protein expression by stable transfection rendered the DLD1/TRAIL-R cell line fully sensitive to TRAIL protein, suggesting that the low level of caspase-8 protein expression might be the culprit in TRAIL resistance in DLD1/TRAIL-R cells. Sequencing analysis of the caspase-8 coding region revealed a missense mutation that is present in both TRAIL-sensitive and TRAIL-resistant DLD1 cells. Subsequent study showed that the degradation of caspase-8 protein was accelerated in DLD1/TRAIL-R cells compared to parental DLD1 cells. Thus, accelerated degradation of caspase-8 protein is one of the mechanisms that lead to TRAIL resistance. PMID:16036110

  9. Plant resistance to fungal infection induced by nontoxic pokeweed antiviral protein mutants.

    PubMed

    Zoubenko, O; Uckun, F; Hur, Y; Chet, I; Tumer, N

    1997-10-01

    Pokeweed antiviral protein (PAP), a 29-kD protein isolated from Phytolacca americana inhibits translation by catalytically removing a specific adenine residue from the large rRNA of the 60S subunit of eukaryotic ribosomes. Transgenic plants expressing PAP are resistant to a broad spectrum of plant viruses. Nontoxic PAP mutants have been isolated by random mutagenesis and selection in yeast. One of these mutants, PAP-X, had a point mutation at the active-site (E176V) that abolished enzymatic activity, and another mutant, delta C25PAP, had a nonsense mutation near the C-terminus (W237stop) that deleted 25 C-terminal amino acids. Unlike the wild-type PAP, expression of neither mutant was toxic to transgenic plants. We show that both class I (basic) and class II (acidic) isoforms of pathogenesis-related (PR) proteins are overexpressed in transgenic plants expressing PAP and the nontoxic PAP mutants. Although PR-proteins are constitutively expressed, no increase in salicylic acid levels was detected. Homozygous progeny of transgenic plants expressing either PAP or the nontoxic PAP mutants displayed resistance to the fungal pathogen Rhizoctonia solani. These results show that expression of PAP or the nontoxic PAP mutants activates multiple plant defense pathways independently of salicylic acid and confers resistance to fungal infection. The C-terminal 25 amino acids of PAP, which are required for toxicity in vivo, are not critical for resistance to viral or fungal infection, indicating that toxicity of PAP can be separated from pathogen resistance.

  10. Resistance to heat radiosensitization and protein damage in thermotolerant and thermoresistant cells.

    PubMed

    Kampinga, H H; Konings, A W; Evers, A J; Brunsting, J F; Misfud, N; Anderson, R L

    1997-03-01

    Recently, randomized phase III trials have indicated that hyperthermia combined with radiation leads to significantly better tumour control of certain malignancies than does radio-therapy alone. Yet, the full capacity of such combined treatments might not have been optimally exploited as in vitro data indicate that repeated beating of cells can result in either the development of a transient heat resistance (thermotolerance) and/or the selection/induction of a stable heat resistant cell population. Although the mechanism of thermotolerance and its effect on thermo-radiotherapy has been studied extensively, little data are available on the mechanism of stable heat resistance and its impact on combined heat and radiation treatments. In the current study, a comprehensive analysis was made of the differences and similarities between thermotolerance (TT) and stable heat resistance (TR) in terms of the mechanism of resistance to the direct toxic action of heat and in terms of the impact on the extent of thermal radiosensitization. Using heat resistant mutants previously derived from a murine radiation-induced fibrosarcoma (RIF-1), it was observed that these cells were resistant to protein denaturation and aggregation in the cytoplasmic/membrane compartment (measured by ESR (electron spin resonance) analysis and by in situ thermal denaturation of the foreign firefly luciferase targeted to the cytoplasm) but not in the nuclear compartment (measured by TX-100 insoluble nuclear proteins and by in situ thermal denaturation of luciferase targeted to the nucleus). RIF-1-TT cells, in contrast, were resistant for a 1 end-points tested. The lack of protection of nuclear heat damage in the RIF-TR cells could not be explained by a failure of one or more of the HSP70 isoforms to enter the nuclei of these cells. In relation to the absence or presence of heat resistance in the nucleus, the extent of heat radiosensitization was reduced in RIF-1-TT but not RIF-TR cells. This implies that

  11. Multidrug resistance protein 1 (MRP1, ABCC1), a "multitasking" ATP-binding cassette (ABC) transporter.

    PubMed

    Cole, Susan P C

    2014-11-01

    The multidrug resistance protein 1 (MRP1) encoded by ABCC1 was originally discovered as a cause of multidrug resistance in tumor cells. However, it is now clear that MRP1 serves a broader role than simply mediating the ATP-dependent efflux of drugs from cells. The antioxidant GSH and the pro-inflammatory cysteinyl leukotriene C4 have been identified as key physiological organic anions effluxed by MRP1, and an ever growing body of evidence indicates that additional lipid-derived mediators are also substrates of this transporter. As such, MRP1 is a multitasking transporter that likely influences the etiology and progression of a host of human diseases.

  12. Protein kinase C-gamma is present in adriamycin resistant HL-60 leukemia cells.

    PubMed

    Aquino, A; Warren, B S; Omichinski, J; Hartman, K D; Glazer, R I

    1990-01-30

    The isoform pattern of protein kinase C (PKC) was examined in wild-type and Adriamycin-resistant (HL-60/AR) HL-60 leukemia cells. Analyses were carried out by immunoblotting with mouse monoclonal antibodies against PKC-alpha and PKC-beta and a rabbit polyclonal antibody against the variable (V3) region of PKC-gamma. HL-60/AR cells contained an equivalent level of PKC-alpha and a lower amount of PKC-beta than HL-60 cells. In contrast, only HL-60/AR cells contained PKC-gamma. These results indicate that the regulation of this family of isoenzymes is altered in drug-resistant cells. PMID:2302237

  13. Increased Systemic Exposure of Methotrexate by a Polyphenol-Rich Herb via Modulation on Efflux Transporters Multidrug Resistance-Associated Protein 2 and Breast Cancer Resistance Protein.

    PubMed

    Yu, Chung-Ping; Hsieh, Yun-Chung; Shia, Chi-Sheng; Hsu, Pei-Wen; Chen, Jen-Yuan; Hou, Yu-Chi; Hsieh, Yo-Wen

    2016-01-01

    Scutellariae radix (SR, roots of Scutellaria baicalensis Georgi), a popular Chinese medicine, contains plenty of flavonoids such as baicalin, wogonoside, baicalein, and wogonin. Methotrexate (MTX), an important immunosuppressant with a narrow therapeutic index, is a substrate of multidrug resistance-associated proteins (MRPs) and breast cancer resistance protein (BCRP). This study investigated the effect of SR on MTX pharmacokinetics and the underlying mechanisms. Rats were orally administered MTX alone and with 1.0 or 2.0 g/kg of SR. The serum concentrations of MTX were determined by a fluorescence polarization immunoassay. Cell models were used to explore the involvement of MRP2 and BCRP in the interaction. The results showed that 1.0 g/kg of SR significantly increased Cmax, AUC(0-30), AUC(0-2880), and mean residence time (MRT) of MTX by 50%, 45%, 501%, and 347%, respectively, and 2.0 g/kg of SR significantly enhanced the AUC(0-2880) and MRT by 242% and 293%, respectively, but decreased AUC(0-30) by 41%. Cell line studies indicated that SR activated the BCRP-mediated efflux transport, whereas the serum metabolites of SR inhibited both the BCRP- and MRP2-mediated efflux transports. In conclusion, SR ingestion increased the systemic exposure and MRT of MTX via modulation on MRP2 and BCRP. PMID:26852865

  14. Increased Systemic Exposure of Methotrexate by a Polyphenol-Rich Herb via Modulation on Efflux Transporters Multidrug Resistance-Associated Protein 2 and Breast Cancer Resistance Protein.

    PubMed

    Yu, Chung-Ping; Hsieh, Yun-Chung; Shia, Chi-Sheng; Hsu, Pei-Wen; Chen, Jen-Yuan; Hou, Yu-Chi; Hsieh, Yo-Wen

    2016-01-01

    Scutellariae radix (SR, roots of Scutellaria baicalensis Georgi), a popular Chinese medicine, contains plenty of flavonoids such as baicalin, wogonoside, baicalein, and wogonin. Methotrexate (MTX), an important immunosuppressant with a narrow therapeutic index, is a substrate of multidrug resistance-associated proteins (MRPs) and breast cancer resistance protein (BCRP). This study investigated the effect of SR on MTX pharmacokinetics and the underlying mechanisms. Rats were orally administered MTX alone and with 1.0 or 2.0 g/kg of SR. The serum concentrations of MTX were determined by a fluorescence polarization immunoassay. Cell models were used to explore the involvement of MRP2 and BCRP in the interaction. The results showed that 1.0 g/kg of SR significantly increased Cmax, AUC(0-30), AUC(0-2880), and mean residence time (MRT) of MTX by 50%, 45%, 501%, and 347%, respectively, and 2.0 g/kg of SR significantly enhanced the AUC(0-2880) and MRT by 242% and 293%, respectively, but decreased AUC(0-30) by 41%. Cell line studies indicated that SR activated the BCRP-mediated efflux transport, whereas the serum metabolites of SR inhibited both the BCRP- and MRP2-mediated efflux transports. In conclusion, SR ingestion increased the systemic exposure and MRT of MTX via modulation on MRP2 and BCRP.

  15. IMP3 Protein Promotes Chemoresistance in Breast Cancer Cells by Regulating Breast Cancer Resistance Protein (ABCG2) Expression*

    PubMed Central

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

    2013-01-01

    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. PMID:23539627

  16. Prediction of ligand binding site by insilico approach in cold resistant protein isolated from cold resistant mutant of Pseudomonas fluorescens.

    PubMed

    Kumar, Amit; Khan, Mahejibin

    2012-09-01

    Cold shock proteins perform vital functions, such as mRNA masking, coupling of transcription to translation and developmental timing and regulation, which aids in survival of microbes in cold stress. Pseudomonas fluorescens is an ecologically important bacterium which helps in plant growth promotion. Since the cold tolerant mutant of the bacterium is able to grow at the temperature ranges from 30 to 4°C, it is of interest to study the process of its survival in the extreme temperatures. Therefore, this study is focused on the three dimensional structure and molecular modeling of cold resistant protein (CRP) from P. fluorescens to predict its molecular mechanism. Investigating the structure of CRP confirmed the presence of a conserved domain characteristic of the cold shock domain (CSD) family and a single nucleotide binding domain. When 3D structure of CRP was compared with the existing cold shock proteins, major deviations were found in the loop regions connecting the β2-β3, β3-β4 and β4-β5 sheets. Docking studies showed that CRP forms a significant clamp like structure at the substrate binding cleft which stabilizes the ligand. Therefore, it can be concluded that CRP has a strong affinity for the poly thymidine (poly T) stretch and can be considered a candidate for transcription regulation. PMID:23099776

  17. 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. PMID:25837301

  18. Transgenic sugarcane resistant to Sorghum mosaic virus based on coat protein gene silencing by RNA interference.

    PubMed

    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.

  19. A Computational Approach towards the Understanding of Plasmodium falciparum Multidrug Resistance Protein 1

    PubMed Central

    Patel, Saumya K.; Prasanth Kumar, Sivakumar; Highland, Hyacinth N.; Jasrai, Yogesh T.; Pandya, Himanshu A.; Desai, Ketaki R.

    2013-01-01

    The emergence of drug resistance in Plasmodium falciparum tremendously affected the chemotherapy worldwide while the intense distribution of chloroquine-resistant strains in most of the endemic areas added more complications in the treatment of malaria. The situation has even worsened by the lack of molecular mechanism to understand the resistance conferred by Plasmodia species. Recent studies have suggested the association of antimalarial resistance with P. falciparum multidrug resistance protein 1 (PfMDR1), an ATP-binding cassette (ABC) transporter and a homologue of human P-glycoprotein 1 (P-gp1). The present study deals about the development of PfMDR1 computational model and the model of substrate transport across PfMDR1 with insights derived from conformations relative to inward- and outward-facing topologies that switch on/off the transportation system. Comparison of ATP docked positions and its structural motif binding properties were found to be similar among other ATPases, and thereby contributes to NBD domains dimerization, a unique structural agreement noticed in Mus musculus Pgp and Escherichia coli MDR transporter homolog (MsbA). The interaction of leading antimalarials and phytochemicals within the active pocket of both wild-type and mutant-type PfMDR1 demonstrated the mode of binding and provided insights of less binding affinity thereby contributing to parasite's resistance mechanism. PMID:25937947

  20. A Novel Family of Cys-Rich Membrane Proteins Mediates Cadmium Resistance in Arabidopsis1

    PubMed Central

    Song, Won-Yong; Martinoia, Enrico; Lee, Joohyun; Kim, Dongwoo; Kim, Do-Young; Vogt, Esther; Shim, Donghwan; Choi, Kwan Sam; Hwang, Inhwan; Lee, Youngsook

    2004-01-01

    Cadmium (Cd) is a widespread pollutant that is toxic to plant growth. However, only a few genes that contribute to Cd resistance in plants have been identified. To identify additional Cd(II) resistance genes, we screened an Arabidopsis cDNA library using a yeast (Saccharomyces cerevisiae) expression system employing the Cd(II)-sensitive yeast mutant ycf1. This screening process yielded a small Cys-rich membrane protein (Arabidopsis plant cadmium resistance, AtPcrs). Database searches revealed that there are nine close homologs in Arabidopsis. Homologs were also found in other plants. Four of the five homologs that were tested also increased resistance to Cd(II) when expressed in ycf1. AtPcr1 localizes at the plasma membrane in both yeast and Arabidopsis. Arabidopsis plants overexpressing AtPcr1 exhibited increased Cd(II) resistance, whereas antisense plants that showed reduced AtPcr1 expression were more sensitive to Cd(II). AtPcr1 overexpression reduced Cd uptake by yeast cells and also reduced the Cd contents of both yeast and Arabidopsis protoplasts treated with Cd. Thus, it appears that the Pcr family members may play an important role in the Cd resistance of plants. PMID:15181212

  1. Hypothetical proteins present during recovery phase of radiation resistant bacterium Deinococcus radiodurans are under purifying selection.

    PubMed

    Das, Anubrata D; Misra, Hari S

    2013-08-01

    Deinococcus radiodurans has an unusual capacity to recover from intense doses of ionizing radiation. The DNA repair proteins of this organism play an important role in repairing the heavily damaged DNA by employing a novel mechanism of DNA double-strand break repair. An earlier report stated that genes of many of these repair proteins are under positive selection implying that these genes have a tendency to mutate, which in turn provides selective advantage to this bacterium. Several "hypothetical proteins" are also present during the recovery phase and some of them have also been shown for their roles in radiation resistance. Therefore, we tested the selection pressure on the genes encoding these poorly characterized proteins. Our results show that a number of "hypothetical proteins" present during the repair phase have structural adaptations compared to their orthologs and the genes encoding them as well as those for the DNA repair proteins present during this phase are under purifying selection. Evidence of purifying selection in these hypothetical proteins suggests that certain novel characteristics among these proteins are conserved and seem to be under functional constraints to perform important functions during recovery process after gamma radiation damage.

  2. Partly replacing meat protein with soy protein alters insulin resistance and blood lipids in postmenopausal women with abdominal obesity.

    PubMed

    van Nielen, Monique; Feskens, Edith J M; Rietman, Annemarie; Siebelink, Els; Mensink, Marco

    2014-09-01

    Increasing protein intake and soy consumption appear to be promising approaches to prevent metabolic syndrome (MetS). However, the effect of soy consumption on insulin resistance, glucose homeostasis, and other characteristics of MetS is not frequently studied in humans. We aimed to investigate the effects of a 4-wk, strictly controlled, weight-maintaining, moderately high-protein diet rich in soy on insulin sensitivity and other cardiometabolic risk factors. We performed a randomized crossover trial of 2 4-wk diet periods in 15 postmenopausal women with abdominal obesity to test diets with 22 energy percent (En%) protein, 27 En% fat, and 50 En% carbohydrate. One diet contained protein of mixed origin (mainly meat, dairy, and bread), and the other diet partly replaced meat with soy meat analogues and soy nuts containing 30 g/d soy protein. For our primary outcome, a frequently sampled intravenous glucose tolerance test (FSIGT) was performed at the end of both periods. Plasma total, LDL, and HDL cholesterol, triglycerides, glucose, insulin, and C-reactive protein were assessed, and blood pressure, arterial stiffness, and intrahepatic lipid content were measured at the start and end of both periods. Compared with the mixed-protein diet, the soy-protein diet resulted in greater insulin sensitivity [FSIGT: insulin sensitivity, 34 ± 29 vs. 22 ± 17 (mU/L)(-1) · min(-1), P = 0.048; disposition index, 4974 ± 2543 vs. 2899 ± 1878, P = 0.038; n = 11]. Total cholesterol was 4% lower after the soy-protein diet than after the mixed-protein diet (4.9 ± 0.7 vs. 5.1 ± 0.6 mmol/L, P = 0.001), and LDL cholesterol was 9% lower (2.9 ± 0.7 vs. 3.2 ± 0.6 mmol/L, P = 0.004; n = 15). Thus, partly replacing meat with soy in a moderately high-protein diet has clear advantages regarding insulin sensitivity and total and LDL cholesterol. Therefore, partly replacing meat products with soy products could be important in preventing MetS. This trial was registered at clinicaltrials

  3. Caspase-resistant VirD2 protein provides enhanced gene delivery and expression in plants.

    PubMed

    Reavy, Brian; Bagirova, Svetlana; Chichkova, Nina V; Fedoseeva, Svetlana V; Kim, Sang Hyon; Vartapetian, Andrey B; Taliansky, Michael E

    2007-08-01

    Agrobacterium tumefaciens VirD2 protein is one of the key elements of Agrobacterium-mediated plant transformation, a process of transfer of T-DNA sequence from the Agrobacterium tumour inducing plasmid into the nucleus of infected plant cells and its integration into the host genome. The VirD2 protein has been shown to be a substrate for a plant caspase-like protease activity (PCLP) in tobacco. We demonstrate here that mutagenesis of the VirD2 protein to prevent cleavage by PCLP increases the efficiency of reporter gene transfer and expression. These results indicate that PCLP cleavage of the Agrobacterium VirD2 protein acts to limit the effectiveness of T-DNA transfer and is a novel resistance mechanism that plants utilise to combat Agrobacterium infection. PMID:17370074

  4. Proteomics-based identification of midgut proteins correlated with Cry1Ac resistance in Plutella xylostella (L.).

    PubMed

    Xia, Jixing; Guo, Zhaojiang; Yang, Zezhong; Zhu, Xun; Kang, Shi; Yang, Xin; Yang, Fengshan; Wu, Qingjun; Wang, Shaoli; Xie, Wen; Xu, Weijun; Zhang, Youjun

    2016-09-01

    The diamondback moth, Plutella xylostella (L.), is a worldwide pest of cruciferous crops and can rapidly develop resistance to many chemical insecticides. Although insecticidal crystal proteins (i.e., Cry and Cyt toxins) derived from Bacillus thuringiensis (Bt) have been useful alternatives to chemical insecticides for the control of P. xylostella, resistance to Bt in field populations of P. xylostella has already been reported. A better understanding of the resistance mechanisms to Bt should be valuable in delaying resistance development. In this study, the mechanisms underlying P. xylostella resistance to Bt Cry1Ac toxin were investigated using two-dimensional differential in-gel electrophoresis (2D-DIGE) and ligand blotting for the first time. Comparative analyses of the constitutive expression of midgut proteins in Cry1Ac-susceptible and -resistant P. xylostella larvae revealed 31 differentially expressed proteins, 21 of which were identified by mass spectrometry. Of these identified proteins, the following fell into diverse eukaryotic orthologous group (KOG) subcategories may be involved in Cry1Ac resistance in P. xylostella: ATP-binding cassette (ABC) transporter subfamily G member 4 (ABCG4), trypsin, heat shock protein 70 (HSP70), vacuolar H(+)-ATPase, actin, glycosylphosphatidylinositol anchor attachment 1 protein (GAA1) and solute carrier family 30 member 1 (SLC30A1). Additionally, ligand blotting identified the following midgut proteins as Cry1Ac-binding proteins in Cry1Ac-susceptible P. xylostella larvae: ABC transporter subfamily C member 1 (ABCC1), solute carrier family 36 member 1 (SLC36A1), NADH dehydrogenase iron-sulfur protein 3 (NDUFS3), prohibitin and Rap1 GTPase-activating protein 1. Collectively, these proteomic results increase our understanding of the molecular resistance mechanisms to Bt Cry1Ac toxin in P. xylostella and also demonstrate that resistance to Bt Cry1Ac toxin is complex and multifaceted. PMID:27521921

  5. 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 Central

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

    2014-01-01

    Abstract 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. PMID:24803409

  6. Establishment of a paclitaxel resistant human breast cancer cell strain (MCF-7/Taxol) and intracellular paclitaxel binding protein analysis.

    PubMed

    Zuo, K-Q; Zhang, X-P; Zou, J; Li, D; Lv, Z-W

    2010-01-01

    Multidrug resistance of tumours is one of the most important factors that leads to chemotherapy failure. A multidrug-resistant breast cancer cell line, MCF-7/Taxol, was established from the drug-sensitive parent cell line MCF-7. The biological properties of MCF-7/Taxol, including its drug resistance profile and profile of paclitaxel binding proteins, were analysed and compared with the parent cell line. A number of paclitaxel binding proteins were present in MCF-7 cells but absent from MCF-7/Taxol cells, namely heat shock protein 90, actinin and dermcidin precursor. The identification of differential paclitaxel binding proteins between the multidrug-resistant MCF-7/Taxol cell line and the parent drug-sensitive cell line MCF-7 provides insight into possible mechanisms involved in resistance to these chemotherapy drugs.

  7. A systemic resistance inducing antiviral protein with N-glycosidase activity from Bougainvillea xbuttiana leaves.

    PubMed

    Narwal, S; Balasubrahmanyam, A; Sadhna, P; Kapoor, H; Lodha, M L

    2001-06-01

    An antiviral protein from Bougainvillea xbuttiana leaves induced systemic resistance in host plants N. glutinosa and Cyamopsis tetragonoloba against TMV and SRV, respectively which was reversed by actinomycin D, when applied immediately or shortly after antiviral protein treatment. When the inhibitor was applied to the host plant leaves post inoculation, it was effective if applied upto 4 h after virus infection. It also delayed the expression of symptoms in systemic hosts of TMV. The inhibitor showed characteristic N-glycosidase activity on 25S rRNA of tobacco ribosomes, suggesting that it could also be interfering with virus multiplication through ribosome-inactivation process. PMID:12562026

  8. Pokeweed antiviral protein: its cytotoxicity mechanism and applications in plant disease resistance.

    PubMed

    Di, Rong; Tumer, Nilgun E

    2015-03-06

    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.

  9. Bisubstrate Inhibitors of Biotin Protein Ligase in Mycobacterium tuberculosis Resistant to Cyclonucleoside Formation

    PubMed Central

    2013-01-01

    Mycobacterium tuberculosis (Mtb), the etiological agent of tuberculosis, is the leading cause of bacterial infectious disease mortality. Biotin protein ligase (BirA) globally regulates lipid metabolism in Mtb through the posttranslational biotinylation of acyl coenzyme A carboxylases (ACCs) involved in lipid biosynthesis and is essential for Mtb survival. We previously developed a rationally designed bisubstrate inhibitor of BirA that displays potent enzyme inhibition and whole-cell activity against multidrug resistant and extensively drug resistant Mtb strains. Here we present the design, synthesis, and evaluation of a focused series of inhibitors, which are resistant to cyclonucleoside formation, a key decomposition pathway of our initial analogue. Improved chemical stability is realized through replacement of the adenosyl N-3 nitrogen and C-5′ oxygen atom with carbon as well as incorporation of a bulky group on the nucleobase to prevent the required syn-conformation necessary for proper alignment of N-3 with C-5′. PMID:24363833

  10. Protein kinase Cα protects against multidrug resistance in human colon cancer cells.

    PubMed

    Lee, Se-Kyoung; Shehzad, Adeeb; Jung, Jae-Chang; Sonn, Jong-Kyung; Lee, Jae-Tae; Park, Jeen-Woo; Lee, Young-Sup

    2012-07-01

    Multidrug resistance is the phenomenon by which, after exposure to a single chemotherapeutic agent, cancer cells evade the agent's cytotoxic effects as well as become resistant to several classes of diverse drugs. ATP-binding cassette (ABC) transporters are a family of transporter proteins that contribute to drug resistance via a n ATP - dependent drug efflux pump. P-glycoprotein (P-gp) is a prominent ABC superfamily protein encoded by the mdr gene which has the ability to mediate the cellular extrusion of xenobiotics and anticancer drugs from tumor cells. Exclusively expressed P-gp cells from the human colon cancer HCT15/DOX line showed resistance to doxorubicin while parental HCT15 cells treated with doxorubicin displayed typical signs of apoptosis. In order to verify the hypothesis that expression of MDR is controlled in part, by protein kinase C (PKC), expression patterns of different PKC isoforms were examined in both cell lines. Of the PKC isoforms evaluated, the membrane translocation and expression levels of PKCα were strikingly increased in HCT15/DOX cells. PKCα reversed doxorubicin-induced apoptosis through the scavenging of ROS as well as inhibition of PARP cleavage. In addition, inhibition of PKCα with Go6976, a specific inhibitor of classical PKC, led to reduced MDR expression and increased doxorubicin-induced apoptosis. Knockdown of PKCα by siRNA diminished the protective effects of PKCα for doxorubicin-induced apoptosis. These results suggested that over-expression and activity of PKCα is closely associated with the regulation of the MDR phenotype in human colon cancer HCT15 cells and provided insight into a new strategy for inhibiting doxorubicin resistance in human cancers. PMID:22639047

  11. The Flagellum of Pseudomonas aeruginosa Is Required for Resistance to Clearance by Surfactant Protein A

    PubMed Central

    Zhang, Shiping; McCormack, Francis X.; Levesque, Roger C.; O'Toole, George A.; Lau, Gee W.

    2007-01-01

    Surfactant protein A (SP-A) is an important lung innate immune protein that kills microbial pathogens by opsonization and membrane permeabilization. We investigated the basis of SP-A-mediated pulmonary clearance of Pseudomonas aeruginosa using genetically-engineered SP-A mice and a library of signature-tagged P. aeruginosa mutants. A mutant with an insertion into flgE, the gene that encodes flagellar hook protein, was preferentially cleared by the SP-A+/+ mice, but survived in the SP-A−/− mice. Opsonization by SP-A did not play a role in flgE clearance. However, exposure to SP-A directly permeabilized and killed the flgE mutant, but not the wild-type parental strain. P. aeruginosa strains with mutation in other flagellar genes, as well as mucoid, nonmotile isolates from cystic fibrosis patients, were also permeabilized by SP-A. Provision of the wild-type fliC gene restored the resistance to SP-A-mediated membrane permeabilization in the fliC-deficient bacteria. In addition, non-mucoid, motile revertants of CF isolates reacquired resistance to SP-A-mediated membrane permeability. Resistance to SP-A was dependent on the presence of an intact flagellar structure, and independent of flagellar-dependent motility. We provide evidence that flagellar-deficient mutants harbor inadequate amounts of LPS required to resist membrane permeabilization by SP-A and cellular lysis by detergent targeting bacterial outer membranes. Thus, the flagellum of P. aeruginosa plays an indirect but important role resisting SP-A-mediated clearance and membrane permeabilization. PMID:17593964

  12. 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

  13. Rituximab in treatment-resistant CIDP with antibodies against paranodal proteins

    PubMed Central

    Querol, Luis; Rojas-García, Ricard; Diaz-Manera, Jordi; Barcena, Joseba; Pardo, Julio; Ortega-Moreno, Angel; Sedano, Maria Jose; Seró-Ballesteros, Laia; Carvajal, Alejandra; Ortiz, Nicolau; Gallardo, Eduard

    2015-01-01

    Objective: To describe the response to rituximab in patients with treatment-resistant chronic inflammatory demyelinating polyneuropathy (CIDP) with antibodies against paranodal proteins and correlate the response with autoantibody titers. Methods: Patients with CIDP and IgG4 anti–contactin-1 (CNTN1) or anti–neurofascin-155 (NF155) antibodies who were resistant to IV immunoglobulin and corticosteroids were treated with rituximab and followed prospectively. Immunocytochemistry was used to detect anti-CNTN1 and anti-NF155 antibodies and ELISA with human recombinant CNTN1 and NF155 proteins was used to determine antibody titers. Results: Two patients had a marked improvement; another patient improved slightly after 10 years of stable, severe disease; and the fourth patient had an ischemic stroke unrelated to treatment and was lost to follow-up. Autoantibodies decreased in all patients after rituximab treatment. Conclusions: Rituximab treatment is an option for patients with CIDP with IgG4 anti-CNTN1/NF155 antibodies who are resistant to conventional therapies. Classification of evidence: This study provides Class IV evidence that rituximab is effective for patients with treatment-resistant CIDP with IgG4 anti-CNTN1 or anti-NF155 antibodies. PMID:26401517

  14. 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.

  15. Genomic structure, gene expression, and promoter analysis of human multidrug resistance-associated protein 7

    SciTech Connect

    Kao, Hsin-Hsin; Chang, Ming-Shi; Cheng, Jan-Fang; Huang, Jin-Ding

    2002-03-15

    The multidrug resistance-associated protein (MRP) subfamily transporters associated with anticancer drug efflux are attributed to the multidrug-resistance of cancer cells. The genomic organization of human multidrug resistance-associated protein 7 (MRP7) was identified. The human MRP7 gene, consisting of 22 exons and 21 introns, greatly differs from other members of the human MRP subfamily. A splicing variant of human MRP7, MRP7A, expressed in most human tissues, was also characterized. The 1.93-kb promoter region of MRP7 was isolated and shown to support luciferase activity at a level 4- to 5-fold greater than that of the SV40 promoter. Basal MRP7 gene expression was regulated by 2 regions in the 5-flanking region at 1,780 1,287 bp, and at 611 to 208 bp. In Madin-Darby canine kidney (MDCK) cells, MRP7 promoter activity was increased by 226 percent by genotoxic 2-acetylaminofluorene and 347 percent by the histone deacetylase inhibitor, trichostatin A. The protein was expressed in the membrane fraction of transfected MDCK cells.

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

    PubMed Central

    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. PMID:26020775

  17. Electrochemical deposition and surface-initiated RAFT polymerization: protein and cell-resistant PPEGMEMA polymer brushes.

    PubMed

    Tria, Maria Celeste R; Grande, Carlos David T; Ponnapati, Ramakrishna R; Advincula, Rigoberto C

    2010-12-13

    This paper introduces a novel and versatile method of grafting protein and cell-resistant poly(poly ethylene glycol methyl ether methacrylate) (PPEGMEMA) brushes on conducting Au surface. The process started with the electrochemical deposition and full characterization of an electro-active chain transfer agent (CTA) on the Au surface. The electrochemical behavior of the CTA was investigated by cyclic voltammetry (CV) while the deposition and stability of the CTA on the surface were confirmed by ellipsometry, contact angle, and X-ray photoelectron spectroscopy (XPS). The capability of the electrodeposited CTA to mediate surface-initiated reversible addition-fragmentation chain transfer (SI-RAFT) polymerization on both the polymethyl methacrylate (PMMA; model polymer) and PPEGMEMA brushes was demonstrated by the increase in thicknesses of the films after polymerization. Contact angles also decreased with the incorporation of the more hydrophilic brushes. Significant changes in the morphologies of the films before and after polymerization were also observed with atomic force microscopy (AFM) analyses. Furthermore, XPS results showed an increase in the O 1s peak intensity relative to C 1s after polymerizations, which confirmed the grafting of polyethyleneglycol (PEG) bearing brushes. The ability of the PPEGMEMA-modified Au surface to resist nonspecific adhesion of proteins and cells was monitored and confirmed by XPS, ellipsometry, contact angle, AFM, and fluorescence imaging. The new method presented has potential application as robust protein and cell-resistant coatings for electrically conducting electrodes and biomedical devices.

  18. 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.

  19. Pyrrolopyrimidine Derivatives as Novel Inhibitors of Multidrug Resistance-Associated Protein 1 (MRP1, ABCC1).

    PubMed

    Schmitt, Sven Marcel; Stefan, Katja; Wiese, Michael

    2016-04-14

    Five series of pyrrolo[3,2-d]pyrimidines were synthesized and evaluated with respect to potency and selectivity toward multidrug resistance-associated protein 1 (MRP1, ABCC1). This transport protein is a major target to overcome multidrug resistance in cancer patients. We investigated differently substituted pyrrolopyrimidines using the doxorubicin selected and MRP1 overexpressing small cell lung cancer cell line H69 AR in a calcein AM and daunorubicin cell accumulation assay. New compounds with high potency and selectivity were identified. Piperazine residues at position 4 bearing large phenylalkyl side chains proved to be beneficial for MRP1 inhibition. Its replacement by an amino group led to decreased activity. Aliphatic and aliphatic-aromatic variations at position 5 and 6 revealed compounds with IC50 values in high nanomolar range. All investigated compounds had low affinity toward P-glycoprotein (P-gp, ABCB1). Pyrrolopyrimidines with small substituents showed moderate inhibition against breast cancer resistance protein (BCRP, ABCG2). PMID:26943020

  20. Arabidopsis resistance protein SNC1 activates immune responses through association with a transcriptional corepressor

    PubMed Central

    Zhu, Zhaohai; Xu, Fang; Zhang, Yaxi; Cheng, Yu Ti; Wiermer, Marcel; Li, Xin; Zhang, Yuelin

    2010-01-01

    In both plants and animals, nucleotide-binding (NB) domain and leucine-rich repeat (LRR)-containing proteins (NLR) function as sensors of pathogen-derived molecules and trigger immune responses. Although NLR resistance (R) proteins were first reported as plant immune receptors more than 15 years ago, how these proteins activate downstream defense responses is still unclear. Here we report that the Toll-like/interleukin-1 receptor (TIR)-NB-LRR R protein, suppressor of npr1-1, constitutive 1 (SNC1) functions through its associated protein, Topless-related 1 (TPR1). Knocking out TPR1 and its close homologs compromises immunity mediated by SNC1 and several other TIR-NB-LRR–type R proteins, whereas overexpression of TPR1 constitutively activates SNC1-mediated immune responses. TPR1 functions as a transcriptional corepressor and associates with histone deacetylase 19 in vivo. Among the target genes of TPR1 are Defense no Death 1 (DND1) and Defense no Death 2 (DND2), two known negative regulators of immunity that are repressed during pathogen infection, suggesting that TPR1 activates R protein-mediated immune responses through repression of negative regulators. PMID:20647385

  1. Efflux by small multidrug resistance proteins is inhibited by membrane-interactive helix-stapled peptides.

    PubMed

    Bellmann-Sickert, Kathrin; Stone, Tracy A; Poulsen, Bradley E; Deber, Charles M

    2015-01-16

    Bacterial cell membranes contain several protein pumps that resist the toxic effects of drugs by efficiently extruding them. One family of these pumps, the small multidrug resistance proteins (SMRs), consists of proteins of about 110 residues that need to oligomerize to form a structural pathway for substrate extrusion. As such, SMR oligomerization sites should constitute viable targets for efflux inhibition, by disrupting protein-protein interactions between helical segments. To explore this proposition, we are using Hsmr, an SMR from Halobacter salinarum that dimerizes to extrude toxicants. Our previous work established that (i) Hsmr dimerization is mediated by a helix-helix interface in Hsmr transmembrane (TM) helix 4 (residues (90)GLALIVAGV(98)); and (ii) a peptide comprised of the full TM4(85-105) sequence inhibits Hsmr-mediated ethidium bromide efflux from bacterial cells. Here we define the minimal linear sequence for inhibitor activity (determined as TM4(88-100), and then "staple" this sequence via Grubbs metathesis to produce peptides typified by acetyl-A-(Sar)3-(88)VVGLXLIZXGVVV(100)-KKK-NH2 (X = 2-(4'-pentenyl)alanine at positions 92 and 96; Z = Val, Gly, or Asn at position 95)). The Asn(95) peptide displayed specific efflux inhibition and resensitization of Hsmr-expressing cells to ethidium bromide; and was non-hemolytic to human red blood cells. Stapling essentially prevented peptide degradation in blood plasma and liver homogenates versus an unstapled counterpart. The overall results confirm that the stapled analog of TM4(88-100) retains the structural complementarity required to disrupt the Hsmr TM4-TM4 locus in Hsmr, and portend the general validity of stapled peptides as therapeutics for the disruption of functional protein-protein interactions in membranes.

  2. Efflux by Small Multidrug Resistance Proteins Is Inhibited by Membrane-interactive Helix-stapled Peptides*

    PubMed Central

    Bellmann-Sickert, Kathrin; Stone, Tracy A.; Poulsen, Bradley E.; Deber, Charles M.

    2015-01-01

    Bacterial cell membranes contain several protein pumps that resist the toxic effects of drugs by efficiently extruding them. One family of these pumps, the small multidrug resistance proteins (SMRs), consists of proteins of about 110 residues that need to oligomerize to form a structural pathway for substrate extrusion. As such, SMR oligomerization sites should constitute viable targets for efflux inhibition, by disrupting protein-protein interactions between helical segments. To explore this proposition, we are using Hsmr, an SMR from Halobacter salinarum that dimerizes to extrude toxicants. Our previous work established that (i) Hsmr dimerization is mediated by a helix-helix interface in Hsmr transmembrane (TM) helix 4 (residues 90GLALIVAGV98); and (ii) a peptide comprised of the full TM4(85–105) sequence inhibits Hsmr-mediated ethidium bromide efflux from bacterial cells. Here we define the minimal linear sequence for inhibitor activity (determined as TM4(88–100), and then “staple” this sequence via Grubbs metathesis to produce peptides typified by acetyl-A-(Sar)3-88VVGLXLIZXGVVV100-KKK-NH2 (X = 2-(4′-pentenyl)alanine at positions 92 and 96; Z = Val, Gly, or Asn at position 95)). The Asn95 peptide displayed specific efflux inhibition and resensitization of Hsmr-expressing cells to ethidium bromide; and was non-hemolytic to human red blood cells. Stapling essentially prevented peptide degradation in blood plasma and liver homogenates versus an unstapled counterpart. The overall results confirm that the stapled analog of TM4(88–100) retains the structural complementarity required to disrupt the Hsmr TM4-TM4 locus in Hsmr, and portend the general validity of stapled peptides as therapeutics for the disruption of functional protein-protein interactions in membranes. PMID:25425644

  3. Mutations in G protein β subunits promote transformation and kinase inhibitor resistance.

    PubMed

    Yoda, Akinori; Adelmant, Guillaume; Tamburini, Jerome; Chapuy, Bjoern; Shindoh, Nobuaki; Yoda, Yuka; Weigert, Oliver; Kopp, Nadja; Wu, Shuo-Chieh; Kim, Sunhee S; Liu, Huiyun; Tivey, Trevor; Christie, Amanda L; Elpek, Kutlu G; Card, Joseph; Gritsman, Kira; Gotlib, Jason; Deininger, Michael W; Makishima, Hideki; Turley, Shannon J; Javidi-Sharifi, Nathalie; Maciejewski, Jaroslaw P; Jaiswal, Siddhartha; Ebert, Benjamin L; Rodig, Scott J; Tyner, Jeffrey W; Marto, Jarrod A; Weinstock, David M; Lane, Andrew A

    2015-01-01

    Activating mutations in genes encoding G protein α (Gα) subunits occur in 4-5% of all human cancers, but oncogenic alterations in Gβ subunits have not been defined. Here we demonstrate that recurrent mutations in the Gβ proteins GNB1 and GNB2 confer cytokine-independent growth and activate canonical G protein signaling. Multiple mutations in GNB1 affect the protein interface that binds Gα subunits as well as downstream effectors and disrupt Gα interactions with the Gβγ dimer. Different mutations in Gβ proteins clustered partly on the basis of lineage; for example, all 11 GNB1 K57 mutations were in myeloid neoplasms, and seven of eight GNB1 I80 mutations were in B cell neoplasms. Expression of patient-derived GNB1 variants in Cdkn2a-deficient mouse bone marrow followed by transplantation resulted in either myeloid or B cell malignancies. In vivo treatment with the dual PI3K-mTOR inhibitor BEZ235 suppressed GNB1-induced signaling and markedly increased survival. In several human tumors, mutations in the gene encoding GNB1 co-occurred with oncogenic kinase alterations, including the BCR-ABL fusion protein, the V617F substitution in JAK2 and the V600K substitution in BRAF. Coexpression of patient-derived GNB1 variants with these mutant kinases resulted in inhibitor resistance in each context. Thus, GNB1 and GNB2 alterations confer transformed and resistance phenotypes across a range of human tumors and may be targetable with inhibitors of G protein signaling. PMID:25485910

  4. 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.

  5. Contributions of Aspergillus fumigatus ATP-Binding Cassette Transporter Proteins to Drug Resistance and Virulence

    PubMed Central

    Paul, Sanjoy; Diekema, Daniel

    2013-01-01

    In yeast cells such as those of Saccharomyces cerevisiae, expression of ATP-binding cassette (ABC) transporter proteins has been found to be increased and correlates with a concomitant elevation in azole drug resistance. In this study, we investigated the roles of two Aspergillus fumigatus proteins that share high sequence similarity with S. cerevisiae Pdr5, an ABC transporter protein that is commonly overproduced in azole-resistant isolates in this yeast. The two A. fumigatus genes encoding the ABC transporters sharing the highest sequence similarity to S. cerevisiae Pdr5 are called abcA and abcB here. We constructed deletion alleles of these two different ABC transporter-encoding genes in three different strains of A. fumigatus. Loss of abcB invariably elicited increased azole susceptibility, while abcA disruption alleles had variable phenotypes. Specific antibodies were raised to both AbcA and AbcB proteins. These antisera allowed detection of AbcB in wild-type cells, while AbcA could be visualized only when overproduced from the hspA promoter in A. fumigatus. Overproduction of AbcA also yielded increased azole resistance. Green fluorescent protein fusions were used to provide evidence that both AbcA and AbcB are localized to the plasma membrane in A. fumigatus. Promoter fusions to firefly luciferase suggested that expression of both ABC transporter-encoding genes is inducible by azole challenge. Virulence assays implicated AbcB as a possible factor required for normal pathogenesis. This work provides important new insights into the physiological roles of ABC transporters in this major fungal pathogen. PMID:24123268

  6. Mutation in ribosomal protein S5 leads to spectinomycin resistance in Neisseria gonorrhoeae.

    PubMed

    Ilina, Elena N; Malakhova, Maya V; Bodoev, Ivan N; Oparina, Nina Y; Filimonova, Alla V; Govorun, Vadim M

    2013-01-01

    Spectinomycin remains a useful reserve option for therapy of gonorrhea. The emergence of multidrug-resistant Neisseria gonorrhoeae strains with decreased susceptibility to cefixime and to ceftriaxone makes it the only medicine still effective for treatment of gonorrhea infection in analogous cases. However, adoption of spectinomycin as a routinely used drug of choice was soon followed by reports of spectinomycin resistance. The main molecular mechanism of spectinomycin resistance in N. gonorrhoeae was C1192T substitution in 16S rRNA genes. Here we reported a Thr-24→Pro mutation in ribosomal protein S5 (RPS5) found in spectinomycin resistant clinical N. gonorrhoeae strain, which carried no changes in 16S rRNA. In a series of experiments, the transfer of rpsE gene allele encoding the mutant RPS5 to the recipient N. gonorrhoeae strains was analyzed. The relatively high rate of transformation [ca. 10(-5) colony-forming units (CFUs)] indicates the possibility of spread of spectinonycin resistance within gonococcal population due to the horizontal gene transfer (HGT). PMID:23847609

  7. Identification of Siah-interacting protein as a potential regulator of apoptosis and curcumin resistance.

    PubMed

    Luo, J; Yang, J; Yu, B-Y; Liu, W; Li, M; Zhuang, S-M

    2010-12-01

    The mechanism underlying curcumin (diferuloylmethane) resistance is still largely unknown. Here we employed proteomic approach to identify the Siah-interacting protein (SIP) as a candidate for detailed study, because the spot intensity of SIP on a two-dimensional gel displayed 70-90% reduction in curcumin-sensitive cells, but remained unchanged in curcumin-resistant sublines, after curcumin treatment. Both gain- and loss-of-function studies revealed that SIP promoted curcumin-induced apoptosis. Moreover, SIP underwent phosphorylation and nuclear translocation in curcumin-sensitive but not resistant cells, upon curcumin exposure. The nuclear translocation of SIP was remarkably impaired when a putative nuclear localization sequence (NLS, amino acid (aa) 143-159) was deleted or the serine 141 was mutated into alanine, whereas truncation of the N-terminal region (aa 1-43) obviously increased the nuclear import of SIP. In accordance with their nuclear localization, N-terminal truncation significantly enhanced the proapoptotic effect of SIP, whereas NLS deletion or Ser141Ala mutation attenuated the apoptosis-promoting activity of both wild-type- and N-terminal truncated-SIP. These data suggest that SIP plays a role in apoptosis and curcumin resistance, and the function of SIP may be regulated by different motifs, such as the NLS, N-terminal region and serine 141. Our findings provide new insights into the biological significance of SIP and the mechanisms of drug resistance.

  8. Short-term muscle disuse lowers myofibrillar protein synthesis rates and induces anabolic resistance to protein ingestion.

    PubMed

    Wall, Benjamin T; Dirks, Marlou L; Snijders, Tim; van Dijk, Jan-Willem; Fritsch, Mario; Verdijk, Lex B; van Loon, Luc J C

    2016-01-15

    Disuse leads to rapid loss of skeletal muscle mass and function. It has been hypothesized that short successive periods of muscle disuse throughout the lifespan play an important role in the development of sarcopenia. The physiological mechanisms underlying short-term muscle disuse atrophy remain to be elucidated. We assessed the impact of 5 days of muscle disuse on postabsorptive and postprandial myofibrillar protein synthesis rates in humans. Twelve healthy young (22 ± 1 yr) men underwent a 5-day period of one-legged knee immobilization (full leg cast). Quadriceps cross-sectional area (CSA) of both legs was assessed before and after immobilization. Continuous infusions of l-[ring-(2)H5]phenylalanine and l-[1-(13)C]leucine were combined with the ingestion of a 25-g bolus of intrinsically l-[1-(13)C]phenylalanine- and l-[1-(13)C]leucine-labeled dietary protein to assess myofibrillar muscle protein fractional synthetic rates in the immobilized and nonimmobilized control leg. Immobilization led to a 3.9 ± 0.6% decrease in quadriceps muscle CSA of the immobilized leg. Based on the l-[ring-(2)H5]phenylalanine tracer, immobilization reduced postabsorptive myofibrillar protein synthesis rates by 41 ± 13% (0.015 ± 0.002 vs. 0.032 ± 0.005%/h, P < 0.01) and postprandial myofibrillar protein synthesis rates by 53 ± 4% (0.020 ± 0.002 vs. 0.044 ± 0.003%/h, P < 0.01). Comparable results were found using the l-[1-(13)C]leucine tracer. Following protein ingestion, myofibrillar protein bound l-[1-(13)C]phenylalanine enrichments were 53 ± 18% lower in the immobilized compared with the control leg (0.007 ± 0.002 and 0.015 ± 0.002 mole% excess, respectively, P < 0.05). We conclude that 5 days of muscle disuse substantially lowers postabsorptive myofibrillar protein synthesis rates and induces anabolic resistance to protein ingestion.

  9. A Lipid Transfer Protein Increases the Glutathione Content and Enhances Arabidopsis Resistance to a Trichothecene Mycotoxin

    PubMed Central

    McLaughlin, John E.; Bin-Umer, Mohamed Anwar; Widiez, Thomas; Finn, Daniel; McCormick, Susan; Tumer, Nilgun E.

    2015-01-01

    Fusarium head blight (FHB) or scab is one of the most important plant diseases worldwide, affecting wheat, barley and other small grains. Trichothecene mycotoxins such as deoxynivalenol (DON) accumulate in the grain, presenting a food safety risk and health hazard to humans and animals. Despite considerable breeding efforts, highly resistant wheat or barley cultivars are not available. We screened an activation tagged Arabidopsis thaliana population for resistance to trichothecin (Tcin), a type B trichothecene in the same class as DON. Here we show that one of the resistant lines identified, trichothecene resistant 1 (trr1) contains a T-DNA insertion upstream of two nonspecific lipid transfer protein (nsLTP) genes, AtLTP4.4 and AtLTP4.5. Expression of both nsLTP genes was induced in trr1 over 10-fold relative to wild type. Overexpression of AtLTP4.4 provided greater resistance to Tcin than AtLTP4.5 in Arabidopsis thaliana and in Saccharomyces cerevisiae relative to wild type or vector transformed lines, suggesting a conserved protection mechanism. Tcin treatment increased reactive oxygen species (ROS) production in Arabidopsis and ROS stain was associated with the chloroplast, the cell wall and the apoplast. ROS levels were attenuated in Arabidopsis and in yeast overexpressing AtLTP4.4 relative to the controls. Exogenous addition of glutathione and other antioxidants enhanced resistance of Arabidopsis to Tcin while the addition of buthionine sulfoximine, an inhibitor of glutathione synthesis, increased sensitivity, suggesting that resistance was mediated by glutathione. Total glutathione content was significantly higher in Arabidopsis and in yeast overexpressing AtLTP4.4 relative to the controls, highlighting the importance of AtLTP4.4 in maintaining the redox state. These results demonstrate that trichothecenes cause ROS accumulation and overexpression of AtLTP4.4 protects against trichothecene-induced oxidative stress by increasing the glutathione

  10. 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

  11. A small RNA controls a protein regulator involved in antibiotic resistance in Staphylococcus aureus

    PubMed Central

    Eyraud, Alex; Tattevin, Pierre; Chabelskaya, Svetlana; Felden, Brice

    2014-01-01

    The emergence of Staphylococcus aureus strains that are resistant to glycopeptides has led to alarming scenarios where serious staphylococcal infections cannot be treated. The bacterium expresses many small regulatory RNAs (sRNAs) that have unknown biological functions for the most part. Here we show that an S. aureus sRNA, SprX (alias RsaOR), shapes bacterial resistance to glycopeptides, the invaluable treatments for Methicillin-resistant staphylococcal infections. Modifying SprX expression levels influences Vancomycin and Teicoplanin glycopeptide resistance. Comparative proteomic studies have identified that SprX specifically downregulates stage V sporulation protein G, SpoVG. SpoVG is produced from the yabJ-spoVG operon and contributes to S. aureus glycopeptide resistance. SprX negatively regulates SpoVG expression by direct antisense pairings at the internal translation initiation signals of the second operon gene, without modifying bicistronic mRNA expression levels or affecting YabJ translation. The SprX and yabJ-spoVG mRNA domains involved in the interaction have been identified, highlighting the importance of a CU-rich loop of SprX in the control of SpoVG expression. We have shown that SpoVG might not be the unique SprX target involved in the glycopeptide resistance and demonstrated that the regulation of glycopeptide sensitivity involves the CU-rich domain of SprX. Here we report the case of a sRNA influencing antibiotic resistance of a major human pathogen. PMID:24557948

  12. A very early induction of major vault protein accompanied by increased drug resistance in U-937 cells.

    PubMed

    Hu, Yi; Stephen, Andrew G; Cao, Jin; Tanzer, Lee R; Slapak, Christopher A; Harrison, Steadman D; Devanarayan, Viswanath; Dantzig, Anne H; Starling, James J; Rome, Leonard H; Moore, Robert E

    2002-01-10

    U-937 human leukemia cells were selected for resistance to doxorubicin in the presence or absence of a specific drug modulator that inhibits the activity of P-glycoprotein (Pgp), encoded by the multidrug-resistance gene (MDR1). Parental cells expressed low basal levels of the multidrug-resistance-associated gene (MRP1) and major vault protein (MVP) mRNAs and no MDR1 mRNA. Two doxorubicin-resistant cell lines were selected. Both drug-resistant cell lines upregulated the MVP mRNA level 1.5-fold within 1 cell passage. The MVP mRNA level continued to increase over time as the doxorubicin selection pressure was increased. MVP protein levels generally paralleled the mRNA levels. The 2 high molecular weight vault protein mRNAs were always expressed at constitutive levels. Fully formed vault particles consisting of the MVP, the 2 high molecular weight proteins and the vault RNA assembled and accumulated to increased levels in drug-selected cells. MVP induction is therefore the rate-limiting step for vault particle formation in U-937 cells. By passage 25 and thereafter, the selected cells were resistant to doxorubicin, etoposide, mitoxantrone and 5-fluorouracil by a pathway that was independent of MDR1, MRP1, MRP2 and breast cancer resistance protein. In summary, U-937 doxorubicin-selected cells are programmed to rapidly upregulate MVP mRNA levels, to accumulate vault particles and to become multidrug resistant.

  13. 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

  14. Hemocompatible mixed-charge copolymer brushes of pseudozwitterionic surfaces resistant to nonspecific plasma protein fouling.

    PubMed

    Chang, Yung; Shu, Shih-Hung; Shih, Yu-Ju; Chu, Chih-Wei; Ruaan, Ruoh-Chyu; Chen, Wen-Yih

    2010-03-01

    In this work, the hemocompatibility of a sulfobetaine-like copolymer brush resulting from a mixed-charge copolymerization of the positively charged 11-mercapto-N,N,N-trimethylammonium chloride (TMA) and negatively charged 11-mercaptoundecylsulfonic acid (SA) was studied. Mixed charge distribution in the prepared poly(TMA-co-SA) copolymer brushes was controlled by the regulation of the reaction rate of the surface-initiated atom transfer radical polymerization (ATRP). The adsorption behavior of plasma proteins on a surface grafted with poly(TMA-co-SA) was measured by a surface plasmon resonance (SPR) sensor. The effects of varying temperature, solution pH, and ionic strength on the antifouling characteristics of the mixed-charge copolymer brushes were systematically evaluated, and the protein-fouling resistance was discussed in detail, especially with respect to the effect of ionic strength on the intra- and intermolecular interactions of the poly(TMA-co-SA) with proteins. The adhesion and activation of blood cells on the poly(TMA-co-SA)-grafted surface in contact with human whole blood was also demonstrated. The results suggest that mixed-charge copolymer brushes of poly(TMA-co-SA), which, like zwitterionic homopolymer brushes, have overall charge neutrality, can be used in similar applications for protein-fouling resistance and have excellent hemocompatibility with human whole blood at physiologic temperatures. PMID:19947616

  15. Investigation of Ligand Binding to the Multidrug Resistance Protein EmrE by Isothermal Titration Calorimetry

    PubMed Central

    Sikora, Curtis W.; Turner, Raymond J.

    2005-01-01

    Escherichia coli multidrug resistance protein E (EmrE) is an integral membrane protein spanning the inner membrane of Escherichia coli that is responsible for this organism's resistance to a variety of lipophilic cations such as quaternary ammonium compounds (QACs) and interchelating dyes. EmrE is a 12-kDa protein of four transmembrane helices considered to be functional as a multimer. It is an efflux transporter that can bind and transport cytoplasmic QACs into the periplasm using the energy of the proton gradient across the inner membrane. Isothermal titration calorimetry provides information about the stoichiometry and thermodynamic properties of protein-ligand interactions, and can be used to monitor the binding of QACs to EmrE in different membrane mimetic environments. In this study the ligand binding to EmrE solubilized in dodecyl maltoside, sodium dodecyl sulfate and reconstituted into small unilamellar vesicles is examined by isothermal titration calorimetry. The binding stoichiometry of EmrE to drug was found to be 1:1, demonstrating that oligomerization of EmrE is not necessary for binding to drug. The binding of EmrE to drug was observed with the dissociation constant (KD) in the micromolar range for each of the drugs in any of the membrane mimetic environments. Thermodynamic properties demonstrated this interaction to be enthalpy-driven with similar enthalpies of 8–12 kcal/mol for each of the drugs in any of the membrane mimetics. PMID:15501941

  16. Investigation of ligand binding to the multidrug resistance protein EmrE by isothermal titration calorimetry.

    PubMed

    Sikora, Curtis W; Turner, Raymond J

    2005-01-01

    Escherichia coli multidrug resistance protein E (EmrE) is an integral membrane protein spanning the inner membrane of Escherichia coli that is responsible for this organism's resistance to a variety of lipophilic cations such as quaternary ammonium compounds (QACs) and interchelating dyes. EmrE is a 12-kDa protein of four transmembrane helices considered to be functional as a multimer. It is an efflux transporter that can bind and transport cytoplasmic QACs into the periplasm using the energy of the proton gradient across the inner membrane. Isothermal titration calorimetry provides information about the stoichiometry and thermodynamic properties of protein-ligand interactions, and can be used to monitor the binding of QACs to EmrE in different membrane mimetic environments. In this study the ligand binding to EmrE solubilized in dodecyl maltoside, sodium dodecyl sulfate and reconstituted into small unilamellar vesicles is examined by isothermal titration calorimetry. The binding stoichiometry of EmrE to drug was found to be 1:1, demonstrating that oligomerization of EmrE is not necessary for binding to drug. The binding of EmrE to drug was observed with the dissociation constant (K(D)) in the micromolar range for each of the drugs in any of the membrane mimetic environments. Thermodynamic properties demonstrated this interaction to be enthalpy-driven with similar enthalpies of 8-12 kcal/mol for each of the drugs in any of the membrane mimetics.

  17. Interaction Forces and Morphology of a Protein-Resistant Poly(ethylene glycol) Layer

    PubMed Central

    Heuberger, M.; Drobek, T.; Spencer, N. D.

    2005-01-01

    The molecular interactions on a protein-resistant surface coated with low-molecular-weight poly(ethylene glycol) (PEG) copolymer brushes are investigated using the extended surface forces apparatus. The observed interaction force is predominantly repulsive and nearly elastic. The chains are extended with respect to the Flory radius, which is in agreement with qualitative predictions of scaling theory. Comparison with theory allows the determination of relevant quantities such as brush length and adsorbed mass. Based on these results, we propose a molecular model for the adsorbed copolymer morphology. Surface-force isotherms measured at high resolution allow distinctive structural forces to be detected, suggesting the existence of a weak equilibrium network between poly(ethylene glycol) and water—a finding in accordance with the remarkable solution properties of PEG. The occurrence of a fine structure is interpreted as a water-induced restriction of the polymer's conformational space. This restriction is highly relevant for the phenomenon of PEG protein resistance. Protein adsorption requires conformational transitions, both in the protein as well as in the PEG layer, which are energetically and kinetically unfavorable. PMID:15501935

  18. A novel inducible protein production system and neomycin resistance as selection marker for Methanosarcina mazei.

    PubMed

    Mondorf, Sebastian; Deppenmeier, Uwe; Welte, Cornelia

    2012-01-01

    Methanosarcina mazei is one of the model organisms for the methanogenic order Methanosarcinales whose metabolism has been studied in detail. However, the genetic toolbox is still limited. This study was aimed at widening the scope of utilizable methods in this group of organisms. (i) Proteins specific to methanogens are oftentimes difficult to produce in E. coli. However, a protein production system is not available for methanogens. Here we present an inducible system to produce Strep-tagged proteins in Ms. mazei. The promoter p1687, which directs the transcription of methyl transferases that demethylate methylamines, was cloned into plasmid pWM321 and its activity was determined by monitoring β-glucuronidase production. The promoter was inactive during growth on methanol but was rapidly activated when trimethylamine was added to the medium. The gene encoding the β-glucuronidase from E. coli was fused to a Strep-tag and was cloned downstream of the p1687 promoter. The protein was overproduced in Ms. mazei and was purified in an active form by affinity chromatography. (ii) Puromycin is currently the only antibiotic used as a selectable marker in Ms. mazei and its relatives. We established neomycin resistance as a second selectable marker by designing a plasmid that confers neomycin resistance in Ms. mazei.

  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. 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.

  2. 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.

  3. Polygalacturonase inhibitor protein from fruits of anthracnose resistant and susceptible varieties of Chilli (Capsicum annuum L).

    PubMed

    Shivashankar, S; Thimmareddy, C; Roy, Tapas K

    2010-08-01

    Chilli fruit is highly susceptible to anthracnose infection at the stage of harvest maturity, due to which the fruit yield in the leading commercial variety Byadgi is severely affected. Field studies on screening of several varieties for resistance to anthracnose have shown that a variety of chilli AR-4/99K is resistant to anthracnose infection. In many crops, resistance to fungal attack has been correlated with PGIP activity in developing fruits based on which transgenic varieties have been developed with resistance to fungi. The present study was carried out to determine whether anthracnose resistance in AR-4/99K was due to the increased levels of PGIP alone and/ or due to differences, if any, in the properties of PGIP. Hence, a comparative study of the properties of polygalacturonase inhibitor protein (PGIP) isolated from fruits of anthracnose resistant chilli var AR-4/99K and a susceptible variety Byadgi was conducted with the objective of utilizing the information in genetic transformation studies. Both the PGIPs from anthracnose resistant and susceptible varieties of chilli exhibited similarities in the elution pattern on Sephadex gel, DEAE cellulose, PAGE and SDS-PAGE. The two PGIPs were active over a wide range of pH and temperature. Both PGIPs showed differential inhibitory activity against polygalacturonase (PG) secreted by Colletotrichum gleosporoides, C. capsici, C. lindemuthianum, Fusarium moniliforme and Sclerotium rolfsii. The inhibitory activity of PGIP from both resistant and susceptible varieties was the highest (82% and 76%, respectively) against the PG from Colletotrichum capsici, a pathogen causing anthracnose rot of chilli, while the activity was lower (1.27 to 12.3%) on the other fungal PGs. Although PGIP activity decreased with fruit maturation in both the varieties, the resistant variety maintained a higher activity at 45 days after flowering (DAF) as compared to the susceptible variety which helped it to overcome the infection by

  4. Polygalacturonase inhibitor protein from fruits of anthracnose resistant and susceptible varieties of Chilli (Capsicum annuum L).

    PubMed

    Shivashankar, S; Thimmareddy, C; Roy, Tapas K

    2010-08-01

    Chilli fruit is highly susceptible to anthracnose infection at the stage of harvest maturity, due to which the fruit yield in the leading commercial variety Byadgi is severely affected. Field studies on screening of several varieties for resistance to anthracnose have shown that a variety of chilli AR-4/99K is resistant to anthracnose infection. In many crops, resistance to fungal attack has been correlated with PGIP activity in developing fruits based on which transgenic varieties have been developed with resistance to fungi. The present study was carried out to determine whether anthracnose resistance in AR-4/99K was due to the increased levels of PGIP alone and/ or due to differences, if any, in the properties of PGIP. Hence, a comparative study of the properties of polygalacturonase inhibitor protein (PGIP) isolated from fruits of anthracnose resistant chilli var AR-4/99K and a susceptible variety Byadgi was conducted with the objective of utilizing the information in genetic transformation studies. Both the PGIPs from anthracnose resistant and susceptible varieties of chilli exhibited similarities in the elution pattern on Sephadex gel, DEAE cellulose, PAGE and SDS-PAGE. The two PGIPs were active over a wide range of pH and temperature. Both PGIPs showed differential inhibitory activity against polygalacturonase (PG) secreted by Colletotrichum gleosporoides, C. capsici, C. lindemuthianum, Fusarium moniliforme and Sclerotium rolfsii. The inhibitory activity of PGIP from both resistant and susceptible varieties was the highest (82% and 76%, respectively) against the PG from Colletotrichum capsici, a pathogen causing anthracnose rot of chilli, while the activity was lower (1.27 to 12.3%) on the other fungal PGs. Although PGIP activity decreased with fruit maturation in both the varieties, the resistant variety maintained a higher activity at 45 days after flowering (DAF) as compared to the susceptible variety which helped it to overcome the infection by

  5. Characterisation of a cell wall-anchored protein of Staphylococcus saprophyticus associated with linoleic acid resistance

    PubMed Central

    2012-01-01

    Background The Gram-positive bacterium Staphylococcus saprophyticus is the second most frequent causative agent of community-acquired urinary tract infections (UTI), accounting for up to 20% of cases. A common feature of staphylococci is colonisation of the human skin. This involves survival against innate immune defenses including antibacterial unsaturated free fatty acids such as linoleic acid which act by disrupting bacterial cell membranes. Indeed, S. saprophyticus UTI is usually preceded by perineal skin colonisation. Results In this study we identified a previously undescribed 73.5 kDa cell wall-anchored protein of S. saprophyticus, encoded on plasmid pSSAP2 of strain MS1146, which we termed S. saprophyticus surface protein F (SssF). The sssF gene is highly prevalent in S. saprophyticus clinical isolates and we demonstrate that the SssF protein is expressed at the cell surface. However, unlike all other characterised cell wall-anchored proteins of S. saprophyticus, we were unable to demonstrate a role for SssF in adhesion. SssF shares moderate sequence identity to a surface protein of Staphylococcus aureus (SasF) recently shown to be an important mediator of linoleic acid resistance. Using a heterologous complementation approach in a S. aureus sasF null genetic background, we demonstrate that SssF is associated with resistance to linoleic acid. We also show that S. saprophyticus strains lacking sssF are more sensitive to linoleic acid than those that possess it. Every staphylococcal genome sequenced to date encodes SssF and SasF homologues. Proteins in this family share similar predicted secondary structures consisting almost exclusively of α-helices in a probable coiled-coil formation. Conclusions Our data indicate that SssF is a newly described and highly prevalent surface-localised protein of S. saprophyticus that contributes to resistance against the antibacterial effects of linoleic acid. SssF is a member of a protein family widely disseminated

  6. A new model for raf kinase inhibitory protein induced chemotherapeutic resistance.

    PubMed

    Al-Mulla, Fahd; Bitar, Milad S; Feng, Jingwei; Park, Sungdae; Yeung, Kam C

    2012-01-01

    Therapeutic resistance remains the most challenging aspect of treating cancer. Raf kinase inhibitory protein (RKIP) emerged as a molecule capable of sensitizing cancerous cells to radio- and chemotherapy. Moreover, this small evolutionary conserved molecule, endows significant resistance to cancer therapy when its expression is reduced or lost. RKIP has been shown to inhibit the Raf-MEK-ERK, NFκB, GRK and activate the GSK3β signaling pathways. Inhibition of Raf-MEK-ERK and NFκB remains the most prominent pathways implicated in the sensitization of cells to therapeutic drugs. Our purpose was to identify a possible link between RKIP-KEAP 1-NRF2 and drug resistance. To that end, RKIP-KEAP 1 association was tested in human colorectal cancer tissues using immunohistochemistry. RKIP miRNA silencing and its inducible overexpression were employed in HEK-293 immortalized cells, HT29 and HCT116 colon cancer cell lines to further investigate our aim. We show that RKIP enhanced Kelch-like ECH-associated protein1 (KEAP 1) stability in colorectal cancer tissues and HT29 CRC cell line. RKIP silencing in immortalized HEK-293 cells (termed HEK-499) correlated significantly with KEAP 1 protein degradation and subsequent NRF2 addiction in these cells. Moreover, RKIP depletion in HEK-499, compared to control cells, bestowed resistance to supra physiological levels of H(2)O(2) and Cisplatin possibly by upregulating NF-E2-related nuclear factor 2 (NRF2) responsive genes. Similarly, we observed a direct correlation between the extent of apoptosis, after treatment with Adriamycin, and the expression levels of RKIP/KEAP 1 in HT29 but not in HCT116 CRC cells. Our data illuminate, for the first time, the NRF2-KEAP 1 pathway as a possible target for personalized therapeutic intervention in RKIP depleted cancers.

  7. Association of serum retinol binding protein 4 and insulin resistance in apparently healthy adolescents.

    PubMed

    Lee, Duk-Chul; Lee, Ji-Won; Im, Jee-Aee

    2007-03-01

    Insulin resistance constitutes a pathophysiologic link between obesity, atherosclerosis, and/or cardiovascular complications. Retinol binding protein 4 (RBP4) is a newly discovered adipocyte product that modulates glucose metabolism and consequently induces insulin resistance. We investigated the association between serum RBP4 levels and insulin resistance in obese and nonobese adolescents. A total of 87 nonobese (60 males and 27 females) and 85 obese (62 males and 23 females) apparently healthy adolescents, 12 to 18 years old, were included in this study. A questionnaire was used to obtain participant medical history and lifestyle information, such as smoking and alcohol ingestion habits. Subjects' anthropometric measurements were taken to calculate for body mass index and waist-to-hip ratio. Serum RBP4 levels were measured by an enzyme immunoassay kit. High-sensitivity C-reactive protein, fasting glucose, total cholesterol, triglycerides, high-density lipoprotein cholesterol, and fasting insulin were measured. Low-density lipoprotein cholesterol level and homeostatic model assessment of insulin resistance (HOMA-IR) were calculated. Males had significantly higher RBP4 levels than females. Serum RBP4 levels were significantly higher in the obese group compared with the nonobese group. In all subjects, RBP4 was positively correlated with adiposity index (body mass index, waist circumference, waist-to-hip ratio), systolic and diastolic blood pressures, glucose tolerance index (fasting glucose, insulin, HOMA-IR), lipid profile (total cholesterol, triglycerides), and inflammatory indices (high-sensitivity C-reactive protein, white blood cell count). In multiple linear regression analysis, RBP4 was independently associated with age, HOMA-IR, and triglyceride levels in the nonobese group and with sex and triglyceride levels in the obese group. These results suggest that serum RBP4 might have clinical implications for lipid metabolism and insulin action in adolescents.

  8. Expression of lung resistance protein in epithelioid sarcoma in vitro and in vivo.

    PubMed

    Kusakabe, H; Iwasaki, H; Sano, K; Kiyokane, K

    2000-06-01

    The incidence of epithelioid sarcoma among patients with malignant soft tissue tumors is small, but the rates of recurrence and metastasis of this type of sarcoma are high. To date, effective chemotherapy for advanced epithelioid sarcoma has not been established and, furthermore, epithelioid sarcoma is known to exhibit multidrug resistance (MDR). The chemosensitivities to anticancer agents of two cell lines established from epithelioid sarcoma were examined in this study. The results showed that the ES-OMC-MN and SFT-8606 cell lines were resistant to vincristine (IC50 1190 nM and 872 nM, respectively) and Adriamycin (IC50 921 nM and 650 nM, respectively), but sensitive to actinomycin D (IC50 < 10 nM). P-glycoprotein (p-Gp) and MDR-associated protein (MRP) were not expressed in these cell lines, but a high expression level of lung resistance protein (LRP) was observed. The original tumor tissues from which the two cell lines were established were also found to be LRP-positive but not to express p-Gp or MRP. Their chemosensitivities to Adriamycin were not significantly altered in the presence of 2.5 microg/ml anti-LRP antibody (LRP-56), but the IC50 of vincristine was much less (IC50 128 nM and 27 nM, respectively) than that for an untreated cell line. It is thus suggested that the vincristine resistance in the two cell lines is LRP-mediated. Since cyclosporin A, known to be a modifier of p-Gp, also induced reversal of vincristine resistance in the ES-OMC-MN and SFT-8606 cell lines (IC50 6.2 nM and 17 nM, respectively), it is suggested that cyclosporin A acts as a modifier of MDR mediated by LRP.

  9. Insights from molecular modeling and dynamics simulation of pathogen resistance (R) protein from brinjal

    PubMed Central

    Shrivastava, Dipty; Nain, Vikrant; Sahi, Shakti; Verma, Anju; Sharma, Priyanka; Sharma, Prakash Chand; Kumar, Polumetla Ananda

    2011-01-01

    Resistance (R) protein recognizes molecular signature of pathogen infection and activates downstream hypersensitive response signalling in plants. R protein works as a molecular switch for pathogen defence signalling and represent one of the largest plant gene family. Hence, understanding molecular structure and function of R proteins has been of paramount importance for plant biologists. The present study is aimed at predicting structure of R proteins signalling domains (CC-NBS) by creating a homology model, refining and optimising the model by molecular dynamics simulation and comparing ADP and ATP binding. Based on sequence similarity with proteins of known structures, CC-NBS domains were initially modelled using CED- 4 (cell death abnormality protein) and APAF-1 (apoptotic protease activating factor) as multiple templates. The final CC-NBS structural model was built and optimized by molecular dynamic simulation for 5 nanoseconds (ns). Docking of ADP and ATP at active site shows that both ligand bind specifically with same residues and with minor difference (1 Kcal/mol) in binding energy. Sharing of binding site by ADP and ATP and low difference in their binding site makes CC-NBS suitable for working as molecular switch. Furthermore, structural superimposition elucidate that CC-NBS and CARD (caspase recruitment domains) domain of CED-4 have low RMSD value of 0.9 A° Availability of 3D structural model for both CC and NBS domains will . help in getting deeper insight in these pathogen defence genes. PMID:21383919

  10. Interplay between unfolded protein response and autophagy promotes tumor drug resistance

    PubMed Central

    YAN, MING-MING; NI, JIANG-DONG; SONG, DEYE; DING, MULIANG; HUANG, JUN

    2015-01-01

    The endoplasmic reticulum (ER) is involved in the quality control of secreted protein via promoting the correct folding of nascent protein and mediating the degradation of unfolded or misfolded protein, namely ER-associated degradation. When the unfolded or misfolded proteins are abundant, the unfolded protein response (UPR) is elicited, an adaptive signaling cascade from the ER to the nucleus, which restores the homeostatic functions of the ER. Autophagy is a conserved catabolic process where cellular long-lived proteins and damaged organelles are engulfed and degraded for recycling to maintain homeostasis. The UPR and autophagy occur simultaneously and are involved in pathological processes, including tumorigenesis, chemoresistance of malignancies and neurodegeneration. Accumulative data has indicated that the UPR may induce autophagy and that autophagy is able to alleviate the UPR. However, the detailed mechanism of interplay between autophagy and UPR remains to be fully understood. The present review aimed to depict the core pathways of the two processes and to elucidate how autophagy and UPR are regulated. Moreover, the review also discusses the molecular mechanism of crosstalk between the UPR and autophagy and their roles in malignant survival and drug resistance. PMID:26622781

  11. An E4 Ligase Facilitates Polyubiquitination of Plant Immune Receptor Resistance Proteins in Arabidopsis[W

    PubMed Central

    Huang, Yan; Minaker, Sean; Roth, Charlotte; Huang, Shuai; Hieter, Philip; Lipka, Volker; Wiermer, Marcel; Li, Xin

    2014-01-01

    Proteins with nucleotide binding and leucine-rich repeat domains (NLRs) serve as immune receptors in animals and plants that recognize pathogens and activate downstream defense responses. As high accumulation of NLRs can result in unwarranted autoimmune responses, their cellular concentrations must be tightly regulated. However, the molecular mechanisms of this process are poorly detailed. The F-box protein Constitutive expressor of PR genes 1 (CPR1) was previously identified as a component of a Skp1, Cullin1, F-box protein E3 complex that targets NLRs, including Suppressor of NPR1, Constitutive 1 (SNC1) and Resistance to Pseudomonas syringae 2 (RPS2), for ubiquitination and further protein degradation. From a forward genetic screen, we identified Mutant, snc1-enhancing 3 (MUSE3), an E4 ubiquitin ligase involved in polyubiquitination of its protein targets. Knocking out MUSE3 in Arabidopsis thaliana results in increased levels of NLRs, including SNC1 and RPS2, whereas overexpressing MUSE3 together with CPR1 enhances polyubiquitination and protein degradation of these immune receptors. This report on the functional role of an E4 ligase in plants provides insight into the scarcely understood NLR degradation pathway. PMID:24449689

  12. Direct interaction between the Arabidopsis disease resistance signaling proteins, EDS1 and PAD4.

    PubMed

    Feys, B J; Moisan, L J; Newman, M A; Parker, J E

    2001-10-01

    The Arabidopsis EDS1 and PAD4 genes encode lipase-like proteins that function in resistance (R) gene-mediated and basal plant disease resistance. Phenotypic analysis of eds1 and pad4 null mutants shows that EDS1 and PAD4 are required for resistance conditioned by the same spectrum of R genes but fulfil distinct roles within the defence pathway. EDS1 is essential for elaboration of the plant hypersensitive response, whereas EDS1 and PAD4 are both required for accumulation of the plant defence-potentiating molecule, salicylic acid. EDS1 is necessary for pathogen-induced PAD4 mRNA accumulation, whereas mutations in PAD4 or depletion of salicylic acid only partially compromise EDS1 expression. Yeast two-hybrid analysis reveals that EDS1 can dimerize and interact with PAD4. However, EDS1 dimerization is mediated by different domains to those involved in EDS1-PAD4 association. Co-immunoprecipitation experiments show that EDS1 and PAD4 proteins interact in healthy and pathogen-challenged plant cells. We propose two functions for EDS1. The first is required early in plant defence, independently of PAD4. The second recruits PAD4 in the amplification of defences, possibly by direct EDS1-PAD4 association.

  13. Trichoderma mitogen-activated protein kinase signaling is involved in induction of plant systemic resistance.

    PubMed

    Viterbo, Ada; Harel, Michal; Horwitz, Benjamin A; Chet, Ilan; Mukherjee, Prasun K

    2005-10-01

    The role of a mitogen-activated protein kinase (MAPK) TmkA in inducing systemic resistance in cucumber against the bacterial pathogen Pseudomonas syringae pv. lacrymans was investigated by using tmkA loss-of-function mutants of Trichoderma virens. In an assay where Trichoderma spores were germinated in proximity to cucumber roots, the mutants were able to colonize the plant roots as effectively as the wild-type strain but failed to induce full systemic resistance against the leaf pathogen. Interactions with the plant roots enhanced the level of tmkA transcript in T. virens and its homologue in Trichoderma asperellum. At the protein level, we could detect the activation of two forms reacting to the phospho-p44/42 MAPK antibody. Biocontrol experiments demonstrated that the tmkA mutants retain their biocontrol potential against Rhizoctonia solani in soil but are not effective against Sclerotium rolfsii in reducing disease incidence. Our results show that, unlike in many plant-pathogen interactions, Trichoderma TmkA MAPK is not involved in limited root colonization. Trichoderma, however, needs MAPK signaling in order to induce full systemic resistance in the plant.

  14. Deletions in a ribosomal protein-coding gene are associated with tigecycline resistance in Enterococcus faecium.

    PubMed

    Niebel, Marc; Quick, Joshua; Prieto, Ana Maria Guzman; Hill, Robert L R; Pike, Rachel; Huber, Damon; David, Miruna; Hornsey, Michael; Wareham, David; Oppenheim, Beryl; Woodford, Neil; van Schaik, Willem; Loman, Nicholas

    2015-11-01

    Enterococcus faecium is an emerging nosocomial pathogen associated with antibiotic therapy in the hospital environment. Whole-genome sequences were determined for three pairs of related, consecutively collected E. faecium clinical isolates to determine putative mechanisms of resistance to tigecycline. The first isolates (1S, 2S and 3S) in each of the three pairs were sensitive to tigecycline [minimum inhibitory concentration (MIC) of 0.125 mg/L]. Following tigecycline therapy, the second isolate in each pair demonstrated increased resistance to tigecycline. Two isolates (1R and 2R) were resistant (MIC of 8 mg/L) and one isolate (3I) demonstrated reduced susceptibility (MIC of 0.5 mg/L). Mutations distinguishing each pair of sensitive and resistant isolates were determined through alignment to a reference genome and variant detection. In addition, a de novo assembly of each isolate genome was constructed to confirm mutations. A total of 16 mutations in eleven coding sequences were determined. Mutations in the rpsJ gene, which encodes a structural protein forming part of the 30S ribosomal subunit, were detected in each of the pairs. Mutations were in regions proximal to the predicted tigecycline-binding site. Predicted amino acid substitutions were detected in 1R and 3I. The resistant strains were additionally associated with deletions of 15 nucleotides (2R) and 3 nucleotides (1R). This study confirms that amino acid substitutions in rpsJ contribute towards reduced susceptibility to tigecycline and suggests that deletions may be required for tigecycline resistance in E. faecium.

  15. 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

  16. Expression of Pokeweed Antiviral Protein in Transgenic Plants Induces Virus Resistance in Grafted Wild-Type Plants Independently of Salicylic Acid Accumulation and Pathogenesis-Related Protein Synthesis.

    PubMed Central

    Smirnov, S.; Shulaev, V.; Tumer, N. E.

    1997-01-01

    Pokeweed antiviral protein (PAP), a 29-kD protein isolated from Phytolacca americana, inhibits translation by catalytically removing a specific adenine residue from the large rRNA of the 60S subunit of eukaryotic ribosomes. Transgenic tobacco (Nicotiana tabacum) plants expressing PAP or a variant (PAP-v) were shown to be resistant to a broad spectrum of plant viruses. Expression of PAP-v in transgenic plants induces synthesis of pathogenesis-related proteins and a very weak (<2-fold) increase in salicylic acid levels. Using reciprocal grafting experiments, we demonstrate here that transgenic tobacco rootstocks expressing PAP-v induce resistance to tobacco mosaic virus infection in both N. tabacum NN and nn scions. Increased resistance to potato virus X was also observed in N. tabacum nn scions grafted on transgenic rootstocks. PAP expression was not detected in the wild-type scions or rootstocks that showed virus resistance, nor was there any increase in salicylic acid levels or pathogenesis-related protein synthesis. Grafting experiments with transgenic plants expressing an inactive PAP mutant demonstrated that an intact active site of PAP is necessary for induction of virus resistance in wild-type scions. These results indicate that enzymatic activity of PAP is responsible for generating a signal that renders wild-type scions resistant to virus infection in the absence of increased salicylic acid levels and pathogenesis-related protein synthesis. PMID:12223762

  17. 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...

  18. Analysis of proteins responsive to acetic acid in Acetobacter: molecular mechanisms conferring acetic acid resistance in acetic acid bacteria.

    PubMed

    Nakano, Shigeru; Fukaya, Masahiro

    2008-06-30

    Acetic acid bacteria are used for industrial vinegar production because of their remarkable ability to oxidize ethanol and high resistance to acetic acid. Although several molecular machineries responsible for acetic acid resistance in acetic acid bacteria have been reported, the entire mechanism that confers acetic acid resistance has not been completely understood. One of the promising methods to elucidate the entire mechanism is global analysis of proteins responsive to acetic acid by two-dimensional gel electrophoresis. Recently, two proteins whose production was greatly enhanced by acetic acid in Acetobacter aceti were identified to be aconitase and a putative ABC-transporter, respectively; furthermore, overexpression or disruption of the genes encoding these proteins affected acetic acid resistance in A. aceti, indicating that these proteins are involved in acetic acid resistance. Overexpression of each gene increased acetic acid resistance in Acetobacter, which resulted in an improvement in the productivity of acetic acid fermentation. Taken together, the results of the proteomic analysis and those of previous studies indicate that acetic acid resistance in acetic acid bacteria is conferred by several mechanisms. These findings also provide a clue to breed a strain having high resistance to acetic acid for vinegar fermentation.

  19. Identification of a protein which differs in lines of barley resistant or susceptible to Erysiphe graminis f. sp. hordei

    SciTech Connect

    Simons, S.P.; Somerville, S.C. )

    1990-05-01

    As yet no resistance genes or gene products have been isolated from a plant. We wish to isolate the Ml-a gene which encodes resistance by barley to Erysiphe graminis f. sp. hordei, the causal agent of the powdery mildew disease. We have utilized near isogenic barley lines, resistant and susceptible to E. g. hordei, race CR3 to provide a homogenous background for examination of protein differences related to resistance. To enrich for epidermal tissue, the site of infection, proteins from {sup 35}S labelled coleoptiles were analyzed by 2D-PAGE. Two major protein differences were observed, one of which is a polymorphism identified by a higher molecular weight form in the susceptible line, with the lower molecular weight form appearing in the resistant line. A partially susceptible mutant line derived from the resistant line shows a 63% reduction in the level of the resistant form of the polypeptide. A likely conclusion from these results is that we have identified the Ml-a gene product. Further characterization of the polymorphic protein and its gene will lead to an understanding of gene-for-gene related resistance processes.

  20. The use of LC-MS to identify differentially expressed proteins in docetaxel-resistant prostate cancer cell lines.

    PubMed

    O'Connell, Kathleen; Prencipe, Maria; O'Neill, Amanda; Corcoran, Claire; Rani, Sweta; Henry, Michael; Dowling, Paul; Meleady, Paula; O'Driscoll, Lorraine; Watson, William; O'Connor, Robert

    2012-07-01

    Docetaxel is a taxane-derived chemotherapy drug that has been approved for treatment of prostate cancer. While docetaxel is frequently used as a treatment for hormone-refractory prostate cancer, a subset of patients either do not respond to this treatment or those that do respond eventually become resistant to the drug over time. Resistance to docetaxel is complex and multi-factoral and further understanding of the cellular biochemistry underlying resistance is vital to improve treatment efficacy. To identify proteins altered in the resistant phenotype, three parental cell lines DU145, 22RV1 and PC-3, as well as their docetaxel resistant sub-lines, were subjected to quantitative label-free LC-MS proteomic profiling. A total of 189 significant (p < 0.05) protein abundance changes were identified in the DU145 resistant sub-lines, 254 in the 22RV1 sub-lines, and 51 and 72 in the 8 and 12 nM resistant PC-3 sub-lines, respectively. From these, 29 proteins demonstrated a significant (p < 0.05) fold change across two or more resistant variants. These included proteins indicative of an epithelial-to-mesenchemyl transition as well as altered heat shock response elements. PMID:22623417

  1. 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

  2. The multidrug resistance-associated protein 1 transports methoxychlor and protects the seminiferous epithelium from injury.

    PubMed

    Tribull, Tiffany E; Bruner, Richard H; Bain, Lisa J

    2003-04-30

    We examined the ability of the multidrug resistance-associated protein 1 (MRP1/ABCC1) to transport pesticides, as this transporter mediates the cellular efflux of a variety of xenobiotics, typically as glucuronide, sulfate, or glutathione conjugates. NIH3T3 cells stably expressing MRP1 were 3.37-fold more resistant to the toxicity of fenitrothion, 3.12-fold more resistant to chlorpropham, and 2.5-fold more resistant to methoxychlor, a pesticide with estrogenic and anti-androgenic metabolites. The cells expressing MRP1 also eliminated methoxychlor two times more rapidly than their mock-transfected counterparts. We then examined whether mrp1 expression could alter the toxicity of methoxychlor in vivo using male FVB/mrp1 knockout mice (FVB/mrp1-/-). Both control and knockout mice were fed 25 mg/kg methoxychlor in honey for 39 days, and its effects on testicular morphology were examined. Methoxychlor treatment did not significantly affect testicular morphology in the FVB mice, but markedly reduced the number of developing spermatocytes in the FVB/mrp1-/- mice. These results suggest that MRPI may play a role in protecting the seminiferous tubules from methoxychlor-induced damage.

  3. 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. PMID:23758273

  4. 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

  5. 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.

  6. Role of spore coat proteins in the resistance of Bacillus subtilis spores to Caenorhabditis elegans predation.

    PubMed

    Laaberki, Maria-Halima; Dworkin, Jonathan

    2008-09-01

    Bacterial spores are resistant to a wide range of chemical and physical insults that are normally lethal for the vegetative form of the bacterium. While the integrity of the protein coat of the spore is crucial for spore survival in vitro, far less is known about how the coat provides protection in vivo against predation by ecologically relevant hosts. In particular, assays had characterized the in vitro resistance of spores to peptidoglycan-hydrolyzing enzymes like lysozyme that are also important effectors of innate immunity in a wide variety of hosts. Here, we use the bacteriovorous nematode Caenorhabditis elegans, a likely predator of Bacillus spores in the wild, to characterize the role of the spore coat in an ecologically relevant spore-host interaction. We found that ingested wild-type Bacillus subtilis spores were resistant to worm digestion, whereas vegetative forms of the bacterium were efficiently digested by the nematode. Using B. subtilis strains carrying mutations in spore coat genes, we observed a correlation between the degree of alteration of the spore coat assembly and the susceptibility to the worm degradation. Surprisingly, we found that the spores that were resistant to lysozyme in vitro can be sensitive to C. elegans digestion depending on the extent of the spore coat structure modifications.

  7. Multidrug resistance protein 1 localization in lipid raft domains and prostasomes in prostate cancer cell lines

    PubMed Central

    Gomà, Alba; Mir, Roser; Martínez-Soler, Fina; Tortosa, Avelina; Vidal, August; Condom, Enric; Pérez–Tomás, Ricardo; Giménez-Bonafé, Pepita

    2014-01-01

    Background One of the problems in prostate cancer (CaP) treatment is the appearance of the multidrug resistance phenotype, in which ATP-binding cassette transporters such as multidrug resistance protein 1 (MRP1) play a role. Different localizations of the transporter have been reported, some of them related to the chemoresistant phenotype. Aim This study aimed to compare the localization of MRP1 in three prostate cell lines (normal, androgen-sensitive, and androgen-independent) in order to understand its possible role in CaP chemoresistance. Methods MRP1 and caveolae protein markers were detected using confocal microscopy, performing colocalization techniques. Lipid raft isolation made it possible to detect these proteins by Western blot analysis. Caveolae and prostasomes were identified by electron microscopy. Results We show that MRP1 is found in lipid raft fractions of tumor cells and that the number of caveolae increases with malignancy acquisition. MRP1 is found not only in the plasma membrane associated with lipid rafts but also in cytoplasmic accumulations colocalizing with the prostasome markers Caveolin-1 and CD59, suggesting that in CaP cells, MRP1 is localized in prostasomes. Conclusion We hypothesize that the presence of MRP1 in prostasomes could serve as a reservoir of MRP1; thus, taking advantage of the release of their content, MRP1 could be translocated to the plasma membrane contributing to the chemoresistant phenotype. The presence of MRP1 in prostasomes could serve as a predictor of malignancy in CaP. PMID:25525371

  8. Structural basis of lantibiotic recognition by the nisin resistance protein from Streptococcus agalactiae

    PubMed Central

    Khosa, Sakshi; Frieg, Benedikt; Mulnaes, Daniel; Kleinschrodt, Diana; Hoeppner, Astrid; Gohlke, Holger; Smits, Sander H. J.

    2016-01-01

    Lantibiotics are potent antimicrobial peptides. Nisin is the most prominent member and contains five crucial lanthionine rings. Some clinically relevant bacteria express membrane-associated resistance proteins that proteolytically inactivate nisin. However, substrate recognition and specificity of these proteins is unknown. Here, we report the first three-dimensional structure of a nisin resistance protein from Streptococcus agalactiae (SaNSR) at 2.2 Å resolution. It contains an N-terminal helical bundle, and protease cap and core domains. The latter harbors the highly conserved TASSAEM region, which lies in a hydrophobic tunnel formed by all domains. By integrative modeling, mutagenesis studies, and genetic engineering of nisin variants, a model of the SaNSR/nisin complex is generated, revealing that SaNSR recognizes the last C-terminally located lanthionine ring of nisin. This determines the substrate specificity of SaNSR and ensures the exact coordination of the nisin cleavage site at the TASSAEM region. PMID:26727488

  9. 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

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

    PubMed

    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

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

    PubMed

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

    2016-06-01

    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

  12. 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

  13. 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.

  14. 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.

  15. Development of resistant transgenic soybeans with inverted repeat-coat protein genes of soybean dwarf virus.

    PubMed

    Tougou, Makoto; Furutani, Noriyuki; Yamagishi, Noriko; Shizukawa, Yoshiaki; Takahata, Yoshihito; Hidaka, Soh

    2006-11-01

    In an attempt to generate soybean plants resistant to soybean dwarf virus (SbDV), we transformed a construct containing inverted repeat-SbDV coat protein (CP) genes spaced by beta-glucuronidase (GUS) sequences into soybean somatic embryos via microprojectile bombardment. Three T(0) plants with an introduced CP gene were obtained, and one generated T(1) seeds. The presence of the transgene in T(1) plants was confirmed by PCR and Southern blot hybridization analysis, but expression of CP was not detected by northern blot hybridization analysis. Two months after inoculation of SbDV by aphid, T(2) plants contained little SbDV-specific RNA and remained symptomless. These plants contained SbDV-CP-specific siRNA. These results suggest that the T(2) plants achieved resistance to SbDV by an RNA-silencing-mediated process.

  16. Molecular basis of glyphosate resistance-different approaches through protein engineering.

    PubMed

    Pollegioni, Loredano; Schonbrunn, Ernst; Siehl, Daniel

    2011-08-01

    Glyphosate (N-phosphonomethyl-glycine) is the most widely 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 attributable to the high specificity of glyphosate for the plant enzyme enolpyruvyl shikimate-3-phosphate synthase in the shikimate pathway, leading to the biosynthesis of aromatic amino acids. Starting in 1996, transgenic glyphosate-resistant plants were introduced, thus allowing application of the herbicide to the crop (post-emergence) to remove emerged weeds without crop damage. This review focuses on 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 a rationale for the means by which the modifications made have had their intended effect.

  17. Protein-resistant polymer coatings obtained by matrix assisted pulsed laser evaporation

    NASA Astrophysics Data System (ADS)

    Rusen, L.; Mustaciosu, C.; Mitu, B.; Filipescu, M.; Dinescu, M.; Dinca, V.

    2013-08-01

    Adsorption of proteins and polysaccharides is known to facilitate microbial attachment and subsequent formation of biofilm on surfaces that ultimately results in its biofouling. Therefore, protein repellent modified surfaces are necessary to block the irreversible attachment of microorganisms. Within this context, the feasibility of using the Poly(ethylene glycol)-block-poly(ɛ-caprolactone) methyl ether (PEG-block-PCL Me) copolymer as potential protein-resistant coating was explored in this work. The films were deposited using Matrix Assisted Pulsed Laser Evaporation (MAPLE), a technique that allows good control of composition, thickness and homogeneity. The chemical and morphological characteristics of the films were examined using Fourier Transform Infrared Spectroscopy (FTIR), contact angle measurements and Atomic Force Microscopy (AFM). The FTIR data demonstrates that the functional groups in the MAPLE-deposited films remain intact, especially for fluences below 0.5 J cm-2. Optical Microscopy and AFM images show that the homogeneity and the roughness of the coatings are related to both laser parameters (fluence, number of pulses) and target composition. Protein adsorption tests were performed on the PEG-block-PCL Me copolymer coated glass and on bare glass surface as a control. The results show that the presence of copolymer as coating significantly reduces the adsorption of proteins.

  18. Identification and bioinformatic characterization of a multidrug resistance associated protein (ABCC) gene in Plasmodium berghei

    PubMed Central

    González-Pons, María; Szeto, Ada C; González-Méndez, Ricardo; Serrano, Adelfa E

    2009-01-01

    Background The ATP-binding cassette (ABC) superfamily is one of the largest evolutionarily conserved families of proteins. ABC proteins play key roles in cellular detoxification of endobiotics and xenobiotics. Overexpression of certain ABC proteins, among them the multidrug resistance associated protein (MRP), contributes to drug resistance in organisms ranging from human neoplastic cells to parasitic protozoa. In the present study, the Plasmodium berghei mrp gene (pbmrp) was partially characterized and the predicted protein was classified using bioinformatics in order to explore its putative involvement in drug resistance. Methods The pbmrp gene from the P. berghei drug sensitive, N clone, was sequenced using a PCR strategy. Classification and domain organization of pbMRP were determined with bioinformatics. The Plasmodium spp. MRPs were aligned and analysed to study their conserved motifs and organization. Gene copy number and organization were determined via Southern blot analysis in both N clone and the chloroquine selected line, RC. Chromosomal Southern blots and RNase protection assays were employed to determine the chromosomal location and expression levels of pbmrp in blood stages. Results The pbmrp gene is a single copy, intronless gene with a predicted open reading frame spanning 5820 nucleotides. Bioinformatic analyses show that this protein has distinctive features characteristic of the ABCC sub-family. Multiple sequence alignments reveal a high degree of conservation in the nucleotide binding and transmembrane domains within the MRPs from the Plasmodium spp. analysed. Expression of pbmrp was detected in asexual blood stages. Gene organization, copy number and mRNA expression was similar in both lines studied. A chromosomal translocation was observed in the chloroquine selected RC line, from chromosome 13/14 to chromosome 8, when compared to the drug sensitive N clone. Conclusion In this study, the pbmrp gene was sequenced and classified as a member of

  19. 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.

  20. Protein supplementation does not alter intramuscular anabolic signaling or endocrine response after resistance exercise in trained men.

    PubMed

    Gonzalez, Adam M; Hoffman, Jay R; Jajtner, Adam R; Townsend, Jeremy R; Boone, Carleigh H; Beyer, Kyle S; Baker, Kayla M; Wells, Adam J; Church, David D; Mangine, Gerald T; Oliveira, Leonardo P; Moon, Jordan R; Fukuda, David H; Stout, Jeffrey R

    2015-11-01

    The mammalian/mechanistic target of rapamycin complex 1 (mTORC1) signaling pathway appears to be the primary regulator of muscle protein synthesis. A variety of stimuli including resistance exercise, amino acids, and hormonal signals activate mTORC1 signaling. The purpose of this study was to investigate the effect of a protein supplement on mTORC1 signaling following a resistance exercise protocol designed to promote elevations in circulating hormone concentrations. We hypothesized that the protein supplement would augment the intramuscular anabolic signaling response. Ten resistance-trained men (age, 24.7 ± 3.4 years; weight, 90.1 ± 11.3 kg; height, 176.0 ± 4.9 cm) received either a placebo or a supplement containing 20 g protein, 6 g carbohydrates, and 1 g fat after high-volume, short-rest lower-body resistance exercise. Blood samples were obtained at baseline, immediately, 30 minutes, 1 hour, 2 hours, and 5 hours after exercise. Fine-needle muscle biopsies were completed at baseline, 1 hour, and 5 hours after exercise. Myoglobin, lactate dehydrogenase, and lactate concentrations were significantly elevated after resistance exercise (P < .0001); however, no differences were observed between trials. Resistance exercise also elicited a significant insulin, growth hormone, and cortisol response (P < .01); however, no differences were observed between trials for insulin-like growth factor-1, insulin, testosterone, growth hormone, or cortisol. Intramuscular anabolic signaling analysis revealed significant elevations in RPS6 phosphorylation after resistance exercise (P = .001); however, no differences were observed between trials for signaling proteins including Akt, mTOR, p70S6k, and RPS6. The endocrine response and phosphorylation status of signaling proteins within the mTORC1 pathway did not appear to be altered by ingestion of supplement after resistance exercise in resistance-trained men. PMID:26428621

  1. Protein supplementation does not alter intramuscular anabolic signaling or endocrine response after resistance exercise in trained men.

    PubMed

    Gonzalez, Adam M; Hoffman, Jay R; Jajtner, Adam R; Townsend, Jeremy R; Boone, Carleigh H; Beyer, Kyle S; Baker, Kayla M; Wells, Adam J; Church, David D; Mangine, Gerald T; Oliveira, Leonardo P; Moon, Jordan R; Fukuda, David H; Stout, Jeffrey R

    2015-11-01

    The mammalian/mechanistic target of rapamycin complex 1 (mTORC1) signaling pathway appears to be the primary regulator of muscle protein synthesis. A variety of stimuli including resistance exercise, amino acids, and hormonal signals activate mTORC1 signaling. The purpose of this study was to investigate the effect of a protein supplement on mTORC1 signaling following a resistance exercise protocol designed to promote elevations in circulating hormone concentrations. We hypothesized that the protein supplement would augment the intramuscular anabolic signaling response. Ten resistance-trained men (age, 24.7 ± 3.4 years; weight, 90.1 ± 11.3 kg; height, 176.0 ± 4.9 cm) received either a placebo or a supplement containing 20 g protein, 6 g carbohydrates, and 1 g fat after high-volume, short-rest lower-body resistance exercise. Blood samples were obtained at baseline, immediately, 30 minutes, 1 hour, 2 hours, and 5 hours after exercise. Fine-needle muscle biopsies were completed at baseline, 1 hour, and 5 hours after exercise. Myoglobin, lactate dehydrogenase, and lactate concentrations were significantly elevated after resistance exercise (P < .0001); however, no differences were observed between trials. Resistance exercise also elicited a significant insulin, growth hormone, and cortisol response (P < .01); however, no differences were observed between trials for insulin-like growth factor-1, insulin, testosterone, growth hormone, or cortisol. Intramuscular anabolic signaling analysis revealed significant elevations in RPS6 phosphorylation after resistance exercise (P = .001); however, no differences were observed between trials for signaling proteins including Akt, mTOR, p70S6k, and RPS6. The endocrine response and phosphorylation status of signaling proteins within the mTORC1 pathway did not appear to be altered by ingestion of supplement after resistance exercise in resistance-trained men.

  2. Involvement of a 43-kilodalton outer membrane protein in beta-lactam resistance of Shigella dysenteriae.

    PubMed Central

    Kar, A K; Ghosh, A S; Chauhan, K; Ahamed, J; Basu, J; Chakrabarti, P; Kundu, M

    1997-01-01

    A beta-lactam-sensitive strain (C152) of Shigella dysenteriae showed two major outer membrane proteins (OMPs) with M(r)s of 43,000 and 38,000, while the clinical isolate M2 lacked the 43,000-Mr OMP, which acted as a channel for beta-lactam antibiotics. Permeability of beta-lactams across the outer membrane (OM) of M2 was lower than that across the OM of C152. Mutants deficient in the 43-kDa OMP could be selected in vitro from strain C152 in the presence of cefoxitin. All beta-lactam-resistant strains were sensitive to imipenem. PMID:9333070

  3. Data for chitin binding activity of Moringa seed resistant protein (MSRP).

    PubMed

    Sandanamudi, Anudeep; Bharadwaj, Kishan R; Cheruppanpullil, Radha

    2016-12-01

    Chitin binding activity of moringa seed resistant protein (MSRP) isolated from defatted moringa seed flour was investigated in the present study "Characterization of soluble dietary fiber from Moringa oleifera seeds and its immunomodulatory effects" (S. Anudeep, V.K. Prasanna, S.M. Adya, C. Radha, 2016) [1]. The assay reaction mixture contained 0.4 mg/ml of MSRP and different amounts (20-100 mg) of chitin. MSRP exhibited binding activity over wide range of chitin concentration. Maximum binding activity was observed at 80 mg of chitin. The property of MSRP to bind chitin can be exploited for its purification.

  4. Data for chitin binding activity of Moringa seed resistant protein (MSRP).

    PubMed

    Sandanamudi, Anudeep; Bharadwaj, Kishan R; Cheruppanpullil, Radha

    2016-12-01

    Chitin binding activity of moringa seed resistant protein (MSRP) isolated from defatted moringa seed flour was investigated in the present study "Characterization of soluble dietary fiber from Moringa oleifera seeds and its immunomodulatory effects" (S. Anudeep, V.K. Prasanna, S.M. Adya, C. Radha, 2016) [1]. The assay reaction mixture contained 0.4 mg/ml of MSRP and different amounts (20-100 mg) of chitin. MSRP exhibited binding activity over wide range of chitin concentration. Maximum binding activity was observed at 80 mg of chitin. The property of MSRP to bind chitin can be exploited for its purification. PMID:27672672

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

    PubMed Central

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

    2015-01-01

    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. PMID:26637599

  6. 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

    2016-02-01

    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. PMID:26637599

  7. A reverse transcriptase-related protein mediates phage resistance and polymerizes untemplated DNA in vitro

    PubMed Central

    Wang, Chen; Villion, Manuela; Semper, Cameron; Coros, Colin; Moineau, Sylvain; Zimmerly, Steven

    2011-01-01

    Reverse transcriptases (RTs) are RNA-dependent DNA polymerases that usually function in the replication of selfish DNAs such as retrotransposons and retroviruses. Here, we have biochemically characterized a RT-related protein, AbiK, which is required for abortive phage infection in the Gram-positive bacterium Lactococcus lactis. In vitro, AbiK does not exhibit the properties expected for an RT, but polymerizes long DNAs of ‘random’ sequence, analogous to a terminal transferase. Moreover, the polymerized DNAs appear to be covalently attached to the AbiK protein, presumably because an amino acid serves as a primer. Mutagenesis experiments indicate that the polymerase activity resides in the RT motifs and is essential for phage resistance in vivo. These results establish a novel biochemical property and a non-replicative biological role for a polymerase. PMID:21676997

  8. Construction of plants resistant to TYLCV by using artificial zinc-finger proteins.

    PubMed

    Koshino-Kimura, Yoshihiro; Takenaka, Kosuke; Domoto, Fumiya; Ohashi, Masayoshi; Miyazaki, Toshihide; Aoyama, Yasuhiro; Sera, Takashi

    2009-01-01

    Previously, we have demonstrated that plant DNA virus replication could be inhibited in Arabidopsis thaliana by using an artificial zinc-finger protein (AZP) and created AZP-based transgenic A. thaliana resistant to DNA virus infection. Here we apply the AZP technology to tomato yellow leaf curl virus (TYLCV) causing serious damage to an important agricultural crop, tomato. An AZP was designed to block binding of the TYLCV replication protein (Rep) to the replication origin. The designed AZP had much higher affinities towards the replication origin than did the Rep, and efficiently blocked Rep binding in vitro. The AZP gene was then introduced into a plant genome with the help of Agrobacterium tumefaciens to generate the transgenic plants. The current status of the construction of the AZP-expressing transgenic plants will be reported.

  9. Candle soot-based super-amphiphobic coatings resist protein adsorption.

    PubMed

    Schmüser, Lars; Encinas, Noemi; Paven, Maxime; Graham, Daniel J; Castner, David G; Vollmer, Doris; Butt, Hans Jürgen; Weidner, Tobias

    2016-01-01

    Super nonfouling surfaces resist protein adhesion and have a broad field of possible applications in implant technology, drug delivery, blood compatible materials, biosensors, and marine coatings. A promising route toward nonfouling surfaces involves liquid repelling architectures. The authors here show that soot-templated super-amphiphobic (SAP) surfaces prepared from fluorinated candle soot structures are super nonfouling. When exposed to bovine serum albumin or blood serum, x-ray photoelectron spectroscopy and time of flight secondary ion mass spectrometry analysis showed that less than 2 ng/cm(2) of protein was adsorbed onto the SAP surfaces. Since a broad variety of substrate shapes can be coated by soot-templated SAP surfaces, those are a promising route toward biocompatible materials design. PMID:27460261

  10. Mutations in an Atypical TIR-NB-LRR-LIM Resistance Protein Confer Autoimmunity

    PubMed Central

    Bi, Dongling; Johnson, Kaeli C. M.; Zhu, Zhaohai; Huang, Yan; Chen, Fang; Zhang, Yuelin; Li, Xin

    2011-01-01

    In order to defend against microbial infection, plants employ a complex immune system that relies partly on resistance (R) proteins that initiate intricate signaling cascades upon pathogen detection. The resistance signaling network utilized by plants is only partially characterized. A genetic screen conducted to identify novel defense regulators involved in this network resulted in the isolation of the snc6-1D mutant. Positional cloning revealed that this mutant contained a molecular lesion in the chilling sensitive 3 (CHS3) gene, thus the allele was renamed chs3-2D. CHS3 encodes a TIR-NB-LRR R protein that contains a C-terminal zinc-binding LIM (Lin-11, Isl-1, Mec-3) domain. Although this protein has been previously implicated in cold stress and defense response, the role of the LIM domain in modulating protein activity is unclear. The chs3-2D allele contains a G to A point mutation causing a C1340 to Y1340 substitution close to the LIM domain. It encodes a dominant gain-of-function mutation. The chs3-2D mutant is severely stunted and displays curled leaf morphology. Additionally, it constitutively expresses PATHOGENESIS-RELATED (PR) genes, accumulates salicylic acid, and shows enhanced resistance to the virulent oomycete isolate Hyaloperonospora arabidopsidis (H.a.) Noco2. Subcellular localization assays using GFP fusion constructs indicate that both CHS3 and chs3-2D localize to the nucleus. A third chs3 mutant allele, chs3-3D, was identified in an unrelated genetic screen in our lab. This allele contains a C to T point mutation resulting in an M1017 to V1017 substitution in the LRR–LIM linker region. Additionally, a chs3-2D suppressor screen identified two revertant alleles containing secondary mutations that abolish the mutant morphology. Analysis of the locations of these molecular lesions provides support for the hypothesis that the LIM domain represses CHS3 R-like protein activity. This repression may occur through either autoinhibition or binding of a

  11. Overexpression of centrosomal protein Nlp confers breast carcinoma resistance to paclitaxel.

    PubMed

    Zhao, Weihong; Song, Yongmei; Xu, Binghe; Zhan, Qimin

    2012-02-01

    Nlp (ninein-like protein), an important molecule involved in centrosome maturation and spindle formation, plays an important role in tumorigenesis and its abnormal expression was recently observed in human breast and lung cancers. In this study, the correlation between overexpression of Nlp and paclitaxel chemosensitivity was investigated to explore the mechanisms of resistance to paclitaxel and to understand the effect of Nlp upon apoptosis induced by chemotherapeutic agents. Nlp expression vector was stably transfected into breast cancer MCF-7 cells. With Nlp overexpression, the survival rates, cell cycle distributions and apoptosis were analyzed in transfected MCF-7 cells by MTT test and FCM approach. The immunofluorescent assay was employed to detect the changes of microtubule after paclitaxel treatment. Immunoblotting analysis was used to examine expression of centrosomal proteins and apoptosis associated proteins. Subsequently, Nlp expression was retrospectively examined with 55 breast cancer samples derived from paclitaxel treated patients. Interestingly, the survival rates of MCF-7 cells with Nlp overexpressing were higher than that of control after paclitaxel treatment. Nlp overexpression promoted G2-M arrest and attenuated apoptosis induced by paclitaxel, which was coupled with elevated Bcl-2 protein. Nlp expression significantly lessened the microtubule polymerization and bundling elicited by paclitaxel attributing to alteration on the structure or dynamics of β-tubulin but not on its expression. The breast cancer patients with high expression of Nlp were likely resistant to the treatment of paclitaxel, as the response rate in Nlp negative patients was 62.5%, whereas was 58.3 and 15.8% in Nlp (+) and Nlp (++) patients respectively (p = 0.015). Nlp expression was positive correlated with those of Plk1 and PCNA. These findings provide insights into more rational chemotherapeutic regimens in clinical practice, and more effective approaches might be

  12. 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

  13. 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.

  14. Increased production of penicillin-binding protein 2, increased detection of other penicillin-binding proteins, and decreased coagulase activity associated with glycopeptide resistance in Staphylococcus aureus.

    PubMed Central

    Moreira, B; Boyle-Vavra, S; deJonge, B L; Daum, R S

    1997-01-01

    The mechanism of glycopeptide resistance in the genus Staphylococcus is unknown. Since these antimicrobial compounds act by binding the peptidoglycan precursor terminus, the target of transglycosylase and transpeptidase enzymes, it was hypothesized that resistance might be mediated in Staphylococcus aureus by increased production or activity of these enzymes, commonly called penicillin-binding proteins (PBPs). To evaluate this possibility, glycopeptide-resistant mutants were prepared by passage of several clinical isolates of this species in nutrient broth containing successively increasing concentrations of the glycopeptide vancomycin or teicoplanin. Decreased coagulase activity and increased resistance to lysostaphin were uniformly present in the vancomycin-resistant mutants. Peptidoglycan cross-linking increased in one resistant isolate and decreased in two resistant isolates. The amounts of radioactive penicillin that bound to each PBP in susceptible and resistant strains were compared; PBP2 production was also evaluated by Western blotting. Increased penicillin labeling and production of PBP2 were found in all resistant derivatives selected by either vancomycin or teicoplanin. Moreover, the increase in PBP2 penicillin labeling occurred early in a series of vancomycin-selected derivatives and was strongly correlated (r > 0.9) with the increase in vancomycin and teicoplanin MIC. An increase in penicillin labeling also occurred, variably, in PBP1, PBP3, and/or PBP4. These data demonstrate a strong correlation between resistance to glycopeptides and increased PBP activity and/or production in S. aureus. Such an increase could allow PBPs to better compete with glycopeptides for the peptidoglycan precursor. PMID:9257762

  15. Transport proteins determine drug sensitivity and resistance in a protozoan parasite, Trypanosoma brucei

    PubMed Central

    Munday, Jane C.; Settimo, Luca; de Koning, Harry P.

    2015-01-01

    Drug resistance in pathogenic protozoa is very often caused by changes to the ‘transportome’ of the parasites. In Trypanosoma brucei, several transporters have been implicated in uptake of the main classes of drugs, diamidines and melaminophenyl arsenicals. The resistance mechanism had been thought to be due to loss of a transporter known to carry both types of agents: the aminopurine transporter P2, encoded by the gene TbAT1. However, although loss of P2 activity is well-documented as the cause of resistance to the veterinary diamidine diminazene aceturate (DA; Berenil®), cross-resistance between the human-use arsenical melarsoprol and the diamidine pentamidine (melarsoprol/pentamidine cross resistance, MPXR) is the result of loss of a separate high affinity pentamidine transporter (HAPT1). A genome-wide RNAi library screen for resistance to pentamidine, published in 2012, gave the key to the genetic identity of HAPT1 by linking the phenomenon to a locus that contains the closely related T. brucei aquaglyceroporin genes TbAQP2 and TbAQP3. Further analysis determined that knockdown of only one pore, TbAQP2, produced the MPXR phenotype. TbAQP2 is an unconventional aquaglyceroporin with unique residues in the “selectivity region” of the pore, and it was found that in several MPXR lab strains the WT gene was either absent or replaced by a chimeric protein, recombined with parts of TbAQP3. Importantly, wild-type AQP2 was also absent in field isolates of T. b. gambiense, correlating with the outcome of melarsoprol treatment. Expression of a wild-type copy of TbAQP2 in even the most resistant strain completely reversed MPXR and re-introduced HAPT1 function and transport kinetics. Expression of TbAQP2 in Leishmania mexicana introduced a pentamidine transport activity indistinguishable from HAPT1. Although TbAQP2 has been shown to function as a classical aquaglyceroporin it is now clear that it is also a high affinity drug transporter, HAPT1. We discuss here a

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

    PubMed

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

    2015-06-01

    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

  17. Heterotrimeric G-proteins facilitate resistance to plant pathogenic viruses in Arabidopsis thaliana (L.) Heynh.

    PubMed

    Brenya, Eric; Trusov, Yuri; Dietzgen, Ralf Georg; Botella, José Ramón

    2016-08-01

    Heterotrimeric G-proteins, consisting of Gα, Gβ and Gγ subunits, are important signal transducers in eukaryotes. In plants, G-protein-mediated signaling contributes to defense against a range of fungal and bacterial pathogens. Here we studied response of G-protein-deficient mutants to ssRNA viruses representing 2 different families: Cucumber mosaic virus (CMV) (Bromoviridae) and Turnip mosaic virus (TuMV) (Potyviridae). We found that development of spreading necrosis on infected plants was suppressed in the Gβ-deficient mutant (agb1-2) compared to wild type and Gα-deficient mutant (gpa1-4). In accordance, ion leakage caused by viral infection was also significantly reduced in agb1-2 compared to wild type and gpa1-4. Nevertheless, both viruses replicated better in agb1-2 plants, while gpa1-4 was similar to wild type. Analysis of pathogenesis-related genes showed that Gβ negatively regulated salicylic acid, jasmonic acid and abscisic acid marker genes during CMV and TuMV infections. Interestingly, analysis of salicylic acid deficient transgenic plants indicated that salicylic acid did not affect resistance against these viruses and did not influence the Gβ-mediated defense response. We conclude that heterotrimeric G-proteins play a positive role in defense against viral pathogens probably by promoting cell death. PMID:27454415

  18. 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

  19. 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.

  20. 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. PMID:25940639

  1. 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.

  2. Drug carriers based on highly protein-resistant materials for prolonged in vivo circulation time

    PubMed Central

    Liu, Ruiyuan; Li, Yan; Zhang, Zhenzhong; Zhang, Xin

    2015-01-01

    Long-circulating drug carriers are highly desirable in drug delivery system. However, nonspecific protein adsorption leaves a great challenge in drug delivery of intravenous administration and significantly affects both the pharmacokinetic profiles of the carrier and drugs, resulting in negatively affect of therapeutic efficiency. Therefore, it is important to make surface modification of drug carriers by protein-resistant materials to prolong the blood circulation time and increase the targeted accumulation of therapeutic agents. In this review, we highlight the possible mechanism of protein resistance and recent progress of the alternative protein-resistant materials and their drug carriers, such as poly(ethylene glycol), oligo(ethylene glycol), zwitterionic materials, and red blood cells adhesion. PMID:26813147

  3. Modulation of proteinase-K resistant prion protein by prion peptide immunization.

    PubMed

    Souan, L; Tal, Y; Felling, Y; Cohen, I R; Taraboulos, A; Mor, F

    2001-08-01

    Prion diseases are caused by conformational alterations in the prion protein (PrP). The immune system has been assumed to be non-responsive to the self-prion protein, therefore, PrP autoimmunity has not been investigated. Here, we immunized various strains of mice with PrP peptides, some selected to fit the MHC class II-peptide binding motif. We found that specific PrP peptides elicited strong immune responses in NOD, C57BL/6 and A/J mice. To test the functional effect of this immunization, we examined the expression of proteinase-K-resistant PrP by a scrapie-infected tumor transplanted to immunized syngeneic A/J mice. PrP peptide vaccination did not affect the growth of the infected tumor transplant, but significantly reduced the level of protease-resistant PrP. Our results demonstrate that self-PrP peptides are immunogenic in mice and suggest that this immune response might affect PrP-scrapie levels in certain conditions.

  4. 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

  5. Multidrug Resistance Proteins (MRPs/ABCCs) in Cancer Chemotherapy and Genetic Diseases

    PubMed Central

    Chen, Zhe-Sheng; Tiwari, Amit K.

    2011-01-01

    The ATP-binding cassette (ABC) transporters are a superfamily of membrane proteins that are best known for their ability to transport a wide variety of exogenous and endogenous substances across membranes against a concentration gradient via ATP hydrolysis. There are seven subfamilies of human ABC transporters, one of the largest being the ‘C’ subfamily (gene symbol ABCC). Nine ABCC subfamily members, the so-called Multidrug Resistance Proteins (MRPs) 1-9, have been implicated in mediating multidrug resistance in tumor cells to varying degrees as the efflux extrude chemotherapeutic compounds (or their metabolites) from malignant cells. Some of the MRPs are also known to either influence drug disposition in normal tissues or modulate the elimination of drugs (or their metabolites) via hepatobiliary or renal excretory pathways. In addition, the cellular efflux of physiologically important organic anions such as leukotriene C4 and cAMP is mediated by one or more of the MRPs. Finally, mutations in several MRPs are associated with human genetic disorders. In this review article, the current biochemical and physiological knowledge of MRP1-MRP9 in cancer chemotherapy and human genetic disease is summarized. The mutations in MRP2/ABCC2 leading to conjugated hyperbilirubinemia (Dubin-Johnson syndrome) and in MRP6/ABCC6 leading to the connective tissue disorder Pseudoxanthoma elasticum are also discussed. PMID:21740521

  6. 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.

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

    PubMed

    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

  8. 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

  9. High-resolution structure of the antibiotic resistance protein NimA from Deinococcus radiodurans.

    PubMed

    Leiros, Hanna Kirsti S; Tedesco, Consiglia; McSweeney, Seán M

    2008-06-01

    Many anaerobic human pathogenic bacteria are treated using 5-nitroimidazole-based (5-Ni) antibiotics, a class of inactive prodrugs that contain a nitro group. The nitro group must be activated in an anaerobic one-electron reduction and is therefore dependent on the redox system in the target cells. Antibiotic resistance towards 5-Ni drugs is found to be related to the nim genes (nimA, nimB, nimC, nimD, nimE and nimF), which are proposed to encode a reductase that is responsible for converting the nitro group of the antibiotic into a nonbactericidal amine. A mechanism for the Nim enzyme has been proposed in which two-electron reduction of the nitro group leads to the generation of nontoxic derivatives and confers resistance against these antibiotics. The cofactor was found to be important in the mechanism and was found to be covalently linked to the reactive His71. In this paper, the 1.2 A atomic resolution crystal structure of the 5-nitroimidazole antibiotic resistance protein NimA from Deinococcus radiodurans (DrNimA) is presented. A planar cofactor is clearly visible and well defined in the electron-density map adjacent to His71, the identification of the cofactor and its properties are discussed.

  10. Development of conformation independent computational models for the early recognition of breast cancer resistance protein substrates.

    PubMed

    Gantner, Melisa Edith; Di Ianni, Mauricio Emiliano; Ruiz, María Esperanza; Talevi, Alan; 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

  11. Role of mitochondrial uncoupling protein 2 in cancer cell resistance to gemcitabine.

    PubMed

    Dalla Pozza, Elisa; Fiorini, Claudia; Dando, Ilaria; Menegazzi, Marta; Sgarbossa, Anna; Costanzo, Chiara; Palmieri, Marta; Donadelli, Massimo

    2012-10-01

    Cancer cells exhibit an endogenous constitutive oxidative stress higher than that of normal cells, which renders tumours vulnerable to further reactive oxygen species (ROS) production. Mitochondrial uncoupling protein 2 (UCP2) can mitigate oxidative stress by increasing the influx of protons into the mitochondrial matrix and reducing electron leakage and mitochondrial superoxide generation. Here, we demonstrate that chemical uncouplers or UCP2 over-expression strongly decrease mitochondrial superoxide induction by the anticancer drug gemcitabine (GEM) and protect cancer cells from GEM-induced apoptosis. Moreover, we show that GEM IC(50) values well correlate with the endogenous level of UCP2 mRNA, suggesting a critical role for mitochondrial uncoupling in GEM resistance. Interestingly, GEM treatment stimulates UCP2 mRNA expression suggesting that mitochondrial uncoupling could have a role also in the acquired resistance to GEM. Conversely, UCP2 inhibition by genipin or UCP2 mRNA silencing strongly enhances GEM-induced mitochondrial superoxide generation and apoptosis, synergistically inhibiting cancer cell proliferation. These events are significantly reduced by the addition of the radical scavenger N-acetyl-l-cysteine or MnSOD over-expression, demonstrating a critical role of the oxidative stress. Normal primary fibroblasts are much less sensitive to GEM/genipin combination. Our results demonstrate for the first time that UCP2 has a role in cancer cell resistance to GEM supporting the development of an anti-cancer therapy based on UCP2 inhibition associated to GEM treatment.

  12. 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

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

    PubMed

    Nyström, Sofie; Hammarström, Per

    2015-01-01

    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. PMID:25960067

  14. 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.

  15. The Ablation of Mitochondrial Protein Phosphatase Pgam5 Confers Resistance Against Metabolic Stress

    PubMed Central

    Sekine, Shiori; Yao, Akari; Hattori, Kazuki; Sugawara, Sho; Naguro, Isao; Koike, Masato; Uchiyama, Yasuo; Takeda, Kohsuke; Ichijo, Hidenori

    2016-01-01

    Phosphoglycerate mutase family member 5 (PGAM5) is a mitochondrial protein phosphatase that has been reported to be involved in various stress responses from mitochondrial quality control to cell death. However, its roles in vivo are largely unknown. Here, we show that Pgam5-deficient mice are resistant to several metabolic insults. Under cold stress combined with fasting, Pgam5-deficient mice better maintained body temperature than wild-type mice and showed an extended survival rate. Serum triglycerides and lipid content in brown adipose tissue (BAT), a center of adaptive thermogenesis, were severely reduced in Pgam5-deficient mice. Moreover, although Pgam5 deficiency failed to maintain proper mitochondrial integrity in BAT, it reciprocally resulted in the dramatic induction of fibroblast growth factor 21 (FGF21) that activates various functions of BAT including thermogenesis. Thus, the enhancement of lipid metabolism and FGF21 may contribute to the cold resistance of Pgam5-deficient mice under fasting condition. Finally, we also found that Pgam5-deficient mice are resistant to high-fat-diet-induced obesity. Our study uncovered that PGAM5 is involved in the whole-body metabolism in response to stresses that impose metabolic challenges on mitochondria. PMID:27077115

  16. The Ablation of Mitochondrial Protein Phosphatase Pgam5 Confers Resistance Against Metabolic Stress.

    PubMed

    Sekine, Shiori; Yao, Akari; Hattori, Kazuki; Sugawara, Sho; Naguro, Isao; Koike, Masato; Uchiyama, Yasuo; Takeda, Kohsuke; Ichijo, Hidenori

    2016-03-01

    Phosphoglycerate mutase family member 5 (PGAM5) is a mitochondrial protein phosphatase that has been reported to be involved in various stress responses from mitochondrial quality control to cell death. However, its roles in vivo are largely unknown. Here, we show that Pgam5-deficient mice are resistant to several metabolic insults. Under cold stress combined with fasting, Pgam5-deficient mice better maintained body temperature than wild-type mice and showed an extended survival rate. Serum triglycerides and lipid content in brown adipose tissue (BAT), a center of adaptive thermogenesis, were severely reduced in Pgam5-deficient mice. Moreover, although Pgam5 deficiency failed to maintain proper mitochondrial integrity in BAT, it reciprocally resulted in the dramatic induction of fibroblast growth factor 21 (FGF21) that activates various functions of BAT including thermogenesis. Thus, the enhancement of lipid metabolism and FGF21 may contribute to the cold resistance of Pgam5-deficient mice under fasting condition. Finally, we also found that Pgam5-deficient mice are resistant to high-fat-diet-induced obesity. Our study uncovered that PGAM5 is involved in the whole-body metabolism in response to stresses that impose metabolic challenges on mitochondria. PMID:27077115

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

    PubMed

    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 Ca(2+)-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. The emerging pharmacotherapeutic significance of the breast cancer resistance protein (ABCG2)

    PubMed Central

    Hardwick, L J A; Velamakanni, S; van Veen, H W

    2007-01-01

    The breast cancer resistance protein (also termed ABCG2) is an ATP-binding cassette transporter, which mediates the extrusion of toxic compounds from the cell, and which was originally identified in relation to the development of multidrug resistance of cancer cells. ABCG2 interacts with a range of substrates including clinical drugs but also substances such as sterols, porphyrins and a variety of dietary compounds. Physiological functions of ABCG2 at both cellular and systemic levels are reviewed. For example, ABCG2 expression in erythrocytes may function in porphyrin homeostasis. In addition, ABCG2 expression at apical membranes of cells such as hepatocytes, enterocytes, endothelial and syncytiotrophoblast cells may correlate to protective barrier or secretory functions against environmental or clinically administered substances. ABCG2 also appears influential in the inter-patient variation and generally poor oral bioavailability of certain chemotherapeutic drugs such as topotecan. As this often precludes an oral drug administration strategy, genotypic and environmental factors altering ABCG2 expression and activity are considered. Finally, clinical modulation of ABCG2 activity is discussed. Some of the more recent strategies include co-administered modulating agents, hammerhead ribozymes or antisense oligonucleotides, and with specificity in cell targeting, these may be used to reduce drug resistance and increase drug bioavailability to improve the profile of chemotherapeutic efficacy versus toxicity. While many such strategies remain in relative infancy at present, increased knowledge of modulators of ABCG2 could hold the key to novel approaches in medical treatment. PMID:17375082

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

    PubMed Central

    Nyström, Sofie; Hammarström, Per

    2015-01-01

    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. PMID:25960067

  20. 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

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

    PubMed

    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

  2. Protein Tyrosine Phosphatase Activity in Insulin-Resistant Rodent Psammomys Obesus

    PubMed Central

    Meyerovitch, Joseph; Balta, Yigal; Ziv, Ehud; Sack, Joseph

    2002-01-01

    Phosphotyrosine phosphatase (PTPase) activity and its regulation by overnight food deprivation were studied in Psammomys obesus (sand rat), a gerbil model of insulin resistance and nutritionally induced diabetes mellitus. PTPase activity was measured using a phosphopeptide substrate containing a sequence identical to that of the major site of insulin receptor (IR) β-subunit autophosphorylation. The PTPase activity in membrane fractions was 3.5-, 8.3-, and 5.9-fold lower in liver, fat, and skeletal muscle, respectively, compared with corresponding tissues of albino rat.Western blotting of tissue membrane fractions in Psammomys showed lower PTPase and IR than in albino rats. The density of PTPase transmembrane protein band was 5.5-fold lower in liver and 12-fold lower in adipose tissue. Leukocyte antigen receptor (LAR) and IR were determined by specific immunoblotting and protein bands densitometry and were also found to be 6.3-fold lower in the liver and 22-fold lower in the adipose tissue in the hepatic membrane fractions. Liver cytosolic PTPase activity after an overnight food deprivation in the nondiabetic Psammomys rose 3.7-fold compared with postprandial PTPase activity, but it did not change significantly in diabetic fasted animals. Similar fasting-related changes were detected in the activity of PTPase derived from membrane fraction. In conclusion, the above data demonstrate that despite the insulin resistance, Psammomys is characterized by low level of PTPase activities in membrane and cytosolic fractions in all 3 major insulin responsive tissues, as well as in liver. PTPase activity does not rise in activity as a result of insulin resistance and nutritionally induced diabetes. PMID:12458662

  3. Hypoxia promotes drug resistance in osteosarcoma cells via activating AMP-activated protein kinase (AMPK) signaling

    PubMed Central

    Zhao, Changfu; Zhang, Qiao; Yu, Tao; Sun, Shudong; Wang, Wenjun; Liu, Guangyao

    2016-01-01

    Purpose Drug resistance has been recognized to be a major obstacle to the chemotherapy for osteosarcoma. And the potential importance of hypoxia as a target to reverse drug resistance in osteosarcoma has been indicated, though the mechanism underlining such role is not clarified. The present study aims to investigate the role of hypoxia in the drug resistance in osteosarcoma cells via activating AMP-activated protein kinase (AMPK) signaling. Experimental design We investigated the promotion of the resistance to doxorubicin of osteosarcoma MG-63 and U2-os cells in vitro, and then determined the role of hypoxia-inducible factor-1 (HIF-1)α and HIF-1β, the activation and regulatory role of AMPK in the osteosarcoma U2-os cells which were treated with doxorubicin under hypoxia. Results It was demonstrated that hypoxia significantly reduced the sensitivity of MG-63 and U2-os cells to doxorubicin, indicating an inhibited viability reduction and a reduced apoptosis promotion. And such reduced sensitivity was not associated with HIF-1α, though it was promoted by hypoxia in U2-os cells. Interestingly, the AMPK signaling was significantly promoted by hypoxia in the doxorubicin-treated U2-os cells, with a marked upregulation of phosphorylated AMPK (Thr 172) and phosphorylated acetyl-CoA carboxylase (ACC) (Ser 79), which were sensitive to the AMPK activator, AICAR and the AMPK inhibitor, Compound C. Moreover, the promoted AMPK activity by AICAR or the downregulated AMPK activity by Compound C significantly reduced or promoted the sensitivity of U2-os cells to doxorubicin. Conclusion The present study confirmed the AMPK signaling activation in the doxorubicin-treated osteosarcoma cells, in response to hypoxia, and the chemical upregulation or downregulation of AMPK signaling reduced or increased the chemo-sensitivity of osteosarcoma U2-os cells in vitro. Our study implies that AMPK inhibition might be a effective strategy to sensitize osteocarcoma cells to chemotherapy. PMID

  4. Overexpression of patA and patB, Which Encode ABC Transporters, Is Associated with Fluoroquinolone Resistance in Clinical Isolates of Streptococcus pneumoniae▿

    PubMed Central

    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. PMID:20937787

  5. Identification of a nitroimidazo-oxazine-specific protein involved in PA-824 resistance in Mycobacterium tuberculosis

    PubMed Central

    Manjunatha, Ujjini H.; Boshoff, Helena; Dowd, Cynthia S.; Zhang, Liang; Albert, Thomas J.; Norton, Jason E.; Daniels, Lacy; Dick, Thomas; Pang, Siew Siew; Barry, Clifton E.

    2006-01-01

    PA-824 is a promising new compound for the treatment of tuberculosis that is currently undergoing human trials. Like its progenitors metronidazole and CGI-17341, PA-824 is a prodrug of the nitroimidazole class, requiring bioreductive activation of an aromatic nitro group to exert an antitubercular effect. We have confirmed that resistance to PA-824 (a nitroimidazo-oxazine) and CGI-17341 (a nitroimidazo-oxazole) is most commonly mediated by loss of a specific glucose-6-phosphate dehydrogenase (FGD1) or its deazaflavin cofactor F420, which together provide electrons for the reductive activation of this class of molecules. Although FGD1 and F420 are necessary for sensitivity to these compounds, they are not sufficient and require additional accessory proteins that directly interact with the nitroimidazole. To understand more proximal events in the reductive activation of PA-824, we examined mutants that were wild-type for both FGD1 and F420 and found that, although these mutants had acquired high-level resistance to PA-824 (and another nitroimidazo-oxazine), they retained sensitivity to CGI-17341 (and a related nitroimidazo-oxazole). Microarray-based comparative genome sequencing of these mutants identified lesions in Rv3547, a conserved hypothetical protein with no known function. Complementation with intact Rv3547 fully restored sensitivity to nitroimidazo-oxazines and restored the ability of Mtb to metabolize PA-824. These results suggest that the sensitivity of Mtb to PA-824 and related compounds is mediated by a protein that is highly specific for subtle structural variations in these bicyclic nitroimidazoles. PMID:16387854

  6. The small heat shock protein 20 RSI2 interacts with and is required for stability and function of tomato resistance protein I-2.

    PubMed

    Van Ooijen, Gerben; Lukasik, Ewa; Van Den Burg, Harrold A; Vossen, Jack H; Cornelissen, Ben J C; Takken, Frank L W

    2010-08-01

    Race-specific disease resistance in plants depends on the presence of resistance (R) genes. Most R genes encode NB-ARC-LRR proteins that carry a C-terminal leucine-rich repeat (LRR). Of the few proteins found to interact with the LRR domain, most have proposed (co)chaperone activity. Here, we report the identification of RSI2 (Required for Stability of I-2) as a protein that interacts with the LRR domain of the tomato R protein I-2. RSI2 belongs to the family of small heat shock proteins (sHSPs or HSP20s). HSP20s are ATP-independent chaperones that form oligomeric complexes with client proteins to prevent unfolding and subsequent aggregation. Silencing of RSI2-related HSP20s in Nicotiana benthamiana compromised the hypersensitive response that is normally induced by auto-active variants of I-2 and Mi-1, a second tomato R protein. As many HSP20s have chaperone properties, the involvement of RSI2 and other R protein (co)chaperones in I-2 and Mi-1 protein stability was examined. RSI2 silencing compromised the accumulation of full-length I-2 in planta, but did not affect Mi-1 levels. Silencing of heat shock protein 90 (HSP90) and SGT1 led to an almost complete loss of full-length I-2 accumulation and a reduction in Mi-1 protein levels. In contrast to SGT1 and HSP90, RSI2 silencing led to accumulation of I-2 breakdown products. This difference suggests that RSI2 and HSP90/SGT1 chaperone the I-2 protein using different molecular mechanisms. We conclude that I-2 protein function requires RSI2, either through direct interaction with, and stabilization of I-2 protein or by affecting signalling components involved in initiation of the hypersensitive response. PMID:20497382

  7. Ribosomal Protein S29 Regulates Metabolic Insecticide Resistance through Binding and Degradation of CYP6N3

    PubMed Central

    Yu, Jing; Hu, Shengli; Ma, Kai; Sun, Linchun; Hu, Hongxia; Zou, Feifei; Guo, Qin; Lei, Zhentao; Zhou, Dan; Sun, Yan; Zhang, Donghui; Ma, Lei; Shen, Bo; Zhu, Changliang

    2014-01-01

    Background Many diseases are transmitted by mosquitoes, including malaria, dengue fever, yellow fever, filariasis, and West Nile fever. Chemical control plays a major role in managing mosquito-borne diseases. However, excessive and continuous application of insecticides has caused the development of insecticide resistance in many species including mosquito, and this has become the major obstacle to controlling mosquito-borne diseases. Insecticide resistance is the result of complex polygenic inheritance, and the mechanisms are not well understood. Ribosomal protein RPS29 was found to be associated with DM resistance in our previous study. In this study, we aim to further investigate the involvement of RPS29 in deltamethrin resistance. Methodology and Principal Findings In this study, tandem affinity purification was used to identify proteins that can interact with RPS29. Among the candidate proteins, CYP6N3, a member of the CYP450 superfamily, was identified, and binding to RPS29 was confirmed in vitro and in vivo by GST pull-down and immunofluorescence. CCK-8 assay was used to investigate the RPS29-CTP6N3 interaction in relation to DM resistance. CYP6N3 overexpression significantly enhanced DM resistance and insect cell viability, but this was reversed by RPS29 overexpression. Western blot was used to study the mechanism of interaction between RPS29 and CYP6N3. RPS29 increases CYP6N3 protein degradation through the proteasome. Conclusions and Significance These observations indicate that CYP6N3, a novel RPS29-interacting partner, could stimulate deltamethrin resistance in mosquito cells and RPS29 overexpression targeted CYP6N3 for proteosomal degradation, abrogating the CYP6N3-associated resistence to deltamethrin. Our findings provide a novel mechanism associated with CYP450s mediated DM resistance. PMID:24728095

  8. Tsw gene-based resistance is triggered by a functional RNA silencing suppressor protein of the Tomato spotted wilt virus.

    PubMed

    de Ronde, Dryas; Butterbach, Patrick; Lohuis, Dick; Hedil, Marcio; van Lent, Jan W M; Kormelink, Richard

    2013-05-01

    As a result of contradictory reports, the avirulence (Avr) determinant that triggers Tsw gene-based resistance in Capsicum annuum against the Tomato spotted wilt virus (TSWV) is still unresolved. Here, the N and NSs genes of resistance-inducing (RI) and resistance-breaking (RB) isolates were cloned and transiently expressed in resistant Capsicum plants to determine the identity of the Avr protein. It was shown that the NSs(RI) protein triggered a hypersensitive response (HR) in Tsw-containing Capsicum plants, but not in susceptible Capsicum, whereas no HR was discerned after expression of the N(RI) (/) (RB) protein, or when NSs(RB) was expressed. Although NSs(RI) was able to suppress the silencing of a functional green fluorescence protein (GFP) construct during Agrobacterium tumefaciens transient assays on Nicotiana benthamiana, NSs(RB) had lost this capacity. The observation that RB isolates suppressed local GFP silencing during an infection indicated a recovery of RNA silencing suppressor activity for the NSs protein or the presence of another RNA interference (RNAi) suppressor. The role of NSs as RNA silencing suppressor and Avr determinant is discussed in the light of a putative interplay between RNAi and the natural Tsw resistance gene.

  9. A novel elicitor protein from Phytophthora parasitica induces plant basal immunity and systemic acquired resistance.

    PubMed

    Chang, Yi-Hsuan; Yan, Hao-Zhi; Liou, Ruey-Fen

    2015-02-01

    The interaction between Phytophthora pathogens and host plants involves the exchange of complex molecular signals from both sides. Recent studies of Phytophthora have led to the identification of various apoplastic elicitors known to trigger plant immunity. Here, we provide evidence that the protein encoded by OPEL of Phytophthora parasitica is a novel elicitor. Homologues of OPEL were identified only in oomycetes, but not in fungi and other organisms. Quantitative reverse transcription-polymerase chain reaction (RT-PCR) revealed that OPEL is expressed throughout the development of P. parasitica and is especially highly induced after plant infection. Infiltration of OPEL recombinant protein from Escherichia coli into leaves of Nicotiana tabacum (cv. Samsun NN) resulted in cell death, callose deposition, the production of reactive oxygen species and induced expression of pathogen-associated molecular pattern (PAMP)-triggered immunity markers and salicylic acid-responsive defence genes. Moreover, the infiltration conferred systemic resistance against a broad spectrum of pathogens, including Tobacco mosaic virus, the bacteria wilt pathogen Ralstonia solanacearum and P. parasitica. In addition to the signal peptide, OPEL contains three conserved domains: a thaumatin-like domain, a glycine-rich protein domain and a glycosyl hydrolase (GH) domain. Intriguingly, mutation of a putative laminarinase active site motif in the predicted GH domain abolished its elicitor activity, which suggests enzymatic activity of OPEL in triggering the defence response.

  10. Influence of cyclophilin D protein expression level on endothelial cell oxidative damage resistance.

    PubMed

    Peng, J Z; Xue, L; Chen, J; Chen, B S; Yang, Y Q

    2015-01-01

    We examined the influence of cyclophilin-D (CypD) protein expression level on endothelial cell oxidative damage resistance. A model of CypD protein expression or high expression in endothelial cells was established through gene silencing or cloning. The comparable groups were normal endothelial cells cultured in phosphate-buffered solution in liquid handling cells containing 500 mM H2O2 for 90 or 120 min, and then the medium was replaced with common nutrient solution and cultured again for 24 h. The apoptosis rate and nitric oxide (NO) levels of each group were tested. The cell apoptosis rate of the CyPD low expression group (32.51 ± 6.6 %) was significantly lower than that of the control group (52.57 ± 5.84%, P = 0.001), and total NO production was 24.06 ± 3 and 13.03 ± 3.55 μM. The apoptosis rate of the CyPD high expression group (24.24 + 3.08%) was significantly higher than that of the control group (7.7 + 0.68%, P < 0.001); total NO production was 3.55 ± 1.53 and 8.46 ± 0.77 μM, which was significantly different (P = 0.008). CypD protein could increase oxidative stress and cause endothelial cell injury and apoptosis.

  11. A single bout of resistance exercise improves memory consolidation and increases the expression of synaptic proteins in the hippocampus.

    PubMed

    Fernandes, Jansen; Soares, Juliana Carlota Kramer; do Amaral Baliego, Luiz Guilherme Zaccaro; Arida, Ricardo Mario

    2016-08-01

    Over the past decade, several studies have indicated that chronic resistance exercise (i.e., strength training, weight lifting, etc.) is beneficial for brain health and cognitive function. However, little is known about the effects of a single bout of resistance exercise on brain function, particularly on memory consolidation. Therefore, the purpose of the present study is to examine the effects of a single bout of resistance exercise applied immediately after the training of fear conditioning on memory consolidation and on the expression of IGF-1 and synaptic proteins in the hippocampus. Male Wistar rats were familiarized with climbing a ladder without a load for 3 days and randomly assigned into control (CTL) and resistance exercise (RES) groups. The RES group was subjected to a single bout of resistance exercise applied immediately after fear conditioning training. Subsequently, the animals were tested for contextual (24 h) and tone (48 h) fear memory. Another group of animals were subjected to a single bout of resistance exercise and euthanized 24 h later for hippocampal analysis of IGF-1 and synaptic proteins (synapsin I, synaptophysin, and PSD-95). The exercised rats improved contextual but not tone fear memory. Hippocampal IGF-1 was not altered by resistance exercise. However, the levels of synapsin I, synaptophysin, and PSD-95 increased significantly in the RES group. The results suggested that a single bout of resistance exercise applied immediately after fear conditioning could improve contextual memory, probably through the activation of pre- and postsynaptic machinery required for memory consolidation. © 2016 Wiley Periodicals, Inc.

  12. Protein expression profile related to cisplatin resistance in bladder cancer cell lines detected by two-dimensional gel electrophoresis.

    PubMed

    Taoka, Yoshinori; Matsumoto, Kazumasa; Ohashi, Kazuya; Minamida, Satoru; Hagiwara, Masahiro; Nagi, Shoji; Saito, Tatsuya; Kodera, Yoshio; Iwamura, Masatsugu

    2015-01-01

    We used a proteomic approach to compare the differentially regulated protein expression profiles of cisplatin-naïve and cisplatin-resistant bladder cancer cell lines to screen candidate molecules related to cisplatin resistance. The cisplatin-resistant cell line T24 was established by the stepwise exposure of T24 cells to up to 40 μM of cisplatin. We performed a comprehensive study of protein expression in bladder cancer cell lines that included cisplatin-naïve (T24) and cisplatin-resistant cells (T24CDDPR) by means of agarose two-dimensional gel electrophoresis followed by analysis of liquid chromatography tandem mass spectroscopy. We identified 25 obviously different spots for T24 and T24 CDDPR. Seven spots had increased expression and 18 spots had decreased expression in T24CDDPR compared to those in T24. Cytoskeletal proteins and enzyme modulators were prominent among differential proteins. Of the 25 proteins, we selected HNRNPA3, PCK2, PPL, PGK1, TKT, SERPINB2, GOT2, and EIF3A for further validation by Western blot. HNRNPA3, PGK1, TKT, and SERPINB2 had more than 1.5-times incremental expression in T24CDDPR compared to that in T24. PCK2 and PPL expressions were decreased less than 20% in T24CDDPR compared to that in T24. The results of 25 new proteins in this study could be valuable and could lead to the development of a new molecular marker. PMID:26299484

  13. Homologous metalloregulatory proteins from both gram-positive and gram-negative bacteria control transcription of mercury resistance operons

    SciTech Connect

    Helmann, J.D.; Walsh, C.T. ); Wang, Ying; Mahler, I. )

    1989-01-01

    The authors report the overexpression, purification, and properties of the regulatory protein, MerR, for a chromosomally encoded mercury resistance determinant from Bacillus strain RC607. This protein is similar in sequence to the metalloregulatory proteins encoded by gram-negative resistance determinants found on transposons Tn21 and Tn501 and to a predicted gene product of a Staphylococcus aureus resistance determinant. In vitro DNA-binding and transcription experiments were used to demonstrate those purified Bacillus MerR protein controls transcription from a promoter-operator site similar in sequence to that found in the transposon resistance determinants. The Bacillus MerR protein bound in vitro to its promoter-operator region in both the presence and absence of mercuric ion and functioned as a negative and positive regulator of transcription. The MerR protein bound less tightly to its operator region (ca. 50- to 100-fold) in the presence of mercuric ion; this reduced affinity was largely accounted for by an increased rate of dissociation of the MerR protein from the DNA. Despite this reduced DNA-binding affinity, genetic and biochemical evidence support a model in which the MerR protein-mercuric ion complex is a positive regulator of operon transcription. Although the Bacillus MerR protein bound only weakly to the heterologous Tn501 operator region, the Tn501 and Tn21 MerR proteins bound with high affinity to the Bacillus promoter-operator region and exhibited negative, but not positive, transcriptional control.

  14. Involvement of miR-326 in chemotherapy resistance of breast cancer through modulating expression of multidrug resistance-associated protein 1.

    PubMed

    Liang, Zhongxing; Wu, Hui; Xia, James; Li, Yuhua; Zhang, Yawei; Huang, Ke; Wagar, Nicholas; Yoon, Younghyoun; Cho, Heidi T; Scala, Stefania; Shim, Hyunsuk

    2010-03-15

    Multidrug resistance-associated protein (MRP-1/ABCC1) transports a wide range of therapeutic agents and may play a critical role in the development of multidrug resistance (MDR) in tumor cells. However, the regulation of MRP-1 remains controversial. To explore whether miRNAs are involved in the regulation of MRP-1 expression and modulate the sensitivity of tumor cells to chemotherapeutic agents, we analyzed miRNA expression levels in VP-16-resistant MDR cell line, MCF-7/VP, in comparison with its parent cell line, MCF-7, using a miRNA microarray. MCF-7/VP overexpressed MRP-1 mRNA and protein not MDR-1 and BCRP. miR-326 was downregulated in MCF-7/VP compared to MCF-7. Additionally, miR-326 was downregulated in a panel of advanced breast cancer tissues and consistent reversely with expression levels of MRP-1. Furthermore, the elevated levels of miR-326 in the mimics-transfected VP-16-resistant cell line, MCF-7/VP, downregulated MRP-1 expression and sensitized these cells to VP-16 and doxorubicin. These findings demonstrate for the first time the involvement of miRNAs in multidrug resistance mediated by MRP-1 and suggest that miR-326 may be an efficient agent for preventing and reversing MDR in tumor cells.

  15. Bifendate-chalcone hybrids: a new class of potential dual inhibitors of P-glycoprotein and breast cancer resistance protein.

    PubMed

    Gu, Xiaoke; Ren, Zhiguang; Peng, Hui; Peng, Sixun; Zhang, Yihua

    2014-12-12

    We previously described bifendate-chalcone hybrids as potent P-glycoprotein inhibitors. In the present work, we determine whether these compounds could reverse breast cancer resistance protein (BCRP, ABCG2)-mediated multidrug resistance using HEK293/BCRP cells which was BCRP-transfected stable HEK293 cells. Results indicated that compounds 8d, 8f, 8g and 8h could significantly enhance mitoxantrone accumulation in HEK293/BCRP cells via inhibiting BCRP drug efflux function. The most active compound 8g exhibited little intrinsic cytotoxicity (IC₅₀>100 μM), and could reverse BCRP-mediated drug resistance independent of decreasing BCRP expression level. Notably, 8g had little inhibitory effect on multidrug resistance-associated protein 1 (MRP1, ABCC1), another drug efflux transporter. The present findings, together with the previous results, suggest that 8g might be act as dual inhibitors of P-gp and BCRP.

  16. 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

  17. Long noncoding RNA MALAT1 promotes hepatic steatosis and insulin resistance by increasing nuclear SREBP-1c protein stability

    PubMed Central

    Yan, Caifeng; Chen, Jinfeng; Chen, Nuoqi

    2016-01-01

    Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) is implicated in liver cell proliferation. However, its role in hepatic steatosis and insulin resistance remain poorly understood. The aim of this study was to investigate the effects of MALAT1 on hepatic lipid accumulation and its potential targets. As expected, MALAT1 expression is increased in hepatocytes exposed to palmitate and livers of ob/ob mice. Knockdown of MALAT1 expression dramatically suppressed palmitate-induced lipid accumulation and the increase of nuclear SREBP-1c protein in HepG2 cells. In addition, RNA immunoprecipitation and RNA pull-down assay confirmed that MALAT1 interacted with SREBP-1c to stabilize nuclear SREBP-1c protein. Finally, injection of si-MALAT1 prevented hepatic lipid accumulation and insulin resistance in ob/ob mice. In conclusion, our observations suggest that MALAT1 promotes hepatic steatosis and insulin resistance by increasing nuclear SREBP-1c protein stability. PMID:26935028

  18. Long noncoding RNA MALAT1 promotes hepatic steatosis and insulin resistance by increasing nuclear SREBP-1c protein stability.

    PubMed

    Yan, Caifeng; Chen, Jinfeng; Chen, Nuoqi

    2016-03-03

    Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) is implicated in liver cell proliferation. However, its role in hepatic steatosis and insulin resistance remain poorly understood. The aim of this study was to investigate the effects of MALAT1 on hepatic lipid accumulation and its potential targets. As expected, MALAT1 expression is increased in hepatocytes exposed to palmitate and livers of ob/ob mice. Knockdown of MALAT1 expression dramatically suppressed palmitate-induced lipid accumulation and the increase of nuclear SREBP-1c protein in HepG2 cells. In addition, RNA immunoprecipitation and RNA pull-down assay confirmed that MALAT1 interacted with SREBP-1c to stabilize nuclear SREBP-1c protein. Finally, injection of si-MALAT1 prevented hepatic lipid accumulation and insulin resistance in ob/ob mice. In conclusion, our observations suggest that MALAT1 promotes hepatic steatosis and insulin resistance by increasing nuclear SREBP-1c protein stability.

  19. 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.

  20. Identification of kernel proteins associated with the resistance to fusarium head blight in winter wheat (Triticum aestivum L.).

    PubMed

    Perlikowski, Dawid; Wiśniewska, Halina; Góral, Tomasz; Kwiatek, Michał; Majka, Maciej; Kosmala, Arkadiusz

    2014-01-01

    Numerous potential components involved in the resistance to Fusarium head blight (FHB) in cereals have been indicated, however, our knowledge regarding this process is still limited and further work is required. Two winter wheat (Triticum aestivum L.) lines differing in their levels of resistance to FHB were analyzed to identify the most crucial proteins associated with resistance in this species. The presented work involved analysis of protein abundance in the kernel bulks of more resistant and more susceptible wheat lines using two-dimensional gel electrophoresis and mass spectrometry identification of proteins, which were differentially accumulated between the analyzed lines, after inoculation with F. culmorum under field conditions. All the obtained two-dimensional patterns were demonstrated to be well-resolved protein maps of kernel proteomes. Although, 11 proteins were shown to have significantly different abundance between these two groups of plants, only two are likely to be crucial and have a potential role in resistance to FHB. Monomeric alpha-amylase and dimeric alpha-amylase inhibitors, both highly accumulated in the more resistant line, after inoculation and in the control conditions. Fusarium pathogens can use hydrolytic enzymes, including amylases to colonize kernels and acquire nitrogen and carbon from the endosperm and we suggest that the inhibition of pathogen amylase activity could be one of the most crucial mechanisms to prevent infection progress in the analyzed wheat line with a higher resistance. Alpha-amylase activity assays confirmed this suggestion as it revealed the highest level of enzyme activity, after F. culmorum infection, in the line more susceptible to FHB.

  1. Identification of Kernel Proteins Associated with the Resistance to Fusarium Head Blight in Winter Wheat (Triticum aestivum L.)

    PubMed Central

    Góral, Tomasz; Kwiatek, Michał; Majka, Maciej; Kosmala, Arkadiusz

    2014-01-01

    Numerous potential components involved in the resistance to Fusarium head blight (FHB) in cereals have been indicated, however, our knowledge regarding this process is still limited and further work is required. Two winter wheat (Triticum aestivum L.) lines differing in their levels of resistance to FHB were analyzed to identify the most crucial proteins associated with resistance in this species. The presented work involved analysis of protein abundance in the kernel bulks of more resistant and more susceptible wheat lines using two-dimensional gel electrophoresis and mass spectrometry identification of proteins, which were differentially accumulated between the analyzed lines, after inoculation with F. culmorum under field conditions. All the obtained two-dimensional patterns were demonstrated to be well-resolved protein maps of kernel proteomes. Although, 11 proteins were shown to have significantly different abundance between these two groups of plants, only two are likely to be crucial and have a potential role in resistance to FHB. Monomeric alpha-amylase and dimeric alpha-amylase inhibitors, both highly accumulated in the more resistant line, after inoculation and in the control conditions. Fusarium pathogens can use hydrolytic enzymes, including amylases to colonize kernels and acquire nitrogen and carbon from the endosperm and we suggest that the inhibition of pathogen amylase activity could be one of the most crucial mechanisms to prevent infection progress in the analyzed wheat line with a higher resistance. Alpha-amylase activity assays confirmed this suggestion as it revealed the highest level of enzyme activity, after F. culmorum infection, in the line more susceptible to FHB. PMID:25340555

  2. Altered protein S-glutathionylation identifies a potential mechanism of resistance to acetaminophen-induced hepatotoxicity.

    PubMed

    McGarry, David J; Chakravarty, Probir; Wolf, C Roland; Henderson, Colin J

    2015-11-01

    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

  3. Biophysical characterization and mutational analysis of the antibiotic resistance protein NimA from Deinococcus radiodurans.

    PubMed

    Leiros, Hanna-Kirsti S; Brandsdal, Bjørn Olav; McSweeney, Seán M

    2010-04-01

    Metronidazole (MTZ) is an antibiotic commonly used to treat anaerobic bacterial infections in humans and animals. Antibiotic resistance toward this class of 5-nitroimidazole (5-Ni) drug derivatives has been related to the Nim genes thought to encode a reductase. Here we report the biophysical characteristics of the NimA protein from Deinococcus radiodurans (DrNimA) binding to MTZ and three other 5-Ni drugs. The interaction energies of the protein and antibiotic are studied by isothermal titration calorimetry (ITC) and with free energy and linear interaction energy (LIE) calculations, where the latter method revealed that the antibiotic binding is mainly of hydrophobic character. ITC measurements further found that one DrNimA dimer has two antibiotic binding sites which were not affected by mutation of the reactive His71. The observed association constants (K(a)) were in the range of 5.1-4910(4)M(-1) and the enthalpy release upon binding to DrNimA for the four drugs studied was relatively low (approximately -1 kJ/mol) but still measurable. The drug binding is mainly entropy driven and along with the hydrophobic drug binding site found by crystallography, this possibly explains the low observed enthalpy values. The effect of the His71 mutation and the presence of MTZ were studied by differential scanning calorimetry (DSC). Native DrNimA is a yellow colored protein where the interaction from His71 to the cofactor is thought to be responsible for the coloring. Mutations of His71 to Ala, Ser, Leu or Asp all gave transparent, colorless protein solutions, and the two mutant crystal structures of DrNimA-H71A and DrNimA-H71S presented revealed no cofactor binding.

  4. The multidrug resistance protein 1: a functionally important activation marker for murine Th1 cells.

    PubMed

    Prechtl, S; Roellinghoff, M; Scheper, R; Cole, S P; Deeley, R G; Lohoff, M

    2000-01-15

    Previously, we described the expression of an energy-dependent pump in resting murine Th2 (but not resting Th1) cells which extruded the fluorescent dye Fluo-3. After stimulation with Ag and APCs, Th1 cells also expressed this pump. Furthermore, expression of the murine multidrug resistance protein 1 (mrp1) correlated with the presence of the pump. In this study, we report that Fluo-3 is indeed transported by murine mrp1 or its human ortholog MRP1, as revealed by transfection of HEK 293 cells with mrp1 or MRP1 cDNA. Like antigenic activation, IL-2 dose-dependently enhanced the Fluo-3-extruding activity in murine Th1 cells. Although TNF-alpha and IL-12 by themselves only weakly enhanced Fluo-3 extrusion, each of them did so in strong synergism with IL-2. An Ab directed against mrp1 was used to quantify the expression of mrp1 protein in T cells at the single-cell level. Like the Fluo-3 pump, mrp1 protein expression was enhanced by IL-2. Immunohistochemical studies using confocal laser microscopy indicated that mrp1 is localized mainly at the plasma membrane. In addition, protein expression of mrp1 was induced in Vbeta8+CD4+ T cells 12 h after in vivo application of Staphylococcal enterotoxin B. Finally, mrp1 was functionally relevant during the activation process of Th1 cells, because T cell activation could be suppressed by exposure of cells to the mrp1 inhibitor MK571. Thus, we present mrp1 as a novel, functionally important activation marker for Th1 cells and short-term in vivo activated CD4+ T cells, whereas its expression seems to be constitutive in Th2 cells.

  5. Two seven-transmembrane domain MILDEW RESISTANCE LOCUS O proteins cofunction in Arabidopsis root thigmomorphogenesis.

    PubMed

    Chen, Zhongying; Noir, Sandra; Kwaaitaal, Mark; Hartmann, H Andreas; Wu, Ming-Jing; Mudgil, Yashwanti; Sukumar, Poornima; Muday, Gloria; Panstruga, Ralph; Jones, Alan M

    2009-07-01

    Directional root expansion is governed by nutrient gradients, positive gravitropism and hydrotropism, negative phototropism and thigmotropism, as well as endogenous oscillations in the growth trajectory (circumnutation). Null mutations in phylogenetically related Arabidopsis thaliana genes MILDEW RESISTANCE LOCUS O 4 (MLO4) and MLO11, encoding heptahelical, plasma membrane-localized proteins predominantly expressed in the root tip, result in aberrant root thigmomorphogenesis. mlo4 and mlo11 mutant plants show anisotropic, chiral root expansion manifesting as tightly curled root patterns upon contact with solid surfaces. The defect in mlo4 and mlo11 mutants is nonadditive and dependent on light and nutrients. Genetic epistasis experiments demonstrate that the mutant phenotype is independently modulated by the Gbeta subunit of the heterotrimeric G-protein complex. Analysis of expressed chimeric MLO4/MLO2 proteins revealed that the C-terminal domain of MLO4 is necessary but not sufficient for MLO4 action in root thigmomorphogenesis. The expression of the auxin efflux carrier fusion, PIN1-green fluorescent protein, the pattern of auxin-induced gene expression, and acropetal as well as basipetal auxin transport are altered at the root tip of mlo4 mutant seedlings. Moreover, addition of auxin transport inhibitors or the loss of EIR1/AGR1/PIN2 function abolishes root curling of mlo4, mlo11, and wild-type seedlings. These results demonstrate that the exaggerated root curling phenotypes of the mlo4 and mlo11 mutants depend on auxin gradients and suggest that MLO4 and MLO11 cofunction as modulators of touch-induced root tropism.

  6. Nuclease-resistant c-di-AMP derivatives that differentially recognize RNA and protein receptors

    PubMed Central

    Meehan, Robert E.; Torgerson, Chad D.; Gaffney, Barbara L.; Jones, Roger A.; Strobel, Scott A.

    2016-01-01

    The ability of bacteria to sense environmental cues and adapt is essential for their survival. The use of second-messenger signaling molecules to translate these cues into a physiological response is a common mechanism employed by bacteria. The second messenger 3’-5’-cyclic diadenosine monophosphate (c-di-AMP) has been linked to a diverse set of biological processes involved in maintaining cell viability and homeostasis, as well as pathogenicity. A complex network of both protein and RNA receptors inside the cell activate specific pathways and mediate phenotypic outputs in response to c-di-AMP. Structural analysis of these RNA and protein receptors has revealed the different recognition elements employed by these effectors to bind the same small molecule. Herein, using a series of c-di-AMP analogs, we probed the interactions made with a riboswitch and a phosphodiesterase protein to identify the features important for c-di-AMP binding and recognition. We found that the ydaO riboswitch binds c-di-AMP in two discrete sites with near identical affinity and a Hill coefficient of 1.6. The ydaO riboswitch distinguishes between c-di-AMP and structurally related second messengers by discriminating against an amine at the C2 position, more than a carbonyl at the C6 position. We also identified phosphate-modified analogs that bind both the ydaO RNA and GdpP protein with high affinity, while symmetrically-modified ribose analogs exhibited a substantial decrease in ydaO affinity, but retained high affinity for GdpP. These ligand modifications resulted in increased resistance to enzyme-catalyzed hydrolysis by the GdpP enzyme. Together, these data suggest that these c-di-AMP analogs could be useful as chemical tools to specifically target subsections of the second-messenger signaling pathways. PMID:26789423

  7. Resistance of mice vaccinated with rabies virus internal structural proteins to lethal infection.

    PubMed

    Takita-Sonoda, Y; Fujii, H; Mifune, K; Ito, Y; Hiraga, M; Nishizono, A; Mannen, K; Minamoto, N

    1993-01-01

    Mice were vaccinated with recombinant vaccinia virus (rVac) expressing the glycoprotein (G), nucleoprotein (N), phosphoprotein (NS) or matrix protein (M) of rabies virus and their resistance to peripheral lethal infection with street rabies virus was examined. Mice vaccinated with rVac-G or rVac-N developed strong antibody responses to the corresponding proteins and essentially all mice survived challenge infection. Mice vaccinated with rVac-NS or rVac-M developed only a slight antibody response, however, a significant protection (59%) was observed in the rVac-NS-vaccinated mice, whereas rVac-M-vaccinated mice were not protected. No anti-G antibodies were detected in the sera of mice which has been vaccinated with rVac-N or rVac-NS and survived challenge infection. Passive transfer of anti-N monoclonal antibodies (MAbs) recognizing an epitope located on amino acids 1-224 of the protein prior to challenge resulted in significant protection, although the protection was not complete even with a high amount of antibodies. In contrast, none of the mice given MAbs recognizing an epitope of amino acids 247-415 or F(ab')2 fragments from a protective MAb IgG were protected. Administration of anti-CD 8 MAb to rVac-N-vaccinated mice showed no significant effect on protection. Our observations suggest that a considerable part of the protection achieved by the vaccination with rVac-N can be ascribed to the intact anti-N antibodies recognizing an epitope located on amino acids 1-224 of the protein.

  8. 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

  9. [The role of Cd-binding proteins and phytochelatins in the formation of cadmium resistance in Nicotiana plumbaginifolia cell lines].

    PubMed

    Fenik, S I; Solodushko, V G; Kaliniak, T B; Blium, Ia B

    2007-01-01

    Nicotiana plumbaginifolia callus lines with the equal resistance to cadmium have been produced under different selective conditions--either without inhibition of the phytochelatin synthesis (line Cd-R) or in the presence of the inhibitor butionine sulfoximine (line Cd-Ri). The level of phytochelatin synthesis in the line Cd-R five-fold exceeded the control value and in the line Cd-Ri it was twice as much as in the control. It was shown that in the control line mainly three cadmium-binding proteins are expressed of the molecular weihgts 41, 34 and 19 kD. The common feature of the both resistant lines is the expression of the cadmium-binding proteins of 40, 37 and 19 kD. The resistant lines differ with respect to the synthesis of relatively low-molecular cadmium-binding proteins. The proteins of the molecular weights 12.5, 11.5 and 9 kD are expressed in the line Cd-R, while the proteins of 13 and 10 kD are expressed in the line Cd-Ri. It was supposed that both the phytochelatins and the Cd-binding proteins contribute to the resisitance of N. plumbaginifolia callus lines to cadmium and the lack of the phytochelatins can be equilibrated by the changes in the low-molecular Cd-binding protein synthesis.

  10. Resistance training with excessive training load and insufficient recovery alters skeletal muscle mass-related protein expression.

    PubMed

    Alves Souza, Rodrigo Wagner; Aguiar, Andreo F; Vechetti-Júnior, Ivan J; Piedade, Warlen Pereira; Rocha Campos, Gerson Eduardo; Dal-Pai-Silva, Maeli

    2014-08-01

    The aim of this study was to investigate the effects of a resistance training program with excessive training load and insufficient recovery time between bouts on muscle hypertrophy- and atrophy-related protein expression. Male Wistar rats were randomly assigned to either a trained (TR, N = 9) or a sedentary (SE, N = 9) group. The TR group was subjected to a 12-week resistance training program with excessive training load and insufficient recovery between bouts that was designed to induce plantaris muscle atrophy. After the 12-week experiment, the plantaris muscle was collected to analyze the cross-sectional area (CSA) of the muscle fibers, and MAFbx, MyoD, myogenin, and IGF-I protein expression (Western blot). The CSA was reduced significantly (-17%, p ≤ 0.05) in the TR group compared with the SE group. Reciprocally, there was a significant (p ≤ 0.05) 20% increase in MAFbx protein expression, whereas the MyoD (-27%), myogenin (-29%), and IGF-I (-43%) protein levels decreased significantly (p ≤ 0.05) in the TR group compared with the SE group. In conclusion, our data indicated that muscle atrophy induced by resistance training with excessive training load and insufficient recovery was associated with upregulation of the MAFbx catabolic protein and downregulation of the MyoD, myogenin, and IGF-I anabolic proteins. These findings suggest that quantitative analysis of these proteins can be important and complementary with other biochemical markers to confirm a possible overtraining diagnosis.

  11. A Secretory Protein of Necrotrophic Fungus Sclerotinia sclerotiorum That Suppresses Host Resistance

    PubMed Central

    Zhu, Wenjun; Wei, Wei; Fu, Yanping; Cheng, Jiasen; Xie, Jiatao; Li, Guoqing; Yi, Xianhong; Kang, Zhensheng; Dickman, Martin B.; Jiang, Daohong

    2013-01-01

    SSITL (SS1G_14133) of Sclerotinia sclerotiorum encodes a protein with 302 amino acid residues including a signal peptide, its secretion property was confirmed with immunolocalization and immunofluorescence techniques. SSITL was classified in the integrin alpha N-terminal domain superfamily, and its 3D structure is similar to those of human integrin α4-subunit and a fungal integrin-like protein. When S. sclerotiorum was inoculated to its host, high expression of SSITL was detected during the initial stages of infection (1.5–3.0 hpi). Targeted silencing of SSITL resulted in a significant reduction in virulence; on the other hand, inoculation of SSITL silenced transformant A10 initiated strong and rapid defense response in Arabidopsis, the highest expressions of defense genes PDF1.2 and PR-1 appeared at 3 hpi which was 9 hr earlier than that time when plants were inoculated with the wild-type strain of S. sclerotiorum. Systemic resistance induced by A10 was detected by analysis of the expression of PDF1.2 and PR-1, and confirmed following inoculation with Botrytis cinerea. A10 induced much larger lesions on Arabidopsis mutant ein2 and jar1, and slightly larger lesions on mutant pad4 and NahG in comparison with the wild-type plants. Furthermore, both transient and constitutive expression of SSITL in Arabidopsis suppressed the expression of PDF1.2 and led to be more susceptible to A10 and the wild-type strain of S. sclerotiorum and B. cinerea. Our results suggested that SSITL is an effector possibly and plays significant role in the suppression of jasmonic/ethylene (JA/ET) signal pathway mediated resistance at the early stage of infection. PMID:23342034

  12. Drug efflux by a small multidrug resistance protein is inhibited by a transmembrane peptide.

    PubMed

    Poulsen, Bradley E; Deber, Charles M

    2012-07-01

    Drug-resistant bacteria use several families of membrane-embedded transporters to remove antibiotics from the cell. One such family is the small multidrug resistance proteins (SMRs) that, because of their relatively small size (ca. 110 residues with four transmembrane [TM] helices), must form (at least) dimers to efflux drugs. Here, we use a Lys-tagged synthetic peptide with exactly the same sequence as TM4 of the full-length SMR Hsmr from Halobacterium salinarum [TM4 sequence: AcA(Sar)(3)-VAGVVGLALIVAGVVVLNVAS-KKK (Sar = N-methylglycine)] to compete with and disrupt the native TM4-TM4 interactions believed to constitute the locus of Hsmr dimerization. Using a cellular efflux assay of the fluorescent SMR substrate ethidium bromide, we determined that bacterial cells containing Hsmr are able to remove cellular ethidium via first-order exponential decay with a rate constant (k) of 10.1 × 10(-3) ± 0.7 × 10(-3) s(-1). Upon treatment of the cells with the TM4 peptide, we observed a saturable ~60% decrease in the efflux rate constant to 3.7 × 10(-3) ± 0.2 × 10(-3) s(-1). In corresponding experiments with control peptides, including scrambled sequences and a sequence with d-chirality, a decrease in ethidium efflux either was not observed or was marginal, likely from nonspecific effects. The designed peptides did not evoke bacterial lysis, indicating that they act via the α-helicity and membrane insertion propensities of the native TM4 helix. Our overall results suggest that this approach could conceivably be used to design hydrophobic peptides for disruption of key TM-TM interactions of membrane proteins and represent a valuable route to the discovery of new therapeutics.

  13. Mutations in the Bacterial Ribosomal Protein L3 and Their Association with Antibiotic Resistance

    PubMed Central

    Klitgaard, Rasmus N.; Ntokou, Eleni; Nørgaard, Katrine; Biltoft, Daniel; Hansen, Lykke H.; Trædholm, Nicolai M.; Kongsted, Jacob

    2015-01-01

    Different groups of antibiotics bind to the peptidyl transferase center (PTC) in the large subunit of the bacterial ribosome. Resistance to these groups of antibiotics has often been linked with mutations or methylations of the 23S rRNA. In recent years, there has been a rise in the number of studies where mutations have been found in the ribosomal protein L3 in bacterial strains resistant to PTC-targeting antibiotics but there is often no evidence that these mutations actually confer antibiotic resistance. In this study, a plasmid exchange system was used to replace plasmid-carried wild-type genes with mutated L3 genes in a chromosomal L3 deletion strain. In this way, the essential L3 gene is available for the bacteria while allowing replacement of the wild type with mutated L3 genes. This enables investigation of the effect of single mutations in Escherichia coli without a wild-type L3 background. Ten plasmid-carried mutated L3 genes were constructed, and their effect on growth and antibiotic susceptibility was investigated. Additionally, computational modeling of the impact of L3 mutations in E. coli was used to assess changes in 50S structure and antibiotic binding. All mutations are placed in the loops of L3 near the PTC. Growth data show that 9 of the 10 mutations were well accepted in E. coli, although some of them came with a fitness cost. Only one of the mutants exhibited reduced susceptibility to linezolid, while five exhibited reduced susceptibility to tiamulin. PMID:25845869

  14. 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

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

    PubMed

    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

    2016-06-28

    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.

  16. Soybean dwarf virus-resistant transgenic soybeans with the sense coat protein gene.

    PubMed

    Tougou, Makoto; Yamagishi, Noriko; Furutani, Noriyuki; Shizukawa, Yoshiaki; Takahata, Yoshihito; Hidaka, Soh

    2007-11-01

    We transformed a construct containing the sense coat protein (CP) gene of Soybean dwarf virus (SbDV) into soybean somatic embryos via microprojectile bombardment to acquire SbDV-resistant soybean plants. Six independent T(0) plants were obtained. One of these transgenic lines was subjected to further extensive analysis. Three different insertion patterns of Southern blot hybridization analysis in T(1) plants suggested that these insertions introduced in T(0) plants were segregated from each other or co-inherited in T(1) progenies. These insertions were classified into two types, which overexpressed SbDV-CP mRNA and accumulated SbDV-CP-specific short interfering RNA (siRNA), or repressed accumulation of SbDV-CP mRNA and siRNA by RNA analysis prior to SbDV inoculation. After inoculation of SbDV by the aphids, most T(2) plants of this transgenic line remained symptomless, contained little SbDV-specific RNA by RNA dot-blot hybridization analysis and exhibited SbDV-CP-specific siRNA. We discuss here the possible mechanisms of the achieved resistance, including the RNA silencing.

  17. Rapeseed protein inhibits the initiation of insulin resistance by a high-saturated fat, high-sucrose diet in rats.

    PubMed

    Mariotti, François; Hermier, Dominique; Sarrat, Charlotte; Magné, Joëlle; Fénart, Evelyne; Evrard, Jacques; Tomé, Daniel; Huneau, Jean François

    2008-11-01

    In contrast to the quality of carbohydrates and lipids, little is known on the influence of the type of dietary protein on the development of the metabolic or insulin resistance syndrome. Cysteine intake has been recently documented to impact insulin sensitivity. The aim of this study was to determine whether rapeseed protein, an emergent cysteine-rich protein, could inhibit the onset of the metabolic syndrome. For 9 weeks, rats were fed a diet rich in saturated fats and sucrose, which also included 20 % protein either as milk protein ('Induction' diet I) or rapeseed protein (diet R). A third, control group received an isoenergetic diet containing milk protein but polyunsaturated fats and starch ('Prudent' diet P). Plasma glucose, insulin, TAG and cholesterol, and blood pressure were monitored during the study, glucose tolerance was tested at week 7 and body composition determined at week 9. Plasma glucose, insulin and TAG increased during the experiment and, at week 9, plasma insulin was significantly 34 % lower in the R group and 56 % lower in P group as compared with the I group. The insulin peak after the glucose load was significantly 28-30 % lower in R and P than in I and the insulin sensitivity index was significantly higher in R than in I. Unexpectedly, peripheral fat deposition was slightly higher in R than in I. In this model, substituting rapeseed protein for milk protein had preventive effects on the early onset of insulin resistance, similar to those achieved by manipulating the types of dietary fat and carbohydrates.

  18. A novel type of detergent-resistant membranes may contribute to an early protein sorting event in epithelial cells.

    PubMed

    Alfalah, Marwan; Wetzel, Gabi; Fischer, Ina; Busche, Roger; Sterchi, Erwin E; Zimmer, Klaus-Peter; Sallmann, Hans-Peter; Naim, Hassan Y

    2005-12-30

    One sorting mechanism of apical and basolateral proteins in epithelial cells is based on their solubility profiles with Triton X-100. Nevertheless, apical proteins themselves are also segregated beyond the trans-Golgi network by virtue of their association or nonassociation with cholesterol/sphingolipid-rich microdomains (Jacob, R., and Naim, H. Y. (2001) Curr. Biol. 11, 1444-1450). Therefore, extractability with Triton X-100 does not constitute an absolute criterion of protein sorting. Here, we investigate the solubility patterns of apical and basolateral proteins with other detergents and demonstrate that the mild detergent Tween 20 is adequate to discriminate between apical and basolateral proteins during early stages in their biosynthesis. Although the mannose-rich forms of the apical proteins sucrase-isomaltase, lactase-phlorizin hydrolase, aminopeptidase N, and dipeptidylpeptidase IV reveal similar solubility profiles comprising soluble and nonsoluble fractions, the basolateral proteins, vesicular stomatitis virus G protein, major histocompatibility complex class I, and CD46 are entirely soluble with this detergent. The insoluble Tween 20 membranes are enriched in phosphatidylinositol and phosphatidylglycerol compatible with their synthesis in the endoplasmic reticulum and the existence of a novel class of detergent-resistant membranes. The association of the mannose-rich biosynthetic forms of the apical proteins, sucraseisomaltase, lactase-phlorizin hydrolase, aminopeptidase N, and dipeptidylpeptidase IV with the Tween 20-resistant membranes suggests an early polarized sorting mechanism prior to maturation in the Golgi apparatus.

  19. Assessing proteinase K resistance of fish prion proteins in a scrapie-infected mouse neuroblastoma cell line.

    PubMed

    Salta, Evgenia; Kanata, Eirini; Ouzounis, Christos A; Gilch, Sabine; Schätzl, Hermann; Sklaviadis, Theodoros

    2014-11-13

    The key event in prion pathogenesis is the structural conversion of the normal cellular protein, PrP(C), into an aberrant and partially proteinase K resistant isoform, PrP(Sc). Since the minimum requirement for a prion disease phenotype is the expression of endogenous PrP in the host, species carrying orthologue prion genes, such as fish, could in theory support prion pathogenesis. Our previous work has demonstrated the development of abnormal protein deposition in sea bream brain, following oral challenge of the fish with natural prion infectious material. In this study, we used a prion-infected mouse neuroblastoma cell line for the expression of three different mature fish PrP proteins and the evaluation of the resistance of the exogenously expressed proteins to proteinase K treatment (PK), as an indicator of a possible prion conversion. No evidence of resistance to PK was detected for any of the studied recombinant proteins. Although not indicative of an absolute inability of the fish PrPs to structurally convert to pathogenic isoforms, the absence of PK-resistance may be due to supramolecular and conformational differences between the mammalian and piscine PrPs.

  20. Enhanced expression of multidrug resistance-associated protein 2 and reduced expression of aquaglyceroporin 3 in an arsenic-resistant human cell line.

    PubMed

    Lee, Te-Chang; Ho, I-Ching; Lu, Wen-Jen; Huang, Jin-ding

    2006-07-01

    Arsenic-resistant cells (R15), derived from a human lung adenocarcinoma cell line (CL3), were 10-fold more resistant to sodium arsenite (As(III)). Because R15 cells accumulated less arsenic than parental CL3 cells, this arsenic resistance may be due to higher efflux and/or lower uptake of As(III). We therefore compared expression of the multidrug resistance-associated proteins MRP1, MRP2, and MRP3 in these two cell lines. MRP2 expression was 5-fold higher in R15 cells than in CL3 cells, whereas MRP1 and MRP3 expression levels were similar. Furthermore, verapamil and cyclosporin A, inhibitors of multidrug resistance transporters, significantly reduced the efflux of arsenic from R15. Thus, increased arsenic extrusion by MRP2 may contribute to arsenic resistance in R15 cells. We also examined the expression of several aquaglyceroporins (AQPs), which mediate As(III) uptake by cells. Little AQP7 or AQP9 mRNA was detected by reverse transcription-PCR in either cell line, whereas AQP3 mRNA expression was 2-fold lower in R15 cells than in CL3 cells. When AQP3 expression in CL3 cells was knocked down by RNA interference, CL3 cells accumulated less arsenic and became more resistant to As(III). Conversely, overexpression of AQP3 in human embryonic kidney 293T cells increased arsenic accumulation, and the cells were more susceptible to As(III) than 293T cells transfected with vector alone. These results suggest that AQP3 is involved in As(III) accumulation. Taken together, our results suggest that enhanced expression of MRP2 and lower expression of AQP3 are responsible for lower arsenic accumulation in arsenic-resistant R15 cells.

  1. Significance of multidrug resistance gene-related proteins in the postoperative chemotherapy of gastric cancer

    PubMed Central

    Yu, Pengfei; Du, Yian; Yang, Litao; Fan, Sunfu; Wu, Jian; Zheng, Shusen

    2014-01-01

    Background: Multidrug resistance (MDR) is a serious problem in chemotherapy and is one of the main reasons for a poor outcome of gastric cancer. Study on the key proteins in multidrug resistance is necessary for the treatment of gastric cancer. Methods: The expression of ToPo II, MRP and GST-π in 119 gastric cancers was retrospectively examined, and the results were analyzed in correlation with clinicopathological data. ToPo II negative, MRP positive and GST-π positive were regarded as three risk factors which may be associated with chemotherapy resistance and poor prognosis. Patients were divided into two groups: high-risk group (≥2 risk factors) and the low-risk group (<2 risk factors), and the tumor recurrence and patients’ survival time of the two groups were also analyzed. Results: The positive rates of ToPo II, MRP and GST-π were 73.9%, 42.9% and 51.3%, respectively. The positively correlation between the expression of MRP and GST-π had been found. A significant correlation was shown between ToPo II expression and the level of differentiation. Significant differences with GST-π expression were also found in relation to the sex and differentiation. In the high-risk group, the 3-year survival rate of patients with/without chemotherapy were 62.1% and 52.0%, 5-year survival rates were 44.8% and 40.0%, but the difference was not statistically significant (P>0.05). In the low-risk group, the 3-year survival rate of patients with/without chemotherapy were 81.2% and 51.5%, 5-year survival rates were 71.9% and 45.5%, and the difference was statistically significant (P<0.05). Conclusions: Combined detection of MDR-related proteins ToPo II, MRP and GST-π may be prospectively valuable for postoperative individualized chemotherapy, and further predict the outcomes of gastric cancer patients. PMID:25550836

  2. The response of muscle protein synthesis following whole-body resistance exercise is greater following 40 g than 20 g of ingested whey protein.

    PubMed

    Macnaughton, Lindsay S; Wardle, Sophie L; Witard, Oliver C; McGlory, Chris; Hamilton, D Lee; Jeromson, Stewart; Lawrence, Clare E; Wallis, Gareth A; Tipton, Kevin D

    2016-08-01

    The currently accepted amount of protein required to achieve maximal stimulation of myofibrillar protein synthesis (MPS) following resistance exercise is 20-25 g. However, the influence of lean body mass (LBM) on the response of MPS to protein ingestion is unclear. Our aim was to assess the influence of LBM, both total and the amount activated during exercise, on the maximal response of MPS to ingestion of 20 or 40 g of whey protein following a bout of whole-body resistance exercise. Resistance-trained males were assigned to a group with lower LBM (≤65 kg; LLBM n = 15) or higher LBM (≥70 kg; HLBM n = 15) and participated in two trials in random order. MPS was measured with the infusion of (13)C6-phenylalanine tracer and collection of muscle biopsies following ingestion of either 20 or 40 g protein during recovery from a single bout of whole-body resistance exercise. A similar response of MPS during exercise recovery was observed between LBM groups following protein ingestion (20 g - LLBM: 0.048 ± 0.018%·h(-1); HLBM: 0.051 ± 0.014%·h(-1); 40 g - LLBM: 0.059 ± 0.021%·h(-1); HLBM: 0.059 ± 0.012%·h(-1)). Overall (groups combined), MPS was stimulated to a greater extent following ingestion of 40 g (0.059 ± 0.020%·h(-1)) compared with 20 g (0.049 ± 0.020%·h(-1); P = 0.005) of protein. Our data indicate that ingestion of 40 g whey protein following whole-body resistance exercise stimulates a greater MPS response than 20 g in young resistance-trained men. However, with the current doses, the total amount of LBM does not seem to influence the response. PMID:27511985

  3. The response of muscle protein synthesis following whole-body resistance exercise is greater following 40 g than 20 g of ingested whey protein.

    PubMed

    Macnaughton, Lindsay S; Wardle, Sophie L; Witard, Oliver C; McGlory, Chris; Hamilton, D Lee; Jeromson, Stewart; Lawrence, Clare E; Wallis, Gareth A; Tipton, Kevin D

    2016-08-01

    The currently accepted amount of protein required to achieve maximal stimulation of myofibrillar protein synthesis (MPS) following resistance exercise is 20-25 g. However, the influence of lean body mass (LBM) on the response of MPS to protein ingestion is unclear. Our aim was to assess the influence of LBM, both total and the amount activated during exercise, on the maximal response of MPS to ingestion of 20 or 40 g of whey protein following a bout of whole-body resistance exercise. Resistance-trained males were assigned to a group with lower LBM (≤65 kg; LLBM n = 15) or higher LBM (≥70 kg; HLBM n = 15) and participated in two trials in random order. MPS was measured with the infusion of (13)C6-phenylalanine tracer and collection of muscle biopsies following ingestion of either 20 or 40 g protein during recovery from a single bout of whole-body resistance exercise. A similar response of MPS during exercise recovery was observed between LBM groups following protein ingestion (20 g - LLBM: 0.048 ± 0.018%·h(-1); HLBM: 0.051 ± 0.014%·h(-1); 40 g - LLBM: 0.059 ± 0.021%·h(-1); HLBM: 0.059 ± 0.012%·h(-1)). Overall (groups combined), MPS was stimulated to a greater extent following ingestion of 40 g (0.059 ± 0.020%·h(-1)) compared with 20 g (0.049 ± 0.020%·h(-1); P = 0.005) of protein. Our data indicate that ingestion of 40 g whey protein following whole-body resistance exercise stimulates a greater MPS response than 20 g in young resistance-trained men. However, with the current doses, the total amount of LBM does not seem to influence the response.

  4. Interactions of the alpha2A-adrenoceptor with multiple Gi-family G-proteins: studies with pertussis toxin-resistant G-protein mutants.

    PubMed Central

    Wise, A; Watson-Koken, M A; Rees, S; Lee, M; Milligan, G

    1997-01-01

    The alpha2A-adrenoceptor is the prototypic example of the family of G-protein-coupled receptors which function by activation of 'Gi-like' pertussis toxin-sensitive G-proteins. A number of members of this subfamily of G-proteins are often co-expressed in a single cell type. To examine the interaction of this receptor with individual Gi-family G-proteins the porcine alpha2A-adrenoceptor was transiently transfected into COS-7 cells either alone or with each of wild-type Gi1alpha, Gi2alpha and Gi3alpha or mutations of each of these G-proteins in which the cysteine residue which is the target for pertussis toxin-catalysed ADP-ribosylation was exchanged for a glycine residue. The alpha2-adrenoceptor agonist UK14304 stimulated both high-affinity GTPase activity and the binding of guanosine 5'-[gamma-35thio]-triphosphate (GTP[35S]), when expressed without any additional G-protein. These effects were greatly reduced by pretreatment of the cells with pertussis toxin. Co-expression of each of the wild-type Gi-like G-protein alpha-subunits resulted in enhanced agonist activation of the cellular G-protein population which was fully prevented by pretreatment with pertussis toxin. Co-expression of the receptor along with the cysteine-to-glycine mutations of Gi1alpha, Gi2alpha and Gi3alpha resulted in agonist stimulation of these G-proteins, which was as great as that of the wild type proteins, but now the agonist stimulation produced over that due to the activation of endogenously expressed Gi-like G-proteins was resistant to pertussis toxin treatment. The Cys --> Gly mutations of Gi1alpha, Gi2alpha and Gi3alpha were each also able to limit agonist-mediated stimulation of adenylate cyclase activity. The degree of agonist-mediated activation of the pertussis toxin-resistant mutant of Gi1alpha was correlated highly both with the level of expression of this G-protein and with the level of expression of the alpha2A-adrenoceptor. Half-maximal stimulation of high-affinity GTPase

  5. Liver pathology in severe multidrug resistant 3 protein deficiency: a series of 10 pediatric cases.

    PubMed

    Vij, Mukul; Safwan, Mohamed; Shanmugam, Naresh P; Rela, Mohamed

    2015-10-01

    Multidrug resistance protein 3 (MDR3) is a hepatocyte canalicular membrane protein encoded by the ABCB4/MDR3 gene located on chromosome 7. Several liver diseases are known to be associated with MDR3 deficiency. The basic defect is reduced secretion of biliary phospholipid causing disturbance in the primary bile composition, leading to injury to biliary epithelium inducing cell death and inflammation. Severe MDR3 deficiency typically presents during the first year of life or early childhood, often progressing to chronic liver disease with cirrhosis and portal hypertension, requiring liver transplantation. Negative MDR3 immunostaining is suggestive of MDR3 deficiency. Herein, we report the clinical and histopathologic features of 10 cases (6 male/4 female) in infants and children with severe MDR3 deficiency (age range of 8 months to 7 years) diagnosed with negative MDR3 immunostaining in hepatic canaliculi. Three cases underwent liver transplantation. The cases showed periportal bridging fibrosis to micronodular cirrhosis, ductular proliferation with bile plugs, and lobular canalicular bile stasis with rosetting. All 3 explant livers demonstrated cystically dilated large ducts with crystallization of cholesterol. One case showed well-differentiated hepatocellular carcinoma. We conclude that MDR3 immunostaining on formalin-fixed and paraffin-embedded sections is a useful tool to diagnose severe MDR3 deficiency in pediatric liver cholestatic disease cases where genetic testing is not available. PMID:26117383

  6. Structural and functional characterization of the Pseudomonas hydroperoxide resistance protein Ohr

    PubMed Central

    Lesniak, Jacob; Barton, William A.; Nikolov, Dimitar B.

    2002-01-01

    Bacteria have developed complex strategies to detoxify and repair damage caused by reactive oxygen species. These compounds, produced during bacterial aerobic respiration as well as by the host immune system cells as a defense mechanism against the pathogenic microorganisms, have the ability to damage nucleic acids, proteins and phospholipid membranes. Here we describe the crystal structure of Pseudomonas aeruginosa Ohr, a member of a recently discovered family of organic hydroperoxide resistance proteins. Ohr is a tightly folded homodimer, with a novel α/β fold, and contains two active sites located at the monomer interface on opposite sides of the molecule. Using in vitro assays, we demonstrate that Ohr functions directly as a hydroperoxide reductase, converting both inorganic and organic hydroperoxides to less toxic metabolites. Site-directed mutagenesis confirms that the two conserved cysteines in each active site are essential for catalytic activity. We propose that the Ohr catalytic mechanism is similar to that of the structurally unrelated peroxiredoxins, directly utilizing highly reactive cysteine thiol groups to elicit hydroperoxide reduction. PMID:12485986

  7. Detection of protease-resistant cervid prion protein in water from a CWD-endemic area

    PubMed Central

    Nichols, TA; Pulford, Bruce; Wyckoff, A Christy; Meyerett, Crystal; Michel, Brady; Gertig, Kevin; Hoover, Edward A; Jewell, Jean E; Telling, Glenn C

    2009-01-01

    Chronic wasting disease (CWD) is the only known transmissible spongiform encephalopathy affecting free-ranging wildlife. Although the exact mode of natural transmission remains unknown, substantial evidence suggests that prions can persist in the environment, implicating components thereof as potential prion reservoirs and transmission vehicles.1–4 CWD-positive animals may contribute to environmental prion load via decomposing carcasses and biological materials including saliva, blood, urine and feces.5–7 Sensitivity limitations of conventional assays hamper evaluation of environmental prion loads in soil and water. Here we show the ability of serial protein misfolding cyclic amplification (sPMCA) to amplify a 1.3 × 10−7 dilution of CWD-infected brain homogenate spiked into water samples, equivalent to approximately 5 × 107 protease resistant cervid prion protein (PrPCWD) monomers. We also detected PrPCWD in one of two environmental water samples from a CWD endemic area collected at a time of increased water runoff from melting winter snow pack, as well as in water samples obtained concurrently from the flocculation stage of water processing by the municipal water treatment facility. Bioassays indicated that the PrPCWD detected was below infectious levels. These data demonstrate detection of very low levels of PrPCWD in the environment by sPMCA and suggest persistence and accumulation of prions in the environment that may promote CWD transmission. PMID:19823039

  8. Histone Acetylation and CREB Binding Protein Are Required for Neuronal Resistance against Ischemic Injury

    PubMed Central

    Yildirim, Ferah; Ji, Shengbo; Kronenberg, Golo; Barco, Angel; Olivares, Roman; Benito, Eva; Dirnagl, Ulrich; Gertz, Karen; Endres, Matthias

    2014-01-01

    Epigenetic transcriptional regulation by histone acetylation depends on the balance between histone acetyltransferase (HAT) and deacetylase activities (HDAC). Inhibition of HDAC activity provides neuroprotection, indicating that the outcome of cerebral ischemia depends crucially on the acetylation status of histones. In the present study, we characterized the changes in histone acetylation levels in ischemia models of focal cerebral ischemia and identified cAMP-response element binding protein (CREB)–binding protein (CBP) as a crucial factor in the susceptibility of neurons to ischemic stress. Both neuron-specific RNA interference and neurons derived from CBP heterozygous knockout mice showed increased damage after oxygen-glucose deprivation (OGD) in vitro. Furthermore, we demonstrated that ischemic preconditioning by a short (5 min) subthreshold occlusion of the middle cerebral artery (MCA), followed 24 h afterwards by a 30 min occlusion of the MCA, increased histone acetylation levels in vivo. Ischemic preconditioning enhanced CBP recruitment and histone acetylation at the promoter of the neuroprotective gene gelsolin leading to increased gelsolin expression in neurons. Inhibition of CBP's HAT activity attenuated neuronal ischemic preconditioning. Taken together, our findings suggest that the levels of CBP and histone acetylation determine stroke outcome and are crucially associated with the induction of an ischemia-resistant state in neurons. PMID:24748101

  9. Emerging Role of Nitric Oxide and Heat Shock Proteins in Insulin Resistance.

    PubMed

    Molina, Marisa Nile; Ferder, León; Manucha, Walter

    2016-01-01

    Insulin resistance (IR) is present in pathologies such as diabetes, obesity, metabolic syndrome, impaired glucose tolerance, hypertension, inflammation, cardiac disease, and dyslipidemias. Population studies show that IR is multifactorial and has genetic components, such as defects in the insulin-signaling pathway (as serine phosphorylation on insulin substrate or decreased activation of signaling molecules) and RAS/MAPK-dependent pathways. IR is connected to mitochondrial dysfunction, overproduction of oxidants, accumulation of fat, and an over-activation of the renin-angiotensin system linked to the NADPH oxidase activity. In addition, nitric oxide (NO), synthesized by nitric oxide synthases (endothelial and inducible), is also associated with IR when both impaired release and reduced bioavailability of all which lead to inflammation and hypertension. However, increased NO may promote vasculoprotection. Moreover, reduced NO release induces heat shock protein 70 kDa (HSP70) expression in IR and diabetes, mediating beneficial effects against oxidative stress injury, inflammation and apoptosis. HSP70 may be used as biomarker of the chronicity of diabetes. Hsp72 (inducible protein) is linked to vascular complications with a high-fat diet by blocking inflammation signaling (cytoprotective and anti-cytotoxicity intracellular role). Elucidating the IR signaling pathways and the roles of NO and HSPs is relevant to the application of new treatments, such as heat shock and thermal therapy, nitrosylated drugs, chemical chaperones or exercise training. PMID:26694820

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

    PubMed

    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; de Gruijl, Tanja D

    2008-09-15

    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 E(2), leukotriene B(4) and D(4), 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.

  11. 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

  12. Transport cycle intermediate in small multidrug resistance protein is revealed by substrate fluorescence.

    PubMed

    Basting, Daniel; Lorch, Mark; Lehner, Ines; Glaubitz, Clemens

    2008-02-01

    Efflux pumps of the small multidrug resistance family bind cationic, lipophilic antibiotics and transport them across the membrane in exchange for protons. The transport cycle must involve various conformational states of the protein needed for substrate binding, translocation, and release. A fluorescent substrate will therefore experience a significant change of environment while being transported, which influences its fluorescence properties. Thus the substrate itself can report intermediate states that form during the transport cycle. We show the existence of such a substrate-transporter complex for the EmrE homolog Mycobacterium tuberculosis TBsmr and its substrate ethidium bromide. The pH gradient needed for antiport has been generated by co-reconstituting TBsmr with bacteriorhodopsin. Sample illumination generates a DeltapH, which results in enhanced ethidium fluorescence intensity, which is abolished when DeltapH or DeltaPsi is collapsed or when the essential residue Glu-13 in TBsmr is exchanged with Ala. This observation shows the formation of a pH-dependent, transient substrate-protein complex between binding and release of ethidium. We have further characterized this state by determining the K(d), by inhibiting ethidium transport through titration with nonfluorescent substrate and by fluorescence anisotropy measurements. Our findings support a model with a single occluded intermediate state in which the substrate is highly immobile.

  13. 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.

  14. 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.

  15. An autoactive mutant of the M flax rust resistance protein has a preference for binding ATP, whereas wild-type M protein binds ADP.

    PubMed

    Williams, Simon J; Sornaraj, Pradeep; deCourcy-Ireland, Emma; Menz, R Ian; Kobe, Bostjan; Ellis, Jeffrey G; Dodds, Peter N; Anderson, Peter A

    2011-08-01

    Resistance (R) proteins are key regulators of the plant innate immune system and are capable of pathogen detection and activation of the hypersensitive cell death immune response. To understand the molecular mechanism of R protein activation, we undertook a phenotypic and biochemical study of the flax nucleotide binding (NB)-ARC leucine-rich repeat protein, M. Using Agrobacterium-mediated transient expression in flax cotyledons, site-directed mutations of key residues within the P-loop, kinase 2, and MHD motifs within the NB-ARC domain of M were shown to affect R protein function. When purified using a yeast expression system and assayed for ATP and ADP, these mutated proteins exhibited marked differences in the quantity and identity of the bound nucleotide. ADP was bound to recombinant wild-type M protein, while the nonfunctional P-loop mutant did not have any nucleotides bound. In contrast, ATP was bound to an autoactive M protein mutated in the highly conserved MHD motif. These data provide direct evidence supporting a model of R protein function in which the "off" R protein binds ADP and activation of R protein defense signaling involves the exchange of ADP for ATP.

  16. 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

  17. 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

  18. 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

  19. 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.

  20. Protein Biomarkers for Insulin Resistance and Type 2 Diabetes Risk in Two Large Community Cohorts.

    PubMed

    Nowak, Christoph; Sundström, Johan; Gustafsson, Stefan; Giedraitis, Vilmantas; Lind, Lars; Ingelsson, Erik; Fall, Tove

    2016-01-01

    Insulin resistance (IR) is a precursor of type 2 diabetes (T2D), and improved risk prediction and understanding of the pathogenesis are needed. We used a novel high-throughput 92-protein assay to identify circulating biomarkers for HOMA of IR in two cohorts of community residents without diabetes (n = 1,367) (mean age 73 ± 3.6 years). Adjusted linear regression identified cathepsin D and confirmed six proteins (leptin, renin, interleukin-1 receptor antagonist [IL-1ra], hepatocyte growth factor, fatty acid-binding protein 4, and tissue plasminogen activator [t-PA]) as IR biomarkers. Mendelian randomization analysis indicated a positive causal effect of IR on t-PA concentrations. Two biomarkers, IL-1ra (hazard ratio [HR] 1.28, 95% CI 1.03-1.59) and t-PA (HR 1.30, 1.02-1.65) were associated with incident T2D, and t-PA predicted 5-year transition to hyperglycemia (odds ratio 1.30, 95% CI 1.02-1.65). Additional adjustment for fasting glucose rendered both coefficients insignificant and revealed an association between renin and T2D (HR 0.79, 0.62-0.99). LASSO regression suggested a risk model including IL-1ra, t-PA, and the Framingham Offspring Study T2D score, but prediction improvement was nonsignificant (difference in C-index 0.02, 95% CI -0.08 to 0.12) over the T2D score only. In conclusion, proteomic blood profiling indicated cathepsin D as a new IR biomarker and suggested a causal effect of IR on t-PA. PMID:26420861

  1. 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. PMID:21661746

  2. 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.

  3. 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.

  4. The Use of Green Fluorescent Protein-Tagged Recombinant Viruses to Test Lettuce mosaic virus Resistance in Lettuce.

    PubMed

    Candresse, T; Le Gall, O; Maisonneuve, B; German-Retana, S; Redondo, E

    2002-02-01

    ABSTRACT Seed certification and the use of cultivars containing one of two, probably allelic, recessive genes, mo1(1) and mo1(2), are the principal control methods for Lettuce mosaic virus (LMV) in lettuce. Although for a few LMV isolates, mo1(2) confers resistance with most isolates, the genes mo1(1) or mo1(2) confer a tolerance, and virus accumulation is readily detected in mo1-carrying plants. This phenotype complicates evaluation of the resistance status, in particular for mo1(1), for which there are no viral strains against which a true resistance is expressed. Two green fluorescent protein (GFP)-tagged viruses were constructed, derived from a non-resistance breaking isolate (LMV-0) and from a resistance-breaking isolate (LMV-E). An evaluation of 101 cultivars of known status was carried out with these recombinant viruses. Using the LMV-0-derived recombinant, identification of mo1-carrying cultivars was simple because, contrary to its wild-type parent, systemic movement of LMV-0-GFP was abolished in resistant plants. This assay detected four cases of misidentification of resistance status. In all these cases, further tests confirmed that the prior resistance status information was incorrect, so that a 100% correlation was observed between LMV-0-GFP behavior and the mo1 resistance status. Similarly, the LMV-E-derived recombinant allowed the identification of mo1(2) lettuce lines because its systemic movement was restricted in mo1(2) lines but not in susceptible or in mo1(1) lines. The tagged viruses were able to systemically invade another host, pea, irrespective of its resistance status against another member of the genus Potyvirus, Pea seed-borne mosaic virus. The use of these recombinant viruses could therefore greatly facilitate LMV resistance evaluation and speed up lettuce breeding programs. PMID:18943090

  5. The Prevalence of Activated Protein C Resistance and F V Leiden in Healthy Population of Edirne, Turkey.

    PubMed

    Vurkun, Mutlu; Vural, Özden; Demir, Muzaffer; Turgut, Burhan; Gürgey, Aytemiz; Parlak, Hülya; Duran, Nesrin

    2002-06-01

    Activated protein C (APC) resistance has been found to be an important cause of venous thrombosis. The prevalence of F V Leiden (FVL) in general population is variable according to the region and the ethnic group. The aim of this study was to determine the prevalence of APC resistance and FVL in healthy population in Edirne as a representative sample of province of Edirne. Total 467 healthy subjects were studied. There were 238 males (50.96%) and 229 females (49.04%). APC resistance was studied by functional and DNA methods. There were a total 22/476 subjects (4.7%) were APC resistance. There were 20/476 subjects (4.28%) who had FVL by DNA test. Of these, there were 18 heterozygous and 2 homozygous FVL and other two subjects have no FVL mutation but have the high levels of FVIII to explain acquired APC resistance. The coexistence of FVL and the deficiencies of protein C (1/22), protein S (2/22) and antithrombin (1/22) were also studied. No one of subjects had prothrombin gene mutation. The data showed that the prevalence of APC resitance and FVL in healthy Turkish population were similar to the previously reported publications in Turkey and Europe. One thing is a keeping in mind to be the coexistence of FVL and the other known thrombophilic risk factors.

  6. 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.

  7. Crystal structures of flax rust avirulence proteins AvrL567-A and -D reveal details of the structural basis for flax disease resistance specificity.

    PubMed

    Wang, Ching-I A; Guncar, Gregor; Forwood, Jade K; Teh, Trazel; Catanzariti, Ann-Maree; Lawrence, Gregory J; Loughlin, Fionna E; Mackay, Joel P; Schirra, Horst Joachim; Anderson, Peter A; Ellis, Jeffrey G; Dodds, Peter N; Kobe, Bostjan

    2007-09-01

    The gene-for-gene mechanism of plant disease resistance involves direct or indirect recognition of pathogen avirulence (Avr) proteins by plant resistance (R) proteins. Flax rust (Melampsora lini) AvrL567 avirulence proteins and the corresponding flax (Linum usitatissimum) L5, L6, and L7 resistance proteins interact directly. We determined the three-dimensional structures of two members of the AvrL567 family, AvrL567-A and AvrL567-D, at 1.4- and 2.3-A resolution, respectively. The structures of both proteins are very similar and reveal a beta-sandwich fold with no close known structural homologs. The polymorphic residues in the AvrL567 family map to the surface of the protein, and polymorphisms in residues associated with recognition differences for the R proteins lead to significant changes in surface chemical properties. Analysis of single amino acid substitutions in AvrL567 proteins confirm the role of individual residues in conferring differences in recognition and suggest that the specificity results from the cumulative effects of multiple amino acid contacts. The structures also provide insights into possible pathogen-associated functions of AvrL567 proteins, with nucleic acid binding activity demonstrated in vitro. Our studies provide some of the first structural information on avirulence proteins that bind directly to the corresponding resistance proteins, allowing an examination of the molecular basis of the interaction with the resistance proteins as a step toward designing new resistance specificities.

  8. Budd-Chiari syndrome during nephrotic relapse in a patient with resistance to activated protein C clotting inhibitor.

    PubMed

    Gambaro, G; Patrassi, G; Pittarello, F; Nardellotto, A; Checchetto, S; D'Angelo, A

    1998-10-01

    It has long been known that patients with nephrotic syndrome have a hypercoagulable state, which explains the association between nephrotic syndrome, renal vein thrombosis, and thromboembolism. However, the Budd-Chiari syndrome has never been reported in nephrotic patients. This is the first report of such an association that, most likely, depended on a primary resistance to activated protein C.

  9. 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.

  10. Characterization of site-specific glycosylation of secreted proteins associated with multi-drug resistance of gastric cancer.

    PubMed

    Wu, Jian; Qin, Hongqiang; 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

    2016-05-01

    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.

  11. 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

  12. Protein Kinase A Activation Promotes Cancer Cell Resistance to Glucose Starvation and Anoikis.

    PubMed

    Palorini, Roberta; Votta, Giuseppina; 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

    2016-03-01

    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.

  13. 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

  14. 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

    2016-09-01

    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.

  15. 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

    2016-09-01

    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

  16. Human Skeletal Muscle Disuse Atrophy: Effects on Muscle Protein Synthesis, Breakdown, and Insulin Resistance-A Qualitative Review.

    PubMed

    Rudrappa, Supreeth S; Wilkinson, Daniel J; Greenhaff, Paul L; Smith, Kenneth; Idris, Iskandar; Atherton, Philip J

    2016-01-01

    The ever increasing burden of an aging population and pandemic of metabolic syndrome worldwide demands further understanding of the modifiable risk factors in reducing disability and morbidity associated with these conditions. Disuse skeletal muscle atrophy (sometimes referred to as "simple" atrophy) and insulin resistance are "non-pathological" events resulting from sedentary behavior and periods of enforced immobilization e.g., due to fractures or elective orthopedic surgery. Yet, the processes and drivers regulating disuse atrophy and insulin resistance and the associated molecular events remain unclear-especially in humans. The aim of this review is to present current knowledge of relationships between muscle protein turnover, insulin resistance and muscle atrophy during disuse, principally in humans. Immobilization lowers fasted state muscle protein synthesis (MPS) and induces fed-state "anabolic resistance." While a lack of dynamic measurements of muscle protein breakdown (MPB) precludes defining a definitive role for MPB in disuse atrophy, some proteolytic "marker" studies (e.g., MPB genes) suggest a potential early elevation. Immobilization also induces muscle insulin resistance (IR). Moreover, the trajectory of muscle atrophy appears to be accelerated in persistent IR states (e.g., Type II diabetes), suggesting IR may contribute to muscle disuse atrophy under these conditions. Nonetheless, the role of differences in insulin sensitivity across distinct muscle groups and its effects on rates of atrophy remains unclear. Multifaceted time-course studies into the collective role of insulin resistance and muscle protein turnover in the setting of disuse muscle atrophy, in humans, are needed to facilitate the development of appropriate countermeasures and efficacious rehabilitation protocols. PMID:27610086

  17. 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

  18. Identification of Up- and Down-Regulated Proteins in Pemetrexed-Resistant Human Lung Adenocarcinoma: Flavin Reductase and Calreticulin Play Key Roles in the Development of Pemetrexed-Associated Resistance.

    PubMed

    Chou, Hsiu-Chuan; Chen, Jing-Yi; Lin, Dai-Ying; Wen, Yueh-Feng; Lin, Chi-Chen; Lin, Sheng-Hao; Lin, Ching-Hsiung; Chung, Ting-Wen; Liao, En-Chi; Chen, Ying-Jen; Wei, Yu-Shan; Tsai, Yi-Ting; Chan, Hong-Lin

    2015-11-01

    Drug resistance is one of the major causes of cancer chemotherapy failure. In the current study, we used a pair of lung adenocarcinoma cell lines, A549 and the pemetrexed-resistant A549/PEM cells, as a model to monitor resistance-dependent cellular responses and identify potential therapeutic targets. By means of 2D differential gel electrophoresis (2D-DIGE) and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), we investigated the global protein expression alterations induced by pemetrexed treatment and resistance. The proteomic result revealed that pemetrexed exposure obviously altered the expression of 81 proteins in the A549 cells, whereas no significant response was observed in the similarly treated A549/PEM cells, hence implying an association between these proteins and the drug-specific response. Moreover, 72 proteins including flavin reductase and calreticulin demonstrated differential expression between the A549 and A549/PEM cells, indicating baseline resistance. Additional tests employed siRNA silencing, protein overexpression, cell viability analysis, and analysis of apoptosis to examine and confirm the potency of flavin reductase and calreticulin proteins in the development of pemetrexed resistance. In summary, by using a proteomic approach, we identified numerous proteins, including flavin reductase and calreticulin, involved in pemetrexed drug resistance-developing mechanisms. Our results provide useful diagnostic markers and therapeutic candidates for pemetrexed-resistant lung cancer treatment.

  19. Adjudin disrupts spermatogenesis by targeting drug transporters: Lesson from the breast cancer resistance protein (BCRP).

    PubMed

    Qian, Xiaojing; Cheng, Yan-Ho; Jenardhanan, Pranitha; Mruk, Dolores D; Mathur, Premendu P; Xia, Weiliang; Silvestrini, Bruno; Cheng, C Yan

    2013-04-01

    For non-hormonal male contraceptives that exert their effects in the testis locally instead of via the hypothalamic-pituitary-testicular axis, such as adjudin that disrupts germ cell adhesion, a major hurdle in their development is to improve their bioavailability so that they can be efficiently delivered to the seminiferous epithelium by transporting across the blood-testis barrier (BTB). If this can be done, it would widen the gap between their efficacy and general toxicity. However, Sertoli cells that constitute the BTB, peritubular myoid cells in the tunica propria, germ cells at different stages of their development, as well as endothelial cells that constitute the microvessels in the interstitium are all equipped with multiple drug transporters, most notably efflux drug transporters, such as P-glycoprotein, multidrug resistance-related protein 1 (MRP1) and breast cancer resistance protein (BCRP) that can actively prevent drugs (e.g., adjudin) from entering the seminiferous epithelium to exert their effects. Recent studies have shown that BCRP is highly expressed by endothelial cells of the microvessels in the interstitium in the testis and also peritubular myoid cells in tunica propria even though it is absent from Sertoli cells at the site of the BTB. Furthermore, BCRP is also expressed spatiotemporally by Sertoli cells and step 19 spermatids in the rat testis and stage-specifically, limiting to stage VII‒VIII of the epithelial cycle, and restricted to the apical ectoplasmic specialization [apical ES, a testis-specific F-actin-rich adherens junction (AJ)]. Interestingly, adjudin was recently shown to be capable of downregulating BCRP expression at the apical ES. In this Opinion article, we critically discuss the latest findings on BCRP; in particular, we provide some findings utilizing molecular modeling to define the interacting domains of BCRP with adjudin. Based on this information, it is hoped that the next generation of adjudin analogs to be

  20. Reduced toxicity and broad spectrum resistance to viral and fungal infection in transgenic plants expressing pokeweed antiviral protein II.

    PubMed

    Wang, P; Zoubenko, O; Tumer, N E

    1998-12-01

    Pokeweed antiviral protein II (PAPII), a 30 kDa protein isolated from leaves of Phytolacca americana, inhibits translation by catalytically removing a specific adenine residue from the large rRNA of the 60S subunit of eukaryotic ribosomes. The protein sequence of PAPII shows only 41% identity to PAP and PAP-S, two other antiviral proteins isolated from pokeweed. We isolated a cDNA corresponding to PAPII and introduced it into tobacco plants. PAPII expressed in transgenic tobacco was correctly processed to the mature form as in pokeweed and accumulated to at least 10-fold higher levels than wild-type PAP. We had previously observed a significant decrease in transformation frequency with PAP and recovered only two transgenic lines expressing 1-2 ng per mg protein. In contrast, eight different transgenic lines expressing up to 250 ng/mg PAPII were recovered, indicating that PAPII is less toxic than PAP. Two symptomless transgenic lines expressing PAPII were resistant to tobacco mosaic virus, potato virus X and the fungal pathogen Rhizoctonia solani. The level of viral and fungal resistance observed correlated well with the amount of PAPII protein accumulated. Pathogenesis-related protein PR1 was constitutively expressed in transgenic lines expressing PAPII. Although PR1 was constitutively expressed, no increase in salicylic acid levels was detected, indicating that PAPII may elicit a salicylic acid-independent signal transduction pathway.

  1. 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.

  2. Quality control autophagy degrades soluble ERAD-resistant conformers of the misfolded membrane protein GnRHR

    PubMed Central

    Houck, Scott A.; Ren, Hong Yu; Madden, Victoria J.; Bonner, Jaclyn N.; Conlin, Michael P.; Janovick, Jo Ann; Conn, P. Michael; Cyr, Douglas M.

    2014-01-01

    Molecular chaperones triage misfolded proteins via action as substrate selectors for quality control (QC) machines that fold or degrade clients. Herein, the endoplasmic reticulum (ER) associated Hsp40 JB12 is reported to participate in partitioning mutant conformers of GnRHR, a G-protein coupled receptor, between ER-associated degradation (ERAD) and a novel ERQC-autophagy pathway for membrane proteins. ERQC-autophagy degrades E90K-GnRHR because pools of its partially folded and detergent soluble degradation intermediates are resistant to ERAD. S168R-GnRHR is globally misfolded and disposed of via ERAD, but inhibition of p97, the protein retrotranslocation motor, shunts S168R-GnRHR from ERAD to ERQC autophagy. Partially folded and grossly misfolded forms of GnRHR associate with JB12 and Hsp70. Elevation of JB12 promotes ERAD of S168R-GnRHR, with E90K-GnRHR being resistant. E90K-GnRHR elicits association of the Vps34 autophagy initiation complex with JB12. Interaction between ERassociated Hsp40s and the Vps34 complex permits the selective degradation of ERAD-resistant membrane proteins via ERQC-autophagy. PMID:24685158

  3. 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

  4. Soluble proteins isolated from Borrelia burgdorferi by extraction with Triton X-114 confer resistance to experimental infection.

    PubMed

    Rao, T D; Frey, A B

    1998-10-01

    Fractionation of Borrelia burgdorferi was made by extraction of infectious spirochetes using the detergent Triton X-114. Gel electrophoresis analysis of hydrophilic and hydrophobic proteins demonstrated that detergent extraction resulted in two populations of proteins with nonoverlapping electrophoretic profiles. Immunoblot analysis with monoclonal antibodies reactive with two abundant membrane proteins demonstrated that hydrophilic proteins were uncontaminated with hydrophobic proteins. In addition, assay of thymidine incorporation into and secretion of tumor necrosis factor-alpha from splenocytes cocultured in vitro with either detergent or aqueous phase proteins showed that lymphocyte mitogenic and macrophage activation activities of B. burgdorferi were completely absent from the hydrophilic phase proteins. The Triton X-114 aqueous and detergent phase proteins were used to immunize BALB/c and separately microMT/microMT (B cell knockout) mice that were subsequently challenged with infectious B. burgdorferi. The hydrophilic phase proteins were able to induce protective resistance to infection in either strain of mice demonstrating that potential candidate vaccine antigens are contained in the biochemical class of antigens which is devoid of both lymphocyte mitogen activity and major outer surface proteins. Furthermore, the ability to vaccinate B cell knockout mice suggests that the humoral antispirochete immune response is not the exclusive basis for protective immunity.

  5. Shotgun Redox Proteomics: Identification and Quantitation of Carbonylated Proteins in the UVB-Resistant Marine Bacterium, Photobacterium angustum S14

    PubMed Central

    Matallana-Surget, Sabine; Cavicchioli, Ricardo; Fauconnier, Charles; Wattiez, Ruddy; Leroy, Baptiste; Joux, Fabien; Raftery, Mark J.; Lebaron, Philippe

    2013-01-01

    UVB oxidizes proteins through the generation of reactive oxygen species. One consequence of UVB irradiation is carbonylation, the irreversible formation of a carbonyl group on proline, lysine, arginine or threonine residues. In this study, redox proteomics was performed to identify carbonylated proteins in the UVB resistant marine bacterium Photobacterium angustum. Mass-spectrometry was performed with either biotin-labeled or dinitrophenylhydrazide (DNPH) derivatized proteins. The DNPH redox proteomics method enabled the identification of 62 carbonylated proteins (5% of 1221 identified proteins) in cells exposed to UVB or darkness. Eleven carbonylated proteins were quantified and the UVB/dark abundance ratio was determined at both the protein and peptide levels. As a result we determined which functional classes of proteins were carbonylated, which residues were preferentially modified, and what the implications of the carbonylation were for protein function. As the first large scale, shotgun redox proteomics analysis examining carbonylation to be performed on bacteria, our study provides a new level of understanding about the effects of UVB on cellular proteins, and provides a methodology for advancing studies in other biological systems. PMID:23874515

  6. Nonstructural 5A Protein of Hepatitis C Virus Regulates Soluble Resistance-Related Calcium-Binding Protein Activity for Viral Propagation

    PubMed Central

    Tran, Giao V. Q.; Luong, Trang T. D.; Park, Eun-Mee; Kim, Jong-Wook; Choi, Jae-Woong; Park, Chorong; Lim, Yun-Sook

    2015-01-01

    ABSTRACT Hepatitis C virus (HCV) is a major cause of chronic liver disease and is highly dependent on cellular proteins for virus propagation. To identify the cellular factors involved in HCV propagation, we recently performed protein microarray assays using the HCV nonstructural 5A (NS5A) protein as a probe. Of 90 cellular protein candidates, we selected the soluble resistance-related calcium-binding protein (sorcin) for further characterization. Sorcin is a calcium-binding protein and is highly expressed in certain cancer cells. We verified that NS5A interacted with sorcin through domain I of NS5A, and phosphorylation of the threonine residue 155 of sorcin played a crucial role in protein interaction. Small interfering RNA (siRNA)-mediated knockdown of sorcin impaired HCV propagation. Silencing of sorcin expression resulted in a decrease of HCV assembly without affecting HCV RNA and protein levels. We further demonstrated that polo-like kinase 1 (PLK1)-mediated phosphorylation of sorcin was increased by NS5A. We showed that both phosphorylation and calcium-binding activity of sorcin were required for HCV propagation. These data indicate that HCV modulates sorcin activity via NS5A protein for its own propagation. IMPORTANCE Sorcin is a calcium-binding protein and regulates intracellular calcium homeostasis. HCV NS5A interacts with sorcin, and phosphorylation of sorcin is required for protein interaction. Gene silencing of sorcin impaired HCV propagation at the assembly step of the HCV life cycle. Sorcin is phosphorylated by PLK1 via protein interaction. We showed that sorcin interacted with both NS5A and PLK1, and PLK1-mediated phosphorylation of sorcin was increased by NS5A. Moreover, calcium-binding activity of sorcin played a crucial role in HCV propagation. These data provide evidence that HCV regulates host calcium metabolism for virus propagation, and thus manipulation of sorcin activity may represent a novel therapeutic target for HCV. PMID:26719254

  7. Effect of protein source on resistive-training-induced changes in body composition and muscle size in older men123

    PubMed Central

    Haub, Mark D; Wells, Amanda M; Tarnopolsky, Mark A; Campbell, Wayne W

    2008-01-01

    Background Aging is associated with reductions in muscle mass and strength, but nutrition and exercise interventions can delay this progression and enhance the quality of life. Objective We examined whether the predominant source of protein consumed by older men influenced measures of muscle size and strength, body composition, resting energy expenditure, and skeletal muscle creatine concentrations in response to 12 wk of resistive training. Design After consuming a lactoovovegetarian (LOV) diet for 2 wk, 21 men aged 65 ± 5 y were randomly assigned to either consume a beef-containing (BC) diet (n = 10) or to continue the LOV diet (n = 11) throughout resistive training. The BC diet included 0.6 g protein · kg−1 · d−1 from beef and the LOV diet included 0.6 g protein · kg−1 · d−1 from textured vegetable protein (soy) sources. The remaining protein in the diets came from self-selected LOV sources. Results The mean total protein intake for both groups ranged from 1.03 to 1.17 g · kg−1 · d−1 during the intervention. Men in both groups had improvements (14–38%) in maximal dynamic strength of all the muscle groups trained with no significant difference between groups. With resistive training, cross-sectional muscle area of the vastus lateralis increased in both groups (4.2 ± 3.0% and 6.0 ± 2.6% for the LOV and BC groups, respectively) with no significant difference between groups. Body composition, resting energy expenditure, and concentrations of muscle creatine, phosphocreatine, and total creatine did not differ significantly between groups or change over time. Conclusions These data suggest that increases in muscle strength and size were not influenced by the predominant source of protein consumed by older men with adequate total protein intake. PMID:12197993

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

    PubMed

    Gaisford, W C; Reynolds, P E

    1989-10-20

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

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

    PubMed

    Gaisford, W C; Reynolds, P E

    1989-10-20

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

  10. Suppression of Poly(rC)-Binding Protein 4 (PCBP4) reduced cisplatin resistance in human maxillary cancer cells.

    PubMed

    Ito, Yumi; Narita, Norihiko; Nomi, Nozomi; Sugimoto, Chizuru; Takabayashi, Tetsuji; Yamada, Takechiyo; Karaya, Kazuhiro; Matsumoto, Hideki; Fujieda, Shigeharu

    2015-07-21

    Cisplatin plays an important role in the therapy for human head and neck cancers. However, cancer cells develop cisplatin resistance, leading to difficulty in treatment and poor prognosis. To analyze cisplatin-resistant mechanisms, a cisplatin-resistant cell line, IMC-3CR, was established from the IMC-3 human maxillary cancer cell line. Flow cytometry revealed that, compared with IMC-3 cells, cisplatin more dominantly induced cell cycle G2/M arrest rather than apoptosis in IMC-3CR cells. That fact suggests that IMC-3CR cells avoid cisplatin-induced apoptosis through induction of G2/M arrest, which allows cancer cells to repair damaged DNA and survive. In the present study, we specifically examined Poly(rC)-Binding Protein 4 (PCBP4), which reportedly induces G2/M arrest. Results showed that suppression of PCBP4 by RNAi reduced cisplatin-induced G2/M arrest and enhanced apoptosis in IMC-3CR cells, resulting in the reduction of cisplatin resistance. In contrast, overexpression of PCBP4 in IMC-3 cells induced G2/M arrest after cisplatin treatment and enhanced cisplatin resistance. We revealed that PCBP4 combined with Cdc25A and suppressed the expression of Cdc25A, resulting in G2/M arrest. PCBP4 plays important roles in the induction of cisplatin resistance in human maxillary cancers. PCBP4 is a novel molecular target for the therapy of head and neck cancers, especially cisplatin-resistant cancers.

  11. BLIMP-1/BLMP-1 and Metastasis-Associated Protein Regulate Stress Resistant Development in Caenorhabditis elegans.

    PubMed

    Hyun, Moonjung; Kim, Jeongho; Dumur, Catherine; Schroeder, Frank C; You, Young-Jai

    2016-08-01

    Environmental stress triggers multilevel adaptations in animal development that depend in part on epigenetic mechanisms. In response to harsh environmental conditions and pheromone signals, Caenorhabditis elegans larvae become the highly stress-resistant and long-lived dauer. Despite extensive studies of dauer formation pathways that integrate specific environmental cues and appear to depend on transcriptional reprogramming, the role of epigenetic regulation in dauer development has remained unclear. Here we report that BLMP-1, the BLIMP-1 ortholog, regulates dauer formation via epigenetic pathways; in the absence of TGF-β signaling (in daf-7 mutants), lack of blmp-1 caused lethality. Using this phenotype, we screened 283 epigenetic factors, and identified lin-40, a homolog of metastasis-associate protein 1 (MTA1) as an interactor of BLMP-1 The interaction between LIN-40 and BLMP-1 is conserved because mammalian homologs for both MTA1 and BLIMP-1 could also interact. From microarray studies, we identified several downstream target genes of blmp-1: npr-3, nhr-23, ptr-4, and sams-1 Among them S-adenosyl methionine synthase (SAMS-1), is the key enzyme for production of SAM used in histone methylation. Indeed, blmp-1 is necessary for controlling histone methylation level in daf-7 mutants, suggesting BLMP-1 regulates the expression of SAMS-1, which in turn may regulate histone methylation and dauer formation. Our results reveal a new interaction between BLMP-1/BLIMP-1 and LIN-40/MTA1, as well as potential epigenetic downstream pathways, whereby these proteins cooperate to regulate stress-specific developmental adaptations. PMID:27334271

  12. Generation and Characterization of a Breast Cancer Resistance Protein Humanized Mouse Model.

    PubMed

    Dallas, Shannon; Salphati, Laurent; Gomez-Zepeda, David; Wanek, Thomas; Chen, Liangfu; Chu, Xiaoyan; Kunta, Jeevan; Mezler, Mario; Menet, Marie-Claude; Chasseigneaux, Stephanie; Declèves, Xavier; Langer, Oliver; Pierre, Esaie; DiLoreto, Karen; Hoft, Carolin; Laplanche, Loic; Pang, Jodie; Pereira, Tony; Andonian, Clara; Simic, Damir; Rode, Anja; Yabut, Jocelyn; Zhang, Xiaolin; Scheer, Nico

    2016-05-01

    Breast cancer resistance protein (BCRP) is expressed in various tissues, such as the gut, liver, kidney and blood brain barrier (BBB), where it mediates the unidirectional transport of substrates to the apical/luminal side of polarized cells. Thereby BCRP acts as an efflux pump, mediating the elimination or restricting the entry of endogenous compounds or xenobiotics into tissues and it plays important roles in drug disposition, efficacy and safety. Bcrp knockout mice (Bcrp(-/-)) have been used widely to study the role of this transporter in limiting intestinal absorption and brain penetration of substrate compounds. Here we describe the first generation and characterization of a mouse line humanized for BCRP (hBCRP), in which the mouse coding sequence from the start to stop codon was replaced with the corresponding human genomic region, such that the human transporter is expressed under control of the murineBcrppromoter. We demonstrate robust human and loss of mouse BCRP/Bcrp mRNA and protein expression in the hBCRP mice and the absence of major compensatory changes in the expression of other genes involved in drug metabolism and disposition. Pharmacokinetic and brain distribution studies with several BCRP probe substrates confirmed the functional activity of the human transporter in these mice. Furthermore, we provide practical examples for the use of hBCRP mice to study drug-drug interactions (DDIs). The hBCRP mouse is a promising model to study the in vivo role of human BCRP in limiting absorption and BBB penetration of substrate compounds and to investigate clinically relevant DDIs involving BCRP.

  13. Stability of plant immune-receptor resistance proteins is controlled by SKP1-CULLIN1-F-box (SCF)-mediated protein degradation

    PubMed Central

    Cheng, Yu Ti; Li, Yingzhong; Huang, Shuai; Huang, Yan; Dong, Xinnian; Zhang, Yuelin; Li, Xin

    2011-01-01

    The nucleotide-binding domain and leucine-rich repeats containing proteins (NLRs) serve as immune receptors in both plants and animals. Overaccumulation of NLRs often leads to autoimmune responses, suggesting that the levels of these immune receptors must be tightly controlled. However, the mechanism by which NLR protein levels are regulated is unknown. Here we report that the F-box protein CPR1 controls the stability of plant NLR resistance proteins. Loss-of-function mutations in CPR1 lead to higher accumulation of the NLR proteins SNC1 and RPS2, as well as autoactivation of immune responses. The autoimmune responses in cpr1 mutant plants can be largely suppressed by knocking out SNC1. Furthermore, CPR1 interacts with SNC1 and RPS2 in vivo, and overexpressing CPR1 results in reduced accumulation of SNC1 and RPS2, as well as suppression of immunity mediated by these two NLR proteins. Our data suggest that SKP1-CULLIN1-F-box (SCF) complex-mediated stability control of plant NLR proteins plays an important role in regulating their protein levels and preventing autoimmunity. PMID:21873230

  14. Over-Expression of Cysteine Leucine Rich Protein Is Related to SAG Resistance in Clinical Isolates of Leishmania donovani

    PubMed Central

    Das, Sanchita; Shah, Priyanka; Tandon, Rati; Yadav, Narendra Kumar; Sahasrabuddhe, Amogh A.; Sundar, Shyam; Siddiqi, Mohammad Imran; Dube, Anuradha

    2015-01-01

    Background Resistance emergence against antileishmanial drugs, particularly Sodium Antimony Gluconate (SAG) has severely hampered the therapeutic strategy against visceral leishmaniasis, the mechanism of resistance being indistinguishable. Cysteine leucine rich protein (CLrP), was recognized as one of the overexpressed proteins in resistant isolates, as observed in differential proteomics between sensitive and resistant isolates of L. donovani. The present study deals with the characterization of CLrP and for its possible connection with SAG resistance. Methodology and Principal Findings In pursuance of deciphering the role of CLrP in SAG resistance, gene was cloned, over-expressed in E. coli system and thereafter antibody was raised. The expression profile of CLrP and was found to be over-expressed in SAG resistant clinical isolates of L. donovani as compared to SAG sensitive ones when investigated by real-time PCR and western blotting. CLrP has been characterized through bioinformatics, immunoblotting and immunolocalization analysis, which reveals its post-translational modification along with its dual existence in the nucleus as well as in the membrane of the parasite. Further investigation using a ChIP assay confirmed its DNA binding potential. Over-expression of CLrP in sensitive isolate of L. donovani significantly decreased its responsiveness to SAG (SbV and SbIII) and a shift towards the resistant mode was observed. Further, a significant increase in its infectivity in murine macrophages has been observed. Conclusion/Significance The study reports the differential expression of CLrP in SAG sensitive and resistant isolates of L. donovani. Functional intricacy of CLrP increases with dual localization, glycosylation and DNA binding potential of the protein. Further over-expressing CLrP in sensitive isolate of L. donovani shows significantly decreased sensitivity towards SAG and increased infectivity as well, thus assisting the parasite in securing a safe niche

  15. Drug Resistance in Cortical and Hippocampal Slices from Resected Tissue of Epilepsy Patients: No Significant Impact of P-Glycoprotein and Multidrug Resistance-Associated Proteins

    PubMed Central

    Sandow, Nora; Kim, Simon; Raue, Claudia; Päsler, Dennis; Klaft, Zin-Juan; Antonio, Leandro Leite; Hollnagel, Jan Oliver; Kovacs, Richard; Kann, Oliver; Horn, Peter; Vajkoczy, Peter; Holtkamp, Martin; Meencke, Heinz-Joachim; Cavalheiro, Esper A.; Pragst, Fritz; Gabriel, Siegrun; Lehmann, Thomas-Nicolas; Heinemann, Uwe

    2015-01-01

    Drug resistant patients undergoing epilepsy surgery have a good chance to become sensitive to anticonvulsant medication, suggesting that the resected brain tissue is responsible for drug resistance. Here, we address the question whether P-glycoprotein (Pgp) and multidrug resistance-associated proteins (MRPs) expressed in the resected tissue contribute to drug resistance in vitro. Effects of anti-epileptic drugs [carbamazepine (CBZ), sodium valproate, phenytoin] and two unspecific inhibitors of Pgp and MRPs [verapamil (VPM) and probenecid (PBN)] on seizure-like events (SLEs) induced in slices from 35 hippocampal and 35 temporal cortex specimens of altogether 51 patients (161 slices) were studied. Although in slice preparations the blood brain barrier is not functional, we found that SLEs predominantly persisted in the presence of anticonvulsant drugs (90%) and also in the presence of VPM and PBN (86%). Following subsequent co-administration of anti-epileptic drugs and drug transport inhibitors, SLEs continued in 63% of 143 slices. Drug sensitivity in slices was recognized either as transition to recurrent epileptiform transients (30%) or as suppression (7%), particularly by perfusion with CBZ in PBN containing solutions (43, 9%). Summarizing responses to co-administration from more than one slice per patient revealed that suppression of seizure-like activity in all slices was only observed in 7% of patients. Patients whose tissue was completely or partially sensitive (65%) presented with higher seizure frequencies than those with resistant tissue (35%). However, corresponding subgroups of patients do not differ with respect to expression rates of drug transporters. Our results imply that parenchymal MRPs and Pgp are not responsible for drug resistance in resected tissue. PMID:25741317

  16. Effect of timing of protein and carbohydrate intake after resistance exercise on nitrogen balance in trained and untrained young men

    PubMed Central

    2014-01-01

    Background Resistance exercise alters the post-exercise response of anabolic and catabolic hormones. A previous study indicated that the turnover of muscle protein in trained individuals is reduced due to alterations in endocrine factors caused by resistance training, and that muscle protein accumulation varies between trained and untrained individuals due to differences in the timing of protein and carbohydrate intake. We investigated the effect of the timing of protein and carbohydrate intake after resistance exercise on nitrogen balance in trained and untrained young men. Methods Subjects were 10 trained healthy men (mean age, 23 ± 4 years; height, 173.8 ± 3.1 cm; weight, 72.3 ± 4.3 kg) and 10 untrained healthy men (mean age, 23 ± 1 years; height, 171.8 ± 5.0 cm; weight, 64.5 ± 5.0 kg). All subjects performed four sets of 8 to 10 repetitions of a resistance exercise (comprising bench press, shoulder press, triceps pushdown, leg extension, leg press, leg curl, lat pulldown, rowing, and biceps curl) at 80% one-repetition maximum. After each resistance exercise session, subjects were randomly divided into two groups with respect to intake of protein (0.3 g/kg body weight) and carbohydrate (0.8 g/kg body weight) immediately after (P0) or 6 h (P6) after the session. All subjects were on an experimental diet that met their individual total energy requirement. We assessed whole-body protein metabolism by measuring nitrogen balance at P0 and P6 on the last 3 days of exercise training. Results The nitrogen balance was significantly lower in the trained men than in the untrained men at both P0 (P <0.05) and P6 (P <0.01). The nitrogen balance in trained men was significantly higher at P0 than at P6 (P <0.01), whereas that in the untrained men was not significantly different between the two periods. Conclusion The timing of protein and carbohydrate intake after resistance exercise influences nitrogen balance differently in trained and

  17. A novel approach for synthesis of zwitterionic polyurethane coating with protein resistance.

    PubMed

    Wang, Chunhua; Ma, Chunfeng; Mu, Changdao; Lin, Wei

    2014-11-01

    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. PMID:25310180

  18. Progesterone acts via progesterone receptors A and B to regulate breast cancer resistance protein expression.

    PubMed

    Vore, Mary; Leggas, Markos

    2008-03-01

    The breast cancer resistance protein (BCRP; ABCG2) is an ATP-dependent efflux multidrug transporter that belongs to the G family of half-transporters that consist of six transmembrane-spanning domains and must homodimerize to form the active membrane transporter. It is expressed in the apical plasma membrane domain of the small intestine, endothelium, and liver, where it has been shown to play an important role in limiting drug absorption and distribution and in enhancing drug clearance, respectively. BCRP is also expressed in the apical membrane of mammary alveolar epithelia, where it mediates efflux of substrates into milk, and in the placental syncytiotro-phoblasts, where it reduces fetal exposure to these substrates. BCRP substrates include numerous drugs (topotecan, nitrofurantoin, cimetidine) as well as food carcinogens (2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine) and the vitamins riboflavin and folic acid. BCRP expression is regulated by a number of nuclear transcription factors, including the peroxisome proliferator-activated receptor-gamma and Hif-1. This issue of Molecular Pharmacology includes a study (p. 845) now conclusively demonstrating that progesterone acts via the progesterone A and B receptors to regulate BCRP expression in a placental cell line.

  19. Biliary excretion of curcumin is mediated by multidrug resistance-associated protein 2.

    PubMed

    Lee, Joo Hyun; Oh, Ju-Hee; Lee, Young-Joo

    2012-01-01

    Curcumin has a wide spectrum of pharmacological activities, including antioxidant, anti-inflammatory, antimicrobial, and anticancer properties. Recently, its potential as effective chemoprevention against cholangiocarcinoma, a highly malignant tumor of the bile duct with limited therapeutic options, was reported. The purpose of the present study was to investigate the contribution of multidrug resistance-associated protein 2 (Mrp2) to the biliary excretion of curcumin using Sprague-Dawley rats (SDR) and Eisai hyperbilirubinemic rats (EHBR). After intravenous administration of curcumin with a loading dose of 4.5 mg/kg, followed by a constant infusion of 18 mg/kg/h to the SDR and EHBR, the pharmacokinetic parameters of curcumin were estimated. In EHBR, the total area under the bile concentration-time curve from 0 to 80 min following curcumin administration was dramatically decreased (0.094%) compared to that in SDR. In addition, the plasma-to-bile and liver-to-bile clearances were both significantly decreased compared to SDR. These results provide the first evidence that Mrp2 mediates the biliary excretion of curcumin and thus may be a major factor in the control of exposure of curcumin to the bile duct. This study may be helpful to the potential use of curcumin as a treatment for bile duct cancer, and to understanding the genetic polymorphism of Mrp2 for clinical trials of curcumin.

  20. The bacterial DNA repair protein Mfd confers resistance to the host nitrogen immune response.

    PubMed

    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.

  1. 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.

  2. Family business: the multidrug-resistance related protein (MRP) ABC transporter genes in Arabidopsis thaliana.

    PubMed

    Kolukisaoglu, H Uner; Bovet, Lucien; Klein, Markus; Eggmann, Thomas; Geisler, Markus; Wanke, Dierk; Martinoia, Enrico; Schulz, Burkhard

    2002-11-01

    Despite the completion of the sequencing of the entire genome of Arabidopsis thaliana (L.) Heynh., the exact determination of each single gene and its function remains an open question. This is especially true for multigene families. An approach that combines analysis of genomic structure, expression data and functional genomics to ascertain the role of the members of the multidrug-resistance-related protein ( MRP) gene family, a subfamily of the ATP-binding cassette (ABC) transporters from Arabidopsis is presented. We used cDNA sequencing and alignment-based re-annotation of genomic sequences to define the exact genic structure of all known AtMRP genes. Analysis of promoter regions suggested different induction conditions even for closely related genes. Expression analysis for the entire gene family confirmed these assumptions. Phylogenetic analysis and determination of segmental duplication in the regions of AtMRP genes revealed that the evolution of the extraordinarily high number of ABC transporter genes in plants cannot solely be explained by polyploidisation during the evolution of the Arabidopsis genome. Interestingly MRP genes from Oryza sativa L. (rice; OsMRP) show very similar genomic structures to those from Arabidopsis. Screening of large populations of T-DNA-mutagenised lines of A. thaliana resulted in the isolation of AtMRP insertion mutants. This work opens the way for the defined analysis of a multigene family of important membrane transporters whose broad variety of functions expands their traditional role as cellular detoxifiers. PMID:12430019

  3. 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.

  4. A novel approach for synthesis of zwitterionic polyurethane coating with protein resistance.

    PubMed

    Wang, Chunhua; Ma, Chunfeng; Mu, Changdao; Lin, Wei

    2014-11-01

    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. The bacterial DNA repair protein Mfd confers resistance to the host nitrogen immune response.

    PubMed

    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

  6. 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

  7. Identification of resistance-associated proteins in closely-related maize lines varying in aflatoxin accumulation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Aspergillus flavus infection of maize and subsequent contamination with carcinogenic aflatoxins poses serious health concerns, especially in developing countries. Maize lines resistant to A. flavus infection have been identified; however, the development of commercially-useful aflatoxin-resistant ma...

  8. Mutations in potato virus Y genome-linked protein determine virulence toward recessive resistances in Capsicum annuum and Lycopersicon hirsutum.

    PubMed

    Moury, Benoît; Morel, Caroline; Johansen, Elisabeth; Guilbaud, Laurent; Souche, Sylvie; Ayme, Valérie; Caranta, Carole; Palloix, Alain; Jacquemond, Mireille

    2004-03-01

    The recessive resistance genes pot-1 and pvr2 in Lycopersicon hirsutum and Capsicum annuum, respectively, control Potato virus Y (PVY) accumulation in the inoculated leaves. Infectious cDNA molecules from two PVY isolates differing in their virulence toward these resistances were obtained using two different strategies. Chimeras constructed with these cDNA clones showed that a single nucleotide change corresponding to an amino acid substitution (Arg119His) in the central part of the viral protein genome-linked (VPg) was involved in virulence toward the pot-1 resistance. On the other hand, 15 nucleotide changes corresponding to five putative amino acid differences in the same region of the VPg affected virulence toward the pvr2(1) and pvr2(2) resistances. Substitution models identified six and five codons within the central and C terminal parts of the VPg for PVY and for the related potyvirus Potato virus A, respectively, which undergo positive selection. This suggests that the role of the VPg-encoding region is determined by the protein and not by the viral RNA apart from its protein-encoding capacity.

  9. Discovery of a novel Toxoplasma gondii conoid-associated protein important for parasite resistance to reactive nitrogen intermediates.

    PubMed

    Skariah, Sini; Bednarczyk, Robert B; McIntyre, Matthew K; Taylor, Gregory A; Mordue, Dana G

    2012-04-01

    Toxoplasma gondii modifies its host cell to suppress its ability to become activated in response to IFN-γ and TNF-α and to develop intracellular antimicrobial effectors, including NO. Mechanisms used by T. gondii to modulate activation of its infected host cell likely underlie its ability to hijack monocytes and dendritic cells during infection to disseminate to the brain and CNS where it converts to bradyzoites contained in tissue cysts to establish persistent infection. To identify T. gondii genes important for resistance to the effects of host cell activation, we developed an in vitro murine macrophage infection and activation model to identify parasite insertional mutants that have a fitness defect in infected macrophages following activation but normal invasion and replication in naive macrophages. We identified 14 independent T. gondii insertional mutants out of >8000 screened that share a defect in their ability to survive macrophage activation due to macrophage production of reactive nitrogen intermediates (RNIs). These mutants have been designated counter-immune mutants. We successfully used one of these mutants to identify a T. gondii cytoplasmic and conoid-associated protein important for parasite resistance to macrophage RNIs. Deletion of the entire gene or just the region encoding the protein in wild-type parasites recapitulated the RNI-resistance defect in the counter-immune mutant, confirming the role of the protein in resistance to macrophage RNIs.

  10. 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

  11. 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.

  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. PMID:26220356

  13. Discovery and Characterization of Proteins Associated with Aflatoxin-Resistance: Evaluating Their Potential as Breeding Markers

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Host resistance has become a viable approach to eliminating aflatoxin contamination of maize since the discovery of several maize lines with natural resistance. However, to derive commercial benefit from this resistance and develop lines that can aid growers, markers need to be identified to facilit...

  14. Blue light photoreceptors are required for the stability and function of a resistance protein mediating viral defense in Arabidopsis

    PubMed Central

    Jeong, Rae-Dong; Kachroo, Aardr

    2010-01-01

    This light-perceiving ability of plants requires the activities of proteins termed photoreceptors. In addition to various growth and developmental processes, light also plays a role in plant defense against pathogens and is required for activation of several defense genes and regulation of the cell death response. However, the molecular or biochemical basis of light modulated regulation of defense signaling is largely unclear. We demonstrate a direct role for blue-light photoreceptors in resistance (R) protein-mediated plant defense against Turnip Crinkle Virus (TCV) in Arabidopsis. The blue-light photoreceptors, cryptochrome (CRY) 2 and phototropin (PHOT) 2, are specifically required for maintaining the stability of the R protein HRT, and thereby resistance to TCV. Exogenous application of the phytohormone salicylic acid elevates HRT levels in phot2 but not in cry2 background. These data indicate that CRY2 and PHOT2 function distinctly in maintaining post-transcriptional stability of HRT. HRT-mediated resistance is also dependent on CRY1 and PHOT1 proteins, but these do not contribute to the stability of HRT. HRT interacts with the CRY2/PHOT2-interacting protein COP1, a E3 ubiquitin ligase. Exogenous application of a proteasome inhibitor prevents blue-light-dependent degradation of HRT, suggesting that HRT is degraded via the 26S proteasome. These and the fact that PHOT2 interacts directly with the R protein RPS2 suggest that blue-light photoreceptors might be involved in regulation and/or signaling mediated by several R proteins. PMID:21057210

  15. Mitogen-activated protein kinase-activated protein kinase 2 mediates resistance to hydrogen peroxide-induced oxidative stress in human hepatobiliary cancer cells.

    PubMed

    Nguyen Ho-Bouldoires, Thanh Huong; Clapéron, Audrey; Mergey, Martine; Wendum, Dominique; Desbois-Mouthon, Christèle; Tahraoui, Sylvana; Fartoux, Laetitia; Chettouh, Hamza; Merabtene, Fatiha; Scatton, Olivier; Gaestel, Matthias; Praz, Françoise; Housset, Chantal; Fouassier, Laura

    2015-12-01

    The development and progression of liver cancer are characterized by increased levels of reactive oxygen species (ROS). ROS-induced oxidative stress impairs cell proliferation and ultimately leads to cell death. Although liver cancer cells are especially resistant to oxidative stress, mechanisms of such resistance remain understudied. We identified the MAPK-activated protein kinase 2 (MK2)/heat shock protein 27 (Hsp27) signaling pathway mediating defenses against oxidative stress. In addition to MK2 and Hsp27 overexpression in primary liver tumors compared to adjacent nontumorous tissues, the MK2/Hsp27 pathway is activated by hydrogen peroxide-induced oxidative stress in hepatobiliary cancer cells. MK2 inactivation or inhibition of MK2 or Hsp27 expression increases caspase-3 and PARP cleavage and DNA breaks and therefore cell death. Interestingly, MK2/Hsp27 inhibition decreases antioxidant defenses such as heme oxygenase 1 through downregulation of the transcription factor nuclear factor erythroid-derived 2-like 2. Moreover, MK2/Hsp27 inhibition decreases both phosphorylation of epidermal growth factor receptor (EGFR) and expression of its ligand, heparin-binding EGF-like growth factor. A new identified partner of MK2, the scaffold PDZ protein EBP50, could facilitate these effects through MK2/Hsp27 pathway regulation. These findings demonstrate that the MK2/Hsp27 pathway actively participates in resistance to oxidative stress and may contribute to liver cancer progression.

  16. 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

  17. Insect Resistance to Bacillus thuringiensis Toxin Cry2Ab Is Conferred by Mutations in an ABC Transporter Subfamily A Protein.

    PubMed

    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

    2015-11-01

    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

  18. 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.

  19. Acquired resistance to acetaminophen hepatotoxicity is associated with induction of multidrug resistance-associated protein 4 (Mrp4) in proliferating hepatocytes.

    PubMed

    Aleksunes, Lauren M; Campion, Sarah N; Goedken, Michael J; Manautou, José E

    2008-08-01

    Treatment with hepatotoxicants such as acetaminophen (APAP) causes resistance to a second, higher dose of the same toxicant (autoprotection). APAP induces hepatic mRNA and protein levels of the multidrug resistance-associated proteins (Mrp) transporters in mice and humans. Basolateral efflux transporters Mrp3 and Mrp4 are the most significantly induced. We hypothesized that upregulation of Mrp3 and Mrp4 is one mechanism by which hepatocytes become resistant to a subsequent higher dose of APAP by limiting accumulation of xeno-, endobiotics, and byproducts of hepatocellular injury. The purpose of this study was to evaluate Mrp3 and Mrp4 expression in proliferating hepatocytes in a mouse model of APAP autoprotection. Plasma and livers were collected from male C57BL/6J mice treated with APAP 400 mg/kg for determination of hepatotoxicity and protein expression. Maximal Mrp3 and Mrp4 induction occurred 48 h after APAP. Mrp4 upregulation occurred selectively in proliferating hepatocytes. Additional groups of APAP-pretreated mice were challenged 48 h later with a second, higher dose of APAP. APAP-pretreated mice had reduced hepatotoxicity after APAP challenge compared to those pretreated with vehicle. A more rapid recovery of glutathione (GSH) in APAP-pretreated mice corresponded with increases in GSH synthetic enzymes. Interestingly, mice pretreated and challenged with APAP had dramatic increases in Mrp4 expression as well as enhanced hepatocyte proliferation. Inhibition of hepatocyte replication with colchicine not only restored sensitivity of APAP-pretreated mice to injury, but also blocked Mrp4 induction. Mrp4 overexpression may be one phenotypic property of proliferating hepatocytes that protects against subsequent hepatotoxicant exposure by mechanisms that are presently unknown. PMID:18468992

  20. 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.

  1. Crystal structures of penicillin-binding protein 2 from penicillin-susceptible and -resistant strains of Neisseria gonorrhoeae reveal an unexpectedly subtle mechanism for antibiotic resistance.

    PubMed

    Powell, Ailsa J; Tomberg, Joshua; Deacon, Ashley M; Nicholas, Robert A; Davies, Christopher

    2009-01-01

    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. PMID:18986991

  2. Tomato 14-3-3 protein TFT7 interacts with a MAP kinase kinase to regulate immunity-associated programmed cell death mediated by diverse disease resistance proteins.

    PubMed

    Oh, Chang-Sik; Martin, Gregory B

    2011-04-22

    Programmed cell death (PCD) associated with immunity is triggered when a plant disease resistance (R) protein recognizes a corresponding pathogen virulence protein. In tomato, detection by the host Pto kinase of the Pseudomonas syringae proteins AvrPto or AvrPtoB causes localized PCD. Previously, we reported that both MAPKKKα (mitogen-activated protein kinase kinase kinase) and the tomato 14-3-3 protein 7 (TFT7) positively regulate Pto-mediated PCD in tomato and Nicotiana benthamiana. In addition, in contrast to MAPKKKα, TFT7 is required for PCD mediated by four other R proteins. Here we investigate why TFT7 is required for PCD induced by diverse R proteins in plants. We discovered that a MAPKK, SlMKK2, which acts downstream of SlMAPKKKα, also interacts with TFT7 in plant cells. Gene silencing experiments revealed that the orthologous genes of both SlMKK2 and TFT7 in N. benthamiana are required for PCD mediated by the same set of R proteins. SlMKK2 and its orthologs contain a 14-3-3 binding site in their N terminus, and Thr(33) in this site is required for interaction with TFT7 in vivo. Like the structurally similar human 14-3-3ε protein, TFT7 forms a homodimer in vivo. Because TFT7 interacts with both SlMAPKKKα and SlMKK2 and also forms a homodimer, we propose that TFT7 may coordinately recruit these client proteins for efficient signal transfer, leading to PCD induction. PMID:21378171

  3. 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

  4. Plant Ribosomal Proteins, RPL12 and RPL19, Play a Role in Nonhost Disease Resistance against Bacterial Pathogens

    PubMed Central

    Nagaraj, Satish; Senthil-Kumar, Muthappa; Ramu, Vemanna S.; Wang, Keri; Mysore, Kirankumar S.

    2016-01-01

    Characterizing the molecular mechanism involved in nonhost disease resistance is important to understand the adaptations of plant-pathogen interactions. In this study, virus-induced gene silencing (VIGS)-based forward genetics screen was utilized to identify genes involved in nonhost resistance in Nicotiana benthamiana. Genes encoding ribosomal proteins, RPL12 and RPL19, were identified in the screening. These genes when silenced in N. benthamiana caused a delay in nonhost bacteria induced hypersensitive response (HR) with concurrent increase in nonhost bacterial multiplication. Arabidopsis mutants of AtRPL12 and AtRPL19 also compromised nonhost resistance. The studies on NbRPL12 and NbRPL19 double silenced plants suggested that both RPL12 and RPL19 act in the same pathway to confer nonhost resistance. Our work suggests a role for RPL12 and RPL19 in nonhost disease resistance in N. benthamiana and Arabidopsis. In addition, we show that these genes also play a minor role in basal resistance against virulent pathogens. PMID:26779226

  5. Plant Ribosomal Proteins, RPL12 and RPL19, Play a Role in Nonhost Disease Resistance against Bacterial Pathogens.

    PubMed

    Nagaraj, Satish; Senthil-Kumar, Muthappa; Ramu, Vemanna S; Wang, Keri; Mysore, Kirankumar S

    2015-01-01

    Characterizing the molecular mechanism involved in nonhost disease resistance is important to understand the adaptations of plant-pathogen interactions. In this study, virus-induced gene silencing (VIGS)-based forward genetics screen was utilized to identify genes involved in nonhost resistance in Nicotiana benthamiana. Genes encoding ribosomal proteins, RPL12 and RPL19, were identified in the screening. These genes when silenced in N. benthamiana caused a delay in nonhost bacteria induced hypersensitive response (HR) with concurrent increase in nonhost bacterial multiplication. Arabidopsis mutants of AtRPL12 and AtRPL19 also compromised nonhost resistance. The studies on NbRPL12 and NbRPL19 double silenced plants suggested that both RPL12 and RPL19 act in the same pathway to confer nonhost resistance. Our work suggests a role for RPL12 and RPL19 in nonhost disease resistance in N. benthamiana and Arabidopsis. In addition, we show that these genes also play a minor role in basal resistance against virulent pathogens. PMID:26779226

  6. Riboflavin-Induced Disease Resistance Requires the Mitogen-Activated Protein Kinases 3 and 6 in Arabidopsis thaliana.

    PubMed

    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

  7. BEACH-domain proteins act together in a cascade to mediate vacuolar protein trafficking and disease resistance in Arabidopsis.

    PubMed

    Teh, Ooi-kock; Hatsugai, Noriyuki; Tamura, Kentaro; Fuji, Kentaro; Tabata, Ryo; Yamaguchi, Katsushi; Shingenobu, Shuji; Yamada, Masashi; Hasebe, Mitsuyasu; Sawa, Shinichiro; Shimada, Tomoo; Hara-Nishimura, Ikuko

    2015-03-01

    Membrane trafficking to the protein storage vacuole (PSV) is a specialized process in seed plants. However, this trafficking mechanism to PSV is poorly understood. Here, we show that three types of Beige and Chediak-Higashi (BEACH)-domain proteins contribute to both vacuolar protein transport and effector-triggered immunity (ETI). We screened a green fluorescent seed (GFS) library of Arabidopsis mutants with defects in vesicle trafficking and isolated two allelic mutants gfs3 and gfs12 with a defect in seed protein transport to PSV. The gene responsible for the mutant phenotype was found to encode a putative protein belonging to group D of BEACH-domain proteins, which possess kinase domains. Disruption of other BEACH-encoding loci in the gfs12 mutant showed that BEACH homologs acted in a cascading manner for PSV trafficking. The epistatic genetic interactions observed among BEACH homologs were also found in the ETI responses of the gfs12 and gfs12 bchb-1 mutants, which showed elevated avirulent bacterial growth. The GFS12 kinase domain interacted specifically with the pleckstrin homology domain of BchC1. These results suggest that a cascade of multiple BEACH-domain proteins contributes to vacuolar protein transport and plant defense.

  8. BEACH-domain proteins act together in a cascade to mediate vacuolar protein trafficking and disease resistance in Arabidopsis.

    PubMed

    Teh, Ooi-kock; Hatsugai, Noriyuki; Tamura, Kentaro; Fuji, Kentaro; Tabata, Ryo; Yamaguchi, Katsushi; Shingenobu, Shuji; Yamada, Masashi; Hasebe, Mitsuyasu; Sawa, Shinichiro; Shimada, Tomoo; Hara-Nishimura, Ikuko

    2015-03-01

    Membrane trafficking to the protein storage vacuole (PSV) is a specialized process in seed plants. However, this trafficking mechanism to PSV is poorly understood. Here, we show that three types of Beige and Chediak-Higashi (BEACH)-domain proteins contribute to both vacuolar protein transport and effector-triggered immunity (ETI). We screened a green fluorescent seed (GFS) library of Arabidopsis mutants with defects in vesicle trafficking and isolated two allelic mutants gfs3 and gfs12 with a defect in seed protein transport to PSV. The gene responsible for the mutant phenotype was found to encode a putative protein belonging to group D of BEACH-domain proteins, which possess kinase domains. Disruption of other BEACH-encoding loci in the gfs12 mutant showed that BEACH homologs acted in a cascading manner for PSV trafficking. The epistatic genetic interactions observed among BEACH homologs were also found in the ETI responses of the gfs12 and gfs12 bchb-1 mutants, which showed elevated avirulent bacterial growth. The GFS12 kinase domain interacted specifically with the pleckstrin homology domain of BchC1. These results suggest that a cascade of multiple BEACH-domain proteins contributes to vacuolar protein transport and plant defense. PMID:25618824

  9. Overexpression of heat shock protein 70 in stomach of stress-induced gastric ulcer-resistant rats.

    PubMed

    Shichijo, Kazuko; Ihara, Makoto; Matsuu, Mutsumi; Ito, Masahiro; Okumura, Yutaka; Sekine, Ichiro

    2003-02-01

    Expression of heat shock protein 70, induced by an antiulcer drug, provides protection against gastric ulcers. However, the mechanisms responsible for this protection are not known. The expression in ulcer-resistant, spontaneously hypertensive rats was 2.8-fold higher than in normotensive rats. One hour after restraint and water immersion stress, strong nuclear immunoreactivity was observed in nuclei of surface epithelial cells at the crest of gastric mucosal folds, the first site of ulceration, only in spontaneously hypertensive rats. Heat shock cognate protein 70, which is expressed in mucus-secreting cells, was not overexpressed in spontaneously hypertensive rats. Heat shock protein 70 expression was attenuated by chemical sympathectomy, which also resulted in abolition of the increase of mucosal blood flow and aggravation of ulcers. Our results indicate that overexpression of heat shock protein 70 in the stomach seems to protect against gastric ulcers through its cytoprotective effects on gastric mucosa by increasing mucosal blood flow. PMID:12643613

  10. The Enterococcus hirae R40 penicillin-binding protein 5 and the methicillin-resistant Staphylococcus aureus penicillin-binding protein 2' are similar.

    PubMed

    el Kharroubi, A; Jacques, P; Piras, G; Van Beeumen, J; Coyette, J; Ghuysen, J M

    1991-12-01

    The penicillin-resistant Enterococcus hirae R40 has a typical profile of membrane-bound penicillin-binding proteins (PBPs) except that the 71 kDa PBP5 of low penicillin affinity represents about 50% of all the PBPs present. Water-soluble tryptic-digest peptides were selectively produced from PBP5, their N-terminal regions were sequenced and synthetic oligonucleotides were used as primers to generate a 476 bp DNA fragment by polymerase chain reaction. On the basis of these data, the PBP5-encoding gene was cloned in Escherichia coli by using pBR322 as vector. The gene, included in a 7.1 kb insert, had the information for a 678-amino acid-residue protein. PBP5 shows similarity, in the primary structure, with the high-molecular-mass PBPs of class B. In particular, amino acid alignment of the enterococcal PBP5 and the methicillin-resistant staphylococcal PBP2' generates scores that are 30, for the N-terminal domains, and 53, for the C-terminal domains, standard deviations above that expected for a run of 20 randomized pairs of proteins having the same amino acid compositions as the two proteins under consideration. PMID:1747121

  11. 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 .

  12. Role of Protein Farnesylation in Burn-Induced Metabolic Derangements and Insulin Resistance in Mouse Skeletal Muscle

    PubMed Central

    Tanaka, Tomokazu; Kramer, Joshua; Yu, Yong-Ming; Fischman, Alan J.; Martyn, J. A. Jeevendra; Tompkins, Ronald G.; Kaneki, Masao

    2015-01-01

    Objective Metabolic derangements, including insulin resistance and hyperlactatemia, are a major complication of major trauma (e.g., burn injury) and affect the prognosis of burn patients. Protein farnesylation, a posttranslational lipid modification of cysteine residues, has been emerging as a potential component of inflammatory response in sepsis. However, farnesylation has not yet been studied in major trauma. To study a role of farnesylation in burn-induced metabolic aberration, we examined the effects of farnesyltransferase (FTase) inhibitor, FTI-277, on burn-induced insulin resistance and metabolic alterations in mouse skeletal muscle. Methods A full thickness burn (30% total body surface area) was produced under anesthesia in male C57BL/6 mice at 8 weeks of age. After the mice were treated with FTI-277 (5 mg/kg/day, IP) or vehicle for 3 days, muscle insulin signaling, metabolic alterations and inflammatory gene expression were evaluated. Results Burn increased FTase expression and farnesylated proteins in mouse muscle compared with sham-burn at 3 days after burn. Simultaneously, insulin-stimulated phosphorylation of insulin receptor (IR), insulin receptor substrate (IRS)-1, Akt and GSK-3β was decreased. Protein expression of PTP-1B (a negative regulator of IR-IRS-1 signaling), PTEN (a negative regulator of Akt-mediated signaling), protein degradation and lactate release by muscle, and plasma lactate levels were increased by burn. Burn-induced impaired insulin signaling and metabolic dysfunction were associated with increased inflammatory gene expression. These burn-induced alterations were reversed or ameliorated by FTI-277. Conclusions Our data demonstrate that burn increased FTase expression and protein farnesylation along with insulin resistance, metabolic alterations and inflammatory response in mouse skeletal muscle, all of which were prevented by FTI-277 treatment. These results indicate that increased protein farnesylation plays a pivotal role in burn

  13. 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

  14. 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

  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. The Nuclear Factor (Erythroid-derived 2)-like 2 and Proteasome Maturation Protein Axis Mediate Bortezomib Resistance in Multiple Myeloma.

    PubMed

    Li, Bingzong; Fu, Jinxiang; Chen, Ping; Ge, Xueping; Li, Yali; Kuiatse, Isere; Wang, Hua; Wang, Huihan; Zhang, Xingding; Orlowski, Robert Z

    2015-12-11

    Resistance to the proteasome inhibitor bortezomib is an emerging clinical problem whose mechanisms have not been fully elucidated. We considered the possibility that this could be associated with enhanced proteasome activity in part through the action of the proteasome maturation protein (POMP). Bortezomib-resistant myeloma models were used to examine the correlation between POMP expression and bortezomib sensitivity. POMP expression was then modulated using genetic and pharmacologic approaches to determine the effects on proteasome inhibitor sensitivity in cell lines and in vivo models. Resistant cell lines were found to overexpress POMP, and while its suppression in cell lines enhanced bortezomib sensitivity, POMP overexpression in drug-naive cells conferred resistance. Overexpression of POMP was associated with increased levels of nuclear factor (erythroid-derived 2)-like (NRF2), and NRF2 was found to bind to and activate the POMP promoter. Knockdown of NRF2 in bortezomib-resistant cells reduced POMP levels and proteasome activity, whereas its overexpression in drug-naive cells increased POMP and proteasome activity. The NRF2 inhibitor all-trans-retinoic acid reduced cellular NRF2 levels and increased the anti-proliferative and pro-apoptotic activities of bortezomib in resistant cells, while decreasing proteasome capacity. Finally, the combination of all-trans-retinoic acid with bortezomib showed enhanced activity against primary patient samples and in a murine model of bortezomib-resistant myeloma. Taken together, these studies validate a role for the NRF2/POMP axis in bortezomib resistance and identify NRF2 and POMP as potentially attractive targets for chemosensitization to this proteasome inhibitor.

  18. Efficient secretion of human lysozyme fused to the Sh ble phleomycin resistance protein by the fungus Tolypocladium geodes.

    PubMed

    Baron, M; Tiraby, G; Calmels, T; Parriche, M; Durand, H

    1992-07-01

    Tolypocladium geodes strain NC50 was transformed by different integrating vectors bearing both a synthetic gene encoding human lysozyme (HLz) and the Sh ble phleomycin resistance marker, either in separate expression cassettes or in transcriptional or translational fusion configurations. Clones derived from all vectors were able to secrete HLz. The highest productivities in shake flasks (up to 150 mg l-1 in 5 days) were obtained when HLz was fused at the C-terminal end of the Sh ble protein. The fusion protein is efficiently secreted and release of active lysozyme occurs by extracellular proteolytic cleavage in the junction peptide.

  19. Activated protein C resistance among postmenopausal women using transdermal estrogens: importance of progestogen

    PubMed Central

    Canonico, Marianne; Alhenc-Gelas, Martine; Plu-Bureau, Geneviève; Olié, Valérie; Scarabin, Pierre-Yves

    2010-01-01

    Introduction While the route of estrogen administration is known to be an important determinant of the thrombotic risk among postmenopausal women using hormone therapy, recent data have shown that norpregnane derivatives but not micronized progesterone would increase venous thromboembolism risk among transdermal estrogens users. However, differential effects of progesterone and norpregnanes on haemostasis have not yet been investigated. Methods We set up a cross-sectional study among healthy postmenopausal women aged 45 to 70 years. The impact of Activated Protein C (APC) on endogenous thrombin potential was investigated in plasma samples of 108 women who did not use any hormone therapy (n=40) or who were treated by transdermal estrogens combined with micronized progesterone (n=30) or norpregnane derivatives (n=38). Results After exclusion of women with factor V Leiden and/or G20210A prothrombin gene mutations, there was no significant change in APC sensitivity among women who used transdermal estrogens combined with micronized progesterone compared to non-users. Women using transdermal estrogens combined with norpregnanes were less sensitive to APC than were non-users (p=0.003) or users of transdermal estrogens combined with micronized progesterone (p=0.004). In addition, prothrombin fragment 1+2 concentration was higher in users of transdermal estrogens plus norpregnanes than in non-users (p=0.004). Other haemostatic parameters did not vary significantly across the different subgroups. Conclusion Transdermal estrogens combined with norpregnanes may induce an APC resistance and activate blood coagulation. These results provide a biological support to epidemiological data regarding the potential thrombogenic effects of norpregnanes. However, these findings need to be confirmed in a randomized trial. PMID:20613675

  20. 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. PMID:26746389

  1. Modulation of expression and activity of intestinal multidrug resistance-associated protein 2 by xenobiotics.

    PubMed

    Tocchetti, Guillermo Nicolás; Rigalli, Juan Pablo; Arana, Maite Rocío; Villanueva, Silvina Stella Maris; Mottino, Aldo Domingo

    2016-07-15

    The multidrug resistance-associated protein 2 (MRP2/ABCC2) is a transporter that belongs to the ATP-binding cassette (ABC) superfamily. In the intestine, it is localized to the apical membrane of the enterocyte and plays a key role in limiting the absorption of xenobiotics incorporated orally. MRP2 may also play a role in systemic clearance of xenobiotics available from the serosal side of the intestine. MRP2 transports a wide range of substrates, mainly organic anions conjugated with glucuronic acid, glutathione and sulfate and its expression can be modulated by xenobiotics at transcriptional- and post-transcriptional levels. Transcriptional regulation is usually mediated by a group of nuclear receptors. The pregnane X receptor (PXR) is a major member of this group. Relevant drugs described to up-regulate intestinal MRP2 via PXR are rifampicin, spironolactone and carbamazepine, among others. The constitutive androstane receptor (CAR, NR1I3) was also reported to modulate MRP2 expression, phenobarbital being a typical activator. Dietary compounds, including micronutrients and other natural products, are also capable of regulating intestinal MRP2 expression transcriptionally. We have given them particular attention since the composition of the food ingested daily is not necessarily supervised and may result in interactions with therapeutic drugs. Post-transcriptional regulation of MRP2 activity by xenobiotics, e.g. as a consequence of inhibitory actions, is also described in this review. Unfortunately, only few studies report on drug-drug or nutrient-drug interactions as a consequence of modulation of intestinal MRP2 activity by xenobiotics. Future clinical studies are expected to identify additional interactions resulting in changes in efficacy or safety of therapeutic drugs. PMID:27155371

  2. The sulfated conjugate of biochanin A is a substrate of breast cancer resistant protein (ABCG2).

    PubMed

    An, Guohua; Morris, Marilyn E

    2011-11-01

    The aim of the study was to investigate the role of breast cancer resistance protein (BCRP, ABCG2) in the transport of biochanin A and its metabolites. Transport studies were carried out in MDCK/bcrp1 as well as in control cells, and samples were analysed for biochanin A aglycone and metabolites using LC/MS/MS. In bidirectional transport studies biochanin A sulfate was detected in both apical and basolateral chambers after the addition of biochanin A. Analysis by RT-PCR revealed that the enzyme sulfotransferase 1A1 is expressed in Madin-Darby canine kidney (MDCK)-II cells. After its intracellular formation, biochanin A sulfate was preferentially transported to the basolateral side in MDCK/Mock cells, whereas apical transport of biochanin A sulfate was predominant in MDCK/Bcrp1 cells. Genistein, an additional metabolite of biochanin A formed intracellularly, was also found to be a bcrp1 substrate. Studies with MDCK/MRP2 (ABCC2) cells demonstrated that both genistein and biochanin A sulfate are not MRP2 substrates. In contrast, biochanin A aglycone was not transported by murine or human BCRP; nor is it a substrate of MRP2 or P-glycoprotein. Therefore, BCRP may play an important role in the enteric cycling of biochanin A sulfate and through this mechanism may alter the bioavailability of its non-substrate parent compound biochanin A. Moreover, MDCK-II cells might be a suitable model to investigate the synergistic role of sulfotransferase enzymes with efflux transporters. PMID:21910126

  3. 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

  4. Identification of a cell envelope protein (MtrF) involved in hydrophobic antimicrobial resistance in Neisseria gonorrhoeae.

    PubMed

    Veal, Wendy L; Shafer, William M

    2003-01-01

    The mtrCDE-encoded efflux pump of Neisseria gonorrhoeae provides gonococci with a mechanism to resist structurally diverse antimicrobial hydrophobic agents (HAs). Strains of N. gonorrhoeae that display hypersusceptibility to HAs often contain mutations in the efflux pump genes, mtrCDE. Such strains frequently contain a phenotypically suppressed mutation in mtrR, a gene that encodes a repressor (MtrR) of mtrCDE gene expression, and one that would normally result in HA resistance. We have recently examined HA-hypersusceptible clinical isolates of gonococci that contain such phenotypically suppressed mtrR mutations, in order to determine whether genes other than mtrCDE are involved in HA resistance. These studies led to the discovery of a gene that we have designated mtrF, located downstream of the mtrR gene, that is predicted to encode a 56.1 kDa cytoplasmic membrane protein containing 12 transmembrane domains. Expression of mtrF was enhanced in a strain deficient in MtrR production, indicating that this gene, together with the closely linked mtrCDE operon, is subject to MtrR-dependent transcriptional control. Orthologues of mtrF were identified in a number of diverse bacteria. Except for the AbgT protein of Escherichia coli, their products have been identified as hypothetical proteins with unknown function(s). Genetic evidence is presented that MtrF is important in the expression of high-level detergent resistance by gonococci. We propose that MtrF acts in conjunction with the MtrC-MtrD-MtrE efflux pump, to confer on gonococci high-level resistance to certain HAs. PMID:12493784

  5. Supplementation of a suboptimal protein dose with leucine or essential amino acids: effects on myofibrillar protein synthesis at rest and following resistance exercise in men.

    PubMed

    Churchward-Venne, Tyler A; Burd, Nicholas A; Mitchell, Cameron J; West, Daniel W D; Philp, Andrew; Marcotte, George R; Baker, Steven K; Baar, Keith; Phillips, Stuart M

    2012-06-01

    Leucine is a nutrient regulator of muscle protein synthesis by activating mTOR and possibly other proteins in this pathway. The purpose of this study was to examine the role of leucine in the regulation of human myofibrillar protein synthesis (MPS). Twenty-four males completed an acute bout of unilateral resistance exercise prior to consuming either: a dose (25 g) of whey protein (WHEY); 6.25 g whey protein with total leucine equivalent to WHEY (LEU); or 6.25 g whey protein with total essential amino acids (EAAs) equivalent to WHEY for all EAAs except leucine (EAA-LEU). Measures of MPS, signalling through mTOR, and amino acid transporter (AAT) mRNA abundance were made while fasted (FAST), and following feeding under rested (FED) and post-exercise (EX-FED) conditions. Leucinaemia was equivalent between WHEY and LEU and elevated compared to EAA-LEU (P=0.001). MPS was increased above FAST at 1–3 h post-exercise in both FED (P <0.001) and EX-FED (P <0.001) conditions with no treatment effect.At 3–5 h, only WHEY remained significantly elevated above FAST in EX-FED(WHEY 184% vs. LEU 55% and EAA-LEU 35%; P =0.036). AAT mRNA abundance was increased above FAST after feeding and exercise with no effect of leucinaemia. In summary, a low dose of whey protein supplemented with leucine or all other essential amino acids was as effective as a complete protein (WHEY) in stimulating postprandial MPS; however only WHEY was able to sustain increased rates of MPS post-exercise and may therefore be most suited to increase exercise-induced muscle protein accretion.

  6. Synergistic activity of BET protein antagonist-based combinations in mantle cell lymphoma cells sensitive or resistant to ibrutinib

    PubMed Central

    Sun, Baohua; Shah, Bhavin; Fiskus, Warren; Qi, Jun; Rajapakshe, Kimal; Coarfa, Cristian; Li, Li; Devaraj, Santhana G. T.; Sharma, Sunil; Zhang, Liang; Wang, Michael L.; Saenz, Dyana T.; Krieger, Stephanie; Bradner, James E.

    2015-01-01

    Mantle cell lymphoma (MCL) cells exhibit increased B-cell receptor and nuclear factor (NF)-κB activities. The bromodomain and extra-terminal (BET) protein bromodomain 4 is essential for the transcriptional activity of NF-κB. Here, we demonstrate that treatment with the BET protein bromodomain antagonist (BA) JQ1 attenuates MYC and cyclin-dependent kinase (CDK)4/6, inhibits the nuclear RelA levels and the expression of NF-κB target genes, including Bruton tyrosine kinase (BTK) in MCL cells. Although lowering the levels of the antiapoptotic B-cell lymphoma (BCL)2 family proteins, BA treatment induces the proapoptotic protein BIM and exerts dose-dependent lethality against cultured and primary MCL cells. Cotreatment with BA and the BTK inhibitor ibrutinib synergistically induces apoptosis of MCL cells. Compared with each agent alone, cotreatment with BA and ibrutinib markedly improved the median survival of mice engrafted with the MCL cells. BA treatment also induced apoptosis of the in vitro isolated, ibrutinib-resistant MCL cells, which overexpress CDK6, BCL2, Bcl-xL, XIAP, and AKT, but lack ibrutinib resistance-conferring BTK mutation. Cotreatment with BA and panobinostat (pan-histone deacetylase inhibitor) or palbociclib (CDK4/6 inhibitor) or ABT-199 (BCL2 antagonist) synergistically induced apoptosis of the ibrutinib-resistant MCL cells. These findings highlight and support further in vivo evaluation of the efficacy of the BA-based combinations with these agents against MCL, including ibrutinib-resistant MCL. PMID:26254443

  7. Virulent Diuraphis noxia Aphids Over-Express Calcium Signaling Proteins to Overcome Defenses of Aphid-Resistant Wheat Plants.

    PubMed

    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.

  8. 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.

  9. Mutations in the Nonstructural Protein 3A Confer Resistance to the Novel Enterovirus Replication Inhibitor TTP-8307▿

    PubMed Central

    De Palma, Armando M.; Thibaut, Hendrik Jan; van der Linden, Lonneke; Lanke, Kjerstin; Heggermont, Ward; Ireland, Stephen; Andrews, Robert; Arimilli, Murty; Al-Tel, Taleb H.; De Clercq, Erik; van Kuppeveld, Frank; Neyts, Johan

    2009-01-01

    A novel compound, TTP-8307, was identified as a potent inhibitor of the replication of several rhino- and enteroviruses. TTP-8307 inhibits viral RNA synthesis in a dose-dependent manner, without affecting polyprotein synthesis and/or processing. Drug-resistant variants of coxsackievirus B3 were all shown to carry at least one amino acid mutation in the nonstructural protein 3A. In particular, three mutations located in a nonstructured region preceding the hydrophobic domain (V45A, I54F, and H57Y) appeared to contribute to the drug-resistant phenotype. This region has previously been identified as a hot sport for mutations that resulted in resistance to enviroxime, the sole 3A-targeting enterovirus inhibitor reported thus far. This was corroborated by the fact that TTP-8307 and enviroxime proved cross-resistant. It is hypothesized that TTP-8307 and enviroxime disrupt proper interactions of 3A(B) with other viral or cellular proteins that are required for efficient replication. PMID:19237651

  10. Virulent Diuraphis noxia Aphids Over-Express Calcium Signaling Proteins to Overcome Defenses of Aphid-Resistant Wheat Plants.

    PubMed

    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

  11. Isolation of a strawberry gene fragment encoding an actin depolymerizing factor-like protein from genotypes resistant to Colletotrichum acutatum.

    PubMed

    Ontivero, Marta; Zamora, Gustavo Martínez; Salazar, Sergio; Ricci, Juan Carlos Díaz; Castagnaro, Atilio Pedro

    2011-12-01

    Actin depolymerizing factors (ADFs) have been recently implicated in plant defense against pathogenic fungi, associated with the cytoskeletal rearrangements that contribute to establish an effective barrier against fungal ingress. In this work, we identified a DNA fragment corresponding to a part of a gene predicted to encode an ADF-like protein in genotypes of Fragaria ananassa resistant to the fungus Colletotrichum acutatum. Bulked segregant analysis combined with AFLP was used to identify polymorphisms linked to resistance in hybrids derived from the cross between the resistant cultivar 'Sweet Charlie' and the susceptible cultivar 'Pájaro'. The sequence of one out of three polymorphic bands detected showed significant BLASTX hits to ADF proteins from other plants. Two possible exons were identified and bioinformatic analysis revealed the presence of the ADF homology domain with two actin-binding sites, an N-terminal phosphorylation site, and a nuclear localization signal. In addition to its possible application in strawberry breeding programs, these finding may contribute to investigate the role of ADFs in plant resistance against fungi. PMID:22107362

  12. 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

  13. 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. PMID:25545960

  14. SGT1 interacts with the Prf resistance protein and is required for Prf accumulation and Prf-mediated defense signaling.

    PubMed

    Kud, Joanna; Zhao, Zhulu; Du, Xinran; Liu, Yule; Zhao, Yun; Xiao, Fangming

    2013-02-15

    The highly conserved eukaryotic co-chaperone SGT1 (suppressor of the G2 allele of skp1) is an important signaling component of plant defense responses and positively regulates disease resistance conferred by many resistance (R) proteins. In this study, we investigated the contribution of SGT1 in the Prf-mediated defense responses in both Nicotiana benthamiana and tomato (Solanum lycopersicum). SGT1 was demonstrated to interact with Prf in plant cells by co-immunoprecipitation. The requirement of SGT1 in the accumulation of Prf or autoactive Prf(D1416V) was determined by the degradation of these proteins in N. benthamiana, in which SGT1 was repressed by virus-induced gene silencing (VIGS). Pseudomonas pathogen assay on the SGT1-silenced tomato plants implicates SGT1 is required for the Prf-mediated full resistance to Pseudomonas syringae pv. tomato (Pst). These results suggest that, in both N. benthamiana and tomato, SGT1 contributes to the Prf-mediated defense responses by stabilizing Prf protein via its co-chaperone activity.

  15. 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

  16. CROP/Luc7A, a novel serine/arginine-rich nuclear protein, isolated from cisplatin-resistant cell line.

    PubMed

    Nishii, Y; Morishima, M; Kakehi, Y; Umehara, K; Kioka, N; Terano, Y; Amachi, T; Ueda, K

    2000-01-14

    A novel putative SR protein, designated cisplatin resistance-associated overexpressed protein (CROP), has been cloned from cisplatin-resistant cell lines by differential display. The N-half of the deduced amino acid sequence of 432 amino acids of CROP contains cysteine/histidine motifs and leucine zipper-like repeats. The C-half consists mostly of charged and polar amino acids: arginine (58 residues or 25%), glutamate (36 residues or 16%), serine (35 residues or 15%), lysine (30 residues, 13%), and aspartate (20 residues or 9%). The C-half is extremely hydrophilic and comprises domains rich in lysine and glutamate residues, rich in alternating arginine and glutamate residues, and rich in arginine and serine residues. The arginine/serine-rich domain is dominated by a series of 8 amino acid imperfect repetitive motif (consensus sequence, Ser-Arg-Ser-Arg-Asp/Glu-Arg-Arg-Arg), which has been found in RNA splicing factors. The RNase protection assay and Western blotting analysis indicate that the expression of CROP is about 2-3-fold higher in mRNA and protein levels in cisplatin-resistant ACHN/CDDP cells than in host ACHN cells. CROP is the human homologue of yeast Luc7p, which is supposed to be involved in 5'-splice site recognition and is essential for vegetative growth. PMID:10631324

  17. 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.

  18. Transgenic plants expressing ω-ACTX-Hv1a and snowdrop lectin (GNA) fusion protein show enhanced resistance to aphids.

    PubMed

    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.

  19. 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

    2016-11-01

    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. PMID:27485973

  20. 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.

  1. 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

  2. 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

  3. 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.

  4. 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

  5. Genetic mapping of Bt-toxin binding proteins in a Cry1A-toxin resistant strain of diamondback moth Plutella xylostella.

    PubMed

    Baxter, Simon W; Zhao, Jian-Zhou; Shelton, Anthony M; Vogel, Heiko; Heckel, David G

    2008-02-01

    A major mechanism of resistance to Bacillus thuringiensis (Bt) toxins in Lepidoptera is a reduction of toxin binding to sites in the midgut membrane. Genetic studies of three different species have shown that mutations in a candidate Bt receptor, a 12-cadherin-domain protein, confer Cry1A toxin resistance. Despite a similar resistance profile in a fourth lepidopteran species, Plutella xylostella, we have previously shown that the cadherin orthologue maps to a different linkage group (LG8) than Cry1Ac resistance (LG22). Here we tested the hypothesis that mutations in other genes encoding candidate Bt-binding targets could be responsible for Bt resistance, by mapping eight aminopeptidases, an alkaline phosphatase (ALP), an intestinal mucin, and a P252 glycoprotein with respect to the 29 AFLP marked linkage groups in a P. xylostella cross segregating for Cry1Ac resistance. A homologue of the Caenorhabditis elegans Bt resistance gene bre-2 was also mapped. None of the genes analysed were on the same chromosome containing the Cry1Ac resistance locus, eliminating them as candidate resistance genes in the parental resistant strain SC1. Although this finding excludes cis-acting mutations in these genes as causing resistance in this strain, one or more of the expressed proteins may still bind Cry1Ac toxin, and post-translational modifications could affect this binding and thereby exert a trans-acting effect on resistance. PMID:18207074

  6. Functional differentiation in the leucine-rich repeat domains of closely related plant virus-resistance proteins that recognize common avr proteins.

    PubMed

    Sekine, Ken-Taro; Tomita, Reiko; Takeuchi, Shigeharu; Atsumi, Go; Saitoh, Hiromasa; Mizumoto, Hiroyuki; Kiba, Akinori; Yamaoka, Naoto; Nishiguchi, Masamichi; Hikichi, Yasufumi; Kobayashi, Kappei

    2012-09-01

    The N' gene of Nicotiana sylvestris and L genes of Capsicum plants confer the resistance response accompanying the hypersensitive response (HR) elicited by tobamovirus coat proteins (CP) but with different viral specificities. Here, we report the identification of the N' gene. We amplified and cloned an N' candidate using polymerase chain reaction primers designed from L gene sequences. The N' candidate gene was a single 4143 base pairs fragment encoding a coiled-coil nucleotide-binding leucine-rich repeat (LRR)-type resistance protein of 1,380 amino acids. The candidate gene induced the HR in response to the coexpression of tobamovirus CP with the identical specificity as reported for N'. Analysis of N'-containing and tobamovirus-susceptible N. tabacum accessions supported the hypothesis that the candidate is the N' gene itself. Chimera analysis between N' and L(3) revealed that their LRR domains determine the spectrum of their tobamovirus CP recognition. Deletion and mutation analyses of N' and L(3) revealed that the conserved sequences in their C-terminal regions have important roles but contribute differentially to the recognition of common avirulence proteins. The results collectively suggest that Nicotiana N' and Capsicum L genes, which most likely evolved from a common ancestor, differentiated in their recognition specificity through changes in the structural requirements for LRR function.

  7. Overexpression, purification, crystallization and preliminary X-ray diffraction of the nisin resistance protein from Streptococcus agalactiae.

    PubMed

    Khosa, Sakshi; Hoeppner, Astrid; Kleinschrodt, Diana; Smits, Sander H J

    2015-06-01

    Nisin is a 34-amino-acid antimicrobial peptide produced by Lactococcus lactis belonging to the class of lantibiotics. Nisin displays a high bactericidal activity against various Gram-positive bacteria, including some human-pathogenic strains. However, there are some nisin-non-producing strains that are naturally resistant owing to the presence of the nsr gene within their genome. The encoded protein, NSR, cleaves off the last six amino acids of nisin, thereby reducing its bactericidal efficacy. An expression and purification protocol has been established for the NSR protein from Streptococcus agalactiae COH1. The protein was successfully crystallized using the vapour-diffusion method in hanging and sitting drops, resulting in crystals that diffracted X-rays to 2.8 and 2.2 Å, respectively. PMID:26057793

  8. Overexpression, purification, crystallization and preliminary X-ray diffraction of the nisin resistance protein from Streptococcus agalactiae.

    PubMed

    Khosa, Sakshi; Hoeppner, Astrid; Kleinschrodt, Diana; Smits, Sander H J

    2015-06-01

    Nisin is a 34-amino-acid antimicrobial peptide produced by Lactococcus lactis belonging to the class of lantibiotics. Nisin displays a high bactericidal activity against various Gram-positive bacteria, including some human-pathogenic strains. However, there are some nisin-non-producing strains that are naturally resistant owing to the presence of the nsr gene within their genome. The encoded protein, NSR, cleaves off the last six amino acids of nisin, thereby reducing its bactericidal efficacy. An expression and purification protocol has been established for the NSR protein from Streptococcus agalactiae COH1. The protein was successfully crystallized using the vapour-diffusion method in hanging and sitting drops, resulting in crystals that diffracted X-rays to 2.8 and 2.2 Å, respectively.

  9. UCP-3 uncoupling protein confers hypoxia resistance to renal epithelial cells and is upregulated in renal cell carcinoma

    PubMed Central

    Braun, Norbert; Klumpp, Dominik; Hennenlotter, Jörg; Bedke, Jens; Duranton, Christophe; Bleif, Martin; Huber, Stephan M.

    2015-01-01

    Tumor cells can adapt to a hostile environment with reduced oxygen supply. The present study aimed to identify mechanisms that confer hypoxia resistance. Partially hypoxia/reoxygenation (H/R)-resistant proximal tubular (PT) cells were selected by exposing PT cultures to repetitive cycles of H/R. Thereafter, H/R-induced changes in mRNA and protein expression, inner mitochondrial membrane potential (ΔΨm), formation of superoxide, and cell death were compared between H/R-adapted and control PT cultures. As a result, H/R-adapted PT cells exhibited lower H/R-induced hyperpolarization of ΔΨm and produced less superoxide than the control cultures. Consequently, H/R triggered ΔΨm break-down and DNA degradation in a lower percentage of H/R-adapted than control PT cells. Moreover, H/R induced upregulation of mitochondrial uncoupling protein-3 (UCP-3) in H/R-adapted PT but not in control cultures. In addition, ionizing radiation killed a lower percentage of H/R-adapted as compared to control cells suggestive of an H/R-radiation cross-resistance developed by the selection procedure. Knockdown of UCP-3 decreased H/R- and radioresitance of the H/R-adapted cells. Finally, UCP-3 protein abundance of PT-derived clear cell renal cell carcinoma and normal renal tissue was compared in human specimens indicating upregulation of UCP-3 during tumor development. Combined, our data suggest functional significance of UCP-3 for H/R resistance. PMID:26304588

  10. The Expression of Lung Resistance Protein in Saliva: A Novel Prognostic Indicator Protein for Carcinoma of the Breast.

    PubMed

    Wood, Nelson; Streckfus, Charles F

    2015-01-01

    Considering that saliva is a fluid inundated with proteins, it is possible that solubilized oncogenic proteins may be present in saliva and may be useful in differentiating between healthy and diseased individuals. As a consequence, the purpose of this study was to determine if the solubilized form of LRP was present in stimulated whole saliva and could differentiate between 16 healthy women and 16 women with confirmed Stage I breast cancer. LRP levels were determined using gel electrophoresis and Western blot technology. The results showed LRP at significantly higher concentrations among breast cancer subjects as compared to healthy women.

  11. A nuclear fraction of turnip crinkle virus capsid protein is important for elicitation of the host resistance response.

    PubMed

    Kang, Sung-Hwan; Qu, Feng; Morris, T Jack

    2015-12-01

    The N-terminal 25 amino acids (AAs) of turnip crinkle virus (TCV) capsid protein (CP) are recognized by the resistance protein HRT to trigger a hypersensitive response (HR) and systemic resistance to TCV infection. This same region of TCV CP also contains a motif that interacts with the transcription factor TIP, as well as a nuclear localization signal (NLS). However, it is not yet known whether nuclear localization of TCV CP is needed for the induction of HRT-mediated HR and resistance. Here we present new evidence suggesting a tight correlation between nuclear inclusions formed by CP and the manifestation of HR. We show that a fraction of TCV CP localized to cell nuclei to form discrete inclusion-like structures, and a mutated CP (R6A) known to abolish HR failed to form nuclear inclusions. Notably, TIP-CP interaction augments the inclusion-forming activity of CP by tethering inclusions to the nuclear membrane. This TIP-mediated augmentation is also critical for HR resistance, as another CP mutant (R8A) known to elicit a less restrictive HR, though still self-associated into nuclear inclusions, failed to direct inclusions to the nuclear membrane due to its inability to interact with TIP. Finally, exclusion of CP from cell nuclei abolished induction of HR. Together, these results uncovered a strong correlation between nuclear localization and nuclear inclusion formation by TCV CP and induction of HR, and suggest that CP nuclear inclusions could be the key trigger of the HRT-dependent, yet TIP-reinforced, resistance to TCV.

  12. 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

  13. A nuclear fraction of turnip crinkle virus capsid protein is important for elicitation of the host resistance response.

    PubMed

    Kang, Sung-Hwan; Qu, Feng; Morris, T Jack

    2015-12-01

    The N-terminal 25 amino acids (AAs) of turnip crinkle virus (TCV) capsid protein (CP) are recognized by the resistance protein HRT to trigger a hypersensitive response (HR) and systemic resistance to TCV infection. This same region of TCV CP also contains a motif that interacts with the transcription factor TIP, as well as a nuclear localization signal (NLS). However, it is not yet known whether nuclear localization of TCV CP is needed for the induction of HRT-mediated HR and resistance. Here we present new evidence suggesting a tight correlation between nuclear inclusions formed by CP and the manifestation of HR. We show that a fraction of TCV CP localized to cell nuclei to form discrete inclusion-like structures, and a mutated CP (R6A) known to abolish HR failed to form nuclear inclusions. Notably, TIP-CP interaction augments the inclusion-forming activity of CP by tethering inclusions to the nuclear membrane. This TIP-mediated augmentation is also critical for HR resistance, as another CP mutant (R8A) known to elicit a less restrictive HR, though still self-associated into nuclear inclusions, failed to direct inclusions to the nuclear membrane due to its inability to interact with TIP. Finally, exclusion of CP from cell nuclei abolished induction of HR. Together, these results uncovered a strong correlation between nuclear localization and nuclear inclusion formation by TCV CP and induction of HR, and suggest that CP nuclear inclusions could be the key trigger of the HRT-dependent, yet TIP-reinforced, resistance to TCV. PMID:26299399

  14. Identification and characterization of functionally important elements in the multidrug resistance protein 1 COOH-terminal region.

    PubMed

    Westlake, Christopher J; Payen, Lea; Gao, Mian; Cole, Susan P C; Deeley, Roger G

    2004-12-17

    The ATP binding cassette (ABC) transporter, multidrug resistance protein 1 (MRP1/ABCC1), transports a broad spectrum of conjugated and unconjugated compounds, including natural product chemotherapeutic agents. In this study, we have investigated the importance of the COOH-terminal region of MRP1 for transport activity and basolateral plasma membrane trafficking. The COOH-terminal regions of some ABCC proteins have been implicated in protein trafficking, but the function of this region of MRP1 has not been defined. In contrast to results obtained with other ABCC proteins, we found that the COOH-proximal 30 amino acids of MRP1 can be removed without affecting trafficking to basolateral membranes. However, the truncated protein is inactive. Furthermore, removal of as few as 4 COOH-terminal amino acids profoundly decreases transport activity. Although amino acid sequence conservation of the COOH-terminal regions of ABC proteins is low, secondary structure predictions indicate that they consist of a broadly conserved helix-sheet-sheet-helix-helix structure. Consistent with a conservation of secondary and tertiary structure, MRP1 hybrids containing the COOH-terminal regions of either the homologous MRP2 or the distantly related P-glycoprotein were fully active and trafficked normally. Using mutated proteins, we have identified structural elements containing five conserved hydrophobic amino acids that are required for activity. We show that these are important for binding and hydrolysis of ATP by nucleotide binding domain 2. Based on crystal structures of several ABC proteins, we suggest that the conserved amino acids may stabilize a helical bundle formed by the COOH-terminal three helices and may contribute to interactions between the COOH-terminal region and the protein's two nucleotide binding domains.

  15. 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.

  16. Implementing bacterial acid resistance into cell-free protein synthesis for buffer-free expression and screening of enzymes.

    PubMed

    Kim, Ho-Cheol; Kim, Kwang-Soo; Kang, Taek-Jin; Choi, Jong Hyun; Song, Jae Jun; Choi, Yun Hee; Kim, Byung-Gee; Kim, Dong-Myung

    2015-12-01

    Cell-free protein synthesis utilizes translational machinery isolated from the cells for in vitro expression of template genes. Because it produces proteins without gene cloning and cell cultivation steps, cell-free protein synthesis can be used as a versatile platform for high-throughput expression of enzyme libraries. Furthermore, the open nature of cell-free protein synthesis allows direct integration of enzyme synthesis with subsequent screening steps. However, the presence of high concentration of chemical buffers in the conventional reaction mixture makes it difficult to streamline cell-free protein synthesis with pH-based assay of the synthesized enzymes. In this study, we have implemented an enzyme-assisted bacterial acid resistance mechanism into an Escherichia coli (E.coli) extract-based cell-free protein synthesis system in place of chemical buffers. When deployed in the reaction mixture for cell-free synthesis of enzymes, through proton-consuming conversion of glutamate into γ-aminobutyric acid (GABA), an engineered glutamate decarboxylase (GADβ) was able to maintain the pH of reaction mixture during enzyme synthesis. Because the reaction mixture becomes free of buffering capacity upon the depletion of glutamate, synthesized enzyme could be directly assayed without purification steps. The designed method was successfully applied to the screening of mutant library of sialyltransferase genes to identify mutants with improved enzymatic activity.

  17. 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...

  18. A Single Amino Acid Substitution in the Core Protein of West Nile Virus Incre