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Sample records for acriflavine resistance protein

  1. Methanol and acriflavine resistance in Dictyostelium are caused by loss of catalase.

    PubMed

    Garcia, Ma Xenia U; Roberts, Catherine; Alexander, Hannah; Stewart, A Michael; Harwood, Adrian; Alexander, Stephen; Insall, Robert H

    2002-01-01

    Various chemicals with harmful effects are not themselves toxic, but are metabolized in vivo to produce toxic products. One example is methanol in Dictyostelium, which is lethal to cells containing the acrA gene, but relatively harmless to acrA mutants. This makes methanol resistance one of the tightest genetic selections in DICTYOSTELIUM: Loss of acrA also confers cross-resistance to unrelated compounds such as acriflavine and thiabendazole. We have used insertional mutagenesis to demonstrate that the acrA locus encodes the peroxisomal catalase A enzyme. Disruption of the catA gene results in parallel resistance to acriflavine. Molecular and biochemical studies of several previously characterized methanol-resistant strains reveal that each lacks catalase activity. One allele, acrA2, contains a 13 bp deletion which introduces a frameshift in the middle of the gene. The involvement of catalase in methanol resistance in Dictyostelium compares with its role in methanol metabolism in yeast and rodents. However, this is the first study to show that catalase is required for the toxicity of acriflavine. Our results imply that acriflavine and thiabendazole are precursors which must be oxidized to generate biologically active species. The catA/acrA gene is also a potentially invaluable negative selectable marker for Dictyostelium molecular genetics. PMID:11782526

  2. Acriflavine-binding Capacity of Escherichia coli in Relation to Acriflavine Sensitivity and Metabolic Activity

    PubMed Central

    Nakamura, Hakobu

    1966-01-01

    Nakamura, Hakobu (Konan University, Kobe, Japan). Acriflavine-binding capacity of Escherichia coli in relation to acriflavine sensitivity and metabolic activity. J. Bacteriol. 92:1447–1452. 1966.—Inheritance of the acriflavine resistance gene by an acriflavine-sensitive strain of Escherichia coli K-12 resulted in a reduction in the cellular accumulation of acriflavine and other basic dyes, but had no effect on the accumulation of acid dyes. In both acriflavine-resistant and -sensitive strains, the cooling of cells from 37 to 0 C increased acriflavine accumulation. This increased accumulation was released from the cells after restoration to 37 C. Acriflavine accumulation was increased by carbon shortage and by metabolic disturbance caused by potassium cyanide, arsenite, puromycin, chloramphenicol, 2-thiouracil, and 8-azaguanine. The functional relation of acrifiavine accumulation to the acriflavine concentration of the medium suggests that adsorption is involved in the accumulation. PMID:5332403

  3. 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

  4. 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

  5. Acriflavine treatment promotes dyskinetoplasty in Trypanosoma cruzi as revealed by ultrastructural analysis.

    PubMed

    Manchester, Thiago; Cavalcanti, Danielle Pereira; Zogovich, Marcelo; DE Souza, Wanderley; Motta, Maria Cristina Machado

    2013-09-01

    Trypanosomatid mitochondrial DNA is structured as a giant network of thousands of interlocked DNA molecules enclosed within the kinetoplast. The structure and replication mechanism of kinetoplast DNA (kDNA) is unique, thereby making it an excellent chemotherapeutic target. Alteration in the structural organization of kDNA can give rise to dyskinetoplastic (Dk) strains. In Dk cells, the kDNA is dispersed in clumps throughout the mitochondrial matrix and not organized into a network. In this work, Trypanosoma cruzi epimastigotes were treated with acriflavine, a DNA intercalating drug, which promoted a decrease in cell proliferation and induced the appearance of Dk protozoa. In treated cells, the kinetoplast lost its normal disc-shaped structure because the fibrillar arrangement was reduced to a compact, amorphous mass within the mitochondrion. Moreover, basic proteins associated with kDNA were redistributed throughout the Dk protozoal kinetoplast. We sought to understand how the disruption of the kDNA leads to the emergence of the Dk phenotype with atomic force microscopy (AFM) analysis of isolated networks. Our results demonstrate that the detachment of minicircles from the kDNA disk promotes the disassembly of the network, thereby generating Dk cells. Our data strongly suggest that acriflavine inhibits T. cruzi multiplication by interfering with kDNA replication. PMID:23965822

  6. [Proteins in cancer multidrug resistance].

    PubMed

    Popęda, Marta; Płuciennik, Elżbieta; Bednarek, Andrzej K

    2014-01-01

    Multidrug Resistance (MDR) is defined as insensitivity to administered medicines that are structurally unrelated and have different molecular targets. Cancers possess numerous mechanisms of drug resistance, involving various aspects of cell biology. A pivotal role in this phenomenon is played by proteins--enzymatic or structural parts of the cell. Membrane transporters, including the main members of ABC protein family--P-gp, MRP1 and BCRP, as well as LRP, which builds structure of vaults, determine the multidrug-resistant phenotype by decreasing drug concentration within the cell or modifying its distribution to intracellular compartments. The π isoform of protein enzyme--glutathione S-transferase (GSTP-1), is responsible for excessive intensity of detoxification of cytostatics. A common example of altered drug target site that does not respond to chemotherapy is topoisomerase II α (TopoIIa). Alterations of programmed cell death result from expression of metallothionein (MT)--inhibitor of the process, and cytokeratin 18 (CK18), which, if in high concentration, also prevents apoptosis of cells. Several methods of decreasing activity of these proteins have been developed, aiming to overcome MDR in cancer cells. However, for a variety of reasons, their clinical suitability is still very low, leading to continuous increase in death rate among patients. This paper presents current state of knowledge on the most important examples of proteins responsible for MDR of cancer cells and molecular mechanisms of their action. PMID:24864112

  7. Resistive Switching Memory Devices Based on Proteins.

    PubMed

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

    2015-12-01

    Resistive switching memory constitutes a prospective candidate for next-generation data storage devices. Meanwhile, naturally occurring biomaterials are promising building blocks for a new generation of environmentally friendly, biocompatible, and biodegradable electronic devices. Recent progress in using proteins to construct resistive switching memory devices is highlighted. The protein materials selection, device engineering, and mechanism of such protein-based resistive switching memory are discussed in detail. Finally, the critical challenges associated with protein-based resistive switching memory devices are presented, as well as insights into the future development of resistive switching memory based on natural biomaterials. PMID:25753764

  8. 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.

  9. 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

  10. Yeast ABC proteins involved in multidrug resistance.

    PubMed

    Piecuch, Agata; Obłąk, Ewa

    2014-03-01

    Pleiotropic drug resistance is a complex phenomenon that involves many proteins that together create a network. One of the common mechanisms of multidrug resistance in eukaryotic cells is the active efflux of a broad range of xenobiotics through ATP-binding cassette (ABC) transporters. Saccharomyces cerevisiae is often used as a model to study such activity because of the functional and structural similarities of its ABC transporters to mammalian ones. Numerous ABC transporters are found in humans and some are associated with the resistance of tumors to chemotherapeutics. Efflux pump modulators that change the activity of ABC proteins are the most promising candidate drugs to overcome such resistance. These modulators can be chemically synthesized or isolated from natural sources (e.g., plant alkaloids) and might also be used in the treatment of fungal infections. There are several generations of synthetic modulators that differ in specificity, toxicity and effectiveness, and are often used for other clinical effects. PMID:24297686

  11. Antiseptic and antibiotic resistance plasmid in Staphylococcus aureus that possesses ability to confer chlorhexidine and acrinol resistance.

    PubMed Central

    Yamamoto, T; Tamura, Y; Yokota, T

    1988-01-01

    Plasmid pSAJ1 from a methicillin- and gentamicin-resistant strain of Staphylococcus aureus had am molecular size of 50 kilobases and conferred resistance not only to kanamycin, gentamicin, tobramycin, amikacin, benzalkonium chloride, acriflavin, and ethidium bromide but also to chlorhexidine. In addition, the cloned antiseptic resistance gene(s) manifested acrinol resistance in Escherichia coli. Images PMID:3415214

  12. Potential of Tetracycline Resistance Proteins To Evolve Tigecycline Resistance

    PubMed Central

    Linkevicius, Marius; Sandegren, Linus

    2015-01-01

    Tigecycline is a glycylcycline antibiotic active against multidrug-resistant bacterial pathogens. The objectives of our study were to examine the potential of the Tet(A), Tet(K), Tet(M), and Tet(X) tetracycline resistance proteins to acquire mutations causing tigecycline resistance and to determine how this affects resistance to earlier classes of tetracyclines. Mutations in all four tet genes caused a significant increase in the tigecycline MIC in Escherichia coli, and strains expressing mutant Tet(A) and Tet(X) variants reached clinically relevant MICs (2 mg/liter and 3 mg/liter, respectively). Mutations predominantly accumulated in transmembrane domains of the efflux pumps, most likely increasing the accommodation of tigecycline as a substrate. All selected Tet(M) mutants contained at least one mutation in the functionally most important loop III of domain IV. Deletion of leucine 505 of this loop led to the highest increase of the tigecycline MIC (0.5 mg/liter) among Tet(M) mutants. It also caused collateral sensitivity to earlier classes of tetracyclines. A majority of the Tet(X) mutants showed increased activity against all three classes of tetracylines. All tested Tet proteins have the potential to acquire mutations leading to increased MICs of tigecycline. As tet genes are widely found in pathogenic bacteria and spread easily by horizontal gene transfer, resistance development by alteration of existing Tet proteins might compromise the future medical use of tigecycline. We predict that Tet(X) might become the most problematic future Tet determinant, since its weak intrinsic tigecycline activity can be mutationally improved to reach clinically relevant levels without collateral loss in activity to other tetracyclines. PMID:26596936

  13. 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.

  14. 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

  15. Spectral, kinetic and polarization characteristics of luminescence of acriflavine in polymeric matrix under pulsed excitation with different durations and intensities

    NASA Astrophysics Data System (ADS)

    Kaputskaya, I. A.; Ermilov, E. A.; Tannert, S.; Röder, B.; Gorbatsevich, S. K.

    2006-08-01

    Spectral, kinetic and polarization characteristics of fluorescence and thermally activated delayed fluorescence (TADF) of dye solid solutions have been investigated. It was shown that the increasing of the excitation pulse duration results in rise of TADF decay time, but an increasing of the long pulse excitation intensity results in a faster TADF decay. In the presence of Förster resonance energy transfer (FRET) the fluorescence spectrum is shifting non-monotonically with time when the intensity of the excitation pulse is high. At the time moment when the excitation is switched off the polarization degree of luminescence of the concentrated dye solutions strongly reduces. The energy transfer from the molecules in the S 1 state to the molecules in the T 1 state reduces the depolarization of luminescence caused by FRET. Numerical simulations were made by means of Monte-Carlo integrations and results were compared with experimental data obtained for acriflavine in polyvinyl alcohol films.

  16. Mammalian multidrug-resistance proteins (MRPs).

    PubMed

    Slot, Andrew J; Molinski, Steven V; Cole, Susan P C

    2011-09-01

    Subfamily C of the human ABC (ATP-binding cassette) superfamily contains nine proteins that are often referred to as the MRPs (multidrug-resistance proteins). The 'short' MRP/ABCC transporters (MRP4, MRP5, MRP8 and ABCC12) have a typical ABC structure with four domains comprising two membrane-spanning domains (MSD1 and MSD2) each followed by a nucleotide-binding domain (NBD1 and NBD2). The 'long' MRP/ABCCs (MRP1, MRP2, MRP3, ABCC6 and MRP7) have five domains with the extra domain, MSD0, at the N-terminus. The proteins encoded by the ABCC6 and ABCC12 genes are not known to transport drugs and are therefore referred to as ABCC6 and ABCC12 (rather than MRP6 and MRP9) respectively. A large number of molecules are transported across the plasma membrane by the MRPs. Many are organic anions derived from exogenous sources such as conjugated drug metabolites. Others are endogenous metabolites such as the cysteinyl leukotrienes and prostaglandins which have important signalling functions in the cell. Some MRPs share a degree of overlap in substrate specificity (at least in vitro), but differences in transport kinetics are often substantial. In some cases, the in vivo substrates for some MRPs have been discovered aided by studies in gene-knockout mice. However, the molecules that are transported in vivo by others, including MRP5, MRP7, ABCC6 and ABCC12, still remain unknown. Important differences in the tissue distribution of the MRPs and their membrane localization (apical in contrast with basolateral) in polarized cells also exist. Together, these differences are responsible for the unique pharmacological and physiological functions of each of the nine ABCC transporters known as the MRPs. PMID:21967058

  17. Comparing The Efficacy of Hematoxylin and Eosin, Periodic Acid Schiff and Fluorescent Periodic Acid Schiff-Acriflavine Techniques for Demonstration of Basement Membrane in Oral Lichen Planus: A Histochemical Study

    PubMed Central

    Pujar, Ashwini; Pereira, Treville; Tamgadge, Avinash; Bhalerao, Sudhir; Tamgadge, Sandhya

    2015-01-01

    Background: Basement membrane (BM) is a thick sheet of extracellular matrix molecules, upon which epithelial cells attach. Various immunohistochemical studies in the past have been carried out but these advanced staining techniques are expensive and not feasible in routine laboratories. Although hematoxylin and eosin (H-E) is very popular among pathologists for looking at biopsies, the method has some limitations. This is where special stains come handy. Aims and Objectives: The aim of the present study was to demonstrate and compare the efficacy of H-E, periodic acid Schiff (PAS) and fluorescent periodic acid–acriflavine staining techniques for the basement membrane and to establish a histochemical stain which could be cost effective, less time consuming, and unambiguous for observation of the basement membrane zone. Materials and Methods: A total number of 40 paraffin-embedded tissue sections of known basement membrane containing tissues including 10 – Normal oral mucosa (NOM) and 30 – oral lichen planus (OLP) were considered in the study. Four-micron-thick sections of each block were cut and stained with H-E stain, PAS and fluorescent periodic acid–acriflavine stain. Sections were evaluated by three oral pathologists independently for continuity, contrast and pattern. Results: Though all the three stains showed favorable features at different levels, acriflavine stain was better than the other stains in demonstrating BM continuity, contrast and also the pattern followed by PAS stain. Acriflavine stain was the better in demonstrating a fibrillar pattern of a BM. Acriflavine stains a BM distinctly and is less time consuming and easy to carry out using readily available dyes as compared to other stains. Conclusion: The continuity and contrast along with the homogenous pattern and the afibrillar pattern of the BM was better demonstrated by acriflavine followed by the PAS stain. PMID:26538690

  18. Physically Transient Resistive Switching Memory Based on Silk Protein.

    PubMed

    Wang, Hong; Zhu, Bowen; Ma, Xiaohua; Hao, Yue; Chen, Xiaodong

    2016-05-01

    Physically transient resistive switching devices based on silk protein are successfully demonstrated. The devices can be absolutely dissolved in deionized water or in phosphate-buffered saline in 2 h. At the same time, a reasonable resistance OFF/ON ratio of larger than 10(2) and a retention time of more than 10(4) s are achieved for nonvolatile memory applications. PMID:27028213

  19. Resistive random access memory utilizing ferritin protein with Pt nanoparticles

    NASA Astrophysics Data System (ADS)

    Uenuma, Mutsunori; Kawano, Kentaro; Zheng, Bin; Okamoto, Naofumi; Horita, Masahiro; Yoshii, Shigeo; Yamashita, Ichiro; Uraoka, Yukiharu

    2011-05-01

    This study reports controlled single conductive paths found in resistive random access memory (ReRAM) formed by embedding Pt nanoparticles (Pt NPs) in NiO film. Homogeneous Pt NPs produced and placed by ferritin protein produce electric field convergence which leads to controlled conductive path formation. The ReRAM with Pt NPs shows stable switching behavior. A Pt NP density decrease results in an increase of OFF state resistance and decrease of forming voltage, whereas ON resistance was independent of the Pt NP density, which indicates that a single metal NP in a memory cell will achieve low power and stable operation.

  20. 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

  1. Targeting protein kinases to reverse multidrug resistance in sarcoma.

    PubMed

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

    2016-02-01

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

  2. 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

  3. Resistance of platelet proteins to effects of ionizing radiation

    SciTech Connect

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

    1990-12-01

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

  4. Impaired protein metabolism: interlinks between obesity, insulin resistance and inflammation.

    PubMed

    Guillet, C; Masgrau, A; Walrand, S; Boirie, Y

    2012-12-01

    Metabolic and structural changes in skeletal muscle that accompany obesity are often associated with the development of insulin resistance. The first events in the pathogenesis of this disorder are considered as an accumulation of lipids within skeletal muscle due to blunted muscle capacity to oxidize fatty acids. Fat infiltration is also associated with muscle fibre typology modification, decrease in muscle mass and impairments in muscle strength. Thus, as a result of obesity, mobility and quality of life are affected, and this is in part due to quantitative and qualitative impairments in skeletal muscle. In addition, the insulin resistance related to obesity results not only in defective insulin-stimulated glucose disposal but has also detrimental consequences on protein metabolism at the skeletal muscle level and whole-body level. This review highlights the involvement of fat accumulation and insulin resistance in metabolic disorders occurring in skeletal muscle during the development of obesity, and the impairments in the regulation of protein metabolism and protein turnover in the links between obesity, metabolic inflammation and insulin resistance. PMID:23107259

  5. Marine natural products as breast cancer resistance protein inhibitors.

    PubMed

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

    2015-04-01

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

  6. Marine Natural Products as Breast Cancer Resistance Protein Inhibitors

    PubMed Central

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

    2015-01-01

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

  7. Structural Basis of Protein Oxidation Resistance: A Lysozyme Study

    PubMed Central

    Girod, Marion; Enjalbert, Quentin; Brunet, Claire; Antoine, Rodolphe; Lemoine, Jérôme; Lukac, Iva; Radman, Miroslav; Krisko, Anita; Dugourd, Philippe

    2014-01-01

    Accumulation of oxidative damage in proteins correlates with aging since it can cause irreversible and progressive degeneration of almost all cellular functions. Apparently, native protein structures have evolved intrinsic resistance to oxidation since perfectly folded proteins are, by large most robust. Here we explore the structural basis of protein resistance to radiation-induced oxidation using chicken egg white lysozyme in the native and misfolded form. We study the differential resistance to oxidative damage of six different parts of native and misfolded lysozyme by a targeted tandem/mass spectrometry approach of its tryptic fragments. The decay of the amount of each lysozyme fragment with increasing radiation dose is found to be a two steps process, characterized by a double exponential evolution of their amounts: the first one can be largely attributed to oxidation of specific amino acids, while the second one corresponds to further degradation of the protein. By correlating these results to the structural parameters computed from molecular dynamics (MD) simulations, we find the protein parts with increased root-mean-square deviation (RMSD) to be more susceptible to modifications. In addition, involvement of amino acid side-chains in hydrogen bonds has a protective effect against oxidation Increased exposure to solvent of individual amino acid side chains correlates with high susceptibility to oxidative and other modifications like side chain fragmentation. Generally, while none of the structural parameters alone can account for the fate of peptides during radiation, together they provide an insight into the relationship between protein structure and susceptibility to oxidation. PMID:24999730

  8. A yeast metal resistance protein similar to human cystic fibrosis transmembrane conductance regulator (CFTR) and multidrug resistance-associated protein.

    PubMed

    Szczypka, M S; Wemmie, J A; Moye-Rowley, W S; Thiele, D J

    1994-09-01

    Members of the ATP binding cassette (ABC) protein superfamily transport a variety of substances across biological membranes, including drugs, ions, and peptides. The yeast cadmium factor (YCF1) gene from Saccharomyces cerevisiae is required for cadmium resistance and encodes a 1,515 amino acid protein with extensive homology to both the human multidrug resistance-associated protein (MRP1) and the cystic fibrosis transmembrane conductance regulator (hCFTR). S. cerevisiae cells harboring a deletion of the YCF1 gene are hypersensitive to cadmium compared with wild type cells. Mutagenesis experiments demonstrate that conserved amino acid residues, functionally critical in hCFTR, play a vital role in YCF1-mediated cadmium resistance. Mutagenesis of phenylalanine 713 in the YCF1 nucleotide binding fold 1, which correlates with the delta F508 mutation found in the most common form of cystic fibrosis, completely abolished YCF1 function in cadmium detoxification. Furthermore, substitution of a serine to alanine residue in a potential protein kinase A phosphorylation site in a central region of YCF1, which displays sequence similarity to the central regulatory domain of hCFTR, also rendered YCF1 nonfunctional. These results suggest that YCF1 is composed of modular domains found in human proteins which function in drug and ion transport. PMID:7521334

  9. Nuclear export of proteins and drug resistance in cancer

    PubMed Central

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

    2015-01-01

    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, p21CIP, p27KIP1, 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. PMID:22209898

  10. Mutagenesis Mapping of the Protein-Protein Interaction Underlying FusB-Type Fusidic Acid Resistance

    PubMed Central

    Cox, Georgina; Edwards, Thomas A.

    2013-01-01

    FusB-type proteins represent the predominant mechanism of resistance to fusidic acid in staphylococci and act by binding to and modulating the function of the drug target (elongation factor G [EF-G]). To gain further insight into this antibiotic resistance mechanism, we sought to identify residues important for the interaction of FusB with EF-G and thereby delineate the binding interface within the FusB–EF-G complex. Replacement with alanine of any one of four conserved residues within the C-terminal domain of FusB (F156, K184, Y187, and F208) abrogated the ability of the protein to confer resistance to fusidic acid; the purified mutant proteins also lost the ability to bind S. aureus EF-G in vitro. E. coli EF-G, which is not ordinarily able to bind FusB-type proteins, was rendered competent for binding to FusB following deletion of a 3-residue tract (529SNP531) from domain IV of the protein. This study has identified key regions of both FusB and EF-G that are important for the interaction between the proteins, findings which corroborate our previous in silico prediction for the architecture of the complex formed between the resistance protein and the drug target (G. Cox, G. S. Thompson, H. T. Jenkins, F. Peske, A. Savelsbergh, M. V. Rodnina, W. Wintermeyer, S. W. Homans, T. A. Edwards, and A. J. O'Neill, Proc. Natl. Acad. Sci. U. S. A. 109:2102-2107, 2012). PMID:23836182

  11. Mutagenesis mapping of the protein-protein interaction underlying FusB-type fusidic acid resistance.

    PubMed

    Cox, Georgina; Edwards, Thomas A; O'Neill, Alex J

    2013-10-01

    FusB-type proteins represent the predominant mechanism of resistance to fusidic acid in staphylococci and act by binding to and modulating the function of the drug target (elongation factor G [EF-G]). To gain further insight into this antibiotic resistance mechanism, we sought to identify residues important for the interaction of FusB with EF-G and thereby delineate the binding interface within the FusB-EF-G complex. Replacement with alanine of any one of four conserved residues within the C-terminal domain of FusB (F156, K184, Y187, and F208) abrogated the ability of the protein to confer resistance to fusidic acid; the purified mutant proteins also lost the ability to bind S. aureus EF-G in vitro. E. coli EF-G, which is not ordinarily able to bind FusB-type proteins, was rendered competent for binding to FusB following deletion of a 3-residue tract (529SNP531) from domain IV of the protein. This study has identified key regions of both FusB and EF-G that are important for the interaction between the proteins, findings which corroborate our previous in silico prediction for the architecture of the complex formed between the resistance protein and the drug target (G. Cox, G. S. Thompson, H. T. Jenkins, F. Peske, A. Savelsbergh, M. V. Rodnina, W. Wintermeyer, S. W. Homans, T. A. Edwards, and A. J. O'Neill, Proc. Natl. Acad. Sci. U. S. A. 109:2102-2107, 2012). PMID:23836182

  12. Protein Carbonylation, Mitochondrial Dysfunction, and Insulin Resistance123

    PubMed Central

    Frohnert, Brigitte I.; Bernlohr, David A.

    2013-01-01

    Oxidative stress has been identified as a common mechanism for cellular damage and dysfunction in a wide variety of disease states. Current understanding of the metabolic changes associated with obesity and the development of insulin resistance has focused on the role of oxidative stress and its interaction with inflammatory processes at both the tissue and organismal level. Obesity-related oxidative stress is an important contributing factor in the development of insulin resistance in the adipocyte as well as the myocyte. Moreover, oxidative stress has been linked to mitochondrial dysfunction, and this is thought to play a role in the metabolic defects associated with oxidative stress. Of the various effects of oxidative stress, protein carbonylation has been identified as a potential mechanism underlying mitochondrial dysfunction. As such, this review focuses on the relationship between protein carbonylation and mitochondrial biology and addresses those features that point to either the causal or casual relationship of lipid peroxidation–induced protein carbonylation as a determining factor in mitochondrial dysfunction. PMID:23493532

  13. 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.

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

    PubMed Central

    Pollegioni, Loredano; Schonbrunn, Ernst; Siehl, Daniel

    2011-01-01

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

  15. 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

  16. Protein Kinase Cα Mediates Erlotinib Resistance in Lung Cancer Cells

    PubMed Central

    Abera, Mahlet B.

    2015-01-01

    Overexpression and mutational activation of the epidermal growth factor receptor (EGFR) plays an important role in the pathogenesis of non–small cell lung cancer (NSCLC). EGFR tyrosine-kinase inhibitors (TKIs) are given as a primary therapy for advanced patients with EGFR-activating mutations; however, the majority of these tumors relapse and patients eventually develop resistance to TKIs. To address a potential role of protein kinase C (PKC) isozymes in the resistance to TKIs, we used the isogenic NSCLC H1650 cell line and its erlotinib-resistant derivative H1650-M3, a cell line that displays a mesenchymal-like morphology driven by transforming growth factor-β signaling. We found that H1650-M3 cells display remarkable PKCα upregulation and PKCδ downregulation. Notably, silencing PKCα from H1650-M3 cells using RNA interference caused a significant reduction in the expression of epithelial-to-mesenchymal transition (EMT) markers vimentin, Zeb2, Snail, and Twist. Moreover, pharmacological inhibition or PKCα RNA interference depletion and PKCδ restoring sensitized H1650-M3 cells to erlotinib. Whereas ectopic overexpression of PKCα in parental H1650 cells was not sufficient to alter the expression of EMT genes or to confer resistance to erlotinib, it caused downregulation of PKCδ expression, suggesting a unidirectional crosstalk. Finally, mechanistic studies revealed that PKCα upregulation in H1650-M3 cells is driven by transforming growth factor-β. Our results identified important roles for specific PKC isozymes in erlotinib resistance and EMT in lung cancer cells, and highlight PKCα as a potential target for lung cancer treatment. PMID:25724832

  17. Protein kinase Cα mediates erlotinib resistance in lung cancer cells.

    PubMed

    Abera, Mahlet B; Kazanietz, Marcelo G

    2015-05-01

    Overexpression and mutational activation of the epidermal growth factor receptor (EGFR) plays an important role in the pathogenesis of non-small cell lung cancer (NSCLC). EGFR tyrosine-kinase inhibitors (TKIs) are given as a primary therapy for advanced patients with EGFR-activating mutations; however, the majority of these tumors relapse and patients eventually develop resistance to TKIs. To address a potential role of protein kinase C (PKC) isozymes in the resistance to TKIs, we used the isogenic NSCLC H1650 cell line and its erlotinib-resistant derivative H1650-M3, a cell line that displays a mesenchymal-like morphology driven by transforming growth factor-β signaling. We found that H1650-M3 cells display remarkable PKCα upregulation and PKCδ downregulation. Notably, silencing PKCα from H1650-M3 cells using RNA interference caused a significant reduction in the expression of epithelial-to-mesenchymal transition (EMT) markers vimentin, Zeb2, Snail, and Twist. Moreover, pharmacological inhibition or PKCα RNA interference depletion and PKCδ restoring sensitized H1650-M3 cells to erlotinib. Whereas ectopic overexpression of PKCα in parental H1650 cells was not sufficient to alter the expression of EMT genes or to confer resistance to erlotinib, it caused downregulation of PKCδ expression, suggesting a unidirectional crosstalk. Finally, mechanistic studies revealed that PKCα upregulation in H1650-M3 cells is driven by transforming growth factor-β. Our results identified important roles for specific PKC isozymes in erlotinib resistance and EMT in lung cancer cells, and highlight PKCα as a potential target for lung cancer treatment. PMID:25724832

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

    PubMed Central

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

    2012-01-01

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

  19. 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

  20. Timing and distribution of protein ingestion during prolonged recovery from resistance exercise alters myofibrillar protein synthesis

    PubMed Central

    Areta, José L; Burke, Louise M; Ross, Megan L; Camera, Donny M; West, Daniel W D; Broad, Elizabeth M; Jeacocke, Nikki A; Moore, Daniel R; Stellingwerff, Trent; Phillips, Stuart M; Hawley, John A; Coffey, Vernon G

    2013-01-01

    Quantity and timing of protein ingestion are major factors regulating myofibrillar protein synthesis (MPS). However, the effect of specific ingestion patterns on MPS throughout a 12 h period is unknown. We determined how different distributions of protein feeding during 12 h recovery after resistance exercise affects anabolic responses in skeletal muscle. Twenty-four healthy trained males were assigned to three groups (n= 8/group) and undertook a bout of resistance exercise followed by ingestion of 80 g of whey protein throughout 12 h recovery in one of the following protocols: 8 × 10 g every 1.5 h (PULSE); 4 × 20 g every 3 h (intermediate: INT); or 2 × 40 g every 6 h (BOLUS). Muscle biopsies were obtained at rest and after 1, 4, 6, 7 and 12 h post exercise. Resting and post-exercise MPS (l-[ring-13C6] phenylalanine), and muscle mRNA abundance and cell signalling were assessed. All ingestion protocols increased MPS above rest throughout 1–12 h recovery (88–148%, P < 0.02), but INT elicited greater MPS than PULSE and BOLUS (31–48%, P < 0.02). In general signalling showed a BOLUS>INT>PULSE hierarchy in magnitude of phosphorylation. MuRF-1 and SLC38A2 mRNA were differentially expressed with BOLUS. In conclusion, 20 g of whey protein consumed every 3 h was superior to either PULSE or BOLUS feeding patterns for stimulating MPS throughout the day. This study provides novel information on the effect of modulating the distribution of protein intake on anabolic responses in skeletal muscle and has the potential to maximize outcomes of resistance training for attaining peak muscle mass. PMID:23459753

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

    PubMed

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

    2016-01-01

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

  2. Listeria Phage and Phage Tail Induction Triggered by Components of Bacterial Growth Media (Phosphate, LiCl, Nalidixic Acid, and Acriflavine)

    PubMed Central

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

    2015-01-01

    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. PMID:25595760

  3. 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

  4. Mitomycin resistance in mammalian cells expressing the bacterial mitomycin C resistance protein MCRA.

    PubMed

    Belcourt, M F; Penketh, P G; Hodnick, W F; Johnson, D A; Sherman, D H; Rockwell, S; Sartorelli, A C

    1999-08-31

    The mitomycin C-resistance gene, mcrA, of Streptomyces lavendulae produces MCRA, a protein that protects this microorganism from its own antibiotic, the antitumor drug mitomycin C. Expression of the bacterial mcrA gene in mammalian Chinese hamster ovary cells causes profound resistance to mitomycin C and to its structurally related analog porfiromycin under aerobic conditions but produces little change in drug sensitivity under hypoxia. The mitomycins are prodrugs that are enzymatically reduced and activated intracellularly, producing cytotoxic semiquinone anion radical and hydroquinone reduction intermediates. In vitro, MCRA protects DNA from cross-linking by the hydroquinone reduction intermediate of these mitomycins by oxidizing the hydroquinone back to the parent molecule; thus, MCRA acts as a hydroquinone oxidase. These findings suggest potential therapeutic applications for MCRA in the treatment of cancer with the mitomycins and imply that intrinsic or selected mitomycin C resistance in mammalian cells may not be due solely to decreased bioactivation, as has been hypothesized previously, but instead could involve an MCRA-like mechanism. PMID:10468636

  5. Mitomycin resistance in mammalian cells expressing the bacterial mitomycin C resistance protein MCRA

    PubMed Central

    Belcourt, Michael F.; Penketh, Philip G.; Hodnick, William F.; Johnson, David A.; Sherman, David H.; Rockwell, Sara; Sartorelli, Alan C.

    1999-01-01

    The mitomycin C-resistance gene, mcrA, of Streptomyces lavendulae produces MCRA, a protein that protects this microorganism from its own antibiotic, the antitumor drug mitomycin C. Expression of the bacterial mcrA gene in mammalian Chinese hamster ovary cells causes profound resistance to mitomycin C and to its structurally related analog porfiromycin under aerobic conditions but produces little change in drug sensitivity under hypoxia. The mitomycins are prodrugs that are enzymatically reduced and activated intracellularly, producing cytotoxic semiquinone anion radical and hydroquinone reduction intermediates. In vitro, MCRA protects DNA from cross-linking by the hydroquinone reduction intermediate of these mitomycins by oxidizing the hydroquinone back to the parent molecule; thus, MCRA acts as a hydroquinone oxidase. These findings suggest potential therapeutic applications for MCRA in the treatment of cancer with the mitomycins and imply that intrinsic or selected mitomycin C resistance in mammalian cells may not be due solely to decreased bioactivation, as has been hypothesized previously, but instead could involve an MCRA-like mechanism. PMID:10468636

  6. Role of C Reactive Protein (CRP) in Leptin Resistance

    PubMed Central

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

    2014-01-01

    Increased plasma levels of both leptin and C reactive protein (CRP) have been reported in a number of conditions, including obesity, and have been linked to cardiovascular pathophysiological processes and increased cardiovascular risk; interestingly these two biomarkers appear to be able to reciprocally regulate their bioavailability, through complex mechanisms that have not been completely clarified yet. Here we first review clinical evidence suggesting not only that the circulatory levels of CRP and leptin show an independent correlation, but also that assessing them in tandem may result in an increased ability to predict cardiovascular disease. We summarize also molecular studies showing that leptin is able to promote CRP production from hepatocytes and endothelial cells in vitro and discuss the studies addressing the possibility that in vivo leptin administration may be able to modulate plasma CRP levels. Furthermore, we describe two studies demonstrating that CRP directly binds leptin in extra-cellular settings, thus impairing its biological actions. Finally we report genetic evidence that common variations at the leptin receptor locus are associated with CRP blood levels. Overall, the data reviewed here show that the chronic elevation of CRP observed in obese subjects may worsen leptin resistance, contributing to the pathogenesis of cardiovascular disease, and highlight a potential link between conditions, such as leptin resistance and endothelial dysfunction, that may be amenable of pharmacological treatment targeted to the disruption of leptin-CRP interaction. PMID:23688010

  7. Protein-Resistant Biodegradable Amphiphilic Graft Copolymer Vesicles as Protein Carriers.

    PubMed

    Wang, Yupeng; Yan, Lesan; Li, Bin; Qi, Yanxin; Xie, Zhigang; Jing, Xiabin; Chen, Xuesi; Huang, Yubin

    2015-09-01

    The protein adsorption and self-assembly behavior of biocompatible graft copolymer, poly(lactide-co-diazidomethyl trimethylene carbonate)-g-poly(ethylene glycol) [P(LA-co-DAC)-g-PEG], were systematically studied. The graft copolymers showed enhanced resistance to non-specific protein adsorption compared with their block copolymer counterparts, indicative of the increased effect of PEG density beyond PEG length. Diverse nanostructures including vesicles can be assembled from the amphiphilic graft copolymers with well-defined nano-sizes. Hemoglobin (Hb), as a model protein, can be entrapped in the formed vesicles and keep the gas-binding capacity. The reduced release rate of Hb from graft copolymer vesicles indicated the relatively stable membrane packing compared with block copolymer counterpart. PMID:26036907

  8. 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

  9. [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

  10. Tumor promotion by caspase-resistant retinoblastoma protein

    PubMed Central

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

    2005-01-01

    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

  11. Cutaneous necrosis in pregnancy secondary to activated protein C resistance in hereditary angioedema.

    PubMed

    Perkins, W; Downie, I; Keefe, M; Chisholm, M

    1995-04-01

    A 26-year-old woman with hereditary angineurotic oedema (HAE) presented at 22 weeks gestation with severe cutaneous necrosis similar to that seen in coumarin skin necrosis. Protein S deficiency secondary to HAE and pregnancy was postulated. Treatment with heparin, C1-inhibitor concentrates, systemic steroids and surgical debridement resulted in a successful outcome for both mother and child. Subsequent investigations revealed normal levels of protein C, antithrombin III, total protein S, free protein S but reduced function protein S activity with evidence of activated protein C resistance. Cutaneous necrosis has not been reported in associated with activated protein C resistance previously and the possible mechanisms are discussed. PMID:7745572

  12. 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

  13. Interferon Consensus Sequence Binding Protein Confers Resistance against Yersinia enterocolitica

    PubMed Central

    Hein, Joachim; Kempf, Volkhard A. J.; Diebold, Joachim; Bücheler, Nicole; Preger, Sonja; Horak, Ivan; Sing, Andreas; Kramer, Uwe; Autenrieth, Ingo B.

    2000-01-01

    Interferon consensus sequence binding protein (ICSBP)-deficient mice display enhanced susceptibility to intracellular pathogens. At least two distinct immunoregulatory defects are responsible for this phenotype. First, diminished production of reactive oxygen intermediates in macrophages results in impaired intracellular killing of microorganisms. Second, defective early interleukin-12 (IL-12) production upon microbial challenge leads to a failure in gamma interferon (IFN-γ) induction and subsequently in T helper 1 immune responses. Here, we investigated the role of ICSBP in resistance against the extracellular bacterium Yersinia enterocolitica. ICSBP−/− mice failed to produce IL-12 and IFN-γ, but also IL-4, after Yersinia challenge. In addition, granuloma formation was highly disturbed in infected ICSBP−/− mice, leading to multiple necrotic abscesses in affected organs. Consequently, ICSBP−/− mice rapidly succumbed to acute Yersinia infection. In vitro treatment of spleen cells from ICSBP−/− mice with recombinant IL-12 (rIL-12) or rIL-18 in combination with a second stimulus resulted in IFN-γ induction. In experimental therapy of infected ICSBP−/− mice, we observed that administration of rIL-12 induced IFN-γ production which was associated with improved resistance to Yersinia. In contrast, treatment with rIL-18 failed to enhance endogenous IFN-γ production but nevertheless reduced bacterial burden in ICSBP−/− mice. Although cytokine therapy with rIL-12 or rIL-18 ameliorated the course of Yersinia infection in ICSBP−/− mice, both cytokines failed to completely restore impaired immunity. Taken together, the results indicate that the transcription factor ICSBP is essential for efficient host immune defense against Yersinia. These results are important for understanding the complex host immune responses in bacterial infections. PMID:10678954

  14. IDENTIFICATION OF MAIZE KERNEL ENDOSPERM PROTEINS ASSOCIATED WITH RESISTANCE TO AFLATOXIN CONTAMINATION BY ASPERGILLUS FLAVUS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Aflatoxins are carcinogens produced mainly by Aspergillus flavus during infection of susceptible crops, such as maize (Zea mays L.). Previously, embryo proteins from maize genotypes resistant or susceptible to A. flavus infection were compared using proteomics and resistance-associated proteins wer...

  15. 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

  16. 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

  17. 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

  18. The major vault protein is related to the toxic anion resistance protein (TelA) family.

    PubMed

    Suprenant, Kathy A; Bloom, Nathan; Fang, Jianwen; Lushington, Gerald

    2007-03-01

    Vaults are barrel-shaped ribonucleoprotein particles that are abundant in certain tumors and multidrug resistant cancer cells. Prokaryotic relatives of the major vault protein, MVP, have not been identified. We used sequence analysis and molecular modeling to show that MVP and the toxic anion resistance protein, TelA of Rhodobacter sphaeroides strain 2.4.1, share a novel fold that consists of a three-stranded antiparallel beta-sheet. Because of this strong structural correspondence, we examined whether mammalian cell vaults respond to tellurite treatment. In the presence of the oxyanion tellurite, large vault aggregates, or vaultosomes, appear at the cell periphery in 15 min or less. Vaultosome formation is temperature-dependent, reversible, and occurs in normal human umbilical vein endothelial cells as well as transformed HeLa cervical cancer cells. Vaultosome formation is not restricted to tellurite and occurs in the presence of other toxic oxyanions (selenate, selinite, arsenate, arsenite, vanadate). In addition, vaultosomes form independently from other stress-induced ribonucleoprotein complexes, stress granules and aggresomes. Vaultosome formation is therefore a unique cellular response to an environmental toxin. PMID:17337707

  19. 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

    2012-01-01

    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. PMID:22869077

  20. Protein resistance of surfaces modified with oligo(ethylene glycol) aryl diazonium derivatives.

    PubMed

    Fairman, Callie; Ginges, Joshua Z; Lowe, Stuart B; Gooding, J Justin

    2013-07-22

    Anti-fouling surfaces are of great importance for reducing background interference in biosensor signals. Oligo(ethylene glycol) (OEG) moieties are commonly used to confer protein resistance on gold, silicon and carbon surfaces. Herein, we report the modification of surfaces using electrochemical deposition of OEG aryl diazonium salts. Using electrochemical and contact angle measurements, the ligand packing density is found to be loose, which supports the findings of the fluorescent protein labelling that aryl diazonium OEGs confer resistance to nonspecific adsorption of proteins albeit lower than alkane thiol-terminated OEGs. In addition to protein resistance, aryl diazonium attachment chemistry results in stable modification. In common with OEG species on gold electrodes, OEGs with distal hydroxyl moieties do confer superior protein resistance to those with a distal methoxy group. This is especially the case for longer derivatives where superior coiling of the OEG chains is possible. PMID:23650106

  1. Differentially Expressed Proteins Associated with Fusarium Head Blight Resistance in Wheat

    PubMed Central

    Zhang, Xianghui; Fu, Jianming; Hiromasa, Yasuaki; Pan, Hongyu; Bai, Guihua

    2013-01-01

    Background Fusarium head blight (FHB), mainly caused by Fusarium graminearum, substantially reduces wheat grain yield and quality worldwide. Proteins play important roles in defense against the fungal infection. This study characterized differentially expressed proteins between near-isogenic lines (NILs) contrasting in alleles of Fhb1, a major FHB resistance gene in wheat, to identify proteins underlining FHB resistance of Fhb1. Methods The two-dimensional protein profiles were compared between the Fusarium-inoculated spikes of the two NILs collected 72 h after inoculation. The protein profiles of mock- and Fusarium-inoculated Fhb1+NIL were also compared to identify pathogen-responsive proteins. Results Eight proteins were either induced or upregulated in inoculated Fhb1+NIL when compared with mock-inoculated Fhb1+NIL; nine proteins were either induced or upregulated in the Fusarium-inoculated Fhb1+NIL when compared with Fusarium-inoculated Fhb1−NIL. Proteins that were differentially expressed in the Fhb1+NIL, not in the Fhb1−NIL, after Fusarium inoculation included wheat proteins for defending fungal penetration, photosynthesis, energy metabolism, and detoxification. Conclusions Coordinated expression of the identified proteins resulted in FHB resistance in Fhb1+NIL. The results provide insight into the pathway of Fhb1-mediated FHB resistance. PMID:24376514

  2. 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

  3. Innate resistance to avian influenza: Of MHC's and Mx proteins

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Avian influenza (AI) is an economically important virus of poultry that has significant impact on global trade. Recently, increased attention to animal genomics has been applied to enhance innate resistance to infectious diseases in poultry. Two known contributors to innate resistance are the host m...

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

    PubMed

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

    2014-08-01

    A proteomic approach combining two-dimensional polyacrylamide gel electrophoresis and tandem mass spectrometry was used to compare hemolymph expression profiles of a beta-cypermethrin-resistant Blattella germanica L. strain and a beta-cypermethrin-susceptible strain. Twenty-eight hemolymph proteins were differentially expressed in the resistant cockroach strain; 19 proteins were upregulated and 9 proteins were downregulated compared with the susceptible strain. Protein identification indicated that expression of putative cuticular protein, nitric oxide synthase, triosephosphate isomerase, alpha-amylase, ABC transporter, and Per a 3 allergen was elevated, and expression of arginine kinase and glycosidase was reduced. The differential expression of these proteins reflects the overall change in cellular structure and metabolism related to the resistance of pyrethroid insecticides. PMID:25182623

  5. 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. PMID:16948480

  6. 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

  7. 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

  8. 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.

  9. Polyglycerol coatings of glass vials for protein resistance.

    PubMed

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

    2013-11-01

    Proteins are surface active molecules which undergo non-specific adsorption when getting in contact with surfaces such as the primary packaging material. This process is critical as it may cause a loss of protein content or protein aggregation. To prevent unspecific adsorption, protein repellent coatings are of high interest. We describe the coating of industrial relevant borosilicate glass vials with linear methoxylated polyglycerol, hyperbranched polyglycerol, and hyperbranched methoxylated polyglycerol. All coatings provide excellent protein repellent effects. The hyperbranched, non-methoxylated coating performed best. The protein repellent properties were maintained also after applying industrial relevant sterilization methods (≥200 °C). Marginal differences in antibody stability between formulations stored in bare glass vials and coated vials were detected after 3 months storage; the protein repellent effect remained largely stable. Here, we describe a new material suitable for the coating of primary packaging material of proteins which significantly reduces the protein adsorption and thus could present an interesting new possibility for biomedical applications. PMID:23624376

  10. 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

  11. Skeletal muscle protein metabolism in the elderly: Interventions to counteract the 'anabolic resistance' of ageing

    PubMed Central

    2011-01-01

    Age-related muscle wasting (sarcopenia) is accompanied by a loss of strength which can compromise the functional abilities of the elderly. Muscle proteins are in a dynamic equilibrium between their respective rates of synthesis and breakdown. It has been suggested that age-related sarcopenia is due to: i) elevated basal-fasted rates of muscle protein breakdown, ii) a reduction in basal muscle protein synthesis (MPS), or iii) a combination of the two factors. However, basal rates of muscle protein synthesis and breakdown are unchanged with advancing healthy age. Instead, it appears that the muscles of the elderly are resistant to normally robust anabolic stimuli such as amino acids and resistance exercise. Ageing muscle is less sensitive to lower doses of amino acids than the young and may require higher quantities of protein to acutely stimulate equivalent muscle protein synthesis above rest and accrue muscle proteins. With regard to dietary protein recommendations, emerging evidence suggests that the elderly may need to distribute protein intake evenly throughout the day, so as to promote an optimal per meal stimulation of MPS. The branched-chain amino acid leucine is thought to play a central role in mediating mRNA translation for MPS, and the elderly should ensure sufficient leucine is provided with dietary protein intake. With regards to physical activity, lower, than previously realized, intensity high-volume resistance exercise can stimulate a robust muscle protein synthetic response similar to traditional high-intensity low volume training, which may be beneficial for older adults. Resistance exercise combined with amino acid ingestion elicits the greatest anabolic response and may assist elderly in producing a 'youthful' muscle protein synthetic response provided sufficient protein is ingested following exercise. PMID:21975196

  12. 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

  13. 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.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  15. Fixation-resistant photoactivatable fluorescent proteins for CLEM.

    PubMed

    Paez-Segala, Maria G; Sun, Mei G; Shtengel, Gleb; Viswanathan, Sarada; Baird, Michelle A; Macklin, John J; Patel, Ronak; Allen, John R; Howe, Elizabeth S; Piszczek, Grzegorz; Hess, Harald F; Davidson, Michael W; Wang, Yalin; Looger, Loren L

    2015-03-01

    Fluorescent proteins facilitate a variety of imaging paradigms in live and fixed samples. However, they lose their fluorescence after heavy fixation, hindering applications such as correlative light and electron microscopy (CLEM). Here we report engineered variants of the photoconvertible Eos fluorescent protein that fluoresce and photoconvert normally in heavily fixed (0.5-1% OsO4), plastic resin-embedded samples, enabling correlative super-resolution fluorescence imaging and high-quality electron microscopy. PMID:25581799

  16. 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

  17. Evaluation of the clinical relevance of the expression and function of P-glycoprotein, multidrug resistance protein and lung resistance protein in patients with primary acute myelogenous leukemia.

    PubMed

    Tsimberidou, Apostolia Maria; Paterakis, George; Androutsos, George; Anagnostopoulos, Nikolaos; Galanopoulos, Athanasios; Kalmantis, Themistoklis; Meletis, John; Rombos, Yiannis; Sagriotis, Alexandros; Symeonidis, Argyrios; Tiniakou, Maria; Zoumbos, Nikolaos; Yataganas, Xenophon

    2002-02-01

    The multidrug resistance (MDR) transporter-proteins P-glycoprotein (Pgp), multidrug resistance protein (MRP) and lung resistance protein (LRP) have been associated with treatment failure. The aim of this study was to investigate prospectively the clinical significance of expression and function of the MDR proteins, considering other prognostic factors, such as age, immunophenotype, and cytogenetics. Mononuclear cells of peripheral blood or bone marrow from 61 patients with de novo acute myelogenous leukemia (AML) were analyzed. The monoclonal antibodies JSB1, MRPm6 and LRP56 were used for expression studies. Accumulation and retention studies were performed using the substrates Daunorubicin, Calcein-AM, Rhodamine-123 and DiOC(2) in the presence or absence of the modifiers Verapamil, Genistein, Probenecid, BIBW22S and PSC833. Induction treatment consisted of a 3+7 combination of Ida/Ara-C for patients < or = 60 years of age and a 3+5 Ida/VP-16 combination per OS for patients >60. MDR function was expressed as the ratio of mean fluorescence intensity substrate in the presence of modifier over the substrate alone (resistance index, RI). Patients with advanced age, low CD15 expression and high RI for accumulation of DiOC(2) in the presence of BIBW22S had significantly lower complete remission (CR) rates. No factor was prognostic for event-free survival analysis, which was limited to remitters only. Overall survival was shorter in patients with advanced age, poor prognosis cytogenetics, high CD7 expression, and high RI for Daunorubicin efflux modulated by Verapamil. These results suggest that MDR transporter-proteins have a limited role in the treatment failure of patients treated with Idarubicin-based regimens. PMID:11755464

  18. 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

  19. Analysis Of Proteins Differentially Accumulated During Potato Late Blight Resistance Mediated by the RB Resistance Gene

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The RB gene cloned from the wild diploid potato species Solanum bulbocastanum confers resistance against the late blight pathogen, Phytophthora infestans. Two-dimensional gel electrophoresis followed by mass spectrometry was used to examine for the first time the changes of the proteome pattern of ...

  20. Resistive Switching: Physically Transient Resistive Switching Memory Based on Silk Protein (Small 20/2016).

    PubMed

    Wang, Hong; Zhu, Bowen; Ma, Xiaohua; Hao, Yue; Chen, Xiaodong

    2016-05-01

    On page 2715, physically transient resistive switching memory based on silk fibroin is demonstrated by Y. Hao, X. Chen, and co-workers. The memory devices can be absolutely dissolved in deionized water or phosphate-buffered saline in 2 hours. The transient devices have the potential for application in secure data storage systems and biocompatible electronics. PMID:27198963

  1. 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

  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. Context-dependent resistance to proteolysis of intrinsically disordered proteins

    PubMed Central

    Suskiewicz, Marcin J; Sussman, Joel L; Silman, Israel; Shaul, Yosef

    2011-01-01

    Intrinsically disordered proteins (IDPs), also known as intrinsically unstructured proteins (IUPs), lack a well-defined 3D structure in vitro and, in some cases, also in vivo. Here, we discuss the question of proteolytic sensitivity of IDPs, with a view to better explaining their in vivo characteristics. After an initial assessment of the status of IDPs in vivo, we briefly survey the intracellular proteolytic systems. Subsequently, we discuss the evidence for IDPs being inherently sensitive to proteolysis. Such sensitivity would not, however, result in enhanced degradation if the protease-sensitive sites were sequestered. Accordingly, IDP access to and degradation by the proteasome, the major proteolytic complex within eukaryotic cells, are discussed in detail. The emerging picture appears to be that IDPs are inherently sensitive to proteasomal degradation along the lines of the “degradation by default” model. However, available data sets of intracellular protein half-lives suggest that intrinsic disorder does not imply a significantly shorter half-life. We assess the power of available systemic half-life measurements, but also discuss possible mechanisms that could protect IDPs from intracellular degradation. Finally, we discuss the relevance of the proteolytic sensitivity of IDPs to their function and evolution. PMID:21574196

  4. Expression of lung resistance-related protein, LRP, and multidrug resistance-related protein, MRP1, in normal human lung cells in long-term cultures.

    PubMed

    Lehmann, Thomas; Torky, Abdel-Rahman Wageeh; Stehfest, Ekkehard; Hofmann, Stefan; Foth, Heidi

    2005-10-01

    Transport processes form part of the body's defense mechanism, and they determine the intracellular levels of many endogenous and exogenous compounds. The multidrug resistance-related protein MRP1 and the lung resistance-related protein LRP are associated with drug resistance against chemotherapeutics; they protect cells against toxic compounds. There is much experimental evidence to suggest that both of these transporter proteins serve important physiological functions. The expression of LRP and MRP1 was studied in normal human bronchial epithelial cells (NHBEC) and peripheral lung cells (PLC) obtained from explant cultures from morphologically-normal human lung tissue taken from patients with lung cancer. LRP (mRNA and protein) was detected in the cells of the bronchi as well as the peripheral lung with low (a factor of 2.6) inter-individual variation in the first generation. No significant alterations were noted for LRP within three-to-four generations in the same patient. LRP expression was not substantially different between cultures from different topographic regions of the human lung. MRP1 protein and MRP1 mRNA could also be detected in all of the NHBEC and PLC cultures studied, but with substantially higher (a factor of 7.7) intra-individual variation in the first generation than for LRP. MRP expression was the same for bronchial cells and PLC when the material was obtained from both sites. The level of mRNA for MRP1 was, in general, less stable than that for LRP. In multigeneration explant cultures, the levels of LRP mRNA and protein and MRP1 protein did not fluctuate greatly, but the level of MRP1 mRNA dropped to about 25% of the reference value within four generations (after about 8-10 weeks of culture). In one case, NHBEC subpassages were followed over a period of 20 weeks. In this system MRP mRNA levels increased by more than threefold, while levels of MRP1 protein and LRP mRNA and protein were expressed at almost constant rates. PMID:15986202

  5. Absolute immunoquantification of the expression of ABC transporters P-glycoprotein, breast cancer resistance protein and multidrug resistance-associated protein 2 in human liver and duodenum.

    PubMed

    Tucker, Theodora G H A; Milne, Alison M; Fournel-Gigleux, Sylvie; Fenner, Katherine S; Coughtrie, Michael W H

    2012-01-15

    The ATP-binding cassette (ABC) transporters breast cancer resistance protein (BCRP), multidrug resistance-associated protein 2 (MRP2), and P-glycoprotein (Pgp) are important in the distribution and elimination of many drugs and endogenous metabolites. Due to their membrane location and hydrophobicity it is difficult to generate purified protein standards to quantify these transporters in human tissues. The present study generated transporter proteins fused with the S-peptide of ribonuclease for use as standards in immunoquantification in human liver and small intestine. Quantification of the S•tag™, a 15 amino acid peptide, is based on the formation of a functional ribonuclease activity upon its high affinity reconstitution with ribonuclease S-protein. S-tagged transporters were used as full-length protein standards in the immunoquantification of endogenous BCRP, MRP2, and Pgp levels in 14 duodenum and 13 liver human tissue samples. Expression levels in the duodenum were 305±248 (BCRP), 66±70 (MRP2), and 275±205 (Pgp) fmoles per cm(2). Hepatic levels were 2.6±0.9 (BCRP), 19.8±10.5 (MRP2), and 26.1±10.1 (total Pgp) pmoles per g of liver. The mean hepatic scaling factor was 35.8mg crude membrane per g of liver, and the mean duodenal scaling factor was 1.3mg crude membrane per cm(2) mucosal lining. Interindividual variability was greater in duodenal samples than liver samples. It is hoped that this innovative method of quantifying these transporters (and other membrane proteins) will improve in vivo-in vitro extrapolation and in silico prediction of drug absorption and elimination, thus supporting drug development. PMID:22062654

  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

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

    2016-03-18

    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. 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

  9. Abnormal protein turnover and anabolic resistance to exercise in sarcopenic obesity.

    PubMed

    Nilsson, Mats I; Dobson, Justin P; Greene, Nicholas P; Wiggs, Michael P; Shimkus, Kevin L; Wudeck, Elyse V; Davis, Amanda R; Laureano, Marissa L; Fluckey, James D

    2013-10-01

    Obesity may impair protein synthesis rates and cause anabolic resistance to growth factors, hormones, and exercise, ultimately affecting skeletal muscle mass and function. To better understand muscle wasting and anabolic resistance with obesity, we assessed protein 24-h fractional synthesis rates (24-h FSRs) in selected hind-limb muscles of sedentary and resistance-exercised lean and obese Zucker rats. Despite atrophied hind-limb muscles (-28% vs. lean rats), 24-h FSRs of mixed proteins were significantly higher in quadriceps (+18%) and red or white gastrocnemius (+22 or +38%, respectively) of obese animals when compared to lean littermates. Basal synthesis rates of myofibrillar (+8%) and mitochondrial proteins (-1%) in quadriceps were not different between phenotypes, while manufacture of cytosolic proteins (+12%) was moderately elevated in obese cohorts. Western blot analyses revealed a robust activation of p70S6k (+178%) and a lower expression of the endogenous mTOR inhibitor DEPTOR (-28%) in obese rats, collectively suggesting that there is an obesity-induced increase in net protein turnover favoring degradation. Lastly, the protein synthetic response to exercise of mixed (-7%), myofibrillar (+6%), and cytosolic (+7%) quadriceps subfractions was blunted compared to the lean phenotype (+34, +40, and +17%, respectively), indicating a muscle- and subfraction-specific desensitization to the anabolic stimulus of exercise in obese animals. PMID:23804240

  10. Total Protein Profile and Drug Resistance in Candida albicans Isolated from Clinical Samples

    PubMed Central

    Thawani, Vijay; Mehra, Arti

    2016-01-01

    This study was done to assess the antifungal susceptibility of clinical isolates of Candida albicans and to evaluate its total protein profile based on morphological difference on drug resistance. Hundred and twenty clinical isolates of C. albicans from various clinical specimens were tested for susceptibility against four antifungal agents, namely, fluconazole, itraconazole, amphotericin B, and ketoconazole. A significant increase of drug resistance in clinical isolates of C. albicans was observed. The study showed 50% fluconazole and itraconazole resistance at 32 μg mL−1 with a MIC50 and MIC90 values at 34 and 47 and 36 and 49 μg mL−1, respectively. All isolates were sensitive to amphotericin B and ketoconazole. The SDS-PAGE protein profile showed a prevalent band of ~52.5 kDa, indicating overexpression of gene in 72% strains with fluconazole resistance. Since the opportunistic infections of Candida spp. are increasing along with drug resistance, the total protein profile will help in understanding the evolutionary changes in drug resistance and also to characterize them. PMID:27478638

  11. 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

  12. Protein profiling of mefloquine resistant Plasmodium falciparum using mass spectrometry-based proteomics

    PubMed Central

    Reamtong, Onrapak; Srimuang, Krongkan; Saralamba, Naowarat; Sangvanich, Polkit; Day, Nicholas P.J.; White, Nicholas J.; Imwong, Mallika

    2015-01-01

    Malaria is a mosquito borne infectious disease caused by protozoa of genus Plasmodium. There are five species of Plasmodium that are found to infect humans. Plasmodium falciparum can cause severe malaria leading to higher morbidity and mortality of malaria than the other four species. Antimalarial resistance is the major obstacle to control malaria. Mefloquine was used in combination with Artesunate for uncomplicated P. falciparum in South East Asia and it has developed and established mefloquine resistance in this region. Here, gel-enhanced liquid chromatography/tandem mass spectrometry (GeLC–MS/MS)-based proteomics and label-free quantification were used to explore the protein profiles of mefloquine-sensitive and -induced resistant P. falciparum. A Thai P. falciparum isolate (S066) was used as a model in this research. Our data revealed for the first time that 69 proteins exhibited at least 2-fold differences in their expression levels between the two parasite lines. Of these, 36 were up-regulated and 33 were down-regulated in the mefloquine-resistant line compared with the mefloquine-sensitive line. These findings are consistent with those of past studies, where the multidrug resistance protein Pgh1 showed an up-regulation pattern consistent with that expected from its average 3-copy pfmdr1 gene number. Pgh1 and eight other up-regulated proteins (i.e., histo-aspartyl protease protein, exportin 1, eukaryotic translation initiation factor 3 subunit 8, peptidyl-prolyl cis-trans isomerase, serine rich protein homologue, exported protein 1, ATP synthase beta chain and phospholipid scramblase 1) were further validated for their expression levels using reverse transcriptase quantitative real-time PCR. The data support the up-regulation status in the mefloquine-resistant parasite line of all the candidate genes referred to above. Therefore, GeLC–MS/MS-based proteomics combined with label-free quantification is a reliable approach for exploring mefloquine resistance

  13. 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.

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

    PubMed

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

    2013-03-01

    It is well known that carbapenem-resistant mutations in penicillin-binding proteins (PBPs) are not observed in most Gram-negative bacteria under either clinical or experimental conditions. To understand the mechanisms involved in carbapenem resistance, this study constructed a mutS- and tolC-deficient Escherichia coli strain, which was expected to have elevated mutation frequencies and to lack drug efflux. Using this mutant, carbapenem-resistant strains with target mutations were successfully and efficiently isolated. The mutations T547I/A, M574I and G601D were identified in the PBP2 gene. Meropenem (MEPM)-resistant strains with the PBP2 T547I mutation showed fourfold increased resistance to 1-β-methyl-substituted carbapenems, such as doripenem, MEPM and biapenem, but not to non-substituted carbapenems such as imipenem and panipenem and other β-lactams. In addition, resistance resulting from the G601D mutation was limited to MEPM, whilst the M574I mutation conferred resistance to MEPM, imipenem and panipenem. This is the first report, to the best of our knowledge, that E. coli also has a carbapenem-resistance mechanism as a result of PBP2 mutations, and it provides insight into the resistance profiles of PBP2 mutations to carbapenems with and without the 1-β-methyl group. PMID:23222859

  15. 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

  16. 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

  17. 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

  18. A Nucleotide Phosphatase Activity in the Nucleotide Binding Domain of an Orphan Resistance Protein from Rice*

    PubMed Central

    Fenyk, Stepan; de San Eustaquio Campillo, Alba; Pohl, Ehmke; Hussey, Patrick J.; Cann, Martin J.

    2012-01-01

    Plant resistance proteins (R-proteins) are key components of the plant immune system activated in response to a plethora of different pathogens. R-proteins are P-loop NTPase superfamily members, and current models describe their main function as ATPases in defense signaling pathways. Here we show that a subset of R-proteins have evolved a new function to combat pathogen infection. This subset of R-proteins possesses a nucleotide phosphatase activity in the nucleotide-binding domain. Related R-proteins that fall in the same phylogenetic clade all show the same nucleotide phosphatase activity indicating a conserved function within at least a subset of R-proteins. R-protein nucleotide phosphatases catalyze the production of nucleoside from nucleotide with the nucleotide monophosphate as the preferred substrate. Mutation of conserved catalytic residues substantially reduced activity consistent with the biochemistry of P-loop NTPases. Kinetic analysis, analytical gel filtration, and chemical cross-linking demonstrated that the nucleotide-binding domain was active as a multimer. Nuclear magnetic resonance and nucleotide analogues identified the terminal phosphate bond as the target of a reaction that utilized a metal-mediated nucleophilic attack by water on the phosphoester. In conclusion, we have identified a group of R-proteins with a unique function. This biochemical activity appears to have co-evolved with plants in signaling pathways designed to resist pathogen attack. PMID:22157756

  19. Identification of proteins responsible for adriamycin resistance in breast cancer cells using proteomics analysis

    PubMed Central

    Wang, Zhipeng; Liang, Shuang; Lian, Xin; Liu, Lei; Zhao, Shu; Xuan, Qijia; Guo, Li; Liu, Hang; Yang, Yuguang; Dong, Tieying; Liu, Yanchen; Liu, Zhaoliang; Zhang, Qingyuan

    2015-01-01

    Chemoresistance is a poor prognostic factor in breast cancer and is a major obstacle to the successful treatment of patients receiving chemotherapy. However, the precise mechanism of resistance remains unclear. In this study, a pair of breast cancer cell lines, MCF-7 and its adriamycin-resistant counterpart MCF-7/ADR was used to examine resistance-dependent cellular responses and to identify potential therapeutic targets. We applied nanoflow liquid chromatography (nLC) and tandem mass tags (TmT) quantitative mass spectrometry to distinguish the differentially expressed proteins (DEPs) between the two cell lines. Bioinformatics analyses were used to identify functionally active proteins and networks. 80 DEPs were identified with either up- or down-regulation. Basing on the human protein-protein interactions (PPI), we have retrieved the associated functional interaction networks for the DEPs and analyzed the biological functions. Six different signaling pathways and most of the DEPs strongly linked to chemoresistance, invasion, metastasis development, proliferation, and apoptosis. The identified proteins in biological networks served to resistant drug and to select critical candidates for validation analyses by western blot. The glucose-6-phosphate dehydrogenase (G6PD), gamma-glutamyl cyclotransferase (GGCT), isocitrate dehydrogenase 1 (NADP+,soluble)(IDH1), isocitrate dehydrogenase 2 (NADP+,mitochondrial) (IDH2) and glutathione S-transferase pi 1(GSTP1), five of the critical components of GSH pathway, contribute to chemoresistance. PMID:25818003

  20. 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

  1. Circulating growth arrest-specific protein 6 levels are associated with erythropoietin resistance in hemodialysis patients.

    PubMed

    Chen, Miao-Pei; Chen, Chien-Wen; Chen, Jin-Shuen; Mao, Hung-Chung; Chou, Chu-Lin

    2016-01-01

    Growth arrest-specific protein 6 (Gas6) works synergistically with erythropoietin (EPO) to increase the proliferation and maturation of erythroblasts. However, the role of Gas 6 levels on EPO resistance in hemodialysis (HD) patients remains unclear. Therefore, the objective of this study was the first to examine the correlation between plasma Gas6 levels and EPO resistance in HD patients. We enrolled 134 HD patients and 85 healthy individuals. The HD patients were divided into 2 groups: 98 non-EPO-resistant patients and 36 EPO-resistant patients. Plasma levels of Gas6, interleukin 6 (IL-6), high-sensitivity C-reactive protein (hs-CRP), and albumin were quantified. Compared with non-EPO-resistant patients, EPO-resistant patients had elevated plasma concentrations of Gas6 (15.4 ± 3.3 vs. 13.7 ± 3.2 ng/mL, P = 0.006), IL-6 (3.1 ± 3.1 vs. 2.1 ± 1.5 pg/mL, P = 0.009), and hs-CRP (12.7 ± 25.2 vs. 4.5 ± 5.5 mg/L, P = 0.002). In EPO-resistant HD patients, plasma Gas6 levels were negatively correlated with albumin levels (r = -0.388, P < 0.021). Elevated Gas6 levels are associated with EPO resistance in HD patients. Also, EPO resistance is related to inflammation and malnutrition. Thus, circulating Gas6 levels could be used as the potential marker in HD patients with EPO resistance. PMID:26788441

  2. Myofibrillar protein synthesis following ingestion of soy protein isolate at rest and after resistance exercise in elderly men

    PubMed Central

    2012-01-01

    Background Increased amino acid availability stimulates muscle protein synthesis, however, aged muscle appears less responsive to the anabolic effects of amino acids when compared to the young. We aimed to compare changes in myofibrillar protein synthesis (MPS) in elderly men at rest and after resistance exercise following ingestion of different doses of soy protein and compare the responses to those we previously observed with ingestion of whey protein isolate. Methods Thirty elderly men (age 71 ± 5 y) completed a bout of unilateral knee-extensor resistance exercise prior to ingesting no protein (0 g), or either 20 g or 40 g of soy protein isolate (0, S20, and S40 respectively). We compared these responses to previous responses from similar aged men who had ingested 20 g and 40 g of whey protein isolate (W20 and W40). A primed constant infusion of L-[1-13 C]leucine and L-[ring-13 C6]phenylalanine and skeletal muscle biopsies were used to measure whole-body leucine oxidation and MPS over 4 h post-protein consumption in both exercised and non-exercised legs. Results Whole-body leucine oxidation increased with protein ingestion and was significantly greater for S20 vs. W20 (P = 0.003). Rates of MPS for S20 were less than W20 (P = 0.02) and not different from 0 g (P = 0.41) in both exercised and non-exercised leg muscles. For S40, MPS was also reduced compared with W40 under both rested and post-exercise conditions (both P < 0.005); however S40 increased MPS greater than 0 g under post-exercise conditions (P = 0.04). Conclusions The relationship between protein intake and MPS is both dose and protein source-dependent, with isolated soy showing a reduced ability, as compared to isolated whey protein, to stimulate MPS under both rested and post-exercise conditions. These differences may relate to the lower postprandial leucinemia and greater rates of amino acid oxidation following ingestion of soy versus whey protein. PMID

  3. Broad distribution of the multidrug resistance-related vault lung resistance protein in normal human tissues and tumors.

    PubMed

    Izquierdo, M A; Scheffer, G L; Flens, M J; Giaccone, G; Broxterman, H J; Meijer, C J; van der Valk, P; Scheper, R J

    1996-03-01

    Multidrug resistance (MDR) to anticancer drugs is a major cause of treatment failure in cancer. The lung resistance protein LRP is a newly described protein related to MDR in several in vitro models. LRP has been shown to be a strong predictor of poor response to chemotherapy and prognosis in acute myeloid leukemia and in ovarian carcinoma patients. Recently, based on a 57% and 88% amino acid identity with major vault proteins from Dictyostelium discoideum and Rattus norvegicus, respectively, we identified LRP as the human major vault protein, the main component of highly conserved cellular organelles named vaults. We have studied the immunohistochemical expression of LRP in freshly frozen normal human tissues and 174 cancer specimens of 28 tumor types. LRP was broadly distributed in normal and malignant cells, but distinct patterns of expression were noticed. High LRP expression was seen in bronchus, digestive tract, renal proximal tubules, keratinocytes, macrophages, and adrenal cortex whereas varying ing levels were observed in other organs. LRP was detected in all tumor types examined, but its frequency varied, fairly reflecting the chemosensitivity of different cancers. For example, low rates of LRP positivity were seen in testicular cancer, neuroblastoma, and acute myeloid leukemia; intermediate in ovarian cancer; and high in colon, renal, and pancreatic carcinomas. The wide occurrence of LRP in normal and transformed cells in humans, its similar distribution to that of vaults in other species, as well as the high level of conservation among eukaryotic cells of both the amino acid sequence of the major vault protein and the composition and structure of vaults, suggest that vault function is important to eukaryotic cells. PMID:8774142

  4. 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

  5. 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

  6. 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

    2015-01-01

    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. PMID:26345948

  7. 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

  8. Prediction of antibiotic resistance proteins from sequence-derived properties irrespective of sequence similarity.

    PubMed

    Zhang, H L; Lin, H H; Tao, L; Ma, X H; Dai, J L; Jia, J; Cao, Z W

    2008-09-01

    Increasing antibiotic resistance has become a worldwide challenge to the clinical treatment of infectious diseases. The identification of antibiotic resistance proteins (ARPs) would be helpful in the discovery of new therapeutic targets and the design of novel drugs to control the potential spread of antibiotic resistance. In this work, a support vector machine (SVM)-based ARP prediction system was developed using 1308 ARPs and 15587 non-ARPs. Its performance was evaluated using 313 ARPs and 7156 non-ARPs. The computed prediction accuracy was 88.5% for ARPs and 99.2% for non-ARPs. A potential application of this method is the identification of ARPs non-homologous to proteins of known function. Further genome screening found that ca. 3.5% and 3.2% of proteins in Escherichia coli and Staphylococcus aureus, respectively, are potential ARPs. These results suggest the usefulness of SVMs for facilitating the identification of ARPs. The software can be accessed at SARPI (Server for Antibiotic Resistance Protein Identification). PMID:18583101

  9. 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...

  10. Understanding Interactions between Phytopathogenic Phytophthora Effector IpiO and the Host Resistance Protein RB

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Species of phytopathogenic Phytophthora are well known for their ability to cause disease on economically important crops, with almost 100 recognized species targeting close to 300 different hosts. The host resistance protein RB, isolated from wild potato, specifically recognizes the P. infestans Ip...

  11. Nutritional regulation of muscle protein synthesis with resistance exercise: strategies to enhance anabolism

    PubMed Central

    2012-01-01

    Provision of dietary amino acids increases skeletal muscle protein synthesis (MPS), an effect that is enhanced by prior resistance exercise. As a fundamentally necessary process in the enhancement of muscle mass, strategies to enhance rates of MPS would be beneficial in the development of interventions aimed at increasing skeletal muscle mass particularly when combined with chronic resistance exercise. The purpose of this review article is to provide an update on current findings regarding the nutritional regulation of MPS and highlight nutrition based strategies that may serve to maximize skeletal muscle protein anabolism with resistance exercise. Such factors include timing of protein intake, dietary protein type, the role of leucine as a key anabolic amino acid, and the impact of other macronutrients (i.e. carbohydrate) on the regulation of MPS after resistance exercise. We contend that nutritional strategies that serve to maximally stimulate MPS may be useful in the development of nutrition and exercise based interventions aimed at enhancing skeletal muscle mass which may be of interest to elderly populations and to athletes. PMID:22594765

  12. 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

  13. Knocked-out and still walking: prion protein-deficient cattle are resistant to prion disease

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background: Transmissible spongiform encephalopathies (TSEs) or prion diseases are caused by the propagation of a misfolded form (PrP**d) of the normal cellular prion protein, PrP**c. Disruption of PrP**c expression in the mouse results in resistance to PrP-propagation and disease. However, the impa...

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  15. A bile‐inducible membrane protein mediates bifidobacterial bile resistance

    PubMed Central

    Ruiz, Lorena; O'Connell‐Motherway, Mary; Zomer, Aldert; de los Reyes‐Gavilán, Clara G.; Margolles, Abelardo; van Sinderen, Douwe

    2012-01-01

    Summary Bbr_0838 from Bifidobacterium breve UCC2003 is predicted to encode a 683 residue membrane protein, containing both a permease domain that displays similarity to transporters belonging to the major facilitator superfamily, as well as a CBS (cystathionine beta synthase) domain. The high level of similarity to bile efflux pumps from other bifidobacteria suggests a significant and general role for Bbr_0838 in bile tolerance. Bbr_0838 transcription was shown to be monocistronic and strongly induced upon exposure to bile. Further analysis delineated the transcriptional start site and the minimal region required for promoter activity and bile regulation. Insertional inactivation of Bbr_0838 in B. breve UCC2003 resulted in a strain, UCC2003:838800, which exhibited reduced survival upon cholate exposure as compared with the parent strain, a phenotype that was reversed when a functional, plasmid‐encoded Bbr_0838 gene was introduced into UCC2003:838800. Transcriptome analysis of UCC2003:838800 grown in the presence or absence of bile demonstrated that transcription of Bbr_0832, which is predicted to encode a macrolide efflux transporter gene, was significantly increased in the presence of bile, representing a likely compensatory mechanism for bile removal in the absence of Bbr_0838. This study represents the first in‐depth analysis of a bile‐inducible locus in bifidobacteria, identifying a key gene relevant for bifidobacterial bile tolerance. PMID:22296641

  16. Daytime pattern of post-exercise protein intake affects whole-body protein turnover in resistance-trained males

    PubMed Central

    2012-01-01

    Background The pattern of protein intake following exercise may impact whole-body protein turnover and net protein retention. We determined the effects of different protein feeding strategies on protein metabolism in resistance-trained young men. Methods Participants were randomly assigned to ingest either 80g of whey protein as 8x10g every 1.5h (PULSE; n=8), 4x20g every 3h (intermediate, INT; n=7), or 2x40g every 6h (BOLUS; n=8) after an acute bout of bilateral knee extension exercise (4x10 repetitions at 80% maximal strength). Whole-body protein turnover (Q), synthesis (S), breakdown (B), and net balance (NB) were measured throughout 12h of recovery by a bolus ingestion of [15N]glycine with urinary [15N]ammonia enrichment as the collected end-product. Results PULSE Q rates were greater than BOLUS (~19%, P<0.05) with a trend towards being greater than INT (~9%, P=0.08). Rates of S were 32% and 19% greater and rates of B were 51% and 57% greater for PULSE as compared to INT and BOLUS, respectively (P<0.05), with no difference between INT and BOLUS. There were no statistical differences in NB between groups (P=0.23); however, magnitude-based inferential statistics revealed likely small (mean effect±90%CI; 0.59±0.87) and moderate (0.80±0.91) increases in NB for PULSE and INT compared to BOLUS and possible small increase (0.42±1.00) for INT vs. PULSE. Conclusion We conclude that the pattern of ingested protein, and not only the total daily amount, can impact whole-body protein metabolism. Individuals aiming to maximize NB would likely benefit from repeated ingestion of moderate amounts of protein (~20g) at regular intervals (~3h) throughout the day. PMID:23067428

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

    PubMed

    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

  18. 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

  19. Cell-Specific Establishment of Poliovirus Resistance to an Inhibitor Targeting a Cellular Protein

    PubMed Central

    Viktorova, Ekaterina G.; Nchoutmboube, Jules; Ford-Siltz, Lauren A.

    2015-01-01

    ABSTRACT It is hypothesized that targeting stable cellular factors involved in viral replication instead of virus-specific proteins may raise the barrier for development of resistant mutants, which is especially important for highly adaptable small (+)RNA viruses. However, contrary to this assumption, the accumulated evidence shows that these viruses easily generate mutants resistant to the inhibitors of cellular proteins at least in some systems. We investigated here the development of poliovirus resistance to brefeldin A (BFA), an inhibitor of the cellular protein GBF1, a guanine nucleotide exchange factor for the small cellular GTPase Arf1. We found that while resistant viruses can be easily selected in HeLa cells, they do not emerge in Vero cells, in spite that in the absence of the drug both cultures support robust virus replication. Our data show that the viral replication is much more resilient to BFA than functioning of the cellular secretory pathway, suggesting that the role of GBF1 in the viral replication is independent of its Arf activating function. We demonstrate that the level of recruitment of GBF1 to the replication complexes limits the establishment and expression of a BFA resistance phenotype in both HeLa and Vero cells. Moreover, the BFA resistance phenotype of poliovirus mutants is also cell type dependent in different cells of human origin and results in a fitness loss in the form of reduced efficiency of RNA replication in the absence of the drug. Thus, a rational approach to the development of host-targeting antivirals may overcome the superior adaptability of (+)RNA viruses. IMPORTANCE Compared to the number of viral diseases, the number of available vaccines is miniscule. For some viruses vaccine development has not been successful after multiple attempts, and for many others vaccination is not a viable option. Antiviral drugs are needed for clinical practice and public health emergencies. However, viruses are highly adaptable and can

  20. 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

  1. 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

  2. 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

  3. 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

  4. 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. PMID:25805107

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

    PubMed

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

    2016-08-01

    Targeting surface receptors overexpressed on cancer cells is one way to specifically treat cancer versus normal cells. Vintafolide (EC145), which consists of folate linked to a cytotoxic small molecule, desacetylvinblastine hydrazide (DAVLBH), takes advantage of the overexpression of folate receptor (FR) on cancer cells. Once bound to FR, vintafolide enters the cell by endocytosis, and the reducing environment of the endosome cleaves the linker, releasing DAVLBH to destabilize microtubules. Vintafolide has shown efficacy and improved tolerability compared with DAVLBH in FR-positive preclinical models. As the first FR-targeting drug to reach the clinic, vintafolide has achieved favorable responses in phase II clinical trials in FR-positive ovarian and lung cancer. However, some FR-positive patients in these clinical trials do not respond to vintafolide. We sought to identify potential biomarkers of resistance to aid in the future development of this and other FR-targeting drugs. Here, we confirm that high P-glycoprotein (P-gp) expression was the strongest predictor of resistance to DAVLBH in a panel of 359 cancer cell lines. Furthermore, targeted delivery of DAVLBH via the FR, as in vintafolide, fails to overcome P-gp-mediated efflux of DAVLBH in both in vitro and in vivo preclinical models. Therefore, we suggest that patients whose tumors express high levels of P-gp be excluded from future clinical trials for vintafolide as well as other FR-targeted therapeutics bearing a P-gp substrate. Mol Cancer Ther; 15(8); 1998-2008. ©2016 AACR. PMID:27256377

  6. Implication of Unfolded Protein Response and Autophagy in the Treatment of BRAF Inhibitor Resistant Melanoma.

    PubMed

    Meng, Xiao-Xiao; Xu, Hong-Xi; Yao, Mu; Dong, Qihan; Zhang, Xu Dong

    2016-01-01

    The continuous activation of the mitogen-activated protein kinase signaling cascade, typified by the BRAFV600E mutation, is one of the key alterations in melanoma. Accordingly, two BRAF inhibitors (BRAFi), vemurafenib and dabrafenib are utilized to treat melanoma and resulted in an excellent clinical outcome. However, the clinical success is not long-lasting, and the BRAFi resistance and disease progression inevitably occurs in nearly all patients. Endoplasmic reticulum stress-induced unfolded protein response and autophagy have emerged as potential pro-survival mechanisms adopted by melanoma cells in response to BRAFi. In this review, we discuss the role of unfolded protein response and autophagy that are implicated in the development of BRAFi-resistant melanoma and the corresponding strategy aiming at overcoming the intractable clinical problem. PMID:26419469

  7. 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

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

    PubMed Central

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

    2014-01-01

    Bacterial wilt phytopathogen Ralstonia solanacearum is a serious soil-borne disease that attacks several economically important plants worldwide, including Brassicaceae. Previous studies indicate that recognition of avirulence (Avr)-effector PopP2 by resistance (R) protein, RRS1-R, and physical interaction between RRS1-R and PopP2 in the nucleus are required for resistance. Of late, we showed that a pair of Arabidopsis thaliana TIR-NLR proteins, RRS1 and RPS4, function together in disease resistance against multiple pathogen isolates. Here, we report that dual R proteins, RRS1 and RPS4, from A. thaliana ecotype Wassilewskija confer resistance to bacterial wilt in transgenic Brassica crops. For practical applications, this finding may provide a new strategy for developing disease resistant plants that express R genes from other plants. PMID:25763492

  9. Comparative analysis of zinc finger proteins involved in plant disease resistance.

    PubMed

    Gupta, Santosh Kumar; Rai, Amit Kumar; Kanwar, Shamsher Singh; Sharma, Tilak R

    2012-01-01

    A meta-analysis was performed to understand the role of zinc finger domains in proteins of resistance (R) genes cloned from different crops. We analyzed protein sequences of seventy R genes of various crops in which twenty six proteins were found to have zinc finger domains along with nucleotide binding sites - leucine rice repeats (NBS-LRR) domains. We identified thirty four zinc finger domains in the R proteins of nine crops and were grouped into 19 types of zinc fingers. The size of individual zinc finger domain within the R genes varied from 11 to 84 amino acids, whereas the size of proteins containing these domains varied from 263 to 1305 amino acids. The biophysical analysis revealed that molecular weight of Pi54 zinc finger was lowest whereas the highest one was found in rice Pib zinc finger named as Transposes Transcription Factor (TTF). The instability (R(2) =0.95) and the aliphatic (R(2) =0.94) indices profile of zinc finger domains follows the polynomial distribution pattern. The pairwise identity analysis showed that the Lin11, Isl-1 & Mec-3 (LIM) zinc finger domain of rice blast resistance protein pi21 have 12.3% similarity with the nuclear transcription factor, X-box binding-like 1 (NFX) type zinc finger domain of Pi54 protein. For the first time, we reported that Pi54 (Pi-k(h)-Tetep), a rice blast resistance (R) protein have a small zinc finger domain of NFX type located on the C-terminal in between NBS and LRR domains of the R-protein. Compositional analysis depicted by the helical wheel diagram revealed the presence of a hydrophobic region within this domain which might help in exposing the LRR region for a possible R-Avr interaction. This domain is unique among all other cloned plant disease resistance genes and might play an important role in broad-spectrum nature of rice blast resistance gene Pi54. PMID:22916136

  10. Comparative Analysis of Zinc Finger Proteins Involved in Plant Disease Resistance

    PubMed Central

    Gupta, Santosh Kumar; Rai, Amit Kumar; Kanwar, Shamsher Singh; Sharma, Tilak R.

    2012-01-01

    A meta-analysis was performed to understand the role of zinc finger domains in proteins of resistance (R) genes cloned from different crops. We analyzed protein sequences of seventy R genes of various crops in which twenty six proteins were found to have zinc finger domains along with nucleotide binding sites - leucine rice repeats (NBS-LRR) domains. We identified thirty four zinc finger domains in the R proteins of nine crops and were grouped into 19 types of zinc fingers. The size of individual zinc finger domain within the R genes varied from 11 to 84 amino acids, whereas the size of proteins containing these domains varied from 263 to 1305 amino acids. The biophysical analysis revealed that molecular weight of Pi54 zinc finger was lowest whereas the highest one was found in rice Pib zinc finger named as Transposes Transcription Factor (TTF). The instability (R2 = 0.95) and the aliphatic (R2 = 0.94) indices profile of zinc finger domains follows the polynomial distribution pattern. The pairwise identity analysis showed that the Lin11, Isl-1 & Mec-3 (LIM) zinc finger domain of rice blast resistance protein pi21 have 12.3% similarity with the nuclear transcription factor, X-box binding-like 1 (NFX) type zinc finger domain of Pi54 protein. For the first time, we reported that Pi54 (Pi-kh-Tetep), a rice blast resistance (R) protein have a small zinc finger domain of NFX type located on the C-terminal in between NBS and LRR domains of the R-protein. Compositional analysis depicted by the helical wheel diagram revealed the presence of a hydrophobic region within this domain which might help in exposing the LRR region for a possible R-Avr interaction. This domain is unique among all other cloned plant disease resistance genes and might play an important role in broad-spectrum nature of rice blast resistance gene Pi54. PMID:22916136

  11. 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

  12. An inhibitory interaction between viral and cellular proteins underlies the resistance of tomato to nonadapted tobamoviruses.

    PubMed

    Ishibashi, Kazuhiro; Naito, Satoshi; Meshi, Tetsuo; Ishikawa, Masayuki

    2009-05-26

    Any individual virus can infect only a limited range of hosts, and most plant species are "nonhosts" to a given virus; i.e., all members of the species are insusceptible to the virus. In nonhost plants, the factors that control virus resistance are not genetically tractable, and how the host range of a virus is determined remains poorly understood. Tomato (Solanum lycopersicum) is a nonhost species for Tobacco mild green mosaic virus (TMGMV) and Pepper mild mottle virus (PMMoV), members of the genus Tobamovirus. Previously, we identified Tm-1, a resistance gene of tomato to another tobamovirus, Tomato mosaic virus (ToMV), and found that Tm-1 binds to ToMV replication proteins to inhibit RNA replication. Tm-1 is derived from a wild tomato species, S. habrochaites, and ToMV-susceptible tomato cultivars have the allelic gene tm-1. The tm-1 protein can neither bind to ToMV replication proteins nor inhibit ToMV multiplication. Here, we show that transgenic tobacco plants expressing tm-1 exhibit resistance to TMGMV and PMMoV. The tm-1 protein bound to the replication proteins of TMGMV and PMMoV and inhibited their RNA replication in vitro. In one of the tm-1-expressing tobacco plants, a tm-1-insensitive TMGMV mutant emerged. In tomato protoplasts, this mutant TMGMV multiplied as efficiently as ToMV. However, in tomato plants, the mutant TMGMV multiplied with lower efficiency compared to ToMV and caused systemic necrosis. These results suggest that an inhibitory interaction between the replication proteins and tm-1 underlies a multilayered resistance mechanism to TMGMV in tomato. PMID:19423673

  13. An inhibitory interaction between viral and cellular proteins underlies the resistance of tomato to nonadapted tobamoviruses

    PubMed Central

    Ishibashi, Kazuhiro; Naito, Satoshi; Meshi, Tetsuo; Ishikawa, Masayuki

    2009-01-01

    Any individual virus can infect only a limited range of hosts, and most plant species are “nonhosts” to a given virus; i.e., all members of the species are insusceptible to the virus. In nonhost plants, the factors that control virus resistance are not genetically tractable, and how the host range of a virus is determined remains poorly understood. Tomato (Solanum lycopersicum) is a nonhost species for Tobacco mild green mosaic virus (TMGMV) and Pepper mild mottle virus (PMMoV), members of the genus Tobamovirus. Previously, we identified Tm-1, a resistance gene of tomato to another tobamovirus, Tomato mosaic virus (ToMV), and found that Tm-1 binds to ToMV replication proteins to inhibit RNA replication. Tm-1 is derived from a wild tomato species, S. habrochaites, and ToMV-susceptible tomato cultivars have the allelic gene tm-1. The tm-1 protein can neither bind to ToMV replication proteins nor inhibit ToMV multiplication. Here, we show that transgenic tobacco plants expressing tm-1 exhibit resistance to TMGMV and PMMoV. The tm-1 protein bound to the replication proteins of TMGMV and PMMoV and inhibited their RNA replication in vitro. In one of the tm-1-expressing tobacco plants, a tm-1-insensitive TMGMV mutant emerged. In tomato protoplasts, this mutant TMGMV multiplied as efficiently as ToMV. However, in tomato plants, the mutant TMGMV multiplied with lower efficiency compared to ToMV and caused systemic necrosis. These results suggest that an inhibitory interaction between the replication proteins and tm-1 underlies a multilayered resistance mechanism to TMGMV in tomato. PMID:19423673

  14. 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

  15. 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

  16. Molecular ecology of tetracycline resistance: development and validation of primers for detection of tetracycline resistance genes encoding ribosomal protection proteins.

    PubMed

    Aminov, R I; Garrigues-Jeanjean, N; Mackie, R I

    2001-01-01

    Phylogenetic analysis of tetracycline resistance genes encoding the ribosomal protection proteins (RPPs) revealed the monophyletic origin of these genes. The most deeply branching class, exemplified by tet and otrA, consisted of genes from the antibiotic-producing organisms Streptomyces rimosus and Streptomyces lividans. With a high degree of confidence, the corresponding genes of the other seven classes (Tet M, Tet S, Tet O, Tet W, Tet Q, Tet T, and TetB P) formed phylogenetically distinct separate clusters. Based on this phylogenetic analysis, a set of PCR primers for detection, retrieval, and sequence analysis of the corresponding gene fragments from a variety of bacterial and environmental sources was developed and characterized. A pair of degenerate primers targeted all tetracycline resistance genes encoding RPPs except otrA and tet, and seven other primer pairs were designed to target the specific classes. The primers were used to detect the circulation of these genes in the rumina of cows, in swine feed and feces, and in swine fecal streptococci. Classes Tet O and Tet W were found in the intestinal contents of both animals, while Tet M was confined to pigs and Tet Q was confined to the rumen. The tet(O) and tet(W) genes circulating in the microbiota of the rumen and the gastrointestinal tract of pigs were identical despite the differences in animal hosts and antibiotic use regimens. Swine fecal streptococci uniformly possessed the tet(O) gene, and 22% of them also carried tet(M). This population could be considered one of the main reservoirs of these two resistance genes in the pig gastrointestinal tract. All classes of RPPs except Tet T and TetB P were found in the commercial components of swine feed. This is the first demonstration of the applicability of molecular ecology techniques to estimation of the gene pool and the flux of antibiotic resistance genes in production animals. PMID:11133424

  17. Effects of resistance training and protein supplementation on bone turnover in young adult women

    PubMed Central

    Mullins, Nicole M; Sinning, Wayne E

    2005-01-01

    Background The strength of aging bone depends on the balance between the resorption and formation phases of the remodeling process. The purpose of this study was to examine the interaction of two factors with the potential to exert opposing influences on bone turnover, resistance exercise training and high dietary protein intake. It was hypothesized that resistance training by young, healthy, untrained women with protein intakes near recommended levels (0.8 g·kg-1·d-1) would promote bone formation and/or inhibit bone resorption, and that subsequent supplementation to provide 2.4 g protein·kg-1·d-1 would reverse these effects. Methods Bone formation was assessed with serum bone-specific alkaline phosphatase (BAP) and osteocalcin (OC), and bone resorption with urinary calcium and deoxypyridinoline (DPD). Biochemical, strength, anthropometric, dietary, and physical activity data were obtained from 24 healthy, untrained, eumenorrheic women (18–29y) at baseline, after eight weeks of resistance training (3 d·wk-1, ~1 hr·d-1; 3 sets, 6–10 repetitions, 13 exercises, 75–85% maximum voluntary contraction), and after 12 weeks of resistance training and 10 days of protein/placebo supplementation. Subjects were randomized (double-blind) to either a high protein (HP) or training control (TC) group and, during the final 10 days, consumed either enough purified whey protein to bring daily protein intake to 2.4 g·kg-1·d-1, or an equivalent dose of isoenergetic, carbohydrate placebo. Results Strength, lean tissue mass, and DPD increased significantly in both groups over time, while percent body fat and BAP decreased (repeated measures ANOVA, p ≤ 0.05, Bonferroni correction). No significant changes were observed for serum OC or urinary calcium, and no significant group (TC, HP) × time (baseline, week 8, week 12) interactions emerged for any of the biochemical measures. Conclusion (1) Twelve weeks of high-intensity resistance training did not appear to enhance bone

  18. 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

  19. 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

  20. Outer membrane protein shifts in biocide-resistant Pseudomonas aeruginosa PAO1.

    PubMed

    Winder, C L; Al-Adham, I S; Abdel Malek, S M; Buultjens, T E; Horrocks, A J; Collier, P J

    2000-08-01

    Benzisothiazolone (BIT), N-methylisothiazolone (MIT) and 5-chloro-N-methylisothiazolone (CMIT) are highly effective biocidal agents and are used as preservatives in a variety of cosmetic preparations. The isothiazolones have proven efficacy against many fungal and bacterial species including Pseudomonas aeruginosa. However, some species are beginning to exhibit resistance towards this group of compounds after extended exposure. This experiment induced resistance in cultures of Ps. aeruginosa exposed to incrementally increasing sub-minimum inhibitory concentrations (MICs) of the isothiazolones in their pure chemical forms. The induced resistance was observed as a gradual increase in MIC with each new passage. The MICs for all three test isothiazolones and a thiol-interactive control compound (thiomersal) increased by approximately twofold during the course of the experiment. The onset of resistance was also observed by reference to the altered presence of an outer membrane protein, designated the T-OMP, in SDS-PAGE preparations. T-OMP was observed to disappear from the biocide-exposed preparations and reappear when the resistance-induced cultures were passaged in the absence of biocide. This reappearance of T-OMP was not accompanied by a complete reversal of induced resistance, but by a small decrease in MIC. The induction of resistance towards one biocide resulted in the development of cross-resistance towards other members of the group and the control, thiomersal. It has been suggested that the disappearance of T-OMP from these preparations is associated with the onset of resistance to the isothiazolones in their Kathon form (CMIT and MIT). PMID:10971761

  1. Involvement of outer membrane proteins and peroxide-sensor genes in Burkholderia cepacia resistance to isothiazolone.

    PubMed

    Zhou, Gang; Shi, Qing-shan; Ouyang, You-sheng; Chen, Yi-ben

    2014-04-01

    Isothiazolones are used as preservatives in various modern industrial products. Although microorganisms that exhibit resistance towards these biocides have been identified, the underlying resistance mechanisms are still unclear. Therefore, we investigated the resistance properties of the following Burkholderia cepacia strains to Kathon (a representative of isothiazolones): a wild-type (WT) strain; a laboratory resistance strain (BC-IR) induced from WT; and an isolated strain (BC-327) screened from industrial contamination samples. The bacterial cell structure was disrupted by 50 μg ml⁻¹ Kathon treatment. BC-IR and BC-327 did not display resistance in the presence of 1 ml L⁻¹ Tween 80, 1 ml L⁻¹ Triton X-100, 0.1 % sodium dodecyl sulfate or 1 mmol L⁻¹ EDTA-2Na. Additionally, BC-IR and BC-327 exhibited lower relative conductivity from 10 to 180 min. The types as well as the levels of outer-membrane proteins (OMPs) were altered among WT, BC-IR and BC-327. Finally, the two Kathon-resistance strains BC-IR and BC-327 presented higher resistance capacity to H₂O₂. We measured the levels of peroxide-sensor genes and observed that the transcriptional activator oxyR, superoxide dismutase sod1, sod2, catalase cat1 and cat3 were all up-regulated under oxidative conditions for all strains. Taken together, OMPs and peroxide-sensor genes in B. cepacia contributed to isothiazolone resistance; However, the laboratory strain BC-IR exhibited a different resistance mechanism and properties compared to the isolated strain BC-327. PMID:24197783

  2. 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

  3. 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

  4. Molecular cloning and pharmacological characterization of rat multidrug resistance protein 1 (mrp1).

    PubMed

    Nunoya, Kenichi; Grant, Caroline E; Zhang, Dawei; Cole, Susan P C; Deeley, Roger G

    2003-08-01

    Multidrug resistance protein 1 (MRP1) transports a wide range of structurally diverse conjugated and nonconjugated organic anions and some peptides, including oxidized and reduced glutathione (GSH). The protein confers resistance to certain heavy metal oxyanions and a variety of natural product-type chemotherapeutic agents. Elevated levels of MRP1 have been detected in many human tumors, and the protein is a candidate therapeutic target for drug resistance reversing agents. Previously, we have shown that human MRP1 (hMRP1) and murine MRP1 (mMRP1) differ in their substrate specificity despite a high degree of structural conservation. Since rat models are widely used in the drug discovery and development stage, we have cloned and functionally characterized rat MRP1 (rMRP1). Like mMRP1 and in contrast to hMRP1, rMRP1 confers no, or very low, resistance to anthracyclines and transports the two estrogen conjugates, 17beta-estradiol-17-(beta-d-glucuronide) (E217betaG) and estrone 3-sulfate, relatively poorly. Mutational studies combined with vesicle transport assays identified several amino acids conserved between rat and mouse, but not hMRP1, that make major contributions to these differences in substrate specificity. Despite the fact that the rodent proteins transport E217betaG poorly and the GSH-stimulated transport of estrone 3-sulfate is low compared with hMRP1, site-directed mutagenesis studies indicate that different nonconserved amino acids are involved in the low efficiency with which each of the two estrogen conjugates is transported. Our studies also suggest that although rMRP1 and mMRP1 are 95% identical in primary structure, their substrate specificities may be influenced by amino acids that are not conserved between the two rodent proteins. PMID:12867490

  5. A protein kinase Cβ inhibitor attenuates multidrug resistance of neuroblastoma cells

    PubMed Central

    Svensson, Karin; Larsson, Christer

    2003-01-01

    Background The acquisition of drug resistance is a major reason for poor outcome of neuroblastoma. Protein kinase C (PKC) has been suggested to influence drug resistance in cancer cells. The aim of this study was to elucidate whether inhibition of PKCβ isoforms influences drug-resistance of neuroblastoma cells. Methods The effect of the PKCβ inhibitor LY379196 on the growth-suppressing effects of different chemotherapeutics on neuroblastoma cells was analyzed with MTT assays. The effect of LY379196 on the accumulation of [3H]vincristine was also investigated Results The PKCβ inhibitor LY379196 suppressed the growth of three neuroblastoma cell lines. LY379196 also augmented the growth-suppressive effect of doxorubicin, etoposide, paclitaxel, and vincristine, but not of carboplatin. The effect was most marked for vincristine and for the cell-line (SK-N-BE(2)) that was least sensitive to vincristine. No effect was observed on the non-resistant IMR-32 cells. Two other PKC inhibitors, Gö6976 and GF109203X, also enhanced the vincristine effect. The PKC inhibitors caused an increased accumulation of [3H]vincristine in SK-N-BE(2) cells. Conclusions This indicates that inhibition of PKCβ could attenuate multidrug resistance in neuroblastoma cells by augmenting the levels of natural product anticancer drugs in resistant cells. PMID:12697075

  6. 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

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

    PubMed

    Patel, Saumya K; George, Linz-Buoy; 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

  8. 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

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

    PubMed

    Gougeon, Réjeanne

    2013-04-01

    Evidence shows that the metabolism of protein is altered in type 2 diabetes mellitus and insulin resistance not only applies to glucose and lipid but protein metabolism as well. Population surveys report greater susceptibility to loss of lean tissue and muscle strength with aging in diabetes. Prevention of sarcopenia requires that protein receives more attention in dietary prescriptions. Protein intake of 1-1.2 g/kg of body weight (with weight at a body mass index of 25 kg/m(2))/day may be distributed equally among 3 meals a day, including breakfast, to optimize anabolism. Adopting a dietary pattern that provides a high plant-to-animal ratio and greater food volume favouring consumption of vegetables, legumes, fruits, complemented with fish, low fat dairy and meat (preferably cooked slowly in moisture), soy and nuts may assist with metabolic and weight control. Depending on the magnitude of energy restriction, usual protein intake should be maintained or increased, and the caloric deficit taken from fat and carbohydrate foods. Exercise before protein-rich meals improves skeletal muscle protein anabolism. Because high levels of amino acids lower glucose uptake in individuals without diabetes, the challenge remains to define the optimal protein intake and exercise regimen to protect from losses of muscle mass and strength while maintaining adequate glucose control in type 2 diabetes. PMID:24070802

  10. 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

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

    PubMed Central

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

    2015-01-01

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

  12. A Salmonella protein that is required for resistance to antimicrobial peptides and transport of potassium.

    PubMed Central

    Parra-Lopez, C; Lin, R; Aspedon, A; Groisman, E A

    1994-01-01

    The ability of invading pathogens to proliferate within host tissues requires the capacity to resist the killing effects of a wide variety of host defense molecules. sap mutants of the facultative intracellular parasite Salmonella typhimurium exhibit hypersensitivity to antimicrobial peptides, cannot survive within macrophages in vitro and are attenuated for mouse virulence in vivo. We conducted a molecular genetic analysis of the sapG locus and showed that it encodes a product that is 99% identical to the NAD+ binding protein TrkA, a component of a low-affinity K+ uptake system in Escherichia coli. SapG exhibits similarity with other E. coli proteins implicated in K+ transport including KefC, a glutathione-regulated efflux protein, and Kch, a putative transporter similar to eukaryotic K+ channel proteins, sapG mutants were killed by the antimicrobial peptide protamine in the presence of both high and low K+, indicating that protamine hypersensitivity is not due to K+ starvation. Strains with mutations in sapG and either sapJ or the sapABCDF operon were as susceptible as sapG single mutants, suggesting that the proteins encoded by these loci participate in the same resistance pathway. SapG may modulate the activities of SapABCDF and SapJ to mediate the transport of peptides and potassium. Images PMID:8076592

  13. Ribosome clearance by FusB-type proteins mediates resistance to the antibiotic fusidic acid

    PubMed Central

    Cox, Georgina; Thompson, Gary S.; Jenkins, Huw T.; Peske, Frank; Savelsbergh, Andreas; Rodnina, Marina V.; Wintermeyer, Wolfgang; Homans, Steve W.; Edwards, Thomas A.; O'Neill, Alexander J.

    2012-01-01

    Resistance to the antibiotic fusidic acid (FA) in the human pathogen Staphylococcus aureus usually results from expression of FusB-type proteins (FusB or FusC). These proteins bind to elongation factor G (EF-G), the target of FA, and rescue translation from FA-mediated inhibition by an unknown mechanism. Here we show that the FusB family are two-domain metalloproteins, the C-terminal domain of which contains a four-cysteine zinc finger with a unique structural fold. This domain mediates a high-affinity interaction with the C-terminal domains of EF-G. By binding to EF-G on the ribosome, FusB-type proteins promote the dissociation of stalled ribosome⋅EF-G⋅GDP complexes that form in the presence of FA, thereby allowing the ribosomes to resume translation. Ribosome clearance by these proteins represents a highly unusual antibiotic resistance mechanism, which appears to be fine-tuned by the relative abundance of FusB-type protein, ribosomes, and EF-G. PMID:22308410

  14. Ribosome clearance by FusB-type proteins mediates resistance to the antibiotic fusidic acid.

    PubMed

    Cox, Georgina; Thompson, Gary S; Jenkins, Huw T; Peske, Frank; Savelsbergh, Andreas; Rodnina, Marina V; Wintermeyer, Wolfgang; Homans, Steve W; Edwards, Thomas A; O'Neill, Alexander J

    2012-02-01

    Resistance to the antibiotic fusidic acid (FA) in the human pathogen Staphylococcus aureus usually results from expression of FusB-type proteins (FusB or FusC). These proteins bind to elongation factor G (EF-G), the target of FA, and rescue translation from FA-mediated inhibition by an unknown mechanism. Here we show that the FusB family are two-domain metalloproteins, the C-terminal domain of which contains a four-cysteine zinc finger with a unique structural fold. This domain mediates a high-affinity interaction with the C-terminal domains of EF-G. By binding to EF-G on the ribosome, FusB-type proteins promote the dissociation of stalled ribosome⋅EF-G⋅GDP complexes that form in the presence of FA, thereby allowing the ribosomes to resume translation. Ribosome clearance by these proteins represents a highly unusual antibiotic resistance mechanism, which appears to be fine-tuned by the relative abundance of FusB-type protein, ribosomes, and EF-G. PMID:22308410

  15. microRNA-16 Is Downregulated During Insulin Resistance and Controls Skeletal Muscle Protein Accretion.

    PubMed

    Lee, David E; Brown, Jacob L; Rosa, Megan E; Brown, Lemuel A; Perry, Richard A; Wiggs, Michael P; Nilsson, Mats I; Crouse, Stephen F; Fluckey, James D; Washington, Tyrone A; Greene, Nicholas P

    2016-08-01

    Insulin resistant diabetes, currently at epidemic levels in developed countries, begins in the skeletal muscle and is linked to altered protein turnover. microRNAs downregulate targeted mRNA translation decreasing the amount of translated protein, thereby regulating many cellular processes. Regulation of miRNAs and their function in skeletal muscle insulin resistance is largely unexplored. The purpose of this study was to identify the effects of insulin resistance on contents of skeletal muscle miRNAs with potential functions in protein turnover. We examined miRs -1, -16, -23, -27, -133a, -133b, and -206 in muscles of Zucker rats. miR-1 was 5- to 10-fold greater in obesity, whereas miRs-16 and -133b were repressed ∼50% in obese compared to lean rats, with no other alterations in miRNA contents. miR-16 correlated to protein synthesis in lean, but not obese rats. miR-16 reduction by lipid overload was verified in-vivo by diet-induced obesity and in-vitro using a diacylglycerol analog. A role for miR-16 in protein turnover of skeletal myocytes was established using transient overexpression and anti-miR inhibition. miR-16 overexpression resulted in lower protein synthesis (puromycin incorporation, ∼25-50%), mTOR (∼25%), and p70S6K1 (∼40%) in starved and insulin stimulated myoblasts. Conversely, anti-miR-16 increased basal protein synthesis (puromycin incorporation, ∼75%), mTOR (∼100%), and p70S6K1 (∼100%). Autophagy was enhanced by miR-16 overexpression (∼50% less BCL-2, ∼100% greater LC3II/I, ∼50% less p62) and impaired with miR-16 inhibition (∼45% greater BCL-2, ∼25% less total LC3, ∼50% greater p62). This study demonstrates reduced miR-16 during insulin resistance and establishes miR-16 control of protein accretion in skeletal muscle. J. Cell. Biochem. 117: 1775-1787, 2016. © 2015 Wiley Periodicals, Inc. PMID:26683117

  16. Use of a yeast-based membrane protein expression technology to overexpress drug resistance efflux pumps.

    PubMed

    Lamping, Erwin; Cannon, Richard D

    2010-01-01

    Azole antifungal drugs are used widely to treat people with oral fungal infections. Unfortunately, fungi can develop resistance to these drugs. This resistance can be due to the overexpression or mutation of cytochrome P450 14alpha-lanosterol demethylase, also known as ERG11 or CYP51, and/or the overexpression of membrane-located multidrug efflux pumps. We have developed a heterologous membrane protein expression system that can be used to study the structure and function of these proteins in the non-pathogenic, genetically stable, and versatile eukaryotic model organism, Saccharomyces cerevisiae. In this chapter we describe the techniques used to express the Candida albicans efflux pump Cdr1p in S. cerevisiae. PMID:20717788

  17. Novel quinolone resistance mutations of the Escherichia coli DNA gyrase A protein: enzymatic analysis of the mutant proteins.

    PubMed Central

    Hallett, P; Maxwell, A

    1991-01-01

    Using the techniques of gap misrepair mutagenesis and site-directed mutagenesis, we have generated two novel quinolone resistance mutations of the Escherichia coli DNA gyrase A protein. DNA sequencing showed these mutations to be Ser-83----Ala and Gln-106----Arg. The mutant proteins were overproduced and purified, and their enzymatic properties were analyzed and compared with those of the wild-type enzyme. With ciprofloxacin and other quinolones, the inhibition of DNA supercoiling, relaxation, and decatenation and the induction of DNA cleavage were investigated for both wild-type and mutant enzymes. In each assay, the mutant enzymes were found to require approximately 10 times more drug to inhibit the reaction or induce cleavage than was the wild-type enzyme. However, the Ca2(+)-directed DNA cleavage reaction was indistinguishable for wild-type and mutant gyrases. We discuss models for the gyrase-mediated bactericidal effects of quinolone drugs. Images PMID:1850970

  18. Macrophage Replication Screen Identifies a Novel Francisella Hydroperoxide Resistance Protein Involved in Virulence

    PubMed Central

    Llewellyn, Anna C.; Bina, James E.; Weiss, David S.

    2011-01-01

    Francisella tularensis is a Gram-negative facultative intracellular pathogen and the causative agent of tularemia. Recently, genome-wide screens have identified Francisella genes required for virulence in mice. However, the mechanisms by which most of the corresponding proteins contribute to pathogenesis are still largely unknown. To further elucidate the roles of these virulence determinants in Francisella pathogenesis, we tested whether each gene was required for replication of the model pathogen F. novicida within macrophages, an important virulence trait. Fifty-three of the 224 genes tested were involved in intracellular replication, including many of those within the Francisella pathogenicity island (FPI), validating our results. Interestingly, over one third of the genes identified are annotated as hypothetical, indicating that F. novicida likely utilizes novel virulence factors for intracellular replication. To further characterize these virulence determinants, we selected two hypothetical genes to study in more detail. As predicted by our screen, deletion mutants of FTN_0096 and FTN_1133 were attenuated for replication in macrophages. The mutants displayed differing levels of attenuation in vivo, with the FTN_1133 mutant being the most attenuated. FTN_1133 has sequence similarity to the organic hydroperoxide resistance protein Ohr, an enzyme involved in the bacterial response to oxidative stress. We show that FTN_1133 is required for F. novicida resistance to, and degradation of, organic hydroperoxides as well as resistance to the action of the NADPH oxidase both in macrophages and mice. Furthermore, we demonstrate that F. holarctica LVS, a strain derived from a highly virulent human pathogenic species of Francisella, also requires this protein for organic hydroperoxide resistance as well as replication in macrophages and mice. This study expands our knowledge of Francisella's largely uncharacterized intracellular lifecycle and demonstrates that FTN_1133 is

  19. Dehydrothyrsiferol does not modulate multidrug resistance-associated protein 1 resistance: a functional screening system for MRP1 substrates.

    PubMed

    Pec, Martina K; Aguirre, Amable; Fernández, Javier J; Souto, Maria L; Dorta, Javier F; Villar, Jesús

    2002-11-01

    We had shown previously that the novel, marine, anticancer compound dehydrothyrsiferol (DHT) does not modulate P-glycoprotein (P-gp) dependent drug efflux. Many chemotherapeutics with clinical impact are substrates for the structurally distant related membrane transport protein MRP1 (multidrug resistance-associated protein 1). Thus, we were interested in analysing the behaviour of DHT and control compounds in specific drug transport of MRP1 overexpressing cells. We established a fluorescence based drug efflux system for specific, functional detection of interference of a test compound in MRP1 mediated drug extrusion. Briefly, MRP1 overexpressing HL60/Adr cells were incubated to uptake and then efflux fluorescent 5(6)-carboxyfluorescein diacetate (CFDA), rhodamine 123 (Rh123), or 3,3-diethylocarbocyanine iodide (DiOC2), respectively. Changes in cell fluorescence intensity after coincubation with the compound of interest were determined by flow cytometry. MRP1 mediated efflux of CFDA was analysed in the presence of DHT, the known substrates genistein, probenecid, and the specific inhibitor MK-571. To exclude unknown P-gp related interference in drug transport, efflux of the fluorescent P-gp substrate DiOC2 and specific inhibition by cyclosporin A (CsA) were analysed. Cytotoxicity of DHT in resistant HL60/Adr cells was found to be even superior to that in the parental HL60 leukaemia cell line. Consequently, DHT did not interfere in MRP1 mediated drug transport. In contrast to DiOC2, rhodamine 123 was not specifically effluxed by P-gp but also by MRP1. Therefore, we propose the MRP1 specific CFDA efflux model as a screening and/or excluding system for MRP1 substrates. Together with previous data our results suggest DHT to be an interesting candidate for further investigation directed towards a drug development regimen. PMID:12373300

  20. Hypoxia-inducible factor-1α induces multidrug resistance protein in colon cancer.

    PubMed

    Lv, Yingqian; Zhao, Shan; Han, Jinzhu; Zheng, Likang; Yang, Zixin; Zhao, Li

    2015-01-01

    Multidrug resistance is the major cause of chemotherapy failure in many solid tumors, including colon cancer. Hypoxic environment is a feature for all solid tumors and is important for the development of tumor resistance to chemotherapy. Hypoxia-inducible factor (HIF)-1α is the key transcription factor that mediates cellular response to hypoxia. HIF-1α has been shown to play an important role in tumor resistance; however, the mechanism is still not fully understood. Here, we found that HIF-1α and the drug resistance-associated gene multidrug resistance associated protein 1 (MRP1) were induced by treatment of colon cancer cells with the hypoxia-mimetic agent cobalt chloride. Inhibition of HIF-1α by RNA interference and dominant-negative protein can significantly reduce the induction of MRP1 by hypoxia. Bioinformatics analysis showed that a hypoxia response element is located at -378 to -373 bp upstream of the transcription start site of MRP1 gene. Luciferase reporter assay combined with mutation analysis confirmed that this element is essential for hypoxia-mediated activation of MRP gene. Furthermore, RNA interference revealed that HIF-1α is necessary for this hypoxia-driven activation of MRP1 promoter. Importantly, chromatin immunoprecipitation analysis demonstrated that HIF-1α could directly bind to this HRE site in vivo. Together, these data suggest that MRP1 is a downstream target gene of HIF-1α, which provides a potential novel mechanism for HIF-1α-mediated drug resistance in colon cancer and maybe other solid tumors as well. PMID:26251616

  1. Hypoxia-inducible factor-1α induces multidrug resistance protein in colon cancer

    PubMed Central

    Lv, Yingqian; Zhao, Shan; Han, Jinzhu; Zheng, Likang; Yang, Zixin; Zhao, Li

    2015-01-01

    Multidrug resistance is the major cause of chemotherapy failure in many solid tumors, including colon cancer. Hypoxic environment is a feature for all solid tumors and is important for the development of tumor resistance to chemotherapy. Hypoxia-inducible factor (HIF)-1α is the key transcription factor that mediates cellular response to hypoxia. HIF-1α has been shown to play an important role in tumor resistance; however, the mechanism is still not fully understood. Here, we found that HIF-1α and the drug resistance-associated gene multidrug resistance associated protein 1 (MRP1) were induced by treatment of colon cancer cells with the hypoxia-mimetic agent cobalt chloride. Inhibition of HIF-1α by RNA interference and dominant-negative protein can significantly reduce the induction of MRP1 by hypoxia. Bioinformatics analysis showed that a hypoxia response element is located at −378 to −373 bp upstream of the transcription start site of MRP1 gene. Luciferase reporter assay combined with mutation analysis confirmed that this element is essential for hypoxia-mediated activation of MRP gene. Furthermore, RNA interference revealed that HIF-1α is necessary for this hypoxia-driven activation of MRP1 promoter. Importantly, chromatin immunoprecipitation analysis demonstrated that HIF-1α could directly bind to this HRE site in vivo. Together, these data suggest that MRP1 is a downstream target gene of HIF-1α, which provides a potential novel mechanism for HIF-1α-mediated drug resistance in colon cancer and maybe other solid tumors as well. PMID:26251616

  2. 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

  3. The human multidrug resistance-associated protein MRP is a plasma membrane drug-efflux pump.

    PubMed Central

    Zaman, G J; Flens, M J; van Leusden, M R; de Haas, M; Mülder, H S; Lankelma, J; Pinedo, H M; Scheper, R J; Baas, F; Broxterman, H J

    1994-01-01

    The multidrug-resistance associated protein MRP is a 180- to 195-kDa membrane protein associated with resistance of human tumor cells to cytotoxic drugs. We have investigated how MRP confers drug resistance in SW-1573 human lung carcinoma cells by generating a subline stably transfected with an expression vector containing MRP cDNA. MRP-overexpressing SW-1573 cells are resistant to doxorubicin, daunorubicin, vincristine, VP-16, colchicine, and rhodamine 123, but not to 4'-(9-acridinylamino)methanesulfon-m-anisidide or taxol. The intracellular accumulation of drug (daunorubicin, vincristine, and VP-16) is decreased and the efflux of drug (daunorubicin) is increased in the transfectant. The decreased accumulation of daunorubicin is abolished by permeabilization of the plasma membrane with digitonin, showing that MRP can lower the intracellular daunorubicin level against a concentration gradient. Anti-MRP antisera predominantly stain the plasma membrane of MRP-overexpressing cells. We conclude that MRP is a plasma membrane drug-efflux pump. Images PMID:7916458

  4. 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

  5. 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. PMID:21028799

  6. Mitomycin Resistance in Streptomyces lavendulae Includes a Novel Drug-Binding-Protein-Dependent Export System

    PubMed Central

    Sheldon, Paul J.; Mao, Yingqing; He, Min; Sherman, David H.

    1999-01-01

    Sequence analysis of Streptomyces lavendulae NRRL 2564 chromosomal DNA adjacent to the mitomycin resistance locus mrd (encoding a previously described mitomycin-binding protein [P. Sheldon, D. A. Johnson, P. R. August, H.-W. Liu, and D. H. Sherman, J. Bacteriol. 179:1796–1804, 1997]) revealed a putative mitomycin C (MC) transport gene (mct) encoding a hydrophobic polypeptide that has significant amino acid sequence similarity with several actinomycete antibiotic export proteins. Disruption of mct by insertional inactivation resulted in an S. lavendulae mutant strain that was considerably more sensitive to MC. Expression of mct in Escherichia coli conferred a fivefold increase in cellular resistance to MC, led to the synthesis of a membrane-associated protein, and correlated with reduced intracellular accumulation of the drug. Coexpression of mct and mrd in E. coli resulted in a 150-fold increase in resistance, as well as reduced intracellular accumulation of MC. Taken together, these data provide evidence that MRD and Mct function as components of a novel drug export system specific to the mitomycins. PMID:10198016

  7. 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. PMID:27249052

  8. 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

  9. 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.

  10. Membrane protein resistance of oligo(ethylene oxide) self-assembled monolayers.

    PubMed

    Vaish, Amit; Vanderah, David J; Vierling, Ryan; Crawshaw, Fay; Gallagher, D Travis; Walker, Marlon L

    2014-10-01

    As part of an effort to develop biointerfaces for structure-function studies of integral membrane proteins (IMPs) a series of oligo(ethylene oxide) self-assembled monolayers (OEO-SAMs) were evaluated for their resistance to protein adsorption (RPA) of IMPs on Au and Pt. Spectroscopic ellipsometry (SE) was used to determine SAM thicknesses and compare the RPA of HS(CH2)3O(CH2CH2O)6CH3 (1), HS(CH2)3O(CH2CH2O)6H (2), [HS(CH2)3]2CHO(CH2CH2O)6CH3 (3) and [HS(CH2)3]2CHO(CH2CH2O)6H (4), assembled from water. For both substrates, SAM thicknesses for 1 to 4 were found to be comparable indicating SAMs with similar surface coverages and OEO chain order and packing densities. Fibrinogen (Fb), a soluble plasma protein, and rhodopsin (Rd), an integral membrane G-protein coupled receptor, adsorbed to the SAMs of 1, as expected from previous reports, but not to the hydroxy-terminated SAMs of 2 and 4. The methoxy-terminated SAMs of 3 were resistant to Fb but, surprisingly, not to Rd. The stark difference between the adsorption of Rd to the SAMs of 3 and 4 clearly indicate that a hydroxy-terminus of the OEO chain is essential for high RPA of IMPs. The similar thicknesses and high RPA of the SAMs of 2 and 4 show the conditions of protein resistance (screening the underlying substrate, packing densities, SAM order, and conformational mobility of the OEO chains) defined from previous studies on Au are applicable to Pt. In addition, the SAMs of 4, exhibiting the highest resistance to Fb and Rd, were placed in contact with undiluted fetal bovine serum for 2h. Low protein adsorption (≈12.4ng/cm(2)), obtained under these more challenging conditions, denote a high potential of the SAMs of 4 for various applications requiring the suppression of non-specific protein adsorption. PMID:25124834

  11. 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

  12. 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

  13. EFFECTS OF SOY PROTEIN AND ISOFLAVONES ON INSULIN RESISTANCE AND ADIPONECTIN IN MALE MONKEYS

    PubMed Central

    Wagner, Janice D.; Zhang, Li; Shadoan, Melanie K.; Kavanagh, Kylie; Chen, Haiying; Trenasari, Kristitianti; Kaplan, Jay R.; Adams, Michael R.

    2008-01-01

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

  14. 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. PMID:26578714

  15. 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

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

    PubMed Central

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

    2007-01-01

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

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

    PubMed

    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

  18. 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

  19. ArsR arsenic-resistance regulatory protein from Cupriavidus metallidurans CH34.

    PubMed

    Zhang, Yian-Biao; Monchy, Sébastien; Greenberg, Bill; Mergeay, Max; Gang, Oleg; Taghavi, Safiyh; van der Lelie, Daniel

    2009-08-01

    The Cupriavidus metallidurans CH34 arsR gene, which is part of the arsRIC(2)BC(1)HP operon, and its putative arsenic-resistance regulatory protein were identified and characterized. The arsenic-induced transcriptome of C. metallidurans CH34 showed that the genes most upregulated in the presence of arsenate were all located within the ars operon, with none of the other numerous heavy metal resistance systems present in CH34 being induced. A transcriptional fusion between the luxCDABE operon and the arsR promoter/operator (P/O) region was used to confirm the in vivo induction of the ars operon by arsenite and arsenate. The arsR gene was cloned into expression vectors allowing for the overexpression of the ArsR protein as either his-tagged or untagged protein. The ability of the purified ArsR proteins to bind to the ars P/O region was analyzed in vitro by gel mobility shift assays. ArsR showed an affinity almost exclusively to its own ars P/O region. Dissociation of ArsR and its P/O region was metal dependent, and based on decreasing degrees of dissociation three groups of heavy metals could be distinguished: As(III), Bi(III), Co(II), Cu(II), Ni(II); Cd(II); Pb(II) and Zn(II), while no dissociation was observed in the presence of As(V). PMID:19238575

  20. ArsR arsenic-resistance regulatory protein from Cupriavidus metallidurans CH34

    SciTech Connect

    Zhang, Y.; van der Lelie, D.; Monchy, S.; Greenberg, B.; Gang, O.; Taghavi, S.

    2009-08-01

    The Cupriavidus metallidurans CH34 arsR gene, which is part of the arsRIC{sub 2}BC{sub 1}HP operon, and its putative arsenic-resistance regulatory protein were identified and characterized. The arsenic-induced transcriptome of C. metallidurans CH34 showed that the genes most upregulated in the presence of arsenate were all located within the ars operon, with none of the other numerous heavy metal resistance systems present in CH34 being induced. A transcriptional fusion between the luxCDABE operon and the arsR promoter/operator (P/O) region was used to confirm the in vivo induction of the ars operon by arsenite and arsenate. The arsR gene was cloned into expression vectors allowing for the overexpression of the ArsR protein as either his-tagged or untagged protein. The ability of the purified ArsR proteins to bind to the ars P/O region was analyzed in vitro by gel mobility shift assays. ArsR showed an affinity almost exclusively to its own ars P/O region. Dissociation of ArsR and its P/O region was metal dependent, and based on decreasing degrees of dissociation three groups of heavy metals could be distinguished: As(III), Bi(III), Co(II), Cu(II), Ni(II); Cd(II); Pb(II) and Zn(II), while no dissociation was observed in the presence of As(V).

  1. 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

  2. Pentapeptide-repeat proteins that act as topoisomerase poison resistance factors have a common dimer interface

    PubMed Central

    Vetting, Matthew W.; Hegde, Subray S.; Zhang, Yong; Blanchard, John S.

    2011-01-01

    The protein AlbG is a self-resistance factor against albicidin, a nonribosomally encoded hybrid polyketide-peptide with antibiotic and phytotoxic properties produced by Xanthomonas albilineans. Primary-sequence analysis indicates that AlbG is a member of the pentapeptide-repeat family of proteins (PRP). The structure of AlbG from X. albilineans was determined at 2.0 Å resolution by SAD phasing using data collected from a single trimethyllead acetate derivative on a home source. AlbG folds into a right-handed quadrilateral β-helix composed of approximately eight semi-regular coils. The regularity of the β-­helix is blemished by a large loop/deviation in the β-helix between coils 4 and 5. The C-terminus of the β-helix is capped by a dimerization module, yielding a dimer with a 110 Å semi-collinear β-helical axis. This method of dimer formation appears to be common to all PRP proteins that confer resistance to topoisomerase poisons and contrasts with most PRP proteins, which are typically monomeric. PMID:21393830

  3. SDS-resistant aggregation of membrane proteins: application to the purification of the vesicular monoamine transporter.

    PubMed Central

    Sagné, C; Isambert, M F; Henry, J P; Gasnier, B

    1996-01-01

    The vesicular monoamine transporter, which catalyses a H+/ monoamine antiport in monoaminergic vesicle membrane, is a very hydrophobic intrinsic membrane protein. After solubilization, this protein was found to have a high tendency to aggregate, as shown by SDS/PAGE, especially when samples were boiled in the classical Laemmli buffer before electrophoresis. This behavior was analysed in some detail. The aggregation was promoted by high temperatures, organic solvents and acidic pH, suggesting that it resulted from the unfolding of structure remaining in SDS. The aggregates were very stable and could be dissociated only by suspension in anhydrous trifluoroacetic acid. This SDS-resistant aggregation behaviour was shared by very few intrinsic proteins of the chromaffin granule membrane. Consequently, a purification procedure was based on this property. A detergent extract of chromaffin granule membranes enriched in monoamine transporter was heated and the aggregates were isolated by size-exclusion HPLC in SDS. The aggregates, containing the transporter, were dissociated in the presence of trifluoroacetic acid and analysed on the same HPLC column. This strategy might be of general interest for the purification of membrane proteins that exhibit SDS-resistant aggregation. PMID:8670158

  4. Differential involvement of glutathione S-transferase mu 1 and multidrug resistance protein 1 in melanoma acquired resistance to vinca alkaloids.

    PubMed

    Attaoua, Chaker; Vincent, Laure-Anaïs; Abdel Jaoued, Aida; Hadj-Kaddour, Kamel; Baï, Qiang; De Vos, John; Vian, Laurence; Cuq, Pierre

    2015-02-01

    On account of its extreme intrinsic resistance to apoptosis and of its strong ability to become chemoresistant after a primary response to drugs, malignant melanoma (MM) is still a therapeutic challenge. We previously showed that glutathione S-transferase mu 1 (GSTM1) acts in synergy with multidrug resistance protein 1 (MRP1) to protect GSTM1-transfected human CAL1 melanoma cells from toxic effects of vincristine (VCR). Herein, we investigated the role of these proteins in the acquired resistance of CAL1 cells to vinca alkaloids (VAs). Resistant lines were established by continuous exposure (>1 year) of parental CAL1-wt cells to VCR, vindesine (VDS), or vinorelbine (VRB): CAL1R-VCR, CAL1R-VDS, CAL1R-VRB, respectively. All resistant lines displayed more than 10-fold increase in resistance to their selection VA, and specifically expressed GSTM1. Suggesting a direct interaction between this protein and VAs, each VA specifically decreased the GSTM1-mediated glutathione conjugation activity in cell lysates. Curcumin (GSTM1 inhibitor), BSO (glutathione synthesis inhibitor), and MK571 (MRP1 inhibitor) considerably reversed the acquired resistance to VCR and VDS, but not to VRB. Microarray data analysis revealed similar gene expression patterns of CAL1R-VCR and CAL1R-VDS, and a distinct one for CAL1R-VRB. These data suggest a differential involvement of GSTM1 and MRP1 in acquired resistance to VAs. A coordinated expression and activity of GSTM1 and MRP1 is required to protect CAL1 cells from VCR and VDS, while the simple expression of GSTM1 is sufficient, possibly by a direct drug/protein interaction, to confer resistance against VRB. PMID:25283245

  5. Ascites Increases Expression/Function of Multidrug Resistance Proteins in Ovarian Cancer Cells.

    PubMed

    Mo, Lihong; Pospichalova, Vendula; Huang, Zhiqing; Murphy, Susan K; Payne, Sturgis; Wang, Fang; Kennedy, Margaret; Cianciolo, George J; Bryja, Vitezslav; Pizzo, Salvatore V; Bachelder, Robin E

    2015-01-01

    Chemotherapy resistance is the major reason for the failure of ovarian cancer treatment. One mechanism behind chemo-resistance involves the upregulation of multidrug resistance (MDR) genes (ABC transporters) that effectively transport (efflux) drugs out of the tumor cells. As a common symptom in stage III/IV ovarian cancer patients, ascites is associated with cancer progression. However, whether ascites drives multidrug resistance in ovarian cancer cells awaits elucidation. Here, we demonstrate that when cultured with ascites derived from ovarian cancer-bearing mice, a murine ovarian cancer cell line became less sensitive to paclitaxel, a first line chemotherapeutic agent for ovarian cancer patients. Moreover, incubation of murine ovarian cancer cells in vitro with ascites drives efflux function in these cells. Functional studies show ascites-driven efflux is suppressible by specific inhibitors of either of two ABC transporters [Multidrug Related Protein (MRP1); Breast Cancer Related Protein (BCRP)]. To demonstrate relevance of our findings to ovarian cancer patients, we studied relative efflux in human ovarian cancer cells obtained from either patient ascites or from primary tumor. Immortalized cell lines developed from human ascites show increased susceptibility to efflux inhibitors (MRP1, BCRP) compared to a cell line derived from a primary ovarian cancer, suggesting an association between ascites and efflux function in human ovarian cancer. Efflux in ascites-derived human ovarian cancer cells is associated with increased expression of ABC transporters compared to that in primary tumor-derived human ovarian cancer cells. Collectively, our findings identify a novel activity for ascites in promoting ovarian cancer multidrug resistance. PMID:26148191

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

    PubMed

    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

  7. Towards identifying Brassica proteins involved in mediating resistance to Leptosphaeria maculans: a proteomics-based approach.

    PubMed

    Sharma, Nidhi; Hotte, Naomi; Rahman, Muhammad H; Mohammadi, Mohsen; Deyholos, Michael K; Kav, Nat N V

    2008-09-01

    To better understand the pathogen-stress response of Brassica species against the ubiquitous hemi-biotroph fungus Leptosphaeria maculans, we conducted a comparative proteomic analysis between blackleg-susceptible Brassica napus and blackleg-resistant Brassica carinata following pathogen inoculation. We examined temporal changes (6, 12, 24, 48 and 72 h) in protein profiles of both species subjected to pathogen-challenge using two-dimensional gel electrophoresis. A total of 64 proteins were found to be significantly affected by the pathogen in the two species, out of which 51 protein spots were identified using tandem mass spectrometry. The proteins identified included antioxidant enzymes, photosynthetic and metabolic enzymes, and those involved in protein processing and signaling. Specifically, we observed that in the tolerant B. carinata, enzymes involved in the detoxification of free radicals increased in response to the pathogen whereas no such increase was observed in the susceptible B. napus. The expression of genes encoding four selected proteins was validated using quantitative real-time PCR and an additional one by Western blotting. Our findings are discussed with respect to tolerance or susceptibility of these species to the pathogen. PMID:18668695

  8. 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

  9. Arabidopsis heterotrimeric G-protein regulates cell wall defense and resistance to necrotrophic fungi.

    PubMed

    Delgado-Cerezo, Magdalena; Sánchez-Rodríguez, Clara; Escudero, Viviana; Miedes, Eva; Fernández, Paula Virginia; Jordá, Lucía; Hernández-Blanco, Camilo; Sánchez-Vallet, Andrea; Bednarek, Pawel; Schulze-Lefert, Paul; Somerville, Shauna; Estevez, José Manuel; Persson, Staffan; Molina, Antonio

    2012-01-01

    The Arabidopsis heterotrimeric G-protein controls defense responses to necrotrophic and vascular fungi. The agb1 mutant impaired in the Gβ subunit displays enhanced susceptibility to these pathogens. Gβ/AGB1 forms an obligate dimer with either one of the Arabidopsis Gγ subunits (γ1/AGG1 and γ2/AGG2). Accordingly, we now demonstrate that the agg1 agg2 double mutant is as susceptible as agb1 plants to the necrotrophic fungus Plectosphaerella cucumerina. To elucidate the molecular basis of heterotrimeric G-protein-mediated resistance, we performed a comparative transcriptomic analysis of agb1-1 mutant and wild-type plants upon inoculation with P. cucumerina. This analysis, together with metabolomic studies, demonstrated that G-protein-mediated resistance was independent of defensive pathways required for resistance to necrotrophic fungi, such as the salicylic acid, jasmonic acid, ethylene, abscisic acid, and tryptophan-derived metabolites signaling, as these pathways were not impaired in agb1 and agg1 agg2 mutants. Notably, many mis-regulated genes in agb1 plants were related with cell wall functions, which was also the case in agg1 agg2 mutant. Biochemical analyses and Fourier Transform InfraRed (FTIR) spectroscopy of cell walls from G-protein mutants revealed that the xylose content was lower in agb1 and agg1 agg2 mutants than in wild-type plants, and that mutant walls had similar FTIR spectratypes, which differed from that of wild-type plants. The data presented here suggest a canonical functionality of the Gβ and Gγ1/γ2 subunits in the control of Arabidopsis immune responses and the regulation of cell wall composition. PMID:21980142

  10. 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

  11. The Mechanism of Herbicide Resistance in Tobacco Cells with a New Mutation in the QB Protein 1

    PubMed Central

    Sigematsu, Yoshio; Sato, Fumihiko; Yamada, Yasuyuki

    1989-01-01

    A new mutant of the psbA gene conferring resistance to 2-chloro-4-(ethylamino)-6-(isopropylamino)-s-triazine (atrazine) was obtained by selection of photomixotrophic tobacco (Nicotiana tabacum cv Samsun NN) cells. The 264th codon AGT (serine) in the wild psbA gene was changed to ACT (threonine) in these mutant tobacco cells. All other higher plants resistant to atrazine exhibit a change to GGT (glycine) in this codon. Measurements of Hill reaction activity and chlorophyll fluorescence showed that the threonine 264-containing plastoquinone serving as secondary stable electron acceptor of PSII (QB protein) had not only strong resistance to triazine-type herbicides but also moderate resistance to substituted urea-type herbicides. Threonine-type QB protein showed especially strong resistance to methoxylamino derivatives of the substituted urea herbicides. The projected secondary structures of the mutant QB proteins indicate that the cross-resistance of threonine 264 QB protein to triazine and urea herbicides is mainly due to a conformational change of the binding site for the herbicides. However, the glycine 264 QB protein is resistant to only triazine herbicides because of the absence of an hydroxyl group and not because of a conformational change. Images Figure 1 PMID:16666653

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

    NASA Astrophysics Data System (ADS)

    Talei, D.; Fotokian, M. H.

    2008-01-01

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

  13. 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...

  14. 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.

  15. 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

  16. Identification of the multidrug resistance-associated protein (mrp) related gene in red mullet (Mullus barbatus).

    PubMed

    Sauerborn, Roberta; Polancec, Darija Stupin; Zaja, Roko; Smital, Tvrtko

    2004-01-01

    Multixenobiotic resistance mechanism (MXR) in aquatic organisms is mediated by the activity of the P-glycoprotein (Pgp) transporter that binds and actively effluxes different chemicals out of cell. In addition to the Pgp, several other, non-Pgp transport proteins have been recently identified in different human and animal tissues. Given their characteristics and tissue distribution we hypothesized that members of the so-called multidrug resistance-associated protein (MRP) family may be expressed in aquatic organisms. This study attempted to identify MRP related genes in different tissues of several marine and freshwater bivalves (Mytilus galloprovincialis, Dreissena polymorpha, Anodonta cygnea) and fish species (Mullus barbatus, Cyprinus carpio, Salmo trutta). Following an alignment of known MRP1 and MRP2 human sequences, as well as the GenBank available mrp2 sequences from different animals, we determined highly conserved regions and used them to design three pairs of consensus primers. Total RNA was isolated, reverse transcribed to cDNA and the obtained cDNAs were PCR amplified with the corresponding primers. The amplified PCR products were sequenced and their homology compared with Pgp and MRP protein sequences from different species. The expression of MRP related mRNA was clearly identified only in liver tissue isolated from red mullet, with homologies at the protein level ranging from 75% to 76%. Described results clearly pointed at the possibility that at least in the red mullet MXR as a general defense mechanism may be mediated by the activities of at least two different types of transport proteins. PMID:15178032

  17. 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

  18. Organic hydroperoxide resistance protein and ergothioneine compensate for loss of mycothiol in Mycobacterium smegmatis mutants.

    PubMed

    Ta, Philong; Buchmeier, Nancy; Newton, Gerald L; Rawat, Mamta; Fahey, Robert C

    2011-04-01

    The mshA::Tn5 mutant of Mycobacterium smegmatis does not produce mycothiol (MSH) and was found to markedly overproduce both ergothioneine and an ~15-kDa protein determined to be organic hydroperoxide resistance protein (Ohr). An mshA(G32D) mutant lacking MSH overproduced ergothioneine but not Ohr. Comparison of the mutant phenotypes with those of the wild-type strain indicated the following: Ohr protects against organic hydroperoxide toxicity, whereas ergothioneine does not; an additional MSH-dependent organic hydroperoxide peroxidase exists; and elevated isoniazid resistance in the mutant is associated with both Ohr and the absence of MSH. Purified Ohr showed high activity with linoleic acid hydroperoxide, indicating lipid hydroperoxides as the likely physiologic targets. The reduction of oxidized Ohr by NADH was shown to be catalyzed by lipoamide dehydrogenase and either lipoamide or DlaT (SucB). Since free lipoamide and lipoic acid levels were shown to be undetectable in M. smegmatis, the bound lipoyl residues of DlaT are the likely source of the physiological dithiol reductant for Ohr. The pattern of occurrence of homologs of Ohr among bacteria suggests that the ohr gene has been distributed by lateral transfer. The finding of multiple Ohr homologs with various sequence identities in some bacterial genomes indicates that there may be multiple physiologic targets for Ohr proteins. PMID:21335456

  19. 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

  20. 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

  1. 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

  2. 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

  3. Host Resistance to Intracellular Infection: Mutation of Natural Resistance-associated Macrophage Protein 1 (Nramp1) Impairs Phagosomal Acidification

    PubMed Central

    Hackam, David J.; Rotstein, Ori D.; Zhang, Wei-jian; Gruenheid, Samantha; Gros, Philippe; Grinstein, Sergio

    1998-01-01

    The mechanisms underlying the survival of intracellular parasites such as mycobacteria in host macrophages remain poorly understood. In mice, mutations at the Nramp1 gene (for natural resistance-associated macrophage protein), cause susceptibility to mycobacterial infections. Nramp1 encodes an integral membrane protein that is recruited to the phagosome membrane in infected macrophages. In this study, we used microfluorescence ratio imaging of macrophages from wild-type and Nramp1 mutant mice to analyze the effect of loss of Nramp1 function on the properties of phagosomes containing inert particles or live mycobacteria. The pH of phagosomes containing live Mycobacterium bovis was significantly more acidic in Nramp1- expressing macrophages than in mutant cells (pH 5.5 ± 0.06 versus pH 6.6 ± 0.05, respectively; P <0.005). The enhanced acidification could not be accounted for by differences in proton consumption during dismutation of superoxide, phagosomal buffering power, counterion conductance, or in the rate of proton “leak”, as these were found to be comparable in wild-type and Nramp1-deficient macrophages. Rather, after ingestion of live mycobacteria, Nramp1-expressing cells exhibited increased concanamycin-sensitive H+ pumping across the phagosomal membrane. This was associated with an enhanced ability of phagosomes to fuse with vacuolar-type ATPase–containing late endosomes and/or lysosomes. This effect was restricted to live M. bovis and was not seen in phagosomes containing dead M. bovis or latex beads. These data support the notion that Nramp1 affects intracellular mycobacterial replication by modulating phagosomal pH, suggesting that Nramp1 plays a central role in this process. PMID:9670047

  4. 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.

  5. Protein and lipid analysis of detergent-resistant membranes isolated from bovine kidney.

    PubMed

    Bonnin, Stéphanie; El Kirat, Karim; Becchi, Michel; Dubois, Madeleine; Grangeasse, Christophe; Giraud, Claire; Prigent, Annie-France; Lagarde, Michel; Roux, Bernard; Besson, Françoise

    2003-12-01

    Detergent-resistant membranes (DRM) were prepared from bovine kidney cortex. The criterion used to test their purification was the increase in the activity of a GPI membrane-anchored protein, the alkaline phosphatase. Its association with specific proteins and lipids was tested. Two successive Triton X-100 treatments followed by purification on sucrose gradient at 4 degrees C were necessary to obtain DRM with a maximum of alkaline phosphatase activity and a typical protein pattern. A third Triton treatment did not alter this DRM composition. Among the enriched protein, we identified, by mass spectrometry, a microsomal dipeptidase, which was GPI membrane-anchored. Protein-kinase activities, mainly serine-kinase, were enriched during the DRM purification. Using the typical FTIR olefinic =C-H bands of the acyl chains, a global decrease in the unsaturation level of DRM lipids was observed as compared with total membranes. Three main phospholipids were identified in DRM. Their fatty acid compositions were determined by gas chromatography and compared with those of total membranes. The most enriched saturated fatty acid was palmitic acid (+44% for phosphatidylethanolamine, +52% for phosphatidylcholine and +49% for sphingomyelin), agreeing with a selection of specific phospholipids among the saturated ones during the DRM purification. PMID:14739076

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

    SciTech Connect

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

    2008-01-04

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

  7. The Giant Protein Ebh Is a Determinant of Staphylococcus aureus Cell Size and Complement Resistance

    PubMed Central

    Cheng, Alice G.; Missiakas, Dominique

    2014-01-01

    Staphylococcus aureus USA300, the clonal type associated with epidemic community-acquired methicillin-resistant S. aureus (MRSA) infections, displays the giant protein Ebh on its surface. Mutations that disrupt the ebh reading frame increase the volume of staphylococcal cells and alter the cross wall, a membrane-enclosed peptidoglycan synthesis and assembly compartment. S. aureus ebh variants display increased sensitivity to oxacillin (methicillin) as well as susceptibility to complement-mediated killing. Mutations in ebh are associated with reduced survival of mutant staphylococci in blood and diminished virulence in mice. We propose that Ebh, following its secretion into the cross wall, contributes to the characteristic cell growth and envelope assembly pathways of S. aureus, thereby enabling complement resistance and the pathogenesis of staphylococcal infections. PMID:24363342

  8. Upper-body obese women are resistant to postprandial stimulation of protein synthesis

    PubMed Central

    Liebau, Felix; Jensen, Michael D.; Nair, K. Sreekumaran; Rooyackers, Olav

    2014-01-01

    Background and Aims Upper-body, i.e. visceral, obesity is associated with insulin resistance and impaired protein synthesis. It is unclear whether postprandial stimulation of protein synthesis is affected by body fat distribution. We investigated the postprandial protein anabolic response in a cohort of obese women. Methods Participants were studied after an overnight fast and after a mixed meal, grouped as upper-body obese (UBO, waist-to-hip ratio, WHR, >0.85, n=6) vs. lower-body obese (LBO, WHR<0.80, n=7). Lipid and carbohydrate metabolism were assessed by measurements of plasma free fatty acids (FFA), insulin and glucose concentrations, and calculation of the Quicki index from fasting glucose and insulin values. Different labels of stable isotopes of phenylalanine were administered intravenously and orally, and leg and whole-body protein breakdown and synthesis were calculated from phenylalanine/tyrosine isotopic enrichments in femoral arterial and venous blood, using equations for steady-state kinetics. Data are denoted as mean±SD. Results Age (38 vs. 40, p=0.549) and body-mass index (33.7±1.9 vs. 35.0±1.8, p=0.241) were similar in both groups. UBO subjects had more visceral fat (p=0.002) and higher fat-free body mass (FFM) (p=0.015). Plasma insulin concentrations were greater in UBO than LBO women (p=0.013), and UBO were less insulin sensitive (Quicki=0.32±0.01 vs. 0.36±0.02, p=0.005). Protein kinetics across the leg were not different between groups. Fasting whole body protein balance was similarly negative in both groups (UBO −6.5±2.4 vs. LBO −7.6±0.9 μmol/kgFFM/h, p=1.0). Postprandially, whole body protein balance became less positive in UBO than in LBO (14.8±3.7 vs. 20.2±3.7 μmol/kgFFM/h, p=0.017). Conclusions Whole-body protein balance following a meal is less positive in upper-body obese, insulin-resistant, women than in lower-body obese women. PMID:24269078

  9. Leucine Supplementation Improves Acquired Growth Hormone Resistance in Rats with Protein-Energy Malnutrition

    PubMed Central

    Wang, Xinying; Zhao, Jie; Wan, Xiao; Zhang, Li; Wu, Chao; Li, Ning; Li, Jieshou

    2015-01-01

    Background Protein-energy malnutrition (PEM) can lead to growth hormone (GH) resistance. Leucine supplementation diets have been shown to increase protein synthesis in muscles. Our study aimed at investigating if long-term leucine supplementation could modulate GH-insulin-like growth factor (IGF)-1 system function and mammalian target of rapamycin (mTOR)-related signal transduction in skeletal muscles in a rat model of severe malnutrition. Methodology/Principal Findings Male Sprague-Dawley rats (n = 50; weight, 302 ± 5 g) were divided into 5 treatment groups, including 2 control groups (a normal control group that was fed chow and ad libitum water [CON, n = 10] and a malnourished control group [MC, n = 10] that was fed a 50% chow diet). After undergoing a weight loss stage for 4 weeks, rats received either the chow diet (MC-CON, n = 10), the chow diet supplemented with low-dose leucine (MC-L, n = 10), or the chow diet supplemented with high-dose leucine (MC-H, n = 10) for 2 weeks. The muscle masses of the gastrocnemius, soleus, and extensor digitorum longus were significantly reduced in the MC group. Re-feeding increased muscle mass, especially in the MC-L and MC-H groups. In the MC group, serum IGF-1, IGF-binding protein (IGFBP)-3, and hepatic growth hormone receptor (GHR) levels were significantly decreased and phosphorylation of the downstream anabolic signaling effectors protein kinase B (Akt), mTOR, and ribosomal protein S6 kinase 1 (S6K1) were significantly lower than in other groups. However, serum IGF-1 and IGF binding protein (IGFBP)-3 concentrations and hepatic growth hormone receptor (GHR) levels were significantly higher in the MC-L and MC-H groups than in the MC-CON group, and serum IGFBP-1 levels was significantly reduced in the MC-L and MC-H groups. These changes were consistent with those observed for hepatic mRNA expression levels. Phosphorylation of the downstream anabolic signaling effectors Akt, mTOR, and S6K1 were also significantly higher in

  10. 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

  11. Reduced membrane protein associated with resistance of human squamous carcinoma cells to methotrexate and cis-platinum.

    PubMed

    Bernal, S D; Speak, J A; Boeheim, K; Dreyfuss, A I; Wright, J E; Teicher, B A; Rosowsky, A; Tsao, S W; Wong, Y C

    1990-06-01

    A membrane protein recognized by monoclonal antibody SQM1 was identified in human squamous carcinomas, including those originating in the head and neck (SqCHN), lung and cervix. Cell lines derived from SqCHN of previously untreated patients expressed high amounts of this protein. In contrast, many cell lines established from SqCHN of patients previously treated with chemotherapy and/or radiation showed diminished amounts of this SQM1 protein. The expression of SQM1 antigen was determined in several SqCHN cell lines made resistant by exposure to methotrexate (MTX) in vitro. The parent cell lines all exhibited strong binding to SQM1 antibody. The MTX-resistant sublines showed much lower membrane binding of SQM1. The lowest SQM1 reactivity was found in cell lines with high resistance to MTX and with diminished rate of MTX transport. Some highly MTX-resistant cell lines which had high levels of dihydrofolate reductase, but which retained a high rate of MTX transport, also retained high levels of SQM1 binding. Reduced SQM1 protein was also found in SqCHN cells which developed resistance to the alkylating drug cis-latinum (CDDP) and which showed reduced membrane transport of CDDP. Cell growth kinetics and non-specific antigenic shifts were not responsible for the differences in SQM1 binding between the parent cell lines and their drug-resistant sublines. The finding of a novel protein which is reduced in cells resistant to MTX and CDDP could contribute to our understanding of the basic mechanisms of drug resistance. By detecting SQM1 protein in clinical specimens, it may be possible to monitor the development of drug resistance in tumors. PMID:2195318

  12. 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.

  13. Regulation of extracellular copper-binding proteins in copper-resistant and copper-sensitive mutants of Vibrio alginolyticus.

    PubMed Central

    Harwood, V J; Gordon, A S

    1994-01-01

    Extracellular proteins of wild-type Vibrio alginolyticus were compared with those of copper-resistant and copper-sensitive mutants. One copper-resistant mutant (Cu40B3) constitutively produced an extracellular protein with the same apparent molecular mass (21 kDa) and chromatographic behavior as copper-binding protein (CuBP), a copper-induced supernatant protein which has been implicated in copper detoxification in wild-type V. alginolyticus. Copper-sensitive V. alginolyticus mutants displayed a range of alterations in supernatant protein profiles. CuBP was not detected in supernatants of one copper-sensitive mutant after cultures had been stressed with 50 microM copper. Increased resistance to copper was not induced by preincubation with subinhibitory levels of copper in the wild type or in the copper-resistant mutant Cu40B3. Copper-resistant mutants maintained the ability to grow on copper-amended agar after 10 or more subcultures on nonselective agar, demonstrating the stability of the phenotype. A derivative of Cu40B3 with wild-type sensitivity to copper which no longer constitutively expressed CuBP was isolated. The simultaneous loss of both constitutive CuBP production and copper resistance in Cu40B3 indicates that constitutive CuBP production is necessary for copper resistance in this mutant. These data support the hypothesis that the extracellular, ca. 20-kDa protein(s) of V. alginolyticus is an important factor in survival and growth of the organism at elevated copper concentrations. The range of phenotypes observed in copper-resistant and copper-sensitive V. alginolyticus indicate that altered sensitivity to copper was mediated by a variety of physiological changes. Images PMID:8031076

  14. Regulation of Multidrug Resistance Proteins by Genistein in a Hepatocarcinoma Cell Line: Impact on Sorafenib Cytotoxicity

    PubMed Central

    Rigalli, Juan Pablo; Ciriaci, Nadia; Arias, Agostina; Ceballos, María Paula; Villanueva, Silvina Stella Maris; Luquita, Marcelo Gabriel; Mottino, Aldo Domingo; Ghanem, Carolina Inés; Catania, Viviana Alicia; Ruiz, María Laura

    2015-01-01

    Hepatocellular carcinoma (HCC) is the fifth most frequent cancer worldwide. Sorafenib is the only drug available that improves the overall survival of HCC patients. P-glycoprotein (P-gp), Multidrug resistance-associated proteins 2 and 3 (MRP2 and 3) and Breast cancer resistance protein (BCRP) are efflux pumps that play a key role in cancer chemoresistance. Their modulation by dietary compounds may affect the intracellular accumulation and therapeutic efficacy of drugs that are substrates of these transporters. Genistein (GNT) is a phytoestrogen abundant in soybean that exerts its genomic effects through Estrogen-Receptors and Pregnane-X-Receptor (PXR), which are involved in the regulation of the above-mentioned transporters. We evaluated the effect of GNT on the expression and activity of P-gp, MRP2, MRP3 and BCRP in HCC-derived HepG2 cells. GNT (at 1.0 and 10 μM) increased P-gp and MRP2 protein expression and activity, correlating well with an increased resistance to sorafenib cytotoxicity as detected by the methylthiazole tetrazolium (MTT) assay. GNT induced P-gp and MRP2 mRNA expression at 10 but not at 1.0 μM concentration suggesting a different pattern of regulation depending on the concentration. Induction of both transporters by 1.0 μM GNT was prevented by cycloheximide, suggesting translational regulation. Downregulation of expression of the miR-379 by GNT could be associated with translational regulation of MRP2. Silencing of PXR abolished P-gp induction by GNT (at 1.0 and 10 μM) and MRP2 induction by GNT (only at 10 μM), suggesting partial mediation of GNT effects by PXR. Taken together, the data suggest the possibility of nutrient-drug interactions leading to enhanced chemoresistance in HCC when GNT is ingested with soy rich diets or dietary supplements. PMID:25781341

  15. Multidrug resistance-associated proteins: Export pumps for conjugates with glutathione, glucuronate or sulfate.

    PubMed

    Homolya, László; Váradi, András; Sarkadi, Balázs

    2003-01-01

    Many endogenous or xenobiotic lipophilic substances are eliminated from the cells by the sequence of oxidation, conjugation to an anionic group (glutathione, glucuronate or sulfate) and transport across the plasma membrane into the extracellular space. The latter step is mediated by integral membrane glycoproteins belonging to the superfamily of ATP-Binding Cassette (ABC) transporters. A subfamily, referred as ABCC, includes the famous/infamous cystic fibrosis transmembrane regulator (CFTR), the sulfonylurea receptors (SUR 1 and 2), and the multidrug resistance-associated proteins (MRPs). The name of the MRPs refers to their potential role in clinical multidrug resistance, a phenomenon that hinders the effective chemotherapy of tumors. The MRPs that have been functionally characterized so far share the property of ATP-dependent export pumps for conjugates with glutathione (GSH), glucuronate or sulfate. MRP1 and MRP2 are also mediating the cotransport of unconjugated amphiphilic compounds together with free GSH. MRP3 preferentially transports glucuronides but not glutathione S-conjugates or free GSH. MRP1 and MRP2 also contribute to the control of the intracellular glutathione disulfide (GSSG) level. Although these proteins are low affinity GSSG transporters, they can play essential role in response to oxidative stress when the activity of GSSG reductase becomes rate limiting. The human MRP4, MRP5 and MRP6 have only partially been characterized. However, it has been revealed that MRP4 can function as an efflux pump for cyclic nucleotides and nucleoside analogues, used as anti-HIV drugs. MRP5 also transports GSH conjugates, nucleoside analogues, and possibly heavy metal complexes. Transport of glutathione S-conjugates mediated by MRP6, the mutation of which causes pseudoxantoma elasticum, has recently been shown. In summary, numerous members of the multidrug resistance-associated protein family serve as export pumps that prevent the accumulation of anionic conjugates

  16. 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

  17. 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

  18. 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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  20. 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.

  1. The Nramp1 protein and its role in resistance to infection and macrophage function.

    PubMed

    Canonne-Hergaux, F; Gruenheid, S; Govoni, G; Gros, P

    1999-01-01

    Susceptibility to infectious diseases is under genetic control in humans. Animal models provide an ideal tool to study the genetic component of susceptibility and to identify candidate genes that can then be tested for association or linkage studies in human populations from endemic areas of disease. The Nramp1 gene was isolated by positional cloning the host resistance locus Bcg/Ity/Lsh, and mutations at this locus impair the resistance of mice to infections with intracellular parasites, such as Salmonella, Leishmania, and Mycobacterium. Allelic variants at the human Nramp1 homologue have recently been found to be associated with susceptibility to tuberculosis and leprosy in humans. The Nramp1 protein is an integral membrane protein expressed exclusively in the lysosomal compartment of monocytes and macrophages. After phagocytosis, Nramp1 is targeted to the membrane of the microbe-containing phagosome, where it may modify the intraphagosomal milieu to affect microbial replication. Although the biochemical mechanism of action of Nramp1 at that site remains unknown, Nramp homologues have been identified in many other animal species and actually define a protein family conserved from bacteria to humans. Some of these homologues have been shown to be divalent cation transporters. Recently, a second member of the mammalian Nramp family, Nramp2, was discovered and shown to be mutated in animal models of iron deficiency. The Nramp2 protein was subsequently shown to be the major transferrin-independent iron uptake system of the intestine. Together, these results suggest that Nramp1 may control intracellular microbial replication by actively removing iron or other divalent cations from the phagosomal space. PMID:10417735

  2. 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

  3. 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

  4. 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

  5. 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

  6. 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

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

    PubMed Central

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

    2002-01-01

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

  8. Hessian fly larval attack triggers elevated expression of disease resistance dirigent-like protein-encoding gene, HfrDrd, in resistant wheat.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Dirigent proteins regulate coupling of monolignol plant phenols to generate the structural cell wall polymers lignins and lignans that are involved in structural fortification of cell wall and defense against pathogens and pests. Microarray expression profiling of resistant wheat (Triticum aestivum)...

  9. 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

  10. Overexpression of the Bacillus thuringiensis (Bt) Cry2Aa2 protein in chloroplasts confers resistance to plants against susceptible and Bt-resistant insects

    PubMed Central

    Kota, Madhuri; Daniell, Henry; Varma, Sam; Garczynski, Stephen F.; Gould, Fred; Moar, William J.

    1999-01-01

    Evolving levels of resistance in insects to the bioinsecticide Bacillus thuringiensis (Bt) can be dramatically reduced through the genetic engineering of chloroplasts in plants. When transgenic tobacco leaves expressing Cry2Aa2 protoxin in chloroplasts were fed to susceptible, Cry1A-resistant (20,000- to 40,000-fold) and Cry2Aa2-resistant (330- to 393-fold) tobacco budworm Heliothis virescens, cotton bollworm Helicoverpa zea, and the beet armyworm Spodoptera exigua, 100% mortality was observed against all insect species and strains. Cry2Aa2 was chosen for this study because of its toxicity to many economically important insect pests, relatively low levels of cross-resistance against Cry1A-resistant insects, and its expression as a protoxin instead of a toxin because of its relatively small size (65 kDa). Southern blot analysis confirmed stable integration of cry2Aa2 into all of the chloroplast genomes (5,000–10,000 copies per cell) of transgenic plants. Transformed tobacco leaves expressed Cry2Aa2 protoxin at levels between 2% and 3% of total soluble protein, 20- to 30-fold higher levels than current commercial nuclear transgenic plants. These results suggest that plants expressing high levels of a nonhomologous Bt protein should be able to overcome or at the very least, significantly delay, broad spectrum Bt-resistance development in the field. PMID:10051556

  11. 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.

  12. 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

  13. Leucine-rich protein 130 contributes to apoptosis resistance of human hepatocarcinoma cells.

    PubMed

    Michaud, Mickaël; Barakat, Stéphane; Magnard, Sandrine; Rigal, Dominique; Baggetto, Loris G

    2011-01-01

    LRP130 is a ubiquitous protein involved in cellular homeostasis, microtubule alteration, and transactivation of a few multidrug resistance genes. Its role in resistance to apoptosis in HepG2 and HUH7 hepatocarcinoma cells was investigated. Using shRNA-producing lentiviruses to down-regulate the LRP130 gene, we showed that i) LRP130 did not affect the capacity of hepatocarcinoma cells to extrude drugs since LRP130 down-regulation was insufficient to significantly reduce P-glycoprotein production in these cells, and ii) the expression of 11 apoptosis-related genes measured by PCR-array was significantly reduced. Interestingly, six of these genes encode extrinsic pathway proapoptotic proteins whose expression was higher in LRP130-non producing than in LRP130-producing HepG2 cells. Fluorescence microscopy confirmed this new anti-apoptotic role of LRP130, which is strengthened by a significantly reduced cytochrome c oxidase activity in LRP130-down-regulated hepatocarcinoma cells. PMID:21109938

  14. 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

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

    PubMed Central

    Azevedo, Hátylas; Moreira-Filho, Carlos Alberto

    2015-01-01

    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. PMID:26582089

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

    PubMed

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

    2015-01-01

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

  17. Multidrug resistance proteins (MRPs/ABCCs) in cancer chemotherapy and genetic diseases.

    PubMed

    Chen, Zhe-Sheng; Tiwari, Amit K

    2011-09-01

    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 C(4) and cAMP is mediated by one or more of the MRPs. Finally, mutations in several MRPs are associated with human genetic disorders. In this minireview, 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

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

    PubMed

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

    2015-01-01

    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

  19. 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

  20. 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. PMID:22705884

  1. 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

  2. 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

  3. 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

  4. 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

  5. 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

  6. Modulation of function of multidrug resistance associated-proteins by Kaempferia parviflora extracts and their components.

    PubMed

    Patanasethanont, Denpong; Nagai, Junya; Matsuura, Chie; Fukui, Kyoko; Sutthanut, Khaetthareeya; Sripanidkulchai, Bung-orn; Yumoto, Ryoko; Takano, Mikihisa

    2007-07-01

    In this study, the effects of extracts and flavone derivatives from the rhizome of Kaempferia parviflora on multidrug resistance associated-proteins (MRP)-mediated transport in A549 cells were examined. The cells employed express MRP1 and MRP2, but not P-glycoprotein. The cellular accumulation of calcein, an MRP substrate, was significantly increased by various MRP inhibitors without being affected by verapamil, a typical P-glycoprotein inhibitor. Ethanol and aqueous extracts from K. parviflora rhizome increased the accumulation of calcein and doxorubicin in A549 cells in a concentration-dependent manner. The inhibitory potency of the ethanol extract for MRP function was greater than that of the aqueous extract. Among six flavone derivatives isolated from K. parviflora rhizome, 5,7-dimethoxyflavone exhibited a maximal stimulatory effect on the accumulation of doxorubicin in A549 cells. The accumulation of doxorubicin was increased by four flavone derivatives without 5-hydroxy group, but not by the other two flavone derivatives with 5-hydroxy group. In addition, 5,7-dimethoxyflavone and 3,5,7,3',4'-pentamethoxyflavone decreased resistance to doxorubicin in A549 cells. These findings indicate that extracts and flavone derivatives from the rhizome of K. parviflora suppress MRP function, and therefore may be useful as modulators of multidrug resistance in cancer cells. PMID:17481606

  7. 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

  8. 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

  9. Perfluorooctane sulfonate-induced insulin resistance is mediated by protein kinase B pathway.

    PubMed

    Qiu, Tianming; Chen, Min; Sun, Xiance; Cao, Jun; Feng, Chang; Li, Dandan; Wu, Wei; Jiang, Liping; Yao, Xiaofeng

    2016-09-01

    Perfluorooctane sulfonate (PFOS), a persistent organic pollutant, is blamed to be associated with the incidence of insulin resistance in the general human population. In this study, we found that PFOS inhibited the phosphorylation and activation of protein kinase B (AKT), a key mediator of cellular insulin sensitivity, in human hepatoma HepG2 cells. The mRNA level of the gluconeogenic gene PEPCK, a downstream target gene of AKT, was increased in PFOS-treated cells. Due to stimulated gluconeogenesis, insulin-stimulated glucose uptake was decreased in HepG2 cells. In our previous study, we found that PFOS disturbed autophagy in HepG2 cells. We proposed that PFOS could inhibit the activation of AKT through inhibiting mTORC2, a key regulator of autophagy. In this study, we found that the levels of triglyceride were increased in HepG2 cells. PFOS-induced accumulation of hepatic lipids also contributed to the inhibition of AKT. Eventually, the inhibition of AKT led to insulin resistance in PFOS-treated cells. Our data would provide new mechanistic insights into PFOS-induced hepatic insulin resistance. PMID:27363333

  10. 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

  11. 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

  12. Molecular pathways: mitogen-activated protein kinase pathway mutations and drug resistance.

    PubMed

    Pritchard, Antonia L; Hayward, Nicholas K

    2013-05-01

    Receptor tyrosine kinases are a diverse family of transmembrane proteins that can activate multiple pathways upon ligation of the receptor, one of which is the series of mitogen-activated protein kinase (MAPK) signaling cascades. The MAPK pathways play critical roles in a wide variety of cancer types, from hematologic malignancies to solid tumors. Aberrations include altered expression levels and activation states of pathway components, which can sometimes be attributable to mutations in individual members. The V600E mutation of BRAF was initially described in 2002 and has been found at particularly high frequency in melanoma and certain subtypes of colorectal cancer. In the relatively short time since this discovery, a family of drugs has been developed that specifically target this mutated BRAF isoform, which, after results from phase I/II and III clinical trials, was granted U.S. Food and Drug Administration approval in August 2011. Although these drugs produce clinically meaningful increases in progression-free and overall survival, due to acquired resistance they have not improved mortality rates. New drugs targeting other members of the MAPK pathways are in clinical trials or advanced stages of development. It is hoped that combination therapies of these new drugs in conjunction with BRAF inhibitors will counteract the mechanisms of resistance and provide cures. The clinical implementation of next-generation sequencing is leading to a greater understanding of the genetic architecture of tumors, along with acquired mechanisms of drug resistance, which will guide the development of tumor-specific inhibitors and combination therapies in the future. PMID:23406774

  13. 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

  14. Resistance of Neisseria meningitidis to Human Serum Depends on T and B Cell Stimulating Protein B

    PubMed Central

    Müller, Maike G.; Moe, Nina E.; Richards, Phillip Q.

    2015-01-01

    The ability of the human bacterial pathogen Neisseria meningitidis to cause invasive disease depends on survival in the bloodstream via mechanisms to suppress complement activation. In this study, we show that prophage genes coding for T and B cell stimulating protein B (TspB), which is an immunoglobulin-binding protein, are essential for survival of N. meningitidis group B strain H44/76 in normal human serum (NHS). H44/76 carries three genes coding for TspB. Mutants having all tspB genes inactivated did not survive in >5% NHS or IgG-depleted NHS. TspB appeared to inhibit IgM-mediated activation of the classical complement pathway, since survival of the tspB triple knockout was the same as that of the parent strain or a complemented mutant when the classical pathway was inactivated by depleting NHS of C1q and was increased in IgM-depleted NHS. A mutant solely carrying tspB gene nmbh4476_0681 was as resistant as the parent strain, while mutants carrying only nmbh4476_0598 or nmbh4476_1698 were killed in ≥5% NHS. The phenotype associated with TspB is formation of a matrix containing TspB, IgG, and DNA that envelopes aggregates of bacteria. Recombinant proteins corresponding to particular subdomains of TspB were found to have human IgG Fcγ- and/or DNA-binding activity, but only TspB derivatives containing both domains formed large, biofilm-like aggregates when combined with purified IgG and DNA. Recognizing the role of TspB in serum resistance may lead to a better understanding of why strains that carry tspB genes are associated with invasive meningococcal disease. PMID:25583528

  15. Modulation of autophagy signaling with resistance exercise and protein ingestion following short-term energy deficit.

    PubMed

    Smiles, William J; Areta, José L; Coffey, Vernon G; Phillips, Stuart M; Moore, Daniel R; Stellingwerff, Trent; Burke, Louise M; Hawley, John A; Camera, Donny M

    2015-09-01

    Autophagy contributes to remodeling of skeletal muscle and is sensitive to contractile activity and prevailing energy availability. We investigated changes in targeted genes and proteins with roles in autophagy following 5 days of energy balance (EB), energy deficit (ED), and resistance exercise (REX) after ED. Muscle biopsies from 15 subjects (8 males, 7 females) were taken at rest following 5 days of EB [45 kcal·kg fat free mass (FFM)(-1)·day(-1)] and 5 days of ED (30 kcal·kg FFM(-1)·day(-1)). After ED, subjects completed a bout of REX and consumed either placebo (PLA) or 30 g whey protein (PRO) immediately postexercise. Muscle biopsies were obtained at 1 and 4 h into recovery in each trial. Resting protein levels of autophagy-related gene protein 5 (Atg5) decreased after ED compared with EB (∼23%, P < 0.001) and remained below EB from 1 to 4 h postexercise in PLA (∼17%) and at 1 h in PRO (∼18%, P < 0.05). In addition, conjugated Atg5 (cAtg12) decreased below EB in PLA at 4 h (∼20, P < 0.05); however, its values were increased above this time point in PRO at 4 h alongside increases in FOXO1 above EB (∼22-26%, P < 0.05). Notably, these changes were subsequent to increases in unc-51-like kinase 1(Ser757) phosphorylation (∼60%) 1 h postexercise in PRO. No significant changes in gene expression of selected autophagy markers were found, but EGR-1 increased above ED and EB in PLA (∼417-864%) and PRO (∼1,417-2,731%) trials 1 h postexercise (P < 0.001). Postexercise protein availability, compared with placebo, can selectively promote autophagic responses to REX in ED. PMID:26136534

  16. Combinatorial protein engineering of proteolytically resistant mesotrypsin inhibitors as candidates for cancer therapy.

    PubMed

    Cohen, Itay; Kayode, Olumide; Hockla, Alexandra; Sankaran, Banumathi; Radisky, Derek C; Radisky, Evette S; Papo, Niv

    2016-05-15

    Engineered protein therapeutics offer advantages, including strong target affinity, selectivity and low toxicity, but like natural proteins can be susceptible to proteolytic degradation, thereby limiting their effectiveness. A compelling therapeutic target is mesotrypsin, a protease up-regulated with tumour progression, associated with poor prognosis, and implicated in tumour growth and progression of many cancers. However, with its unique capability for cleavage and inactivation of proteinaceous inhibitors, mesotrypsin presents a formidable challenge to the development of biological inhibitors. We used a powerful yeast display platform for directed evolution, employing a novel multi-modal library screening strategy, to engineer the human amyloid precursor protein Kunitz protease inhibitor domain (APPI) simultaneously for increased proteolytic stability, stronger binding affinity and improved selectivity for mesotrypsin inhibition. We identified a triple mutant APPIM17G/I18F/F34V, with a mesotrypsin inhibition constant (Ki) of 89 pM, as the strongest mesotrypsin inhibitor yet reported; this variant displays 1459-fold improved affinity, up to 350 000-fold greater specificity and 83-fold improved proteolytic stability compared with wild-type APPI. We demonstrated that APPIM17G/I18F/F34V acts as a functional inhibitor in cell-based models of mesotrypsin-dependent prostate cancer cellular invasiveness. Additionally, by solving the crystal structure of the APPIM17G/I18F/F34V-mesotrypsin complex, we obtained new insights into the structural and mechanistic basis for improved binding and proteolytic resistance. Our study identifies a promising mesotrypsin inhibitor as a starting point for development of anticancer protein therapeutics and establishes proof-of-principle for a novel library screening approach that will be widely applicable for simultaneously evolving proteolytic stability in tandem with desired functionality for diverse protein scaffolds. PMID:26957636

  17. 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

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

    PubMed

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

    2015-01-01

    Powdery mildew fungi form feeding structures called haustoria inside epidermal cells of host plants to extract photosynthates for their epiphytic growth and reproduction. The haustorium is encased by an interfacial membrane termed the extrahaustorial membrane (EHM). The atypical resistance protein RPW8.2 from Arabidopsis is specifically targeted to the EHM where RPW8.2 activates haustorium-targeted (thus broad-spectrum) resistance against powdery mildew fungi. EHM-specific localization of RPW8.2 suggests the existence of an EHM-oriented protein/membrane trafficking pathway during EHM biogenesis. However, the importance of this specific trafficking pathway for host defense has not been evaluated via a genetic approach without affecting other trafficking pathways. Here, we report that expression of EHM-oriented, nonfunctional RPW8.2 chimeric proteins exerts dominant negative effect over functional RPW8.2 and potentially over other EHM-localized defense proteins, thereby compromising both RPW8.2-mediated and basal resistance to powdery mildew. Thus, our results highlight the importance of the EHM-oriented protein/membrane trafficking pathway for host resistance against haustorium-forming pathogens such as powdery mildew fungi. PMID:25830634

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

    PubMed

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

    2016-03-01

    The internalization of some oomycete and fungal pathogen effectors into host plant cells has been reported to be blocked by proteins that bind to the effectors' cell entry receptor, phosphatidylinositol-3-phosphate (PI3P). This finding suggested a novel strategy for disease control by engineering plants to secrete PI3P-binding proteins. In this study, we tested this strategy using the chocolate tree Theobroma cacao. Transient expression and secretion of four different PI3P-binding proteins in detached leaves of T. cacao greatly reduced infection by two oomycete pathogens, Phytophthora tropicalis and Phytophthora palmivora, which cause black pod disease. Lesion size and pathogen growth were reduced by up to 85%. Resistance was not conferred by proteins lacking a secretory leader, by proteins with mutations in their PI3P-binding site, or by a secreted PI4P-binding protein. Stably transformed, transgenic T. cacao plants expressing two different PI3P-binding proteins showed substantially enhanced resistance to both P. tropicalis and P. palmivora, as well as to the fungal pathogen Colletotrichum theobromicola. These results demonstrate that secretion of PI3P-binding proteins is an effective way to increase disease resistance in T. cacao, and potentially in other plants, against a broad spectrum of pathogens. PMID:26214158

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

    PubMed

    Diner, Elie J; Hayes, Christopher S

    2009-02-20

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

  1. 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. PMID:24965864

  2. 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.

  3. 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.

  4. Structure and Function of the E. coli Protein YmgB: a Protein Critical for Biofilm Formation and Acid-resistance

    PubMed Central

    Lee, Jintae; Page, Rebecca; García-Contreras, Rodolfo; Palermino, Jeanne-Marie; Zhang, Xue-Song; Doshi, Ojus; Wood, Thomas K.; Peti, Wolfgang

    2007-01-01

    The Escherichia coli gene cluster ymgABC was identified in transcriptome studies to play a role in biofilm development and stability. In this study we show that YmgB represses biofilm formation in rich medium containing glucose, decreases cellular motility, and protects the cell from acid indicating that YmgB plays a major role in acid-resistance in E. coli. Our data also shows that these phenotypes are potentially mediated through interactions with the important cell signal indole. In addition, gel shift assays suggest that YmgB may be a non-specific DNA-binding protein. Using nickel-enrichment DNA microarrays, we show that YmgB binds, either directly or indirectly via a second protein, 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 Å resolution. YmgB is a biological dimer that is structurally homologous to the E. coli gene regulatory protein Hha, despite its low sequence identity of only 5%. This supports our DNA microarray data 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. PMID:17765265

  5. 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

  6. 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. PMID:25340555

  7. Role of the Outer Membrane Protein OprD2 in Carbapenem-Resistance Mechanisms of Pseudomonas aeruginosa

    PubMed Central

    Shen, Jilu; Pan, Yaping; Fang, Yaping

    2015-01-01

    We investigated the relationship between the outer membrane protein OprD2 and carbapenem-resistance in 141 clinical isolates of Pseudomonas aeruginosa collected between January and December 2013 from the First Affiliated Hospital of Anhui Medical University in China. Agar dilution methods were employed to determine the minimum inhibitory concentration of meropenem (MEM) and imipenem (IMP) for P. aeruginosa. The gene encoding OprD2 was amplified from141 P. aeruginosa isolates and analyzed by PCR and DNA sequencing. Differences between the effects of IMPR and IMPS groups on the resistance of the P. aeruginosa were observed by SDS-poly acrylamide gel electrophoresis (SDS-PAGE). Three resistance types were classified in the 141 carbapenem-resistant P. aeruginosa (CRPA) isolates tested, namely IMPRMEMR (66.7%), IMPRMEMS (32.6%), and IMPRMEMS (0.7%). DNA sequencing revealed significant diverse gene mutations in the OprD2-encoding gene in these strains. Thirty-four strains had large fragment deletions in the OprD2gene, in 6 strains the gene contained fragment inserts, and in 96 resistant strains, the gene featured small fragment deletions or multi-site mutations. Only 4 metallo-β-lactamase strains and 1 imipenem-sensitive (meropenem-resistant) strain showed a normal OprD2 gene. Using SDS-PAGE to detect the outer membrane protein in 16 CRPA isolates, it was found that 10 IMPRMEMR strains and 5 IMPRMEMS strains had lost the OprD2 protein, while the IMPSMEMR strain contained a normal 46-kDa protein. In conclusion, mutation or loss of the OprD2-encoding gene caused the loss of OprD2, which further led to carbapenem-resistance of P. aeruginosa. Our findings provide insights into the mechanism of carbapenem resistance in P. aeruginosa. PMID:26440806

  8. 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

  9. Nuclease-Resistant c-di-AMP Derivatives That Differentially Recognize RNA and Protein Receptors.

    PubMed

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

    2016-02-16

    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 activates specific pathways and mediates 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 analogues, 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 analogues that bind both the ydaO RNA and GdpP protein with high affinity, whereas symmetrically modified ribose analogues 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 analogues could be useful as chemical tools to specifically target subsections of second-messenger signaling pathways. PMID:26789423

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

    PubMed Central

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

    2015-01-01

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

  11. 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

  12. PTEN Protein Loss and Clinical Outcome from Castration-resistant Prostate Cancer Treated with Abiraterone Acetate

    PubMed Central

    Ferraldeschi, Roberta; Nava Rodrigues, Daniel; Riisnaes, Ruth; Miranda, Susana; Figueiredo, Ines; Rescigno, Pasquale; Ravi, Praful; Pezaro, Carmel; Omlin, Aurelius; Lorente, David; Zafeiriou, Zafeiris; Mateo, Joaquin; Altavilla, Amelia; Sideris, Spyridon; Bianchini, Diletta; Grist, Emily; Thway, Khin; Perez Lopez, Raquel; Tunariu, Nina; Parker, Chris; Dearnaley, David; Reid, Alison; Attard, Gerhardt; de Bono, Johann

    2015-01-01

    Background Loss of the tumor suppressor phosphatase and tensin homolog (PTEN) occurs frequently in prostate cancers. Preclinical evidence suggests that activation of PI3K/AKT signaling through loss of PTEN can result in resistance to hormonal treatment in prostate cancer. Objective To explore the antitumor activity of abiraterone acetate (abiraterone) in castration-resistant prostate cancer (CRPC) patients with and without loss of PTEN protein expression. Design, setting, and participants We retrospectively identified patients who had received abiraterone and had hormone-sensitive prostate cancer (HSPC) and/or CRPC tissue available for PTEN immunohistochemical analysis. Outcome measurements and statistical analysis The primary end point was overall survival from initiation of abiraterone treatment. Relationship with outcome was analyzed using multivariate Cox regression and log-rank analyses. Results and limitations A total of 144 patients were identified who had received abiraterone post-docetaxel and had available tumor tissue. Overall, loss of PTEN expression was observed in 40% of patients. Matched HSPC and CRPC tumor biopsies were available for 41 patients. PTEN status in CRPC correlated with HSPC in 86% of cases. Loss of PTEN expression was associated with shorter median overall survival (14 vs 21 mo; hazard ratio [HR]: 1.75; 95% confidence interval [CI], 1.19–2.55; p = 0.004) and shorter median duration of abiraterone treatment (24 vs 28 wk; HR: 1.6; 95% CI, 1.12–2.28; p = 0.009). PTEN protein loss, high lactate dehydrogenase, and the presence of visceral metastases were identified as independent prognostic factors in multivariate analysis. Conclusions Our results indicate that loss of PTEN expression was associated with worse survival and shorter time on abiraterone treatment. Further studies in larger and prospective cohorts are warranted. Patient summary PTEN is a protein often lost in prostate cancer cells. In this study we evaluated if prostate

  13. Expression of multidrug resistance proteins in invasive ductal carcinoma of the breast.

    PubMed

    Li, Weiquan; Song, Maomin

    2014-11-01

    Chemotherapy is commonly used for the treatment of breast cancer. However, the resistance to chemotherapeutic agents, often mediated by multidrug resistance (MDR) mechanisms, is a common occurrence. The present study examined the expression of several MDR-related proteins (MRPs) in invasive ductal carcinoma (IDC) of the breast, and assessed their association with clinicopathological variables and their prognostic significance. In addition, immunohistochemistry was used to measure the expression of MRP, p-glycoprotein (P-gp), topoisomerase 2α (Topo2α), thymidylate synthase (TS) and glutathione-S-transferase π (GST-π) in 156 resected IDCs of the breast. Pearson's χ(2) test and Spearman's correlation coefficient were used to analyze the association between MDR protein expression and several clinicopathological variables. The association between each of the five MDR proteins was also examined. Furthermore, Kaplan-Meier analysis and Cox regression modeling were used to assess overall survival. The expression of MRP, P-gp, Topo2α, TS and GST-π was detected in 20.5% (32/156), 25.0% (39/156), 84.0% (131/156), 41.7% (65/156) and 41.0% (64/156) of cases examined, respectively. No correlation was identified between MRP and Topo-2α and the clinicopathological variables examined. By contrast, P-gp (χ(2)=20.226; P<0.0001) and GST-π (χ(2)=35.032; P<0.0001) were found to positively correlate with tumor grade. In addition, staining for TS was associated with axillary lymph node metastasis (χ(2)=42.281; P<0.0001). The expression levels of P-gp and GST-π were found to be significantly correlated (r= 0.319; P<0.0001). Furthermore, GST-π expression was elevated in estrogen receptor-negative breast cancer (χ(2)=17.407; P<0.0001). Tumor histological grade, in addition to TS and GST-π expression, were significant predictors of a poor survival outcome. TS and GST-π are consequently useful prognostic biomarkers in IDC, therefore, when establishing a personalized

  14. [Sex differences in drug action: the role of multidrug-resistance proteins (MRPs)].

    PubMed

    Smirnova, O V

    2012-01-01

    The mechanisms of participation of multidrug-resistance proteins (MRPs) in sex difference pattern of drug efficiency and side effects have been analyzed. MRPs structure, their tissue and cellular localization, substrate specificity, and functions have been considered. Regulation of expression and activity of MRPs by endogenous metabolites, signal compounds, including sex hormones, as well as by drug agents have been represented. The role of nuclear receptors in the regulation of MRPs expression has been demonstrated. The data on sex differences in hepatic and renal MRPs expression and expression of nuclear receptors, participating in their induction, have been shown. The participation of MRPs in the formation of sex differences of drug pharmacokinetics has been discussed. Sex differences in representation and functional activity of MRPs in such excretory organs as liver and kidney was concluded would play the essential role in sex dependence of pharmacokinetics and efficiency of drug action. PMID:22830252

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

    PubMed

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

    2009-12-15

    Protein-resistant polyurethane (PU) surfaces were prepared by surface-initiated simultaneous normal and reverse atom transfer radical polymerization (s-ATRP) of poly(oligo(ethylene glycol) methacrylate) (poly (OEGMA)). Oxygen plasma treatment was employed for initial activation of the PU surface. The grafted polymer chain length was adjusted by varying the molar ratio of monomer to sacrificial initiator in solution from 5:1 to 200:1. The modified PU surfaces were characterized by water contact angle, X-ray photoelectron spectroscopy (XPS), and atomic force microscopy (AFM). Protein adsorption experiments from tris-buffered saline (TBS) and plasma were carried out to evaluate the protein-resistance of the surfaces. Adsorption from single and binary protein solutions as well as from plasma was significantly reduced after modification. Adsorption decreased with increasing poly(OEGMA) chain length. Fibrinogen (Fg) adsorption on the 200:1 monomer/initiator surface was in the range of 3-33 ng/cm(2) representing 96-99% reduction compared with the unmodified PU. Fg adsorption from 0.01-10% plasma was as low as 1-5 ng/cm(2). Moreover, binary protein adsorption experiments using Fg and lysozyme (Lys) showed that protein size is a factor in the protein resistance of these surfaces. PMID:19148931

  16. 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. PMID:24531430

  17. Mutations in the tobacco mosaic virus 30-kD protein gene overcome Tm-2 resistance in tomato.

    PubMed

    Meshi, T; Motoyoshi, F; Maeda, T; Yoshiwoka, S; Watanabe, H; Okada, Y

    1989-05-01

    A resistance-breaking strain of tobacco mosaic virus (TMV), Ltb1, is able to multiply in tomatoes with the Tm-2 gene, unlike its parent strain, L. Nucleotide sequence analysis of Ltb1 RNA revealed two amino acid changes in the 30-kD protein: from Cys68 to Phe and from Glu133 to Lys (from L to Ltb1). Strains with these two changes generated in vitro multiplied in tomatoes with the Tm-2 gene and induced essentially the same symptoms as those caused by Ltb1. Strains with either one of the two changes did not overcome the resistance as efficiently as Ltb1, although increased levels of multiplication were observed compared with the L strain. Results showed that both mutations are involved in the resistance-breaking property of Ltb1. Sequence analysis indicated that another resistance-breaking strain and its parent strain had two amino acid changes in the 30-kD protein: from Glu52 to Lys and from Glu133 to Lys. The fact that the amino acid changes occurred in or near the well conserved regions in the 30-kD protein suggests that the mechanism of Tm-2 resistance may be closely related to the fundamental function of the 30-kD protein, presumably in cell-to-cell movement. PMID:2535549

  18. The effects of maribavir on the autophosphorylation of ganciclovir resistant mutants of the cytomegalovirus UL97 protein

    PubMed Central

    2010-01-01

    Background The UL97 protein kinase of human cytomegalovirus phosphorylates the antiviral drug ganciclovir and is the target of maribavir action. A detailed enzyme kinetic analysis of maribavir on the various enzymatic functions of wild type and ganciclovir resistant forms of UL97 is required. Methods Wild type and site directed mutant forms of the human cytomegalovirus UL97 gene product were expressed using recombinant baculoviruses and the purified products used to assess the effects of maribavir on the ganciclovir (GCV) kinase and protein kinase (PK) activities. Results Maribavir was a potent inhibitor of the autophosporylation of the wild type and all the major GCV resistant UL97 mutants analysed (M460I, H520Q, A594V and L595F) with a mean IC50 of 35 nM. The M460I mutation resulted in hypersensitivity to maribavir with an IC50 of 4.8 nM. A maribavir resistant mutant of UL97 (L397R) was functionally compromised as both a GCV kinase and a protein kinase (~ 10% of wild type levels). Enzyme kinetic experiments demonstrated that maribavir was a competitive inhibitor of ATP with a Ki of 10 nM. Discussion Maribavir is a potent competitive inhibitor of the UL97 protein kinase function and shows increased activity against the M460I GCV-resistant mutant which may impact on the management of GCV drug resistance in patients. PMID:21429239

  19. Involvement of breast cancer resistance protein (ABCG2) in the biliary excretion mechanism of fluoroquinolones.

    PubMed

    Ando, Tomohiro; Kusuhara, Hiroyuki; Merino, Gracia; Alvarez, Ana I; Schinkel, Alfred H; Sugiyama, Yuichi

    2007-10-01

    Fluoroquinolones are effective antibiotics for the treatment of bile duct infections. It has been shown that the biliary excretion of grepafloxacin is partly accounted for by multidrug resistance-associated protein 2 (MRP2/ABCC2), whereas neither MRP2 nor P-glycoprotein is involved in the biliary excretion of ulifloxacin. In the present study, we examined the involvement of breast cancer resistance protein (BCRP/ABCG2) in the biliary excretion of fluoroquinolones (grepafloxacin, ulifloxacin, ciprofloxacin, and ofloxacin). In Madin-Darby canine kidney II cells expressing human BCRP or mouse Bcrp, the basal-to-apical transport of grepafloxacin and ulifloxacin was greater than that of the mock control, which was inhibited by a BCRP inhibitor, 3-(6-isobutyl-9-methoxy-1,4-dioxo-1,2,3,4,6,7,12,12a-octahydropyrazino[1',2':1,6]pyrido[3,4-b]indol-3-yl)-propionic acid tert-butyl ester (Ko143). Plasma and bile concentrations of fluoroquinolones were determined in wild-type and Bcrp(-/-) mice after i.v. bolus injection. The cumulative biliary excretion of fluoroquinolones was significantly reduced in Bcrp(-/-) mice, resulting in a reduction of the biliary excretion clearances to 86, 50, 40, and 16 of the control values, for ciprofloxacin, grepafloxacin, ofloxacin, and ulifloxacin, respectively. Preinfusion of sulfobromophthalein significantly inhibited the biliary excretion of grepafloxacin in Bcrp(-/-) mice. There was no change in the tissue/plasma concentration ratios of fluoroquinolones in the liver or brain, whereas those in the kidney were increased 3.6- and 1.5-fold for ciprofloxacin and grepafloxacin, respectively, in Bcrp(-/-) mice but were unchanged for ofloxacin and ulifloxacin. The present study shows that BCRP mediates the biliary excretion of fluoroquinolones and suggests that it is also involved in the tubular secretion of ciprofloxacin and grepafloxacin. PMID:17639028

  20. Unique drought resistance functions of the highly ABA-induced clade A protein phosphatase 2Cs.

    PubMed

    Bhaskara, Govinal Badiger; Nguyen, Thao Thi; Verslues, Paul E

    2012-09-01

    Six Arabidopsis (Arabidopsis thaliana) clade A protein phosphatase 2Cs (PP2Cs) have established abscisic acid (ABA) signaling roles; however, phenotypic roles of the remaining three "HAI" PP2Cs, Highly ABA-Induced1 (HAI1), AKT1-Interacting PP2C1/HAI2, and HAI3, have remained unclear. HAI PP2C mutants had enhanced proline and osmoregulatory solute accumulation at low water potential, while mutants of other clade A PP2Cs had no or lesser effect on these drought resistance traits. hai1-2 also had increased expression of abiotic stress-associated genes, including dehydrins and late embryogenesis abundant proteins, but decreased expression of several defense-related genes. Conversely, the HAI PP2Cs had relatively less impact on several ABA sensitivity phenotypes. HAI PP2C single mutants were unaffected in ABA sensitivity, while double and triple mutants were moderately hypersensitive in postgermination ABA response but ABA insensitive in germination. The HAI PP2Cs interacted most strongly with PYL5 and PYL7 to -10 of the PYL/RCAR ABA receptor family, with PYL7 to -10 interactions being relatively little affected by ABA in yeast two-hybrid assays. HAI1 had especially limited PYL interaction. Reduced expression of the main HAI1-interacting PYLs at low water potential when HAI1 expression was strongly induced also suggests limited PYL regulation and a role of HAI1 activity in negatively regulating specific drought resistance phenotypes. Overall, the HAI PP2Cs had greatest effect on ABA-independent low water potential phenotypes and lesser effect on classical ABA sensitivity phenotypes. Both this and their distinct PYL interaction demonstrate a new level of functional differentiation among the clade A PP2Cs and a point of cross talk between ABA-dependent and ABA-independent drought-associated signaling. PMID:22829320

  1. 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

  2. Lysosomes and unfolded protein response, determinants of differential resistance of melanoma cells to vinca alkaloids.

    PubMed

    Vincent, Laure-Anais; Attaoua, Chaker; Bellis, Michel; Rozkydalova, Lucie; Hadj-Kaddour, Kamel; Vian, Laurence; Cuq, Pierre

    2015-04-01

    On account of its strong ability to become chemoresistant after a primary response to drugs, malignant melanoma (MM) remains a therapeutic challenge. This study focuses on acquired resistance to vinca alkaloids (VAs) using VA-resistant MM cell lines (CAL1R-VCR, CAL1R-VDS, and CAL1R-VRB), established by long-term continuous exposure of parental CAL1-wt cells to vincristine (VCR), vindesine (VDS), or vinorelbine (VRB), respectively. Transcriptomic profiling using rma and rdam methods led to distinguish two cell groups: CAL1R-VCR and CAL1R-VDS, CAL1R-VRB, and CAL1-wt. mgsa of the specifically altered genes in the first group evidenced the GO terms 'lysosomal lumen' and 'vacuolar lumen' linked to underexpressed genes, and 'endoplasmic reticulum (ER) stress response' associated with overexpressed genes. A specific reduction of lysosomal enzymes, independent of acidic vacuole organelle (AVO) turnover, was observed (LTG probe) in CAL1R-VCR and CAL1R-VDS cells. It was associated with the specific lowering of cathepsin B and L, known to be involved in the lysosomal pathway of apoptosis. Confirming gene profiling, the same groups (CAL1R-VCR and CAL1R-VDS, CAL1-wt and CAL1R-VRB) could be distinguished regarding the VA-mediated changes on mean size areas and on acidic compartment volumes. These two parameters were reduced in CAL1R-VCR and CAL1R-VDS cells, suggesting a smaller AVO accumulation and thus a reduced sensitivity to lysosomal membrane permeabilization-mediated apoptosis. In addition, 'ER stress response' inhibition by tauroursodeoxycholic acid induced a higher VA sensitization of the first cell group. In conclusion, lysosomes and unfolded protein response could be key determinants of the differential resistance of MM to VAs. PMID:25601431

  3. 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. PMID:27274052

  4. The Pharmacological and Physiological Role of Multidrug-Resistant Protein 4.

    PubMed

    Wen, Jiagen; Luo, Jianquan; Huang, Weihua; Tang, Jie; Zhou, Honghao; Zhang, Wei

    2015-09-01

    Multidrug-resistant protein 4 (MRP4), a member of the C subfamily of ATP-binding cassette transporters, is distributed in a variety of tissues and a number of cancers. As a drug transporter, MRP4 is responsible for the pharmacokinetics and pharmacodynamics of numerous drugs, especially antiviral drugs, antitumor drugs, and diuretics. In this regard, the functional role of MRP4 is affected by a number of factors, such as genetic mutations; tissue-specific transcriptional regulations; post-transcriptional regulations, including miRNAs and membrane internalization; and substrate competition. Unlike other C family members, MRP4 is in a pivotal position to transport cellular signaling molecules, through which it is tightly connected to the living activity and physiologic processes of cells and bodies. In the context of several cancers in which MRP4 is overexpressed, MRP4 inhibition shows striking effects against cancer progression and drug resistance. In this review, we describe the role of MRP4 more specifically in both healthy conditions and disease states, with an emphasis on its potential as a drug target. PMID:26148856

  5. Compartmental modelling of the pharmacokinetics of a breast cancer resistance protein.

    PubMed

    Grandjean, Thomas R B; Chappell, Mike J; Yates, James T W; Jones, Kevin; Wood, Gemma; Coleman, Tanya

    2011-11-01

    A mathematical model for the pharmacokinetics of Hoechst 33342 following administration into a culture medium containing a population of transfected cells (HEK293 hBCRP) with a potent breast cancer resistance protein inhibitor, Fumitremorgin C (FTC), present is described. FTC is reported to almost completely annul resistance mediated by BCRP in vitro. This non-linear compartmental model has seven macroscopic sub-units, with 14 rate parameters. It describes the relationship between the concentration of Hoechst 33342 and FTC, initially spiked in the medium, and the observed change in fluorescence due to Hoechst 33342 binding to DNA. Structural identifiability analysis has been performed using two methods, one based on the similarity transformation/exhaustive modelling approach and the other based on the differential algebra approach. The analyses demonstrated that all models derived are uniquely identifiable for the experiments/observations available. A kinetic modelling software package, namely FACSIMILE (MPCA Software, UK), was used for parameter fitting and to obtain numerical solutions for the system equations. Model fits gave very good agreement with in vitro data provided by AstraZeneca across a variety of experimental scenarios. PMID:20971524

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

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

    2002-01-01

    The breast cancer resistance protein (BCRP/ABCG2) is a member of the ATP-binding cassette family of drug transporters and confers resistance to various anticancer drugs. We show here that mice lacking Bcrp1/Abcg2 become extremely sensitive to the dietary chlorophyll-breakdown product pheophorbide a, resulting in severe, sometimes lethal phototoxic lesions on light-exposed skin. Pheophorbide a occurs in various plant-derived foods and food supplements. Bcrp1 transports pheophorbide a and is highly efficient in limiting its uptake from ingested food. Bcrp1−/− mice also displayed a previously unknown type of protoporphyria. Erythrocyte levels of the heme precursor and phototoxin protoporphyrin IX, which is structurally related to pheophorbide a, were increased 10-fold. Transplantation with wild-type bone marrow cured the protoporphyria and reduced the phototoxin sensitivity of Bcrp1−/− mice. These results indicate that humans or animals with low or absent BCRP activity may be at increased risk for developing protoporphyria and diet-dependent phototoxicity and provide a striking illustration of the importance of drug transporters in protection from toxicity of normal food constituents. PMID:12429862

  8. Molecular Genetics of Cryptopleurine Resistance in Saccharomyces Cerevisiae: Expression of a Ribosomal Protein Gene Family

    PubMed Central

    Paulovich, A. G.; Thompson, J. R.; Larkin, J. C.; Li, Z.; Woolford-Jr., J. L.

    1993-01-01

    The Saccharomyces cerevisiae CRY1 gene encodes the 40S ribosomal subunit protein rp59 and confers sensitivity to the protein synthesis inhibitor cryptopleurine. A yeast strain containing the cry1-δ1::URA3 null allele is viable, cryptopleurine sensitive (Cry(S)), and expresses rp59 mRNA, suggesting that there is a second functional CRY gene. The CRY2 gene has been isolated from a yeast genomic library cloned in bacteriophage λ, using a CRY1 DNA probe. The DNA sequence of the CRY2 gene contains an open reading frame encoding ribosomal protein 59 that differs at five residues from rp59 encoded by the CRY1 gene. The CRY2 gene was mapped to the left arm of chromosome X, centromere-proximal to cdc6 and immediately adjacent to ribosomal protein genes RPS24A and RPL46. Ribosomal protein 59 is an essential protein; upon sporulation of a diploid doubly heterozygous for cry1-δ2::TRP1 cry2-δ1::LEU2 null alleles, no spore clones containing both null alleles were recovered. Several results indicate that CRY2 is expressed, but at lower levels than CRY1: (1) Introduction of CRY2 on high copy plasmids into Cry(R) yeast of genotype cry1 CRY2 confers a Cry(S) phenotype. Transformation of these Cry(R) yeast with CRY2 on a low copy CEN plasmid does not confer a Cry(S) phenotype. (2) Haploids containing the cry1-δ2::TRP1 null allele have a deficit of 40S ribosomal subunits, but cry2-δ1::LEU2 strains have wild-type amounts of 40S ribosomal subunits. (3) CRY2 mRNA is present at lower levels than CRY1 mRNA. (4) Higher levels of β-galactosidase are expressed from a CRY1-lacZ gene fusion than from a CRY2-lacZ gene fusion. Mutations that alter or eliminate the last amino acid of rp59 encoded by either CRY1 or CRY2 result in resistance to cryptopleurine. Because CRY2 (and cry2) is expressed at lower levels than CRY1 (and cry1), the Cry(R) phenotype of cry2 mutants is only expressed in strains containing a cry1-δ null allele. PMID:8293976

  9. Channel Formation by CarO, the Carbapenem Resistance-Associated Outer Membrane Protein of Acinetobacter baumannii

    PubMed Central

    Siroy, Axel; Molle, Virginie; Lemaître-Guillier, Christelle; Vallenet, David; Pestel-Caron, Martine; Cozzone, Alain J.; Jouenne, Thierry; Dé, Emmanuelle

    2005-01-01

    It has been recently shown that resistance to both imipenem and meropenem in multidrug-resistant clinical strains of Acinetobacter baumannii is associated with the loss of a heat-modifiable 25/29-kDa outer membrane protein, called CarO. This study aimed to investigate the channel-forming properties of CarO. Mass spectrometry analyses of this protein band detected another 25-kDa protein (called Omp25), together with CarO. Both proteins presented similar physicochemical parameters (Mw and pI). We overproduced and purified the two polypeptides as His-tagged recombinant proteins. Circular dichroism analyses demonstrated that the secondary structure of these proteins was mainly a β-strand conformation with spectra typical of porins. We studied the channel-forming properties of proteins by reconstitution into artificial lipid bilayers. In these conditions, CarO induced ion channels with a conductance value of 110 pS in 1 M KCl, whereas the Omp25 protein did not form any channels, despite its suggested porin function. The pores formed by CarO showed a slight cationic selectivity and no voltage closure. No specific imipenem binding site was found in CarO, and this protein would rather form unspecific monomeric channels. PMID:16304148

  10. 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

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

    PubMed

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

    2015-03-01

    Systemic acquired resistance (SAR) is a plant defence response that provides immunity to distant uninfected leaves after an initial localised infection. The lipid transfer protein (LTP) Defective in Induced Resistance1 (DIR1) is an essential component of SAR that moves from induced to distant leaves following a SAR-inducing local infection. To understand how DIR1 is transported to distant leaves during SAR, we analysed DIR1 movement in transgenic Arabidopsis lines with reduced cell-to-cell movement caused by the overexpression of Plasmodesmata-Located Proteins PDLP1 and PDLP5. These PDLP-overexpressing lines were defective for SAR, and DIR1 antibody signals were not observed in phloem sap-enriched petiole exudates collected from distant leaves. Our data support the idea that cell-to-cell movement of DIR1 through plasmodesmata is important during long-distance SAR signalling in Arabidopsis. PMID:25296648

  12. 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

  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. Hepatitis B virus-induced calreticulin protein is involved in IFN resistance.

    PubMed

    Yue, Xin; Wang, Hui; Zhao, Fanpeng; Liu, Shi; Wu, Jianguo; Ren, Wendan; Zhu, Ying

    2012-07-01

    IFN-α is a widely used treatment for hepatitis B virus (HBV) infection, and IFN resistance caused by viral and/or host factors is currently a challenging clinical problem. A better understanding of the molecular mechanisms underlying IFN immunotherapy in the treatment of viral infection would be very beneficial clinically and is of immense clinical importance. Calreticulin (CRT) is an endoplasmic reticulum luminal calcium-binding chaperone that is involved in the regulation of calcium homoeostasis, the folding of newly synthesized proteins, and many other cellular functions. However, little is known about the role of CRT in HBV infection. In this study, we observed high levels of CRT expression in the sera and PBMCs of patients with HBV relative to those of healthy individuals. HBV upregulated the expression of CRT at the transcriptional level. Further investigation showed that HBV-induced CRT enhanced HBV replication by antagonizing the IFN pathway. CRT suppressed the production of endogenous IFN-α by reducing the nuclear translocation of IFN regulatory factor-7 but not IFN regulatory factor-3. Furthermore, CRT also suppressed the antiviral activity of IFN-α by inhibiting the phosphorylation of STAT1 and decreasing the expression of two IFN-α downstream effectors, protein kinase R and 2',5'-oligoadenylate synthetase. Our results offer new insights into the pathogenesis of HBV infection and may provide potential targets for anti-HBV therapy. PMID:22661095

  15. 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

  16. 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

  17. Centaurin-like protein Cnt5 contributes to arsenic and cadmium resistance in fission yeast.

    PubMed

    Vashisht, Ajay Amar; Kennedy, Patrick Joseph; Russell, Paul

    2009-03-01

    Arsenic (As) and cadmium (Cd) are two of the most hazardous substances in the environment and have been implicated in a number of human diseases including cancer. Their mechanisms of toxicity and subsequent carcinogenesis are not understood. To identify the genes involved in As/Cd detoxification, we screened a random insertional mutagenesis library of Schizosaccharomyces pombe for mutants that are hypersensitive to As/Cd. Mutations were mapped to spc1(+) (sty1(+)) and SPBC17G9.08c. Spc1 is a stress-activated protein kinase orthologous to human p38. A fragment of SPBC17G9.08c was previously identified as csx2, a high-copy suppressor of cut6 that encodes an acetyl-CoA carboxylase involved in fatty acid biosynthesis. SPBC17G9.08c is a member of the centaurin ADP ribosylation factor GTPase activating protein family found in a variety of fungi, plants and metazoans, but not in Saccharomyces cerevisiae. Cnt5, so named because its closest human homolog is centaurin beta-5, binds to phosphatidic acid and phosphatidyl serine in vitro. Microscopic localization of Cnt5-GFP indicates significant redistribution of Cnt5 from the cytoplasm to the cell membranes in response to As stress. These data suggest a model in which Cnt5 contributes to As/Cd resistance by maintaining membrane integrity or by modulating membrane trafficking. PMID:19076239

  18. Substrates and inhibitors of human multidrug resistance associated proteins and the implications in drug development.

    PubMed

    Zhou, Shu-Feng; Wang, Lin-Lin; Di, Yuan Ming; Xue, Charlie Changli; Duan, Wei; Li, Chun Guang; Li, Yong

    2008-01-01

    Human contains 49 ATP-binding cassette (ABC) transporter genes and the multidrug resistance associated proteins (MRP1/ABCC1, MRP2/ABCC2, MRP3/ABCC3, MRP4/ABCC4, MRP5/ABCC5, MRP6/ABCC6, MRP7/ABCC10, MRP8/ABCC11 and MRP9/ABCC12) belong to the ABCC family which contains 13 members. ABCC7 is cystic fibrosis transmembrane conductance regulator; ABCC8 and ABCC9 are the sulfonylurea receptors which constitute the ATP-sensing subunits of a complex potassium channel. MRP10/ABCC13 is clearly a pseudo-gene which encodes a truncated protein that is highly expressed in fetal human liver with the highest similarity to MRP2/ABCC2 but without transporting activity. These transporters are localized to the apical and/or basolateral membrane of the hepatocytes, enterocytes, renal proximal tubule cells and endothelial cells of the blood-brain barrier. MRP/ABCC members transport a structurally diverse array of important endogenous substances and xenobiotics and their metabolites (in particular conjugates) with different substrate specificity and transport kinetics. The human MRP/ABCC transporters except MRP9/ABCC12 are all able to transport organic anions, such as drugs conjugated to glutathione, sulphate or glucuronate. In addition, selected MRP/ABCC members may transport a variety of endogenous compounds, such as leukotriene C(4) (LTC(4) by MRP1/ABCC1), bilirubin glucuronides (MRP2/ABCC2, and MRP3/ABCC3), prostaglandins E1 and E2 (MRP4/ABCC4), cGMP (MRP4/ABCC4, MRP5/ABCC5, and MRP8/ABCC11), and several glucuronosyl-, or sulfatidyl steroids. In vitro, the MRP/ABCC transporters can collectively confer resistance to natural product anticancer drugs and their conjugated metabolites, platinum compounds, folate antimetabolites, nucleoside and nucleotide analogs, arsenical and antimonial oxyanions, peptide-based agents, and in concert with alterations in phase II conjugating or biosynthetic enzymes, classical alkylating agents, alkylating agents. Several MRP/ABCC members (MRPs 1-3) are

  19. Whey and casein labeled with L-[1-13C]leucine and muscle protein synthesis: effect of resistance exercise and protein ingestion.

    PubMed

    Reitelseder, Søren; Agergaard, Jakob; Doessing, Simon; Helmark, Ida C; Lund, Peter; Kristensen, Niels B; Frystyk, Jan; Flyvbjerg, Allan; Schjerling, Peter; van Hall, Gerrit; Kjaer, Michael; Holm, Lars

    2011-01-01

    Muscle protein turnover following resistance exercise and amino acid availability are relatively well described. By contrast, the beneficial effects of different sources of intact proteins in relation to exercise need further investigation. Our objective was to compare muscle anabolic responses to a single bolus intake of whey or casein after performance of heavy resistance exercise. Young male individuals were randomly assigned to participate in two protein trials (n = 9) or one control trial (n = 8). Infusion of l-[1-(13)C]leucine was carried out, and either whey, casein (0.3 g/kg lean body mass), or a noncaloric control drink was ingested immediately after exercise. l-[1-(13)C]leucine-labeled whey and casein were used while muscle protein synthesis (MPS) was assessed. Blood and muscle tissue samples were collected to measure systemic hormone and amino acid concentrations, tracer enrichments, and myofibrillar protein synthesis. Western blots were used to investigate the Akt signaling pathway. Plasma insulin and branched-chain amino acid concentrations increased to a greater extent after ingestion of whey compared with casein. Myofibrillar protein synthesis was equally increased 1-6 h postexercise after whey and casein intake, both of which were higher compared with control (P < 0.05). Phosphorylation of Akt and p70(S6K) was increased after exercise and protein intake (P < 0.05), but no differences were observed between the types of protein except for total 4E-BP1, which was higher after whey intake than after casein intake (P < 0.05). In conclusion, whey and casein intake immediately after resistance exercise results in an overall equal MPS response despite temporal differences in insulin and amino acid concentrations and 4E-BP1. PMID:21045172

  20. Intestinal ciprofloxacin efflux: the role of breast cancer resistance protein (ABCG2).

    PubMed

    Haslam, I S; Wright, J A; O'Reilly, D A; Sherlock, D J; Coleman, T; Simmons, N L

    2011-12-01

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

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

    PubMed

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

    2005-05-01

    Ascites is a metabolic disorder of modern broilers that is distinguished by cardiopulmonary insufficiency in the face of intense oxygen demands of rapidly growing tissues. Broilers with ascites exhibit sustained elevation of pulmonary arterial pressure and right ventricular hypertrophy, the end result of which is heart failure. It has been shown that mitochondrial function is impaired in broilers with ascites. In the current study, mitochondrial matrix protein levels were compared between ascites-resistant line broilers and ascites-susceptible line broilers with and without ascites using two-dimensional (2-D) gel electrophoresis. One hundred seventy-two protein spots were detected on the gels, and 9 of the spots were present at different levels in the 4 groups of broilers. These 9 protein spots were selected for identification by mass spectrometry. Two of the spots were found to contain single mitochondrial matrix proteins. Both mitochondrial matrix proteins, the dihydrolipoamide succinyltransferase component of the 2-oxoglutarate dehydrogenase complex and the alpha-subunit of mitochondrial trifunctional enzyme, were present at higher levels in ascites-resistant line broilers with ascites in the present study. The elevated levels of 2 key proteins in aerobic metabolism in ascites-resistant line broilers with ascites observed in the present study suggests that the mitochondria of broilers with this disease may respond inappropriately to hypoxia. PMID:15913181

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

    PubMed 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

  3. 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

  4. 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

  5. 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

  6. 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.

  7. THE ANABOLIC RESPONSE TO RESISTANCE EXERCISE AND A PROTEIN-RICH MEAL IS NOT DIMINISHED BY AGE

    PubMed Central

    SYMONS, T. BROCK; SHEFFIELD-MOORE, M.; MAMEROW, M.M.; WOLFE, R.R.; PADDON-JONES, D.

    2012-01-01

    Objectives The synergistic effect of resistance exercise and protein ingestion on muscle protein anabolism in young adults has been well described. However, it is unclear if this relationship is maintained in older adults who are at greater risk of sarcopenic muscle loss. To this end, we sought to determine if the synergistic response to a bout of resistance exercise and a protein-rich lean beef meal was altered by age. Setting The University of Texas Medical Branch, Clinical Research Center, Galveston, Texas. Participants: Healthy young (n=7, 29±3 y) and older (n=7, 67±2 y) adults. Design Mixed muscle fractional synthesis rate (FSR) was calculated during a 3 h post-absorptive/rest period and again during a 5 h period following ingestion of a protein-rich meal (340 g lean beef) and bout of resistance exercise (6 sets of 8 repetitions of isotonic knee extension exercise at 80% one repetition maximum). Measurements Venous blood samples and vastus lateralis muscle biopsy samples were obtained during a primed (2.0 μmol/kg) constant infusion (0.08 μmol·kg−1min−1) of L-[ring-13C6] phenylalanine. Results Mixed muscle FSR increased by approximately 108% in both young [pre: 0.073±0.008; post: 0.156±0.021(SE) %/h, p<0.001] and older adults (pre: 0.075±0.004; post: 0.152±0.017 %/h, p=0.003) following the meal and resistance exercise bout. Conclusion Aging does not diminish the increase in muscle protein synthesis following a high-quality protein rich meal and bout of resistance exercise. PMID:21528164

  8. Crystal Structures of Flax Rust Avirulence Proteins AvrL567-A and -D Reveal Details of the Structural Basis for Flax Disease Resistance Specificity[W

    PubMed Central

    Wang, Ching-I A.; Gunčar, 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, Boštjan

    2007-01-01

    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-Å resolution, respectively. The structures of both proteins are very similar and reveal a β-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. PMID:17873095

  9. 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

  10. Physiological quality and gene expression related to heat-resistant proteins at different stages of development of maize seeds.

    PubMed

    Andrade, T; Von Pinho, E V R; Von Pinho, R G; Oliveira, G E; Andrade, V; Fernandes, J S

    2013-01-01

    We quantified and characterized the expression of heat-resistant proteins during seed development of maize lines with distinct levels of tolerance to high drying temperature. A corn field was planted for multiplication of seeds of different lines, two tolerant and two non-tolerant to high drying temperatures. Harvest of the seeds was carried out at various stages of development and they were then subjected to tests of moisture content, germination, first count of germination, accelerated aging, and cold test. The seeds were stored in a freezer for later analysis of expression of heat-resistant proteins by means of real-time PCR, electrophoresis, and spectrophotometry. We observed that heat-resistant proteins are expressed in a differential manner in seeds from different lines and at different stages of development. The expression of heat-resistant proteins was earlier in lines tolerant to high drying temperatures. Greater germination and vigor values was found for seeds collected at the last stage of development. PMID:24085427

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

    PubMed

    Geng, Shuaifeng; Li, Aili; Tang, Lichuan; Yin, Lingjie; Wu, Liang; Lei, Cailin; Guo, Xiuping; Zhang, Xin; Jiang, Guanghuai; Zhai, Wenxue; Wei, Yuming; Zheng, Youliang; Lan, Xiujin; Mao, Long

    2013-08-01

    Calcium-dependent protein kinases (CPKs) are important Ca2+ signalling components involved in complex immune and stress signalling networks; but the knowledge of CPK gene functions in the hexaploid wheat is limited. Previously, TaCPK2 was shown to be inducible by powdery mildew (Blumeria graminis tritici, Bgt) infection in wheat. Here, its functions in disease resistance are characterized further. This study shows the presence of defence-response and cold-response cis-elements on the promoters of the A subgenome homoeologue (TaCPK2-A) and D subgenome homoeologue (TaCPK2-D), respectively. Their expression patterns were then confirmed by quantitative real-time PCR (qRT-PCR) using genome-specific primers, where TaCPK2-A was induced by Bgt treatment while TaCPK2-D mainly responded to cold treatment. Downregulation of TaCPK2-A by virus-induced gene silencing (VIGS) causes loss of resistance to Bgt in resistant wheat lines, indicating that TaCPK2-A is required for powdery mildew resistance. Furthermore, overexpression of TaCPK2-A in rice enhanced bacterial blight (Xanthomonas oryzae pv. oryzae, Xoo) resistance. qRT-PCR analysis showed that overexpression of TaCPK2-A in rice promoted the expression of OsWRKY45-1, a transcription factor involved in both fungal and bacterial resistance by regulating jasmonic acid and salicylic acid signalling genes. The opposite effect was found in wheat TaCPK2-A VIGS plants, where the homologue of OsWRKY45-1 was significantly repressed. These data suggest that modulation of WRKY45-1 and associated defence-response genes by CPK2 genes may be the common mechanism for multiple disease resistance in grass species, which may have undergone subfunctionalization in promoters before the formation of hexaploid wheat. PMID:23918959

  12. 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.

  13. 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. PMID:26452990

  14. 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

  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. 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

  17. 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

  18. 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. PMID:26978032

  19. [Interrelation of the antibiotic sensitivity (resistance) of staphylococci, clinical forms of the infection and production of protein A].

    PubMed

    Fomenko, G A

    1984-06-01

    Two hundred and thirty-two strains of Staph. aureus isolated from patients with staphylococcal infections were studied. The strains were isolated from the blood of patients with sepsis, from the purulent foci on the skin and in the subcutaneous fat, from the nasopharyngeal mucosa of patients with tonsillitis and inflammation of the upper respiratory tract, from the sputum of patients with the pneumonia signs and from the pus of patients with otitis. The pathogens were identified with the routine methods. The quantitative content of protein A in the strains was determined by the method of indirect hemagglutination with red blood cells sensitized with the hemolytic serum. The data obtained were analysed with regard to the strain group and characteristics of the strain resistance or sensitivity to benzylpenicillin, erythromycin, oleandomycin, chloramphenicol, streptomycin, neomycin, kanamycin, monomycin, ristomycin and furagin K. Statistically significant differences in the protein A content in certain strain groups were observed. These differences might be correlated with the strain antibiotic resistance but not sensitivity. Pronounced changes in the levels of protein A were detected in the staphylococcal hemocultures resistant to erythromycin and streptomycin. The cultures resistant to erythromycin were characterized by decreased content of protein A and those resistant to streptomycin were characterized by increased content of protein A. Comparison of the antibiotic sensitivity of the strains of 5 groups by variation statistics revealed significant differences in the levels of sensitivity to streptomycin, neomycin, kanamycin, monomycin, ristomycin and furagin K but not to erythromycin, oleandomycin and chloramphenicol in the strains of certain groups. The staphylococcal hemocultures isolated from patients with sepsis proved to be the most sensitive to the antibiotics. PMID:6476803

  20. Nanoscale Drug Delivery Platforms Overcome Platinum-Based Resistance in Cancer Cells Due to Abnormal Membrane Protein Trafficking

    PubMed Central

    Xue, Xue; Hall, Matthew D.; Zhang, Qiang; Wang, Paul C.; Gottesman, Michael M.; Liang, Xing-Jie

    2014-01-01

    The development of cellular resistance to platinum-based chemotherapies is often associated with reduced intracellular platinum concentrations. In some models, this reduction is due to abnormal membrane protein trafficking, resulting in reduced uptake by transporters at the cell surface. Given the central role of platinum drugs in the clinic, it is critical to overcome cisplatin resistance by bypassing the plasma membrane barrier to significantly increase the intracellular cisplatin concentration enough to inhibit the proliferation of cisplatin-resistant cells. Therefore, rational design of appropriate nanoscale drug delivery platforms (nDDPs) loaded with cisplatin or other platinum analogs as payloads is a possible strategy to solve this problem. This review will focus on the known mechanism of membrane trafficking in cisplatin-resistant cells, and the development and employment of nDDPs to improve cell uptake of cisplatin. PMID:24219825

  1. 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

  2. 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

  3. 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

  4. Differential expression of sphingolipids in P-glycoprotein or multidrug resistance-related protein 1 expressing human neuroblastoma cell lines.

    PubMed

    Dijkhuis, Anne-Jan; Douwes, Jenny; Kamps, Willem; Sietsma, Hannie; Kok, Jan Willem

    2003-07-31

    The sphingolipid composition and multidrug resistance status of three human neuroblastoma cell lines were established. SK-N-FI cells displayed high expression and functional (efflux) activity of P-glycoprotein, while multidrug resistance-related protein 1 was relatively abundant and most active in SK-N-AS cells. These two cell lines exhibited higher sphingolipid levels, compared to SK-N-DZ, which had the lowest activity of either ATP-binding cassette transporter protein. SK-N-DZ cells also differed in ganglioside composition with predominant expression of b-series gangliosides. In conclusion, these three neuroblastoma cell lines offer a good model system to study sphingolipid metabolism in relation to ATP-binding cassette transporter protein function. PMID:12885402

  5. Detection of the Mr 110,000 lung resistance-related protein LRP/MVP with monoclonal antibodies.

    PubMed

    Schroeijers, A B; Scheffer, G L; Reurs, A W; Pijnenborg, A C; Abbondanza, C; Wiemer, E A; Scheper, R J

    2001-11-01

    The Mr 110,000 lung resistance-related protein (LRP), also termed the major vault protein (MVP), constitutes >70% of subcellular ribonucleoprotein particles called vaults. Overexpression of LRP/MVP and vaults has been linked directly to MDR in cancer cells. Clinically, LRP/MVP expression can be of value to predict response to chemotherapy and prognosis. Monoclonal antibodies (MAbs) against LRP/MVP have played a critical role in determining the relevance of this protein in clinical drug resistance. We compared the applicability of the previously described MAbs LRP-56, LMR-5, LRP, 1027, 1032, and newly isolated MAbs MVP-9, MVP-16, MVP-18, and MVP-37 for the immunodetection of LRP/MVP by immunoblotting analysis and by immunocyto- and histochemistry. The availability of a broader panel of reagents for the specific and sensitive immunodetection of LRP/MVP should greatly facilitate biological and clinical studies of vault-related MDR. PMID:11668191

  6. Two Genes Encoding Structurally Different CC-NB-LRR Proteins are Required for Lr10-Mediated Leaf Rust Resistance in Wheat of Two Ploidy Levels

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The gene pools of crop plant relatives have been proposed as a source of new functional resistance genes to broaden the basis of genetic resistance. Here, we have studied the allelic diversity of the Lr10 leaf rust resistance gene, encoding a CC-NBS-LRR protein originally identified in hexaploid bre...

  7. Increased expression of fibronectin-binding proteins by fluoroquinolone-resistant Staphylococcus aureus exposed to subinhibitory levels of ciprofloxacin.

    PubMed Central

    Bisognano, C; Vaudaux, P E; Lew, D P; Ng, E Y; Hooper, D C

    1997-01-01

    Bacterial adhesion, which plays an important role in Staphylococcus aureus colonization and infection, may be altered by the presence of antibiotics or/and antibiotic resistance determinants. This study evaluated the effect of fluoroquinolone resistance determinants on S. aureus adhesion to solid-phase fibronectin, which is specifically mediated by two surface-located fibronectin-binding proteins. Five isogenic mutants, derived from strain NCTC 8325 and expressing various levels of quinolone resistance, were tested in an in vitro bacterial adhesion assay with polymethylmethacrylate coverslips coated with increasing amounts of fibronectin. These strains contained single or combined mutations in the three major loci contributing to fluoroquinolone resistance, namely, grlA, gyrA, and flqB, which code for altered topoisomerase IV, DNA gyrase, and increased norA-mediated efflux of fluoroquinolones, respectively. Adhesion characteristics of the different quinolone-resistant mutants grown in the absence of fluoroquinolone showed only minor differences from those of parental strains. However, more important changes in adhesion were exhibited by mutants highly resistant to quinolones following their exponential growth in the presence of one-quarter MIC of ciprofloxacin. Increased bacterial adhesion of the highly quinolone-resistant mutants, which contained combined mutations in grlA and gyrA, was associated with and explained by the overexpression of their fibronectin-binding proteins as assessed by Western ligand affinity blotting. These findings contradict the notion that subinhibitory concentrations of antibiotics generally decrease the expression of virulence factors by S. aureus. Perhaps the increased adhesion of S. aureus strains highly resistant to fluoroquinolones contributes in part to that emergence in clinical settings. PMID:9145842

  8. 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. PMID:27008926

  9. 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

  10. Substrates of multidrug resistance-associated proteins block the cystic fibrosis transmembrane conductance regulator chloride channel.

    PubMed

    Linsdell, P; Hanrahan, J W

    1999-03-01

    1. The effects of physiological substrates of multidrug resistance-associated proteins (MRPs) on cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel currents were examined using patch clamp recording from CFTR-transfected mammalian cell lines. 2. Two MRP substrates, taurolithocholate-3-sulphate (TLCS) and beta-estradiol 17-(beta-D-glucuronide) (E217betaG) caused a voltage-dependent block of macroscopic CFTR Cl- currents when applied to the intracellular face of excised membrane patches, with mean apparent dissociation constants (KDs) of 96+/-10 and 563+/-103 microM (at 0 mV) respectively. The unconjugated bile salts taurocholate and cholate were also effective CFTR channel blockers under these conditions, with KDs of 453+/-44 and 3760+/-710 microM (at 0 mV) respectively. 3. Reducing the extracellular Cl- concentration from 154 to 20 mM decreased the KD for block intracellular TLCS to 54+/-1 microM, and also significantly reduced the voltage dependence of block, by suggesting that TLCS blocks Cl- permeation through CFTR by binding within the channel pore. 4. Intracellular TLCS reduced the apparent amplitude of CFTR single channel currents, suggesting that the duration of block is very fast compared to the gating of the channel. 5. The apparent affinity of block by TLCs is comparable to that of other well-known CFTR channel blockers, suggesting that MRP substrates may comprise a novel class of probes of the CFTR channel pore. 6. These results also suggest that the related proteins CFTR and MRP may share a structurally similar anion binding site at the cytoplasmic face of the membrane. PMID:10217542

  11. Substrates of multidrug resistance-associated proteins block the cystic fibrosis transmembrane conductance regulator chloride channel

    PubMed Central

    Linsdell, Paul; Hanrahan, John W

    1999-01-01

    The effects of physiological substrates of multidrug resistance-associated proteins (MRPs) on cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channel currents were examined using patch clamp recording from CFTR-transfected mammalian cell lines. Two MRP substrates, taurolithocholate-3-sulphate (TLCS) and β-estradiol 17-(β-D-glucuronide) (E217βG) caused a voltage-dependent block of macroscopic CFTR Cl− currents when applied to the intracellular face of excised membrane patches, with mean apparent dissociation constants (KDs) of 96±10 and 563±103 μM (at 0 mV) respectively. The unconjugated bile salts taurocholate and cholate were also effective CFTR channel blockers under these conditions, with KDs of 453±44 and 3760±710 μM (at 0 mV) respectively. Reducing the extracellular Cl− concentration from 154 to 20 mM decreased the KD for block intracellular TLCS to 54±1 μM, and also significantly reduced the voltage dependence of block, by suggesting that TLCS blocks Cl− permeation through CFTR by binding within the channel pore. Intracellular TLCS reduced the apparent amplitude of CFTR single channel currents, suggesting that the duration of block is very fast compared to the gating of the channel. The apparent affinity of block by TLCs is comparable to that of other well-known CFTR channel blockers, suggesting that MRP substrates may comprise a novel class of probes of the CFTR channel pore. These results also suggest that the related proteins CFTR and MRP may share a structurally similar anion binding site at the cytoplasmic face of the membrane. PMID:10217542

  12. Heat shock proteins and resistance to desiccation in congeneric land snails.

    PubMed

    Mizrahi, Tal; Heller, Joseph; Goldenberg, Shoshana; Arad, Zeev

    2010-07-01

    Land snails are subject to daily and seasonal variations in temperature and in water availability and depend on a range of behavioral and physiological adaptations for coping with problems of maintaining water, ionic, and thermal balance. Heat shock proteins (HSPs) are a multigene family of proteins whose expression is induced by a variety of stress agents. We used experimental desiccation to test whether adaptation to different habitats affects HSP expression in two closely related Sphincterochila snail species, a desiccation-resistant, desert species Sphincterochila zonata, and a Mediterranean-type, desiccation-sensitive species Sphincterochila cariosa. We examined the HSP response in the foot, hepatopancreas, and kidney tissues of snails exposed to normothermic desiccation. Our findings show variations in the HSP response in both timing and magnitude between the two species. The levels of endogenous Hsp72 in S. cariosa were higher in all the examined tissues, and the induction of Hsp72, Hsp74, and Hsp90 developed earlier than in S. zonata. In contrary, the induction of sHSPs (Hsp25 and Hsp30) was more pronounced in S. zonata compared to S. cariosa. Our results suggest that land snails use HSPs as part of their survival strategy during desiccation and as important components of the aestivation mechanism in the transition from activity to dormancy. Our study underscores the distinct strategy of HSP expression in response to desiccation, namely the delayed induction of Hsp70 and Hsp90 together with enhanced induction of sHSPs in the desert-dwelling species, and suggests that evolution in harsh environments will result in selection for reduced Hsp70 expression. PMID:19953352

  13. Reduced ribosomal protein s6 phosphorylation after progressive resistance exercise in growing adolescent rats.

    PubMed

    Hellyer, Nathan J; Nokleby, Jessica J; Thicke, Bethany M; Zhan, Wen-Zhi; Sieck, Gary C; Mantilla, Carlos B

    2012-06-01

    The purpose of this study was to investigate moderate intensity progressive resistance exercise (PRE) in growing adolescent rats and its effect on muscle hypertrophy (defined as an increase in fiber cross-sectional area [CSA]). We hypothesized that in adolescent animals moderate intensity PRE would increase (a) fiber CSA; (b) myosin heavy chain (MyHC) content; and (c) expression and phosphorylation of cell signaling molecules involved in translational regulation, compared with that in age-matched sedentary (SED) controls. In the PRE group, 3-week-old male rats were trained to climb a vertical ladder as a mode of PRE training such that by 10 weeks all animals in the PRE group had progressed to carry an additional 80% of their body weight per climb. In agreement with our hypotheses, we observed that 10 weeks of moderate PRE in adolescent animals was sufficient to increase the CSA of muscle fibers and increase MyHC content. The average muscle fiber CSA increased by >10%, and the total MyHC content increased by 35% (p < 0.05) in the PRE group compared with that in the SED animals. Concurrently, we investigated sustained changes in the expression and phosphorylation of key signaling molecules that are previously identified regulators of hypertrophy in adult animal models. Contrary to our hypotheses, expression and phosphorylation of the translational regulators mammalian target of rapamycin and Akt were not increased in the PRE group. In addition, we observed that the ratio of phosphorylated-to-unphosphorylated ribosomal protein S6 (rpS6) was reduced over sixfold in PRE animals (p < 0.05) and that total rpS6 protein levels were unchanged between PRE and SED animals (p > 0.05). We conclude that moderate intensity PRE is sufficient to induce muscle hypertrophy in adolescent animals, whereas the signaling mechanisms associated with muscle hypertrophy may differ between growing adolescents and adults. PMID:22614147

  14. 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. PMID:26893303

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

    PubMed Central

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

    2015-01-01

    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. PMID:26196957

  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. 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

  18. 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

  19. A substrate radical intermediate in catalysis by the antibiotic resistance protein Cfr.

    PubMed

    Grove, Tyler L; Livada, Jovan; Schwalm, Erica L; Green, Michael T; Booker, Squire J; Silakov, Alexey

    2013-07-01

    Cfr-dependent methylation of C8 of A2503 in 23S ribosomal RNA confers bacterial resistance to an array of clinically important antibiotics that target the large subunit of the ribosome, including the synthetic oxazolidinone antibiotic linezolid. The key element of the proposed mechanism for Cfr, a radical S-adenosylmethionine enzyme, is the addition of a methylene radical, generated by hydrogen-atom abstraction from the methyl group of an S-methylated cysteine, onto C8 of A2503 to form a protein-nucleic acid crosslinked species containing an unpaired electron. Herein we use continuous-wave and pulsed EPR techniques to provide direct spectroscopic evidence for this intermediate, showing a spin-delocalized radical with maximum spin density at N7 of the adenine ring. In addition, we use rapid freeze-quench EPR to show that the radical forms and decays with rate constants that are consistent with the rate of formation of the methylated product. PMID:23644479

  20. 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. PMID:26153407

  1. 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

  2. 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

  3. Generation and characterization of a novel multidrug resistance protein 2 humanized mouse line.

    PubMed

    Scheer, Nico; Balimane, Praveen; Hayward, Michael D; Buechel, Sandra; Kauselmann, Gunther; Wolf, C Roland

    2012-11-01

    The multidrug resistance protein (MRP) 2 is predominantly expressed in liver, intestine, and kidney, where it plays an important role in the excretion of a range of drugs and their metabolites or endogenous compounds into bile, feces, and urine. Mrp knockout [Mrp2(-/-)] mice have been used recently to study the role of MRP2 in drug disposition. Here, we describe the first generation and initial characterization of a mouse line humanized for MRP2 (huMRP2), which is nulled for the mouse Mrp2 gene and expresses the human transporter in the organs and cell types where MRP2 is normally expressed. Analysis of the mRNA expression for selected cytochrome P450 and transporter genes revealed no major changes in huMRP2 mice compared with wild-type controls. We show that human MRP2 is able to compensate functionally for the loss of the mouse transporter as demonstrated by comparable bilirubin levels in the humanized mice and wild-type controls, in contrast to the hyperbilirubinemia phenotype that is observed in MRP2(-/-) mice. The huMRP2 mouse provides a model to study the role of the human transporter in drug disposition and in assessing the in vivo consequences of inhibiting this transporter by compounds interacting with human MRP2. PMID:22917771

  4. Hypoenergetic diet-induced reductions in myofibrillar protein synthesis are restored with resistance training and balanced daily protein ingestion in older men.

    PubMed

    Murphy, Caoileann H; Churchward-Venne, Tyler A; Mitchell, Cameron J; Kolar, Nathan M; Kassis, Amira; Karagounis, Leonidas G; Burke, Louise M; Hawley, John A; Phillips, Stuart M

    2015-05-01

    Strategies to enhance weight loss with a high fat-to-lean ratio in overweight/obese older adults are important since lean loss could exacerbate sarcopenia. We examined how dietary protein distribution affected muscle protein synthesis during energy balance (EB), energy restriction (ER), and energy restriction plus resistance training (ER + RT). A 4-wk ER diet was provided to overweight/obese older men (66 ± 4 yr, 31 ± 5 kg/m(2)) who were randomized to either a balanced (BAL: 25% daily protein/meal × 4) or skewed (SKEW: 7:17:72:4% daily protein/meal; n = 10/group) pattern. Myofibrillar and sarcoplasmic protein fractional synthetic rates (FSR) were measured during a 13-h primed continuous infusion of l-[ring-(13)C6]phenylalanine with BAL and SKEW pattern of protein intake in EB, after 2 wk ER, and after 2 wk ER + RT. Fed-state myofibrillar FSR was lower in ER than EB in both groups (P < 0.001), but was greater in BAL than SKEW (P = 0.014). In ER + RT, fed-state myofibrillar FSR increased above ER in both groups and in BAL was not different from EB (P = 0.903). In SKEW myofibrillar FSR remained lower than EB (P = 0.002) and lower than BAL (P = 0.006). Fed-state sarcoplasmic protein FSR was reduced similarly in ER and ER + RT compared with EB (P < 0.01) in both groups. During ER in overweight/obese older men a BAL consumption of protein stimulated the synthesis of muscle contractile proteins more effectively than traditional, SKEW distribution. Combining RT with a BAL protein distribution "rescued" the lower rates of myofibrillar protein synthesis during moderate ER. PMID:25738784

  5. Hypoenergetic diet-induced reductions in myofibrillar protein synthesis are restored with resistance training and balanced daily protein ingestion in older men

    PubMed Central

    Murphy, Caoileann H.; Churchward-Venne, Tyler A.; Mitchell, Cameron J.; Kolar, Nathan M.; Kassis, Amira; Karagounis, Leonidas G.; Burke, Louise M.; Hawley, John A.

    2015-01-01

    Strategies to enhance weight loss with a high fat-to-lean ratio in overweight/obese older adults are important since lean loss could exacerbate sarcopenia. We examined how dietary protein distribution affected muscle protein synthesis during energy balance (EB), energy restriction (ER), and energy restriction plus resistance training (ER + RT). A 4-wk ER diet was provided to overweight/obese older men (66 ± 4 yr, 31 ± 5 kg/m2) who were randomized to either a balanced (BAL: 25% daily protein/meal × 4) or skewed (SKEW: 7:17:72:4% daily protein/meal; n = 10/group) pattern. Myofibrillar and sarcoplasmic protein fractional synthetic rates (FSR) were measured during a 13-h primed continuous infusion of l-[ring-13C6]phenylalanine with BAL and SKEW pattern of protein intake in EB, after 2 wk ER, and after 2 wk ER + RT. Fed-state myofibrillar FSR was lower in ER than EB in both groups (P < 0.001), but was greater in BAL than SKEW (P = 0.014). In ER + RT, fed-state myofibrillar FSR increased above ER in both groups and in BAL was not different from EB (P = 0.903). In SKEW myofibrillar FSR remained lower than EB (P = 0.002) and lower than BAL (P = 0.006). Fed-state sarcoplasmic protein FSR was reduced similarly in ER and ER + RT compared with EB (P < 0.01) in both groups. During ER in overweight/obese older men a BAL consumption of protein stimulated the synthesis of muscle contractile proteins more effectively than traditional, SKEW distribution. Combining RT with a BAL protein distribution “rescued” the lower rates of myofibrillar protein synthesis during moderate ER. PMID:25738784

  6. Protein kinase C is involved in resistance to myocardial infarction induced by heat stress.

    PubMed

    Joyeux, M; Baxter, G F; Thomas, D L; Ribuot, C; Yellon, D M

    1997-12-01

    Heat stress (HS) is known to protect against mechanical dysfunction and myocardial necrosis in myocardial ischemia-reperfusion models both in vivo and in vitro. However, the mechanisms involved in this form of cardioprotection remain unclear. Protein kinase C (PKC) and tyrosine kinase activation have both been shown to be involved in the delayed phase of protection following ischemic preconditioning, a phenomenon which appears to be analogous to HS-induced protection. Therefore, we investigated the role of PKC and tyrosine kinase in HS-induced resistance to myocardial infarction, in the isolated rat heart. The selective inhibitors chelerythrine (Che) and genistein (Gen) were used to inhibit PKC and tyrosine kinase, respectively. Rats were treated with Che (5 mg/kg, i.p.) or Gen (5 mg/kg, i.p.) or vehicle before they were either heat stressed (42 degrees C for 15 min) or sham anesthetized. Twenty-four h later their hearts were isolated, retrogradely perfused, and subjected to 35-min occlusion of the left coronary artery followed by 120-min of reperfusion. Infarct-to-risk ratio was significantly reduced in HS (19.9+/-1.1%) compared to sham (43.1+/-1.1%) hearts. This reduction in infarct size was abolished in chelerythrine-treated groups (43.8+/-1.9% in HS+Che v 44.9+/-2.0% in sham+Che), but was conserved in genistein-treated groups (17.7+/-0.9% in HS+Gen v 36.4+/-2.8% in sham+Gen). In order to confirm that genistein at this dose was effectively inhibiting tyrosine kinase activity, we observed the ability of the agent to prevent the hypoglycemic responses to insulin in a separate group of anesthetised rats receiving an i.v. insulin infusion. Western blot analysis of the myocardial hsp72 showed a HS-induced increase of this protein, which was modified by neither the PKC inhibitor, chelerythrine, nor the tyrosine kinase inhibitor, genistein. We conclude that the activation of PKC, but not of tyrosine kinase, appears to play a role in the functional cardioprotection

  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. RTP1 encodes a novel endoplasmic reticulum (ER)-localized protein in Arabidopsis and negatively regulates resistance against biotrophic pathogens.

    PubMed

    Pan, Qiaona; Cui, Beimi; Deng, Fengyan; Quan, Junli; Loake, Gary J; Shan, Weixing

    2016-03-01

    Oomycete pathogens cause serious damage to a wide spectrum of plants. Although host pathogen recognition via pathogen effectors and cognate plant resistance proteins is well established, the genetic basis of host factors that mediate plant susceptibility to oomycete pathogens is relatively unexplored. Here, we report on RTP1, a nodulin-related MtN21 family gene in Arabidopsis that mediates susceptibility to Phytophthora parasitica. RTP1 was identified by screening a T-DNA insertion mutant population and encoded an endoplasmic reticulum (ER)-localized protein. Overexpression of RTP1 rendered Arabidopsis more susceptible, whereas RNA silencing of RTP1 led to enhanced resistance to P. parasitica. Moreover, an RTP1 mutant, rtp1-1, displayed localized cell death, increased reactive oxygen species (ROS) production and accelerated PR1 expression, compared to the wild-type Col-0, in response to P. parasitica infection. rtp1-1 showed a similar disease response to the bacterial pathogen Pseudomonas syringae pv. tomato (Pst) DC3000, including increased disease resistance, cell death and ROS production. Furthermore, rpt1-1 exhibited resistance to the fungal pathogen Golovinomyces cichoracearum, but not to the necrotrophic pathogen Botrytis cinerea. Taken together, these results suggest that RTP1 negatively regulates plant resistance to biotrophic pathogens, possibly by regulating ROS production, cell death progression and PR1 expression. PMID:26484750

  9. Identification of Soybean Proteins and Genes Differentially Regulated in Near Isogenic Lines Differing in Resistance to Aphid Infestation.

    PubMed

    Brechenmacher, Laurent; Nguyen, Tran Hong Nha; Zhang, Ning; Jun, Tae-Hwan; Xu, Dong; Mian, M A Rouf; Stacey, Gary

    2015-10-01

    Soybean aphid is an important pest causing significant yield losses. The Rag2 locus confers resistance to soybean aphid biotypes 1 and 2. Transcriptomic and proteomic analyses were done over a 48 h period after aphid infestation using near isogenic lines (NILs) differing at the Rag2 locus. Comparing the Rag2 and/or rag2 lines identified 3445 proteins, of which 396 were differentially regulated between the two lines, including proteins involved in cell wall metabolism, carbohydrate metabolism, and stress response. RNA-seq transcriptomic analysis identified 2361 genes significantly regulated between the resistant and susceptible lines. Genes upregulated in the Rag2 line were annotated as being involved in cell wall, secondary, and hormone metabolism as well as in stress, signaling, and transcriptional responses. Genes downregulated in the Rag2 line were annotated as being involved in photosynthesis and carbon metabolism. Interestingly, two genes (unknown and mitochondrial protease) located within the defined Rag2 locus were expressed significantly higher in the resistant genotype. The expression of a putative NBS-LRR resistant gene within the Rag2 locus was not different between the two soybean lines, but a second NBL-LRR gene located just at the border of the defined Rag2 locus was. Therefore, this gene may be a candidate R gene controlling aphid resistance. PMID:26350764

  10. 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

  11. Mutation of G234 amino acid residue in Candida albicans drug-resistance-related protein Rta2p is associated with fluconazole resistance and dihydrosphingosine transport

    PubMed Central

    Zhang, Shi-Qun; Miao, Qi; Li, Li-Ping; Zhang, Lu-lu; Yan, Lan; Jia, Yu; Cao, Yong-Bing; Jiang, Yuan-Ying

    2015-01-01

    Widespread and repeated use of azoles has led to the rapid development of drug resistance in Candida albicans. Our previous study found Rta2p, a membrane protein with 7 transmembrane domains, was involved in calcineurin-mediated azole resistance and sphingoid long-chain base release in C. albicans. Conserved amino acids in the transmembrane domain of Rta2p were subjected to site-directed mutagenesis. The sensitivity of C. albicans to fluconazole in vitro was examined by minimum inhibitory concentration and killing assay, and the therapeutic efficacy of fluconazole in vivo was performed by systemic mice candidiasis model. Furthermore, dihydrosphingosine transport activity was detected by NBD labeled D-erythro-dihydrosphingosine uptake and release assay, and the sensitivity to sphingolipid biosynthesis inhibitors. We successfully constructed 14 mutant strains of Rta2p, screened them by minimum inhibitory concentration and found Ca2+ did not completely induce fluconazole resistance with G158E and G234S mutations. Furthermore, we confirmed that G234S mutant enhanced the therapeutic efficacy of fluconazole against systemic candidiasis and significantly increased the accumulation of dihydrosphingosine by decreasing its release. However, G158E mutant didn't affect drug therapeutic efficacy in vivo and dihydrosphingosine transport in C. albicans. G234 of Rta2p in C. albicans is crucial in calcineurin-mediated fluconazole resistance and dihydrosphingosine transport. PMID:26220356

  12. Relationship of LRP-human major vault protein to in vitro and clinical resistance to anticancer drugs.

    PubMed

    Izquierdo, M A; Scheffer, G L; Flens, M J; Shoemaker, R H; Rome, L H; Scheper, R J

    1996-01-01

    Multidrug resistance (MDR) has been related to two members of the ABC-superfamily of transporters, P-glycoprotein (Pgp) and Multidrug Resistance-associated Protein (MRP). We have described a 110 kD protein termed the Lung Resistance-related Protein (LRP) that is overexpressed in several non-Pgp MDR cells lines of different histogenetic origin. Reversal of MDR parallels a decrease in LRP expression. In a panel of 61 cancer cell lines which have not been subjected to laboratory drug selection, LRP was a superior predictor for in vitro resistance to MDR-related drugs when compared to Pgp and MRP, and LRP's predictive value extended to MDR unrelated drugs, such as platinum compounds. LRP is widely distributed in clinical cancer specimens, but the frequency of LRP expression inversely correlates with the known chemosensitivity of different tumour types. Furthermore, LRP expression at diagnosis has been shown to be a strong and independent prognostic factor for response to chemotherapy and outcome in acute myeloid leukemia and ovarian carcinoma (platinum-based treatment) patients. Recently, LRP has been identified as the human major protein. Vaults are novel cellular organelles broadly distributed and highly conserved among diverse eukaryotic cells, suggesting that they play a role in fundamental cell processes. Vaults localise to nuclear pore complexes and may be the central plug of the nuclear pore complexes. Vaults structure and localisation support a transport function for this particle which could involve a variety of substrates. Vaults may therefore play a role in drug resistance by regulating the nucleocytoplasmic transport of drugs. PMID:8862006

  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. 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

  15. 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

  16. Emergence of clonally related multidrug resistant Haemophilus influenzae with penicillin-binding protein 3-mediated resistance to extended-spectrum cephalosporins, Norway, 2006 to 2013.

    PubMed

    Skaare, D; Anthonisen, I L; Kahlmeter, G; Matuschek, E; Natås, O B; Steinbakk, M; Sundsfjord, A; Kristiansen, B E

    2014-01-01

    Resistance to cephalosporins in Haemophilus influenzae is usually caused by characteristic alterations in penicillin-binding protein 3 (PBP3), encoded by the ftsI gene. Resistance to extended-spectrum cephalosporins is associated with high-level PBP3-mediated resistance (high-rPBP3), defined by the second stage S385T substitution in addition to a first stage substitution (R517H or N526K). The third stage L389F substitution is present in some high-rPBP3 strains. High-rPBP3 H. influenzae are considered rare outside Japan and Korea. In this study, 30 high-rPBP3 isolates from Norway, collected between 2006 and 2013, were examined by serotyping, multilocus sequence typing (MLST), ftsI sequencing, detection of beta-lactamase genes and minimum inhibitory concentration (MIC) determination. MICs were interpreted according to clinical breakpoints from the European Committee on Antimicrobial Susceptibility Testing (EUCAST). Respiratory isolates predominated (proportion: 24/30). The 30 isolates included one serotype f isolate, while the remaining 29 lacked polysaccharide capsule genes. Resistance to extended-spectrum cephalosporins (cefixime, 29 isolates/30 isolates; cefepime, 28/30; cefotaxime, 26 /30; ceftaroline, 26/30; ceftriaxone, 14/30), beta-lactamase production (11/30) and co-resistance to non-beta-lactams (trimethoprim-sulfamethoxazole, 13/30; tetracycline, 4/30; chloramphenicol, 4/30; ciprofloxacin, 3/30) was frequent. The N526K substitution in PBP3 was present in 23 of 30 isolates; these included a blood isolate which represents the first invasive S385T + N526K isolate reported from Europe. The L389F substitution, present in 16 of 30 isolates, coincided with higher beta-lactam MICs. Non-susceptibility to meropenem was frequent in S385T + L389F + N526K isolates (8/12). All 11 beta-lactamase positive isolates were TEM-1. Five clonal groups of two to 10 isolates with identical MLST-ftsI allelic profiles were observed, including the first reported high-rPBP3

  17. 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

  18. 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.

  19. Optimal dietary protein level improved growth, disease resistance, intestinal immune and physical barrier function of young grass carp (Ctenopharyngodon idella).

    PubMed

    Xu, Jing; Wu, Pei; Jiang, Wei-Dan; Liu, Yang; Jiang, Jun; Kuang, Sheng-Yao; Tang, Ling; Tang, Wu-Neng; Zhang, Yong-An; Zhou, Xiao-Qiu; Feng, Lin

    2016-08-01

    This study investigated the effects of dietary proteins on the growth, disease resistance, intestinal immune and physical barrier functions of young grass carp (Ctenopharyngodon idella). A total of 540 young grass carp (264.11 ± 0.76 g) were fed six diets containing graded levels of protein (143.1, 176.7, 217.2, 257.5, 292.2 and 322.8 g digestible protein kg(-1) diet) for 8 weeks. After the growth trial, fish were challenged with Aeromonas hydrophila and mortalities were recorded for 14 days. The results indicated that optimal dietary protein levels: increased the production of antibacterial components, up-regulated anti-inflammatory cytokines, inhibitor of κBα, target of rapamycin and ribosomal protein S6 kinases 1 mRNA levels, whereas down-regulated pro-inflammatory cytokines, nuclear factor kappa B (NF-κB) P65, NF-κB P52, c-Rel, IκB kinase β, IκB kinase γ and eIF4E-binding proteins 2 mRNA levels in three intestinal segments of young grass carp (P < 0.05), suggesting that optimal dietary protein level could enhance fish intestinal immune barrier function; up-regulated the mRNA levels of tight junction complexes, B-cell lymphoma protein-2, inhibitor of apoptosis proteins, myeloid cell leukemia-1 and NF-E2-related factor 2, and increased the activities and mRNA levels of antioxidant enzymes, whereas down-regulated myosin light chain kinase, cysteinyl aspartic acid-protease 2, 3, 7, 8, 9, fatty acid synthetase ligand, apoptotic protease activating factor-1, Bcl-2 associated X protein, p38 mitogen-activated protein kinase, c-Jun N-terminal protein kinase and Kelch-like-ECH-associated protein 1b mRNA levels, and decreased reactive oxygen species, malondialdehyde and protein carbonyl contents in three intestinal segments of young grass carp (P < 0.05), indicating that optimal dietary protein level could improve fish intestinal physical barrier function. Finally, the optimal dietary protein levels for the growth performance (PWG) and against enteritis

  20. 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

  1. 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

  2. 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

  3. 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

  4. Systematic analyses of the ultraviolet radiation resistance-associated gene product (UVRAG) protein interactome by tandem affinity purification.

    PubMed

    Son, Ji-Hye; Hwang, Eurim C; Kim, Joungmok

    2016-03-01

    Ultraviolet radiation resistance-associated gene product (UVRAG) was originally identified as a protein involved in cellular responses to UV irradiation. Subsequent studies have demonstrated that UVRAG plays as an important role in autophagy, a lysosome-dependent catabolic program, as a part of a pro-autophagy PIK3C3/VPS34 lipid kinase complex. Several recent studies have shown that UVRAG is also involved in autophagy-independent cellular functions, such as DNA repair/stability and vesicular trafficking/fusion. Here, we examined the UVRAG protein interactome to obtain information about its functional network. To this end, we screened UVRAG-interacting proteins using a tandem affinity purification method coupled with MALDI-TOF/MS analysis. Our results demonstrate that UVRAG interacts with various proteins involved in a wide spectrum of cellular functions, including genome stability, protein translational elongation, protein localization (trafficking), vacuole organization, transmembrane transport as well as autophagy. Notably, the interactome list of high-confidence UVRAG-interacting proteins is enriched for proteins involved in the regulation of genome stability. Our systematic UVRAG interactome analysis should provide important clues for understanding a variety of UVRAG functions. PMID:26590968

  5. 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

  6. 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

  7. Role of acute-phase proteins in interleukin-1-induced nonspecific resistance to bacterial infections in mice.

    PubMed Central

    Vogels, M T; Cantoni, L; Carelli, M; Sironi, M; Ghezzi, P; van der Meer, J W

    1993-01-01

    Treatment with a single low dose (80 to 800 ng) of interleukin-1 (IL-1) 24 h before a lethal bacterial challenge of granulocytopenic and normal mice enhances nonspecific resistance. Since IL-1 induces secretion of acute-phase proteins, liver proteins which possess several detoxifying effects, we investigated the role of these proteins in the IL-1-induced protection. Inhibition of liver protein synthesis with D-galactosamine (GALN) completely inhibited the IL-1-induced synthesis of acute-phase proteins. GALN pretreatment abolished the protective effect of IL-1 on survival completely (neutropenic mice infected with Pseudomonas aeruginosa) or partially (nonneutropenic mice infected with Klebsiella pneumoniae). Pretreatment with IL-6, a cytokine induced by IL-1, did not reproduce the protection offered after IL-1 pretreatment, nor did it enhance or deteriorate the IL-1-enhanced resistance to infection. A protective effect of IL-1 via effects on glucose homeostasis during the acute-phase response was investigated by comparing plasma glucose levels in IL-1-treated mice and control mice before and during infection. Although glucose levels in IL-1-pretreated mice were somewhat higher in the later stages of infection, no significant differences from levels in control mice were present, and the glucose levels in control-treated animals never fell to hypoglycemic values. We conclude that the IL-1-induced nonspecific resistance is mediated neither by the induction of IL-6 nor by the effects of IL-1 on glucose homeostasis. Acute-phase proteins generated after IL-1 pretreatment, however, seem to play a critical role in the IL-1-induced protection to infection. PMID:7509141

  8. Involvement of the Pepper Antimicrobial Protein CaAMP1 Gene in Broad Spectrum Disease Resistance1[C][OA

    PubMed Central

    Lee, Sung Chul; Hwang, In Sun; Choi, Hyong Woo; Hwang, Byung Kook

    2008-01-01

    Pathogen-inducible antimicrobial defense-related proteins have emerged as key antibiotic peptides and enzymes involved in disease resistance in plants. A novel antimicrobial protein gene, CaAMP1 (for Capsicum annuum ANTIMICROBIAL PROTEIN1), was isolated from pepper (C. annuum) leaves infected with Xanthomonas campestris pv vesicatoria. Expression of the CaAMP1 gene was strongly induced in pepper leaves not only during pathogen infection but also after exposure to abiotic elicitors. The purified recombinant CaAMP1 protein possessed broad-spectrum antimicrobial activity against phytopathogenic bacteria and fungi. CaAMP1:smGFP fusion protein was localized mainly in the external and intercellular regions of onion (Allium cepa) epidermal cells. The virus-induced gene silencing technique and gain-of-function transgenic plants were used to determine the CaAMP1 gene function in plant defense. Silencing of CaAMP1 led to enhanced susceptibility to X. campestris pv vesicatoria and Colletotrichum coccodes infection, accompanied by reduced PATHOGENESIS-RELATED (PR) gene expression. In contrast, overexpression of CaAMP1 in Arabidopsis (Arabidopsis thaliana) conferred broad-spectrum resistance to the hemibiotrophic bacterial pathogen Pseudomonas syringae pv tomato, the biotrophic oomycete Hyaloperonospora parasitica, and the fungal necrotrophic pathogens Fusarium oxysporum f. sp. matthiolae and Alternaria brassicicola. CaAMP1 overexpression induced the salicylic acid pathway-dependent genes PR1 and PR5 but not the jasmonic acid-dependent defense gene PDF1.2 during P. syringae pv tomato infection. Together, these results suggest that the antimicrobial CaAMP1 protein is involved in broad-spectrum resistance to bacterial and fungal pathogen infection. PMID:18676663

  9. Electroacupuncture Improves Insulin Resistance by Reducing Neuroprotein Y/Agouti-Related Protein Levels and Inhibiting Expression of Protein Tyrosine Phosphatase 1B in Diet-induced Obese Rats.

    PubMed

    Liu, Xia; He, Jun-Feng; Qu, Ya-Ting; Liu, Zhi-Jun; Pu, Qing-Yang; Guo, Sheng-Tong; Du, Jia; Jiang, Peng-Fei

    2016-04-01

    Electroacupuncture (EA) has been shown to exert beneficial effects on obesity, but the mechanism is unclear. This study investigated the effects of EA on diet-induced obese (DIO) rats. Fifty male Sprague-Dawley rats were randomly divided into low-fat diet (LFD, 10 rats) and high-fat diet (HFD, 40 rats) groups. After the DIO models had been established, successful model rats were randomly divided into HFD, EA, and orlistat (OLST) groups. The EA group received EA at Zusanli (ST36) and Quchi (LI11) for 20 minutes once per day for 28 days. The OLST group was treated with orlistat by gavage. The body weight, homeostasis model assessment-insulin resistance index, adipocyte diameters, and neuroprotein Y/agouti-related protein and protein tyrosine phosphatase 1B levels were significantly lower in the EA group than in the HFD group. The rats of the OLST group showed watery stools and yellow hairs whereas those of the EA group had regular stools and sleek coats. The effect of EA on weight loss may be related to improved insulin resistance caused by changes in the adipocyte size and by reductions in the expressions of neuroprotein Y/agouti-related protein and protein tyrosine phosphatase 1B. This study indicates that EA may be a better method of alternative therapy for treating obesity and other metabolic diseases. PMID:27079226

  10. Elicitin-Induced Distal Systemic Resistance in Plants is Mediated Through the Protein-Protein Interactions Influenced by Selected Lysine Residues.

    PubMed

    Uhlíková, Hana; Obořil, Michal; Klempová, Jitka; Šedo, Ondrej; Zdráhal, Zbyněk; Kašparovský, Tomáš; Skládal, Petr; Lochman, Jan

    2016-01-01

    Elicitins are a family of small proteins with sterol-binding activity that are secreted by Phytophthora and Pythium sp. classified as oomycete PAMPs. Although α- and β-elicitins bind with the same affinity to one high affinity binding site on the plasma membrane, β-elicitins (possessing 6-7 lysine residues) are generally 50- to 100-fold more active at inducing distal HR and systemic resistance than the α-isoforms (with only 1-3 lysine residues). To examine the role of lysine residues in elicitin biological activity, we employed site-directed mutagenesis to prepare a series of β-elicitin cryptogein variants with mutations on specific lysine residues. In contrast to direct infiltration of protein into leaves, application to the stem revealed a rough correlation between protein's charge and biological activity, resulting in protection against Phytophthora parasitica. A detailed analysis of proteins' movement in plants showed no substantial differences in distribution through phloem indicating differences in consequent apoplastic or symplastic transport. In this process, an important role of homodimer formation together with the ability to form a heterodimer with potential partner represented by endogenous plants LTPs is suggested. Our work demonstrates a key role of selected lysine residues in these interactions and stresses the importance of processes preceding elicitin recognition responsible for induction of distal systemic resistance. PMID:26904041

  11. Crystal structure and dimerization equilibria of PcoC, a methionine-rich copper resistance protein from Escherichia coli

    SciTech Connect

    Wernimont, A.K.; Huffman, D.L.; Finney, L.A.; Demeler, B.; O'Halloran, T.V.; Rosenzweig, A.C.

    2010-03-08

    PcoC is a soluble periplasmic protein encoded by the plasmid-born pco copper resistance operon of Escherichia coli. Like PcoA, a multicopper oxidase encoded in the same locus and its chromosomal homolog CueO, PcoC contains unusual methionine rich sequences. Although essential for copper resistance, the functions of PcoC, PcoA, and their conserved methionine-rich sequences are not known. Similar methionine motifs observed in eukaryotic copper transporters have been proposed to bind copper, but there are no precedents for such metal binding sites in structurally characterized proteins. The high-resolution structures of apo PcoC, determined for both the native and selenomethionine-containing proteins, reveal a seven-stranded barrel with the methionines unexpectedly housed on a solvent-exposed loop. Several potential metal-binding sites can be discerned by comparing the structures to spectroscopic data reported for copper-loaded PcoC. In the native structure, the methionine loop interacts with the same loop on a second molecule in the asymmetric unit. In the selenomethionine structure, the methionine loops are more exposed, forming hydrophobic patches on the protein surface. These two arrangements suggest that the methionine motifs might function in protein-protein interactions between PcoC molecules or with other methionine-rich proteins such as PcoA. Analytical ultracentrifugation data indicate that a weak monomer-dimer equilibrium exists in solution for the apo protein. Dimerization is significantly enhanced upon binding Cu(I) with a measured {Delta}({Delta}G{sup o}) {le} -8.0 kJ/mole, suggesting that copper might bind at the dimer interface.

  12. Regulation of Multidrug Resistance-Associated Protein 2 by Calcium Signaling in Mouse Liver

    PubMed Central

    Cruz, Laura N.; Guerra, Mateus T.; Kruglov, Emma; Mennone, Albert; Garcia, Celia R. S.; Chen, Ju; Nathanson, Michael H.

    2011-01-01

    Multidrug resistance associated protein 2 (Mrp2) is a canalicular transporter responsible for organic anion secretion into bile. Mrp2 activity is regulated by insertion into the plasma membrane; however, the factors that control this are not understood. Calcium (Ca2+) signaling regulates exocytosis of vesicles in most cell types, and the type II inositol 1,4,5-triphosphate receptor (InsP3R2) regulates Ca2+ release in the canalicular region of hepatocytes. However, the role of InsP3R2 and of Ca2+ signals in canalicular insertion and function of Mrp2 is not known. The aim of this study was to determine the role of InsP3R2-mediated Ca2+ signals in targeting Mrp2 to the canalicular membrane. Livers, isolated hepatocytes, and hepatocytes in collagen sandwich culture from wild-type (WT) and InsP3R2 knockout (KO) mice were used for western blots, confocal immunofluorescence, and time-lapse imaging of Ca2+ signals and of secretion of a fluorescent organic anion. Plasma membrane insertion of green fluorescent protein (GFP)-Mrp2 expressed in HepG2 cells was monitored by total internal reflection microscopy. InsP3R2 was concentrated in the canalicular region of WT mice but absent in InsP3R2 KO livers, whereas expression and localization of InsP3R1 was preserved, and InsP3R3 was absent from both WT and KO livers. Ca2+ signals induced by either adenosine triphosphate (ATP) or vasopressin were impaired in hepatocytes lacking InsP3R2. Canalicular secretion of the organic anion 5-chloromethylfluorescein diacetate (CMFDA) was reduced in KO hepatocytes, as well as in WT hepatocytes treated with 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA). Moreover, the choleretic effect of tauroursodeoxycholic acid (TUDCA) was impaired in InsP3R2 KO mice. Finally, ATP increased GFP-Mrp2 fluorescence in the plasma membrane of HepG2 cells, and this also was reduced by BAPTA. Conclusion InsP3R2-mediated Ca2+ signals enhance organic anion secretion into bile by targeting Mrp2 to

  13. 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

  14. X-linked inhibitor of apoptosis protein mediates tumor cell resistance to antibody-dependent cellular cytotoxicity.

    PubMed

    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

  15. 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. PMID:26483548

  16. 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 Central

    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

    2012-01-01

    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. PMID:22451437

  17. Role of multi-drug resistance-associated protein-1 transporter in statin-induced myopathy.

    PubMed

    Dorajoo, Rajkumar s o; Pereira, Barry P; Yu, Zou; Gopalakrishnakone, P; Leong, Cheng Chee; Wee, Aileen; Lee, Edmund

    2008-04-01

    This study investigated the effects of probenecid to inhibit the multi-drug resistance-associated protein-1 (MRP-1) in mediating the efflux and myotoxicity in rat skeletal muscles, with administration of rosuvastatin. Male Sprague-Dawley rats were administered daily, for 15 days, with either rosuvastatin (50, 100 or 200 mg/kg) or probenecid (100 mg/kg) alone, or with a combination of rosuvastatin (50, 100 or 200 mg/kg) and probenecid (100 mg/kg). Skeletal muscle toxicity was elevated with probenecid administered with 200 mg/kg/day of rosuvastatin, with the elevation of creatine kinase by 12-fold, alanine aminotrasferase by 10-fold and creatinine by 9-fold at day 15, with no adverse effects observed when probenecid was given alone. Mitochondria ultrastructural damage with enlargement, disruption, cristolysis and vaculation was seen in the soleus and plantaris of animals administered with probenecid and high dosages of statin. These muscles were also expressing more succinic dehydrogenase (SDH)-positive and cytochrome oxidase (CyOX)-positive fibers. Although generally well-tolerated, statins produce a variety of adverse skeletal muscle events. Hydrophilic statins, with reduced levels of non-specific passive diffusion rates into extra-hepatic tissues, are still seen to produce myopathy. This highlights the important roles of transport mechanisms in statin transport at the skeletal muscles. Excessive influx, reduced efflux or the combination of the two could result in elevated cellular levels of statins at the skeletal muscles, resulting in toxicity. This study provides preliminary evidence that the MRP-1 transporter and efflux at skeletal muscles possibly play significant roles in statin-induced myopathy. PMID:18509883

  18. Arachidonic acid pathway activates multidrug resistance related protein in cultured human lung cells.

    PubMed

    Torky, Abdelrahman; Raemisch, Anja; Glahn, Felix; Foth, Heidi

    2008-05-01

    Primary cultures of human lung cells can serve as a model system to study the mechanisms underlying the effects of irritants in air and to get a deeper insight into the (patho)physiological roles of the xenobiotic detoxification systems. For 99 human lung cancer cases the culture duration for bronchial epithelium and peripheral lung cells (PLC) are given in term of generations and weeks. Using this system, we investigated whether and how prostaglandins (PG) modify multidrug resistance related protein (MRP) function in normal human lung cells. PGF2alpha had no effect on MRP function, whereas PGE2 induced MRP activity in cultured NHBECs. The transport activity study of MRP in NHBEC, PLC, and A549 under the effect of exogenously supplied PGF2alpha (10 microM, 1 day) using single cell fluorimetry revealed no alteration in transport activity of MRP. PG concentrations were within the physiological range. COX I and II inhibitors indomethacin (5, 10 microM) and celecoxib (5, 10 microM) could substantially decrease the transport activity of MRP in NHBEC, PLC, and A549 in 1- and 4-day trials. Prostaglandin E2 did not change cadmium-induced caspase 3/7 activation in NHBECs and had no own effect on caspase 3/7 activity. Cadmium chloride (5, 10 microM) was an effective inducer of caspase 3/7 activation in NHBECs with a fivefold and ninefold rise of activity. In primary human lung cells arachidonic acid activates MRP transport function only in primary epithelial lung cells by prostaglandin E2 but not by F2alpha mediated pathways and this effect needs some time to develop. PMID:17943274

  19. 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

  20. 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

  1. Role of Multidrug Resistance Protein 3 in Antifungal-Induced Cholestasis.

    PubMed

    Mahdi, Zainab M; Synal-Hermanns, Uta; Yoker, Aylin; Locher, Kaspar P; Stieger, Bruno

    2016-07-01

    Drug-induced liver injury is an important clinical entity resulting in a considerable number of hospitalizations. While drug-induced cholestasis due to the inhibition of the bile salt export pump (BSEP) is well investigated, only limited information on the interaction of drugs with multidrug resistance protein 3 (MDR3) exists and its role in the pathogenesis of drug-induced cholestasis is poorly understood. Therefore, we aimed to study the interaction of drugs with MDR3 and the effect of drugs on canalicular lipid secretion in a newly established polarized cell line system that serves as a model of canalicular lipid secretion. LLC-PK1 cells were stably transfected with human Na(+)-taurocholate cotransporting polypeptide, BSEP, MDR3, and ABCG5/G8 and grown in the Transwell system. Apical phospholipid secretion and taurocholate transport were assayed to investigate the effect of selected drugs on MDR3-mediated phospholipid secretion as well as inhibition of BSEP. The established cell line displayed vectorial bile salt transport and specific phosphatidylcholine secretion into the apical compartment. The antifungal azoles, posaconazole, itraconazole, and ketoconazole, significantly inhibited MDR3-mediated phosphatidylcholine secretion. In contrast, amoxicillin clavulanate and troglitazone did not interfere with MDR3 activity. Drugs interfering with MDR3 activity did not display a parallel inhibition of BSEP. Our in vitro model for MDR3-mediated phospholipid secretion facilitates parallel screening for MDR3 and BSEP inhibitors. Our data demonstrate that the cholestatic potential of certain drugs may be aggravated by simultaneous inhibition of BSEP and MDR3. PMID:27112167

  2. 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

  3. 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

  4. High-fat diet induced adiposity and insulin resistance in mice lacking the myotonic dystrophy protein kinase.

    PubMed

    Llagostera, Esther; Carmona, Mari Carmen; Vicente, Meritxell; Escorihuela, Rosa María; Kaliman, Perla

    2009-06-18

    Myotonic dystrophy 1 (MD1) is caused by a CTG expansion in the 3'-unstranslated region of the myotonic dystrophy protein kinase (DMPK) gene. MD1 patients frequently present insulin resistance and increased visceral adiposity. We examined whether DMPK deficiency is a genetic risk factor for high-fat diet-induced adiposity and insulin resistance using the DMPK knockout mouse model. We found that high-fat fed DMPK knockout mice had significantly increased body weights, hypertrophic adipocytes and whole-body insulin resistance compared with wild-type mice. This nutrient-genome interaction should be considered by physicians given the cardiometabolic risks and sedentary lifestyle associated with MD1 patients. PMID:19482024

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

    PubMed

    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; Bhalla, Kapil N

    2015-09-24

    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

  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. Lipopolysaccharide-binding protein plasma levels as a biomarker of obesity-related insulin resistance in adolescents

    PubMed Central

    Kim, Ki Eun; Cho, Young Sun; Baek, Kyung Suk; Li, Lan; Baek, Kwang-Hyun; Kim, Jung Hyun; Kim, Ho-Seong

    2016-01-01

    Purpose Lipopolysaccharide-binding protein (LBP) is a 65-kDa acute phase protein, derived from the liver, which is present in high concentrations in plasma. Data regarding the association between circulating plasma LBP levels and obesity-related biomarkers in the pediatric population are scarce. We aimed to determine whether there was a difference in plasma LBP levels between overweight/obese and normal-weight adolescents and to assess the correlation of circulating LBP levels with anthropometric measures and obesity-related biomarkers, including insulin resistance, liver enzyme levels, and lipid profiles. Methods The study included 87 adolescents aged 12–13 years; 44 were overweight/obese and 43 were of normal-weight. We assessed anthropometric and laboratory measures, including body mass index (BMI), blood pressure, insulin resistance, liver enzyme levels, and lipid profiles. Plasma LBP levels were measured using an enzyme-linked immunosorbent assay. Results The mean age of the participants was 12.9±0.3 years. Circulating plasma LBP levels were significantly increased in overweight/obese participants compared with those in normal-weight participants (7.8±1.9 µg/mL vs. 6.0±1.6 µg/mL, P<0.001). LBP levels were significantly and positively associated with BMI, systolic blood pressure, aspartate aminotransferase, alanine aminotransferase, total cholesterol, low density lipoprotein-cholesterol, fasting glucose and insulin, and insulin resistance as indicated by the homeostatic model assessment of insulin resistance (HOMA-IR) (all P<0.05). In multivariate linear regression analysis, BMI and HOMA-IR were independently and positively associated with plasma LBP levels. Conclusion LBP is an inflammatory biomarker associated with BMI and obesity-related insulin resistance in adolescents. The positive correlation between these parameters suggests a potentially relevant pathophysiological mechanism linking LBP to obesity-related insulin resistance in adolescents. PMID

  8. 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

  9. 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

  10. 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

  11. Complete nucleotide sequence of a gene conferring polymyxin B resistance on yeast: similarity of the predicted polypeptide to protein kinases.

    PubMed

    Boguslawski, G; Polazzi, J O

    1987-08-01

    Polymyxin B is an antibiotic that kills sensitive cells by disrupting their membranes. We have cloned a wild-type yeast gene that, when present on a high-copy-number plasmid, renders the cells resistant to the drug. The nucleotide sequence of this gene is presented. A single open reading frame within the sequence has the potential to encode a polypeptide (molecular mass of 77.5 kDa) that shows strong homologies to polypeptides of the protein kinase family. The gene, PBS2, located on chromosome X, is not allelic to the previously described PBS1 gene (where PBS signifies polymyxin B sensitivity). Although pbs1 mutations confer resistance to high levels of polymyxin B, double mutants, pbs1 pbs2, are not resistant to the drug, indicating that PBS2 is essential for pbs1 activity. Models based on the proposed protein kinase activity of the PBS2 gene product are presented to explain the interaction between PBS1 and PBS2 gene products involved in conferring polymyxin B resistance on yeast cells. PMID:3039511

  12. 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

  13. Enhancing the protein resistance of silicone via surface-restructuring PEO-silane amphiphiles with variable PEO length

    PubMed Central

    Rufin, M. A.; Gruetzner, J. A.; Hurley, M. J.; Hawkins, M. L.; Raymond, E. S.; Raymond, J. E.

    2015-01-01

    Silicones with superior protein resistance were produced by bulk-modification with poly(ethylene oxide) (PEO)-silane amphiphiles that demonstrated a higher capacity to restructure to the surface-water interface versus conventional non-amphiphilic PEO-silanes. The PEO-silane amphiphiles were prepared with a single siloxane tether length but variable PEO segment lengths: α-(EtO)3Si(CH2)2-oligodimethylsiloxane13-block-poly(ethylene oxide)n-OCH3 (n = 3, 8, and 16). Conventional PEO-silane analogues (n = 3, 8 and 16) as well as a siloxane tether-silane (i.e. no PEO segment) were prepared as controls. When surface-grafted onto silicon wafer, PEO-silane amphiphiles produced surfaces that were more hydrophobic and thus more adherent towards fibrinogen versus the corresponding PEO-silane. However, when blended into a silicone, PEO-silane amphiphiles exhibited rapid restructuring to the surface-water interface and excellent protein resistance whereas the PEO-silanes did not. Silicones modified with PEO-silane amphiphiles of PEO segment lengths n = 8 and 16 achieved the highest protein resistance. PMID:26339488

  14. Multidrug resistance-associated protein 1 decreases the concentrations of antiepileptic drugs in cortical extracellular fluid in amygdale kindling rats

    PubMed Central

    Chen, Ying-hui; Wang, Cui-cui; Xiao, Xia; Wei, Li; Xu, Guoxiong

    2013-01-01

    Aim: To investigate whether multidrug resistance-associated protein 1 (MRP1) was responsible for drug resistence in refractory epilepsy in amygdale kindling rats. Methods: Rat amygdale kindling was used as a model of refractory epilepsy. The expression of MRP1 mRNA and protein in the brains was examined using RT-PCR and Western blot. MRP1-positive cells in the cortex and hippocampus were studied with immunohistochemical staining. The rats were intraperitoneally injected with phenytoin (50 mg/kg) or carbamazepine (20 mg/kg), and their concentrations in the cortical extracellular fluid were measured using microdialysis and HPLC. Probenecid, a MRP1 inhibitor (40 mmol/L, 50 μL) was administered through an inflow tube into the cortex 30 min before injection of the antiepileptic drugs. Results: The expression of MRP1 mRNA and protein was significantly up-regulated in the cortex and hippocampus in amygdale kindling rats compared with the control group. Furthermore, the number of MRP1-positive cells in the cortex and hippocampus was also significantly increased in amygdale kindling rats. Microdialysis studies showed that the concentrations of phenytoin and carbamazepine in the cortical extracellular fluid were significantly decreased in amygdale kindling rats. Pre-administration of probenecid could restore the concentrations back to their control levels. Conclusion: Up-regulation of MRP1 is responsible for the resistance of brain cells to antiepileptic drugs in the amygdale kindling rats. PMID:23474709

  15. 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

  16. 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.

  17. Modulation of Multidrug Resistance Protein 2 Efflux in the Cisplatin Resistance Human Ovarian Carcinoma Cells A2780/RCIS by Sesquiterpene Coumarins.

    PubMed

    Kasaian, Jamal; Mosaffa, Fatemeh; Behravan, Javad; Masullo, Milena; Piacente, Sonia; Iranshahi, Mehrdad

    2016-01-01

    Recent in vitro studies showed that sesquiterpene coumarins (SCs) can be used as chemosensitizers. In this study, 14 SCs were isolated and purified from roots of four Ferula species and their structures were elucidated by spectroscopic methods. The purified SCs were evaluated for multidrug resistance (MDR) reversal properties in A2780/RCIS cells (cisplatin-resistant derivatives of the human ovarian carcinoma cell line A2780P). Among the tested compounds, mogoltacin, mogoltadone, farnesiferol A, farnesiferol B, farnesiferol C, lehmferin, conferdione, and samarcandin showed significant MDR reversing effects. The combination of nontoxic concentrations of SCs (20 μM) with cisplatin enhanced cisplatin cytotoxicity on A2780/RCIS cells significantly. Flow cytometric efflux assay confirmed that the intracellular accumulation of 5-carboxyfluorescein diacetate (5-CFDA) was significantly increased in A2780/RCIS cells when treated with SCs. Our findings revealed that conferdione and samarcandin possessed the highest inhibitory effects on multidrug resistance-associated protein 2 pump efflux, and therefore, these compounds could be considered as lead scaffolds for further structure modifications. PMID:26503061

  18. Recognition of the Protein Kinase AVRPPHB SUSCEPTIBLE1 by the Disease Resistance Protein RESISTANCE TO PSEUDOMONAS SYRINGAE5 Is Dependent on S-Acylation and an Exposed Loop in AVRPPHB SUSCEPTIBLE11[W][OPEN

    PubMed Central

    Qi, Dong; Dubiella, Ullrich; Kim, Sang Hee; Sloss, D. Isaiah; Dowen, Robert H.; Dixon, Jack E.; Innes, Roger W.

    2014-01-01

    The recognition of pathogen effector proteins by plants is typically mediated by intracellular receptors belonging to the nucleotide-binding leucine-rich repeat (NLR) family. NLR proteins often detect pathogen effector proteins indirectly by detecting modification of their targets. How NLR proteins detect such modifications is poorly understood. To address these questions, we have been investigating the Arabidopsis (Arabidopsis thaliana) NLR protein RESISTANCE TO PSEUDOMONAS SYRINGAE5 (RPS5), which detects the Pseudomonas syringae effector protein Avirulence protein Pseudomonas phaseolicolaB (AvrPphB). AvrPphB is a cysteine protease that specifically targets a subfamily of receptor-like cytoplasmic kinases, including the Arabidopsis protein kinase AVRPPHB Susceptible1 (PBS1). RPS5 is activated by the cleavage of PBS1 at the apex of its activation loop. Here, we show that RPS5 activation requires that PBS1 be localized to the plasma membrane and that plasma membrane localization of PBS1 is mediated by amino-terminal S-acylation. We also describe the development of a high-throughput screen for mutations in PBS1 that block RPS5 activation, which uncovered four new pbs1 alleles, two of which blocked cleavage by AvrPphB. Lastly, we show that RPS5 distinguishes among closely related kinases by the amino acid sequence (SEMPH) within an exposed loop in the C-terminal one-third of PBS1. The SEMPH loop is located on the opposite side of PBS1 from the AvrPphB cleavage site, suggesting that RPS5 associates with the SEMPH loop while leaving the AvrPphB cleavage site exposed. These findings provide support for a model of NLR activation in which NLR proteins form a preactivation complex with effector targets and then sense a conformational change in the target induced by effector modification. PMID:24225654

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

    PubMed Central

    Nakasu, Erich Y. T.; Edwards, Martin G.; Fitches, Elaine; Gatehouse, John A.; Gatehouse, Angharad M. R.

    2014-01-01

    Recombinant fusion proteins containing arthropod toxins have been developed as a new class of biopesticides. The recombinant fusion protein Hv1a/GNA, containing the spider venom toxin ω-ACTX-Hv1a linked to snowdrop lectin (GNA) was shown to reduce survival of the peach-potato aphid Myzus persicae when delivered in artificial diet, with survival <10% after 8 days exposure to fusion protein at 1 mg/ml. Although the fusion protein was rapidly degraded by proteases in the insect, Hv1a/GNA oral toxicity to M. persicae was significantly greater than GNA alone. A construct encoding the fusion protein, including the GNA leader sequence, under control of the constitutive CaMV 35S promoter was transformed into Arabidopsis; the resulting plants contained intact fusion protein in leaf tissues at an estimated level of 25.6 ± 4.1 ng/mg FW. Transgenic Arabidopsis expressing Hv1a/GNA induced up to 40% mortality of M. persicae after 7 days exposure in detached leaf bioassays, demonstrating that transgenic plants can deliver fusion proteins to aphids. Grain aphids (Sitobion avenae) were more susceptible than M. persicae to the Hv1a/GNA fusion protein in artificial diet bioassays (LC50 = 0.73 mg/ml after 2 days against LC50 = 1.81 mg/ml for M. persicae), as they were not able to hydrolyze the fusion protein as readily as M. persicae. Expression of this fusion protein in suitable host plants for the grain aphid is likely to confer higher levels of resistance than that shown with the M. persicae/Arabidopsis model system. PMID:25506351

  20. Low-Load High Volume Resistance Exercise Stimulates Muscle Protein Synthesis More Than High-Load Low Volume Resistance Exercise in Young Men

    PubMed Central

    Burd, Nicholas A.; West, Daniel W. D.; Staples, Aaron W.; Atherton, Philip J.; Baker, Jeff M.; Moore, Daniel R.; Holwerda, Andrew M.; Parise, Gianni; Rennie, Michael J.; Baker, Steven K.; Phillips, Stuart M.

    2010-01-01

    Background We aimed to determine the effect of resistance exercise intensity (% 1 repetition maximum—1RM) and volume on muscle protein synthesis, anabolic signaling, and myogenic gene expression. Methodology/Principal Findings Fifteen men (21±1 years; BMI = 24.1±0.8 kg/m2) performed 4 sets of unilateral leg extension exercise at different exercise loads and/or volumes: 90% of repetition maximum (1RM) until volitional failure (90FAIL), 30% 1RM work-matched to 90%FAIL (30WM), or 30% 1RM performed until volitional failure (30FAIL). Infusion of [ring-13C6] phenylalanine with biopsies was used to measure rates of mixed (MIX), myofibrillar (MYO), and sarcoplasmic (SARC) protein synthesis at rest, and 4 h and 24 h after exercise. Exercise at 30WM induced a significant increase above rest in MIX (121%) and MYO (87%) protein synthesis at 4 h post-exercise and but at 24 h in the MIX only. The increase in the rate of protein synthesis in MIX and MYO at 4 h post-exercise with 90FAIL and 30FAIL was greater than 30WM, with no difference between these conditions; however, MYO remained elevated (199%) above rest at 24 h only in 30FAIL. There was a significant increase in AktSer473 at 24h in all conditions (P = 0.023) and mTORSer2448 phosphorylation at 4 h post-exercise (P = 0.025). Phosporylation of Erk1/2Tyr202/204, p70S6KThr389, and 4E-BP1Thr37/46 increased significantly (P<0.05) only in the 30FAIL condition at 4 h post-exercise, whereas, 4E-BP1Thr37/46 phosphorylation was greater 24 h after exercise than at rest in both 90FAIL (237%) and 30FAIL (312%) conditions. Pax7 mRNA expression increased at 24 h post-exercise (P = 0.02) regardless of condition. The mRNA expression of MyoD and myogenin were consistently elevated in the 30FAIL condition. Conclusions/Significance These results suggest that low-load high volume resistance exercise is more effective in inducing acute muscle anabolism than high-load low volume or work matched resistance exercise modes. PMID

  1. Two Resistance Modes to Clover yellow vein virus in Pea Characterized by a Green Fluorescent Protein-Tagged Virus.

    PubMed

    Andrade, Marcelo; Sato, Masanao; Uyeda, Ichiro

    2007-05-01

    ABSTRACT This study characterized resistance in pea lines PI 347295 and PI 378159 to Clover yellow vein virus (ClYVV). Genetic cross experiments showed that a single recessive gene controls resistance in both lines. Conventional mechanical inoculation did not result in infection; however, particle bombardment with infectious plasmid or mechanical inoculation with concentrated viral inocula did cause infection. When ClYVV No. 30 isolate was tagged with a green fluorescent protein (GFP) and used to monitor infection, viral cell-to-cell movement differed in the two pea lines. In PI 347595, ClYVV replicated at a single-cell level, but did not move to neighboring cells, indicating that resistance operated at a cell-to-cell step. In PI 378159, the virus moved to cells around the infection site and reached the leaf veins, but viral movement was slower than that in the susceptible line. The viruses observed around the infection sites and in the veins were then recovered and inoculated again by a conventional mechanical inoculation method onto PI 378159 demonstrating that ClYVV probably had mutated and newly emerged mutant viruses can move to neighboring cells and systemically infect the plants. Tagging the virus with GFP was an efficient tool for characterizing resistance modes. Implications of the two resistance modes are discussed. PMID:18943572

  2. 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

  3. Characterization of Protein Changes Associated with Sugar Beet (Beta vulgaris) Resistance and Susceptibility to Fusarium oxysporum.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Fusarium oxysporum is serious threat to sugar beet production worldwide. Although certain sugar beet lines appear to have resistance against F. oxysporum, little is understood about the basis for that resistance. Examination of F. oxysporum-induced changes in the sugar beet proteome has the poten...

  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. Structural Studies of Apo Nosl, an Accessory Protein of the Nitrous Oxide Reductase System: Insights from Structural Homology with MerB, a Mercury Resistance Protein

    SciTech Connect

    Taubner, Lara M.; McGuirl, Michele A.; Dooley, David M.; Copie, Valerie

    2006-10-01

    The formation of the unique catalytic tetranuclear copper cluster (CuZ) of nitrous oxide reductase, N2OR, requires the coexpression of a multiprotein assembly apparatus encoded by the nosDFYL operon. NosL, one of the proteins encoded by this transcript, is a 20 kDa lipoprotein of the periplasm that has been shown to bind copper(I), although its function has yet to be detemined. Cu(I) EXAFS data collected on the holo protein demonstrated that features of the copper binding site are consistent with a role for this protein as a metallochaperone, a class of metal ion transporters involved in metal resistance, homeostasis, and metallocluster biosynthesis. To test this hypothesis and to gain insight into other potential functional roles for this protein in the N2OR system, the three-dimensional solution structure of apo NosL has been solved by solution NMR methods. The structure of apo NosL consists of two relatively independent homologous domains that adopt an unusual ββαβ topology. The fold of apo NosL displays structural homology to only one other protein, MerB, an organomercury lyase involved in bacterial mercury resistance. The structural similarity between apo NosL and MerB, together with the absolute conservation of Met109 in all NosL sequences, indicates that this residue may be involved in copper ligation, and that the metal binding site is likely to be solvent-accessible and contiguous with a large binding cleft. The structural observations suggest that NosL is exceptionally adapted for a role in copper and/or sulfur delivery and possibly for metallochaperone function.

  7. Discovery of IL-18 As a Novel Secreted Protein Contributing to Doxorubicin Resistance by Comparative Secretome Analysis of MCF-7 and MCF-7/Dox

    PubMed Central

    Yao, Ling; Zhang, Yan; Chen, Keying; Hu, Xiaofang; Xu, Lisa X.

    2011-01-01

    Background Resistance to chemotherapy is the major cause of failure in breast cancer treatment. Recent studies suggest that secreted proteins may play important roles in chemoresistance. We sought to systematically characterize secreted proteins associated with drug resistance, which may represent potential serum biomarkers or novel drug targets. Methodology/Principal Findings In the present work, we adopted the proteomic strategy of one-dimensional gel electrophoresis followed by liquid chromatography-tandem mass spectrometry to compare the secretome of MCF-7 and doxorubicin-resistant MCF-7/Dox. A total of 2,084 proteins were identified with at least two unique peptides in the conditioned media of two cell lines. By quantification with label-free spectral counting, 89 differentially expressed secreted proteins (DESPs) between the two cell lines were found. Among them, 57 DESPs were first found to be related to doxorubicin resistance in this work, including 24 extracellular matrix related proteins, 2 cytokines and 31 unclassified proteins. We focused on 13 novel DESPs with confirmed roles in tumor metastasis. Among them, the elevated expression of IL-18 in doxorubicin-resistant cell lines and breast tumor tissues was validated and its role in doxorubicin resistance was further confirmed by cell viability experiments in the presence or absence of this protein. Conclusions/Significance Comparative analysis of the secretome of MCF-7 and MCF-7/Dox identified novel secreted proteins related to chemotherapy resistance. IL-18 was further validated to contribute to doxorubicin resistance, in addition to its confirmed role in breast cancer metastasis. Due to its dual roles in both drug resistance and tumor metastasis, IL-18 may represent a useful drug target for breast cancer therapy. PMID:21931812

  8. Differentially expressed proteins associated with Fusarium head blight resistance in wheat

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Fusarium head blight (FHB), mainly caused by Fusarium graminearum, substantially reduces wheat grain yield and quality worldwide. Proteins play important roles in defense against the fungal infection. This study characterized differentially expressed proteins between near-isogenic lines (NILs) contr...

  9. 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

  10. Solute Transport Proteins and the Outer Membrane Protein NmpC Contribute to Heat Resistance of Escherichia coli AW1.7▿

    PubMed Central

    Ruan, Lifang; Pleitner, Aaron; Gänzle, Michael G.; McMullen, Lynn M.

    2011-01-01

    This study aimed to elucidate determinants of heat resistance in Escherichia coli by comparing the composition of membrane lipids, as well as gene expression, in heat-resistant E. coli AW1.7 and heat-sensitive E. coli GGG10 with or without heat shock. The survival of E. coli AW1.7 at late exponential phase was 100-fold higher than that of E. coli GGG10 after incubation at 60°C for 15 min. The cytoplasmic membrane of E. coli AW1.7 contained a higher proportion of saturated and cyclopropane fatty acids than that of E. coli GGG10. Microarray hybridization of cDNA libraries obtained from exponentially growing or heat-shocked cultures was performed to compare gene expression in these two strains. Expression of selected genes from different functional groups was quantified by quantitative PCR. DnaK and 30S and 50S ribosomal subunits were overexpressed in E. coli GGG10 relative to E. coli AW1.7 upon heat shock at 50°C, indicating improved ribosome stability. The outer membrane porin NmpC and several transport proteins were overexpressed in exponentially growing E. coli AW1.7. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of membrane properties confirmed that NmpC is present in the outer membrane of E. coli AW1.7 but not in that of E. coli GGG10. Expression of NmpC in E. coli GGG10 increased survival at 60°C 50- to 1,000-fold. In conclusion, the outer membrane porin NmpC contributes to heat resistance in E. coli AW1.7, but the heat resistance of this strain is dependent on additional factors, which likely include the composition of membrane lipids, as well as solute transport proteins. PMID:21398480

  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. PMID:26299399

  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. 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.

  14. An In Silico Approach for Characterization of an Aminoglycoside Antibiotic-Resistant Methyltransferase Protein from Pyrococcus furiosus (DSM 3638)

    PubMed Central

    Oany, Arafat Rahman; Jyoti, Tahmina Pervin; Ahmad, Shah Adil Ishtiyaq

    2014-01-01

    Pyrococcus furiosus is a hyperthermophilic archaea. A hypothetical protein of this archaea, PF0847, was selected for computational analysis. Basic local alignment search tool and multiple sequence alignment (MSA) tool were employed to search for related proteins. Both the secondary and tertiary structure prediction were obtained for further analysis. Three-dimensional model was assessed by PROCHECK and QMEAN6 programs. To get insights about the physical and functional associations of the protein, STRING network analysis was performed. Binding of the SAM (S-adenosyl-l-methionine) ligand with our protein, fetched from an antibiotic-related methyltransferase (PDB code: 3P2K: D), showed high docking energy and suggested the function of the protein as methyltransferase. Finally, we tried to look for a specific function of the proposed methyltransferase, and binding of the geneticin bound to the eubacterial 16S rRNA A-site (PDB code: 1MWL) in the active site of the PF0847 gave us the indication to predict the protein responsible for aminoglycoside antibiotic resistance. PMID:24683305

  15. Induction of a multixenobiotic resistance protein (MXR) in the Asiatic clam Corbicula fluminea after heavy metals exposure.

    PubMed

    Achard, M; Baudrimont, M; Boudou, A; Bourdineaud, J P

    2004-05-12

    Multixenobiotic resistance mechanisms (MXR) related to the mammalian P-glycoprotein multidrug transporter protein (P-gp) are known to occur in several marine invertebrates. In the present work, we report on the induction of an MXR protein by various heavy metals in the gills of the freshwater clam Corbicula fluminea. The evaluation of the MXR protein level was assessed by Western blot using a specific monoclonal antibody raised against the human P-gp (C219). A field transplantation experiment, where clams were caged in a gradient relative to an industrial site, demonstrated a positive relationship between MXR levels and (a) metal pollution (Cd and Zn) in the environment and (b) metal bioaccumulation in the gills. To establish this correlative relationship, clams were exposed to different levels of cadmium (15-60 microg l(-1)) for up to 15 days in a controlled laboratory experiment. MXR protein levels increased in time for all treatments (including the control). However, the highest levels of MXR protein titer were expressed in clams that had been exposed to the lowest dose of cadmium. The causes for this observed inverse relationship between the exposure dose and the MXR induction is discussed. MXR protein titer was also shown to be induced by other heavy metals (zinc, inorganic mercury, and copper). PMID:15084411

  16. A Single Amino Acid Substitution in the Core Protein of West Nile Virus Increases Resistance to Acidotropic Compounds

    PubMed Central

    Martín-Acebes, Miguel A.; Blázquez, Ana-Belén; de Oya, Nereida Jiménez; Escribano-Romero, Estela; Shi, Pei-Yong; Saiz, Juan-Carlos

    2013-01-01

    West Nile virus (WNV) is a worldwide distributed mosquito-borne flavivirus that naturally cycles between birds and mosquitoes, although it can infect multiple vertebrate hosts including horses and humans. This virus is responsible for recurrent epidemics of febrile illness and encephalitis, and has recently become a global concern. WNV requires to transit through intracellular acidic compartments at two different steps to complete its infectious cycle. These include fusion between the viral envelope and the membrane of endosomes during viral entry, and virus maturation in the trans-Golgi network. In this study, we followed a genetic approach to study the connections between viral components and acidic pH. A WNV mutant with increased resistance to the acidotropic compound NH4Cl, which blocks organelle acidification and inhibits WNV infection, was selected. Nucleotide sequencing revealed that this mutant displayed a single amino acid substitution (Lys 3 to Glu) on the highly basic internal capsid or core (C) protein. The functional role of this replacement was confirmed by its introduction into a WNV infectious clone. This single amino acid substitution also increased resistance to other acidification inhibitor (concanamycin A) and induced a reduction of the neurovirulence in mice. Interestingly, a naturally occurring accompanying mutation found on prM protein abolished the resistant phenotype, supporting the idea of a genetic crosstalk between the internal C protein and the external glycoproteins of the virion. The findings here reported unveil a non-previously assessed connection between the C viral protein and the acidic pH necessary for entry and proper exit of flaviviruses. PMID:23874963

  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. Characterisation of the protein corona using tunable resistive pulse sensing: determining the change and distribution of a particle's surface charge.

    PubMed

    Blundell, Emma L C J; Healey, Matthew J; Holton, Elizabeth; Sivakumaran, Muttuswamy; Manstana, Sarabjit; Platt, Mark

    2016-08-01

    The zeta potential of the protein corona around carboxyl particles has been measured using tunable resistive pulse sensing (TRPS). A simple and rapid assay for characterising zeta potentials within buffer, serum and plasma is presented monitoring the change, magnitude and distribution of proteins on the particle surface. First, we measure the change in zeta potential of carboxyl-functionalised nanoparticles in solutions that contain biologically relevant concentrations of individual proteins, typically constituted in plasma and serum, and observe a significant difference in distributions and zeta values between room temperature and 37 °C assays. The effect is protein dependent, and the largest difference between the two temperatures is recorded for the γ-globulin protein where the mean zeta potential changes from -16.7 to -9.0 mV for 25 and 37 °C, respectively. This method is further applied to monitor particles placed into serum and/or plasma. A temperature-dependent change is again observed with serum showing a 4.9 mV difference in zeta potential between samples incubated at 25 and 37 °C; this shift was larger than that observed for samples in plasma (0.4 mV). Finally, we monitor the kinetics of the corona reorientation for particles initially placed into serum and then adding 5 % (V/V) plasma. The technology presented offers an interesting insight into protein corona structure and kinetics of formation measured in biologically relevant solutions, i.e. high protein, high salt levels, and its particle-by-particle analysis gives a measure of the distribution of particle zeta potential that may offer a better understanding of the behaviour of nanoparticles in solution. Graphical Abstract The relative velocity of a nanoparticle as it traverses a nanopore can be used to determine its zeta potential. Monitoring the changes in translocation speeds can therefore be used to follow changes to the surface chemistry/composition of 210 nm particles that were placed

  19. Resistance to Cucumber mosaic virus in Gladiolus plants transformed with either a defective replicase or coat protein subgroup II gene from Cucumber mosaic virus.

    PubMed

    Kamo, Kathryn; Jordan, Ramon; Guaragna, Mary Ann; Hsu, Hei-Ti; Ueng, Peter

    2010-07-01

    Transgenic Gladiolus plants that contain either Cucumber mosaic virus (CMV) subgroup I coat protein, CMV subgroup II coat protein, CMV replicase, a combination of the CMV subgroups I and II coat proteins, or a combination of the CMV subgroup II coat protein and replicase genes were developed. These plants were multiplied in vitro and challenged with purified CMV isolated from Gladiolus using a hand-held gene gun. Three out of 19 independently transformed plants expressing the replicase gene under control of the duplicated CaMV 35S promoter were found to be resistant to CMV subgroup I. Three out of 21 independently transformed plants with the CMV subgroup II coat protein gene under control of the Arabidopsis UBQ3 promoter were resistant to CMV subgroup II. Eighteen independently transformed plants with either the CMV subgroup I coat protein or a combination of CMV subgroups I and II coat proteins were challenged and found to be susceptible to both CMV subgroups I or II. Virus resistant plants with the CMV replicase transgene expressed much lower RNA levels than resistant plants expressing the CMV subgroup II coat protein. This work will facilitate the evaluation of virus resistance in transgenic Gladiolus plants to yield improved floral quality and productivity. PMID:20411391

  20. The effects of protein and amino acid supplementation on performance and training adaptations during ten weeks of resistance training.

    PubMed

    Kerksick, Chad M; Rasmussen, Christopher J; Lancaster, Stacy L; Magu, Bharat; Smith, Penney; Melton, Charles; Greenwood, Michael; Almada, Anthony L; Earnest, Conrad P; Kreider, Richard B

    2006-08-01

    The purpose of this study was to examine the effects of whey protein supplementation on body composition, muscular strength, muscular endurance, and anaerobic capacity during 10 weeks of resistance training. Thirty-six resistance-trained males (31.0 +/- 8.0 years, 179.1 +/- 8.0 cm, 84.0 +/- 12.9 kg, 17.8 +/- 6.6%) followed a 4 days-per-week split body part resistance training program for 10 weeks. Three groups of supplements were randomly assigned, prior to the beginning of the exercise program, in a double-blind manner to all subjects: 48 g per day (g.d(-1)) carbohydrate placebo (P), 40 g.d(-1) of whey protein + 8 g.d(-1) of casein (WC), or 40 g.d(-1) of whey protein + 3 g.d(-1) branched-chain amino acids + 5 g.d(-1) L-glutamine (WBG). At 0, 5, and 10 weeks, subjects were tested for fasting blood samples, body mass, body composition using dual-energy x-ray absorptiometry (DEXA), 1 repetition maximum (1RM) bench and leg press, 80% 1RM maximal repetitions to fatigue for bench press and leg press, and 30-second Wingate anaerobic capacity tests. No changes (p > 0.05) were noted in all groups for energy intake, training volume, blood parameters, and anaerobic capacity. WC experienced the greatest increases in DEXA lean mass (P = 0.0 +/- 0.9; WC = 1.9 +/- 0.6; WBG = -0.1 +/- 0.3 kg, p < 0.05) and DEXA fat-free mass (P = 0.1 +/- 1.0; WC = 1.8 +/- 0.6; WBG = -0.1 +/- 0.2 kg, p < 0.05). Significant increases in 1RM bench press and leg press were observed in all groups after 10 weeks. In this study, the combination of whey and casein protein promoted the greatest increases in fat-free mass after 10 weeks of heavy resistance training. Athletes, coaches, and nutritionists can use these findings to increase fat-free mass and to improve body composition during resistance training. PMID:16937979

  1. Bovine spongiform encephalopathy induces misfolding of alleged prion-resistant species cellular prion protein without altering its pathobiological features.

    PubMed

    Vidal, Enric; Fernández-Borges, Natalia; Pintado, Belén; Ordóñez, Montserrat; Márquez, Mercedes; Fondevila, Dolors; Torres, Juan María; Pumarola, Martí; Castilla, Joaquín

    2013-05-01

    Bovine spongiform encephalopathy (BSE) prions were responsible for an unforeseen epizootic in cattle which had a vast social, economic, and public health impact. This was primarily because BSE prions were found to be transmissible to humans. Other species were also susceptible to BSE either by natural infection (e.g., felids, caprids) or in experimental settings (e.g., sheep, mice). However, certain species closely related to humans, such as canids and leporids, were apparently resistant to BSE. In vitro prion amplification techniques (saPMCA) were used to successfully misfold the cellular prion protein (PrP(c)) of these allegedly resistant species into a BSE-type prion protein. The biochemical and biological properties of the new prions generated in vitro after seeding rabbit and dog brain homogenates with classical BSE were studied. Pathobiological features of the resultant prion strains were determined after their inoculation into transgenic mice expressing bovine and human PrP(C). Strain characteristics of the in vitro-adapted rabbit and dog BSE agent remained invariable with respect to the original cattle BSE prion, suggesting that the naturally low susceptibility of rabbits and dogs to prion infections should not alter their zoonotic potential if these animals became infected with BSE. This study provides a sound basis for risk assessment regarding prion diseases in purportedly resistant species. PMID:23637170

  2. Implication of protein tyrosine phosphatase 1B in MCF-7 cell proliferation and resistance to 4-OH tamoxifen

    SciTech Connect

    Blanquart, Christophe; Karouri, Salah-Eddine; Issad, Tarik

    2009-10-02

    The protein tyrosine phosphatase 1B (PTP1B) and the T-cell protein tyrosine phosphatase (TC-PTP) were initially thought to be mainly anti-oncogenic. However, overexpression of PTP1B and TC-PTP has been observed in human tumors, and recent studies have demonstrated that PTP1B contributes to the appearance of breast tumors by modulating ERK pathway. In the present work, we observed that decreasing the expression of TC-PTP or PTP1B in MCF-7 cells using siRNA reduced cell proliferation without affecting cell death. This reduction in proliferation was associated with decreased ERK phosphorylation. Moreover, selection of tamoxifen-resistant MCF-7 cells, by long-term culture in presence of 4-OH tamoxifen, resulted in cells that display overexpression of PTP1B and TC-PTP, and concomitant increase in ERK and STAT3 phosphorylation. siRNA experiments showed that PTP1B, but not TC-PTP, is necessary for resistance to 4-OH tamoxifen. Therefore, our work indicates that PTP1B could be a relevant therapeutic target for treatment of tamoxifen-resistant breast cancers.

  3. TaTypA, a Ribosome-Binding GTPase Protein, Positively Regulates Wheat Resistance to the Stripe Rust Fungus

    PubMed Central

    Liu, Peng; Myo, Thwin; Ma, Wei; Lan, Dingyun; Qi, Tuo; Guo, Jia; Song, Ping; Guo, Jun; Kang, Zhensheng

    2016-01-01

    Tyrosine phosphorylation protein A (TypA/BipA) belongs to the ribosome-binding GTPase superfamily. In many bacterial species, TypA acts as a global stress and virulence regulator and also mediates resistance to the antimicrobial peptide bactericidal permeability-increasing protein. However, the function of TypA in plants under biotic stresses is not known. In this study, we isolated and functionally characterized a stress-responsive TypA gene (TaTypA) from wheat, with three copies located on chromosomes 6A, 6B, and 6D, respectively. Transient expression assays indicated chloroplast localization of TaTypA. The transcript levels of TaTypA were up-regulated in response to treatment with methyl viologen, which induces reactive oxygen species (ROS) in chloroplasts through photoreaction, cold stress, and infection by an avirulent strain of the stripe rust pathogen. Knock down of the expression of TaTypA through virus-induced gene silencing decreased the resistance of wheat to stripe rust accompanied by weakened ROS accumulation and hypersensitive response, an increase in TaCAT and TaSOD expression, and an increase in pathogen hyphal growth and branching. Our findings suggest that TaTypA contributes to resistance in an ROS-dependent manner. PMID:27446108

  4. Antibiotic Resistance, Core-Genome and Protein Expression in IncHI1 Plasmids in Salmonella Typhimurium.

    PubMed

    Kubasova, Tereza; Cejkova, Darina; Matiasovicova, Jitka; Sekelova, Zuzana; Polansky, Ondrej; Medvecky, Matej; Rychlik, Ivan; Juricova, Helena

    2016-01-01

    Conjugative plasmids from the IncHI1 incompatibility group play an important role in transferring antibiotic resistance in Salmonella Typhimurium. However, knowledge of their genome structure or gene expression is limited. In this study, we determined the complete nucleotide sequences of four IncHI1 plasmids transferring resistance to antibiotics by two different next generation sequencing protocols and protein expression by mass spectrometry. Sequence data including additional 11 IncHI1 plasmids from GenBank were used for the definition of the IncHI1 plasmid core-genome and pan-genome. The core-genome consisted of approximately 123 kbp and 122 genes while the total pan-genome represented approximately 600 kbp. When the core-genome sequences were used for multiple alignments, the 15 tested IncHI1 plasmids were separated into two main lineages. GC content in core-genome genes was around 46% and 50% in accessory genome genes. A multidrug resistance region present in all 4 sequenced plasmids extended over 20 kbp and, except for tet(B), the genes responsible for antibiotic resistance were those with the highest GC content. IncHI1 plasmids therefore represent replicons that evolved in low GC content bacteria. From their original host, they spread to Salmonella and during this spread these plasmids acquired multiple accessory genes including those coding for antibiotic resistance. Antibiotic-resistance genes belonged to genes with the highest level of expression and were constitutively expressed even in the absence of antibiotics. This is the likely mechanism that facilitates host cell survival when antibiotics suddenly emerge in the environment. PMID:27189997

  5. Crystal Structure of the Zorbamycin-Binding Protein ZbmA, the Primary Self-Resistance Element in Streptomyces flavoviridis ATCC21892.

    PubMed

    Rudolf, Jeffrey D; Bigelow, Lance; Chang, Changsoo; Cuff, Marianne E; Lohman, Jeremy R; Chang, Chin-Yuan; Ma, Ming; Yang, Dong; Clancy, Shonda; Babnigg, Gyorgy; Joachimiak, Andrzej; Phillips, George N; Shen, Ben

    2015-11-17

    The bleomycins (BLMs), tallysomycins (TLMs), phleomycin, and zorbamycin (ZBM) are members of the BLM family of glycopeptide-derived antitumor antibiotics. The BLM-producing Streptomyces verticillus ATCC15003 and the TLM-producing Streptoalloteichus hindustanus E465-94 ATCC31158 both possess at least two self-resistance elements, an N-acetyltransferase and a binding protein. The N-acetyltransferase provides resistance by disrupting the metal-binding domain of the antibiotic that is required for activity, while the binding protein confers resistance by sequestering the metal-bound antibiotic and preventing drug activation via molecular oxygen. We recently established that the ZBM producer, Streptomyces flavoviridis ATCC21892, lacks the N-acetyltransferase resistance gene and that the ZBM-binding protein, ZbmA, is sufficient to confer resistance in the producing strain. To investigate the resistance mechanism attributed to ZbmA, we determined the crystal structures of apo and Cu(II)-ZBM-bound ZbmA at high resolutions of 1.90 and 1.65 Å, respectively. A comparison and contrast with other structurally characterized members of the BLM-binding protein family revealed key differences in the protein-ligand binding environment that fine-tunes the ability of ZbmA to sequester metal-bound ZBM and supports drug sequestration as the primary resistance mechanism in the producing organisms of the BLM family of antitumor antibiotics. PMID:26512730

  6. Crystal Structure of the Zorbamycin-Binding Protein ZbmA, the Primary Self-Resistance Element in Streptomyces flavoviridis ATCC21892

    SciTech Connect

    Rudolf, Jeffrey D.; Bigelow, Lance; Chang, Changsoo; Cuff, Marianne E.; Lohman, Jeremy R.; Chang, Chin-Yuan; Ma, Ming; Yang, Dong; Clancy, Shonda; Babnigg, Gyorgy; Joachimiak, Andrzej; Phillips, George N.; Shen, Ben

    2015-11-17

    The bleomycins (BLMs), tallysomycins (TLMs), phleomycin, and zorbamycin (ZBM) are members of the BLM family of glycopeptide-derived antitumor antibiotics. The BLM-producing Streptomyces verticillus ATCC15003 and the TLM-producing Streptoalloteichus hindustanus E465-94 ATCC31158 both possess at least two self-resistance elements, an N-acetyltransferase and a binding protein. The N-acetyltransferase provides resistance by disrupting the metal-binding domain of the antibiotic that is required for activity, while the binding protein confers resistance by sequestering the metal-bound antibiotic and preventing drug activation via molecular oxygen. We recently established that the ZBM producer, Streptomyces flavoviridis ATCC21892, lacks the N-acetyltransferase resistance gene and that the ZBM-binding protein, ZbmA, is sufficient to confer resistance in the producing strain. To investigate the resistance mechanism attributed to ZbmA, we determined the crystal structures of apo and Cu(II)-ZBM-bound ZbmA at high resolutions of 1.90 and 1.65 angstrom, respectively. A comparison and contrast with other structurally characterized members of the BLM-binding protein family revealed key differences in the protein ligand binding environment that fine-tunes the ability of ZbmA to sequester metal-bound ZBM and supports drug sequestration as the primary resistance mechanism in the producing organisms of the BLM family of antitumor antibiotics.

  7. Computational study of protein specificity: The molecular basis of HIV-1 protease drug resistance

    PubMed Central

    Wang, Wei; Kollman, Peter A.

    2001-01-01

    Drug resistance has sharply limited the effectiveness of HIV-1 protease inhibitors in AIDS therapy. It is critically important to understand the basis of this resistance for designing new drugs. We have evaluated the free energy contribution of each residue in the HIV protease in binding to one of its substrates and to the five FDA-approved protease drugs. Analysis of these free energy profiles and the variability at each sequence position suggests: (i) single drug resistance mutations are likely to occur at not well conserved residues if they interact more favorably with drugs than with the substrate; and (ii) resistance-evading drugs should have a free energy profile similar to the substrate and interact most favorably with well conserved residues. We also propose an empirical parameter, called the free energy/variability value, which combines free energy calculation and sequence analysis to suggest possible drug resistance mutations on the protease. The free energy/variability value is defined as the product of one residue's contribution to the binding free energy and the variability of that residue. This parameter can assist in designing resistance-evading drugs for any target. PMID:11752442

  8. ERK Mutations Confer Resistance to Mitogen-Activated Protein Kinase Pathway Inhibitors

    PubMed Central

    Goetz, Eva M.; Ghandi, Mahmoud; Treacy, Daniel J.; Wagle, Nikhil; Garraway, Levi A.

    2015-01-01

    The use of targeted therapeutics directed against BRAFV600-mutant metastatic melanoma improves progression-free survival in many patients; however, acquired drug resistance remains a major medical challenge. By far, the most common clinical resistance mechanism involves reactivation of the MAPK (RAF/MEK/ERK) pathway by a variety of mechanisms. Thus, targeting ERK itself has emerged as an attractive therapeutic concept, and several ERK inhibitors have entered clinical trials. We sought to preemptively determine mutations in ERK1/2 that confer resistance to either ERK inhibitors or combined RAF/MEK inhibition in BRAFV600-mutant melanoma. Using a random mutagenesis screen, we identified multiple point mutations in ERK1 (MAPK3) and ERK2 (MAPK1) that could confer resistance to ERK or RAF/MEK inhibitors. ERK inhibitor–resistant alleles were sensitive to RAF/ MEK inhibitors and vice versa, suggesting that the future development of alternating RAF/MEK and ERK inhibitor regimens might help circumvent resistance to these agents. PMID:25320010

  9. Thermal stability and flame resistance of cotton fabrics treated with whey proteins.

    PubMed

    Bosco, Francesca; Carletto, Riccardo Andrea; Alongi, Jenny; Marmo, Luca; Di Blasio, Alessandro; Malucelli, Giulio

    2013-04-15

    It is well described in the literature that whey proteins are able to form coatings, which exhibit high mechanical and oxygen barrier properties, notwithstanding a great water vapour adsorption. These peculiarities have been exploited for applying a novel protein-based finishing treatment to cotton and for assessing the protein effect on the thermal and thermo-oxidative stability and on the flame retardant properties of the cellulosic fabric. Indeed, the deposited whey protein coatings have turned out to significantly affect the thermal degradation of cotton in inert and oxidative atmosphere, and to somehow modify its combustion when a flame has been applied. Furthermore, the influence of the secondary and tertiary structure of these proteins on the morphology of the deposited coating, and thus on the thermal and flame retardant properties of the treated fabrics, has been evaluated by performing a denaturation thermal treatment before the protein application. PMID:23544551

  10. Uptake and Degradation of Protease-Sensitive and -Resistant Forms of Abnormal Human Prion Protein Aggregates by Human Astrocytes

    PubMed Central

    Choi, Young Pyo; Head, Mark W.; Ironside, James W.; Priola, Suzette A.

    2015-01-01

    Sporadic Creutzfeldt-Jakob disease is the most common of the human prion diseases, a group of rare, transmissible, and fatal neurologic diseases associated with the accumulation of an abnormal form (PrPSc) of the host prion protein. In sporadic Creutzfeldt-Jakob disease, disease-associated PrPSc is present not only as an aggregated, protease-resistant form but also as an aggregated protease-sensitive form (sPrPSc). Although evidence suggests that sPrPSc may play a role in prion pathogenesis, little is known about how it interacts with cells during prion infection. Here, we show that protease-sensitive abnormal PrP aggregates derived from patients with sporadic Creutzfeldt-Jakob disease are taken up and degraded by immortalized human astrocytes similarly to abnormal PrP aggregates that are resistant to proteases. Our data suggest that relative proteinase K resistance does not significantly influence the astrocyte's ability to degrade PrPSc. Furthermore, the cell does not appear to distinguish between sPrPSc and protease-resistant PrPSc, suggesting that sPrPSc could contribute to prion infection. PMID:25280631

  11. EphB4/ephrinB2 Contributes to Imatinib Resistance in Chronic Myeloid Leukemia Involved in Cytoskeletal Proteins

    PubMed Central

    Li, Lin; Xu, Na; Zhang, Jin-fang; Xu, Lu-lu; Zhou, Xuan; Huang, Bin-tao; Li, Yu-ling; Liu, Xiao-li

    2016-01-01

    Introduction: The mechanism of EphB4/ephrinB2 in the resistance of chronic myelogenous leukemia to imatinib keeps unknown. Methods: The imatinib resistant chronic myelogenous leukemia cell line-K562-R, was established. EphB4 receptor expression was detected in patients and resistant cells. Cell migration and drug sensitivity were tested in the EphB4 knockdown cells and mouse models. Results: The EphB4 receptor was over-expressed in blast crisis patients compared to chronic phase patients. The level of EphB4 receptor expression was associated with a complete cytogenetic response within 12 months. Enhanced expression of the EphB4 receptor was detected in the K562-R cells. EphB4 knockdown inhibited cell migration ability and restored sensitivity to imatinib in vitro and in vivo. Restored sensitivity to imatinib was observed in K562-R cells, along with increased levels of phospho-EphB4 and decreased phosphorylation levels of RhoA, Rac1, and Cdc42. Conclusion: Our study illustrates that aberrant activation of EphB4/ephrinB2 may mediate chronic myeloid leukemia resistance involved in cytoskeletal proteins. PMID:27226777

  12. Role of the breast cancer resistance protein (BCRP/ABCG2) in drug transport--an update.

    PubMed

    Mao, Qingcheng; Unadkat, Jashvant D

    2015-01-01

    The human breast cancer resistance protein (BCRP, gene symbol ABCG2) is an ATP-binding cassette (ABC) efflux transporter. It was so named because it was initially cloned from a multidrug-resistant breast cancer cell line where it was found to confer resistance to chemotherapeutic agents such as mitoxantrone and topotecan. Since its discovery in 1998, the substrates of BCRP have been rapidly expanding to include not only therapeutic agents but also physiological substances such as estrone-3-sulfate, 17β-estradiol 17-(β-D-glucuronide) and uric acid. Likewise, at least hundreds of BCRP inhibitors have been identified. Among normal human tissues, BCRP is highly expressed on the apical membranes of the placental syncytiotrophoblasts, the intestinal epithelium, the liver hepatocytes, the endothelial cells of brain microvessels, and the renal proximal tubular cells, contributing to the absorption, distribution, and elimination of drugs and endogenous compounds as well as tissue protection against xenobiotic exposure. As a result, BCRP has now been recognized by the FDA to be one of the key drug transporters involved in clinically relevant drug disposition. We published a highly-accessed review article on BCRP in 2005, and much progress has been made since then. In this review, we provide an update of current knowledge on basic biochemistry and pharmacological functions of BCRP as well as its relevance to drug resistance and drug disposition. PMID:25236865

  13. THE EFFECTS OF HIV INFECTION ON THE EXPRESSION OF THE DRUG EFFLUX PROTEINS P-GLYCOPROTEIN AND BREAST CANCER RESISTANCE PROTEIN IN A HUMAN INTESTINE MODEL

    PubMed Central

    Ellis, Kelstan; Marlin, Jerry; Taylor, Tracey AH; Fitting, Sylvia; Hauser, Kurt F.; Rice, Greg

    2015-01-01

    Objectives In HIV infection, decreased penetration of antiretroviral drugs is postulated to contribute to HIV persistence within lymphoid rich regions of the gastrointestinal (GI) tract. However, mechanistic explanations for this phenomenon remain unclear. Specifically, investigations of HIV effects on drug efflux proteins within intestinal models are minimal. Methods Using an in vitro co-culture model of the GI tract, effects of HIV infection on drug efflux proteins, P-glycoprotein and Breast Cancer Resistance Protein (BCRP) were evaluated. The influence of the HIV-1 protein, Tat, and oxidative stress on P-glycoprotein and BCRP also was evaluated. Key Findings P-glycoprotein expression demonstrated an HIV-induced upregulation in Caco-2 cells over time for cells grown in co-culture with resting lymphocytes. BCRP overall expression increased with HIV exposure in activated primary human lymphocytes co-cultured with Caco-2 cells. Tat treatment resulted in no significant alterations in P-glycoprotein (43% increase), BCRP expression, or oxidative stress. Conclusions HIV exposure within an in vitro intestinal model resulted in increases in, P-glycoprotein and BCRP in a cell specific manner. Additionally, observed changes were not mediated by Tat. Collectively, these results suggest that alterations in BCRP and P-glycoprotein may contribute, in part, to decreased antiretroviral concentrations within the gastrointestinal tract in HIV infection. PMID:25557407

  14. A novel mechanism of protein thermostability: a unique N-terminal domain confers heat resistance to Fe/Mn-SODs.

    PubMed

    Wang, Wei; Ma, Ting; Zhang, Baoliang; Yao, Nana; Li, Mingchang; Cui, Lianlei; Li, Guoqiang; Ma, Zhenping; Cheng, Jiansong

    2014-01-01

    Superoxide dismutases (SODs), especially thermostable SODs, are widely applied in medical treatments, cosmetics, food, agriculture, and other industries given their excellent antioxidant properties. A novel thermostable cambialistic SOD from Geobacillus thermodenitrificans NG80-2 exhibits maximum activity at 70 °C and high thermostability over a broad range of temperatures (20-80 °C). Unlike other reported SODs, this enzyme contains an extra repeat-containing N-terminal domain (NTD) of 244 residues adjacent to the conserved functional SODA domain. Deletion of the NTD dramatically decreased its optimum active temperature (OAT) to 30 °C and also impaired its thermostability. Conversely, appending the NTD to a mesophilic counterpart from Bacillus subtilis led to a moderately thermophilic enzyme (OAT changed from 30 to 55 °C) with improved heat resistance. Temperature-dependant circular dichroism analysis revealed the enhanced conformational stability of SODs fused with this NTD. Furthermore, the NTD also contributes to the stress resistance of host proteins without altering their metal ion specificity or oligomerisation form except for a slight effect on their pH profile. We therefore demonstrate that the NTD confers outstanding thermostability to the host protein. To our knowledge, this is the first discovery of a peptide capable of remarkably improving protein thermostability and provides a novel strategy for bioengineering thermostable SODs. PMID:25445927

  15. The NB-LRR proteins RGA4 and RGA5 interact functionally and physically to confer disease resistance

    PubMed Central

    Césari, Stella; Kanzaki, Hiroyuki; Fujiwara, Tadashi; Bernoux, Maud; Chalvon, Véronique; Kawano, Yoji; Shimamoto, Ko; Dodds, Peter; Terauchi, Ryohei; Kroj, Thomas

    2014-01-01

    Plant resistance proteins of the class of nucleotide-binding and leucine-rich repeat domain proteins (NB-LRRs) are immune sensors which recognize pathogen-derived molecules termed avirulence (AVR) proteins. We show that RGA4 and RGA5, two NB-LRRs from rice, interact functionally and physically to mediate resistance to the fungal pathogen Magnaporthe oryzae and accomplish different functions in AVR recognition. RGA4 triggers an AVR-independent cell death that is repressed in the presence of RGA5 in both rice protoplasts and Nicotiana benthamiana. Upon recognition of the pathogen effector AVR-Pia by direct binding to RGA5, repression is relieved and cell death occurs. RGA4 and RGA5 form homo- and hetero-complexes and interact through their coiled-coil domains. Localization studies in rice protoplast suggest that RGA4 and RGA5 localize to the cytosol. Upon recognition of AVR-Pia, neither RGA4 nor RGA5 is re-localized to the nucleus. These results establish a model for the interaction of hetero-pairs of NB-LRRs in plants: RGA4 mediates cell death activation, while RGA5 acts as a repressor of RGA4 and as an AVR receptor. PMID:25024433

  16. Elevation of XPA protein level in testis tumor cells without increasing resistance to cisplatin or UV radiation.

    PubMed

    Köberle, Beate; Roginskaya, Vera; Zima, Karen S; Masters, John R W; Wood, Richard D

    2008-08-01

    Most testicular germ cell tumors are curable using cisplatin-based chemotherapy, and cell lines from these tumors are unusually sensitive to cisplatin and other DNA-damaging agents. It has been suggested that this might be caused by a lower-than normal nucleotide excision repair (NER) activity. Previous studies found that cell lines from testicular germ cell tumors have on average about one-third the level of the NER protein XPA in comparison to cell lines from other tumors. We asked whether over-expression of XPA protein would alleviate the cellular sensitivity and increase the DNA repair capacity of a testis tumor cell line. Increasing XPA levels in 833K cells by 10-fold did not increase resistance to UV irradiation. XPA was localized to the cell nucleus in all cell lines, before and after exposure to UV-radiation. 833K cells were proficient in removing UV radiation-induced photoproducts from the genome and increased XPA did not enhance the rate of removal. Further, over-expressing functional XPA protein did not correlate with increased resistance of 833K testis tumor cells to cisplatin. Thus, although the amount of XPA in this testis tumor cell line is lower than normal, it is sufficient for NER in vivo. The relative sensitivity of testis tumor cells to cisplatin, UV radiation, and other DNA damaging agents is likely related not to NER capacity, but to other factors such as the integrity of the p53 pathway in these cells. PMID:18240296

  17. A novel mechanism of protein thermostability: a unique N-terminal domain confers heat resistance to Fe/Mn-SODs

    PubMed Central

    Wang, Wei; Ma, Ting; Zhang, Baoliang; Yao, Nana; Li, Mingchang; Cui, Lianlei; Li, Guoqiang; Ma, Zhenping; Cheng, Jiansong

    2014-01-01

    Superoxide dismutases (SODs), especially thermostable SODs, are widely applied in medical treatments, cosmetics, food, agriculture, and other industries given their excellent antioxidant properties. A novel thermostable cambialistic SOD from Geobacillus thermodenitrificans NG80-2 exhibits maximum activity at 70°C and high thermostability over a broad range of temperatures (20–80°C). Unlike other reported SODs, this enzyme contains an extra repeat-containing N-terminal domain (NTD) of 244 residues adjacent to the conserved functional SODA domain. Deletion of the NTD dramatically decreased its optimum active temperature (OAT) to 30°C and also impaired its thermostability. Conversely, appending the NTD to a mesophilic counterpart from Bacillus subtilis led to a moderately thermophilic enzyme (OAT changed from 30 to 55°C) with improved heat resistance. Temperature-dependant circular dichroism analysis revealed the enhanced conformational stability of SODs fused with this NTD. Furthermore, the NTD also contributes to the stress resistance of host proteins without altering their metal ion specificity or oligomerisation form except for a slight effect on their pH profile. We therefore demonstrate that the NTD confers outstanding thermostability to the host protein. To our knowledge, this is the first discovery of a peptide capable of remarkably improving protein thermostability and provides a novel strategy for bioengineering thermostable SODs. PMID:25445927

  18. GANP protein encoded on human chromosome 21/mouse chromosome 10 is associated with resistance to mammary tumor development.

    PubMed

    Kuwahara, Kazuhiko; Yamamoto-Ibusuki, Mutsuko; Zhang, Zhenhuan; Phimsen, Suchada; Gondo, Naomi; Yamashita, Hiroko; Takeo, Toru; Nakagata, Naomi; Yamashita, Daisuke; Fukushima, Yoshimi; Yamamoto, Yutaka; Iwata, Hiroji; Saya, Hideyuki; Kondo, Eisaku; Matsuo, Keitaro; Takeya, Motohiro; Iwase, Hirotaka; Sakaguchi, Nobuo

    2016-04-01

    Human chromosome 21 is known to be associated with the high risk of hematological malignancy but with resistance to breast cancer in the study of Down syndrome. In human cancers, we previously observed the significant alterations of the protein expression encoded by the ganp/MCM3AP gene on human chromosome 21q22.3. Here, we investigated GANP protein