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Sample records for resistance proteins mrps

  1. Modulation of trabectedin (ET-743) hepatobiliary disposition by multidrug resistance-associated proteins (Mrps) may prevent hepatotoxicity

    International Nuclear Information System (INIS)

    Lee, Jin Kyung; Leslie, Elaine M.; Zamek-Gliszczynski, Maciej J.; Brouwer, Kim L.R.

    2008-01-01

    Trabectedin is a promising anticancer agent, but dose-limiting hepatotoxicity was observed during phase I/II clinical trials. Dexamethasone (DEX) has been shown to significantly reduce trabectedin-mediated hepatotoxicity. The current study was designed to assess the capability of sandwich-cultured primary rat hepatocytes (SCRH) to predict the hepato-protective effect of DEX against trabectedin-mediated cytotoxicity. The role of multidrug resistance-associated protein 2 (Mrp2; Abcc2) in trabectedin hepatic disposition also was examined. In SCRH from wild-type Wistar rats, cytotoxicity was observed after 24-h continuous exposure to trabectedin. SCRH pretreated with additional DEX (1 μM) exhibited a 2- to 3-fold decrease in toxicity at 100 nM and 1000 nM trabectedin. Unexpectedly, toxicity in SCRH from Mrp2-deficient (TR - ) compared to wild-type Wistar rats was markedly reduced. Depletion of glutathione from SCRH using buthionine sulfoximine (BSO) mitigated trabectedin toxicity associated with 100 nM and 1000 nM trabectedin. Western blot analysis demonstrated increased levels of CYP3A1/2 and Mrp2 in SCRH pretreated with DEX; interestingly, Mrp4 expression was increased in SCRH after BSO exposure. Trabectedin biliary recovery in isolated perfused livers from TR - rats was decreased by ∼ 75% compared to wild-type livers. In conclusion, SCRH represent a useful in vitro model to predict the hepatotoxicity of trabectedin observed in vivo. The protection by DEX against trabectedin-mediated cytotoxicity may be attributed, in part, to enhanced Mrp2 biliary excretion and increased metabolism by CYP3A1/2. Decreased trabectedin toxicity in SCRH from TR - rats, and in SCRH pretreated with BSO, may be due to increased basolateral excretion of trabectedin by Mrp3 and/or Mrp4

  2. Antifolate resistance mediated by the multidrug resistance proteins MRP1 and MRP2

    NARCIS (Netherlands)

    Hooijberg, J. H.; Broxterman, H. J.; Kool, M.; Assaraf, Y. G.; Peters, G. J.; Noordhuis, P.; Scheper, R. J.; Borst, P.; Pinedo, H. M.; Jansen, G.

    1999-01-01

    Transfection of multidrug resistance proteins (MRPs) MRP1 and MRP2 in human ovarian carcinoma 2008 cells conferred a marked level of resistance to short-term (1-4 h) exposure to the polyglutamatable antifolates methotrexate (MTX; 21-74-fold), ZD1694 (4-138-fold), and GW1843 (101-156-fold). Evidence

  3. Modifications of hemoglobin and myoglobin by Maillard reaction products (MRPs.

    Directory of Open Access Journals (Sweden)

    Aristos Ioannou

    Full Text Available High performance liquid chromatography (HPLC coupled with a Fraction Collector was employed to isolate Maillard reaction products (MRPs formed in model systems comprising of asparagine and monosaccharides in the 60-180°C range. The primary MRP which is detected at 60°C is important for Acrylamide content and color/aroma development in foods and also in the field of food biotechnology for controlling the extent of the Maillard reaction with temperature. The discrete fractions of the reaction products were reacted with Hemoglobin (Hb and Myoglobin (Mb at physiological conditions and the reaction adducts were monitored by UV-vis and Attenuated Total Reflection-Fourier transform infrared (FTIR spectrophotometry. The UV-vis kinetic profiles revealed the formation of a Soret transition characteristic of a low-spin six-coordinated species and the ATR-FTIR spectrum of the Hb-MRP and Mb-MRP fractions showed modifications in the protein Amide I and II vibrations. The UV-vis and the FTIR spectra of the Hb-MRPs indicate that the six-coordinated species is a hemichrome in which the distal E7 Histidine is coordinated to the heme Fe and blocks irreversibly the ligand binding site. Although the Mb-MRPs complex is a six-coordinated species, the 1608 cm-1 FTIR band characteristic of a hemichrome was not observed.

  4. Modifications of hemoglobin and myoglobin by Maillard reaction products (MRPs).

    Science.gov (United States)

    Ioannou, Aristos; Varotsis, Constantinos

    2017-01-01

    High performance liquid chromatography (HPLC) coupled with a Fraction Collector was employed to isolate Maillard reaction products (MRPs) formed in model systems comprising of asparagine and monosaccharides in the 60-180°C range. The primary MRP which is detected at 60°C is important for Acrylamide content and color/aroma development in foods and also in the field of food biotechnology for controlling the extent of the Maillard reaction with temperature. The discrete fractions of the reaction products were reacted with Hemoglobin (Hb) and Myoglobin (Mb) at physiological conditions and the reaction adducts were monitored by UV-vis and Attenuated Total Reflection-Fourier transform infrared (FTIR) spectrophotometry. The UV-vis kinetic profiles revealed the formation of a Soret transition characteristic of a low-spin six-coordinated species and the ATR-FTIR spectrum of the Hb-MRP and Mb-MRP fractions showed modifications in the protein Amide I and II vibrations. The UV-vis and the FTIR spectra of the Hb-MRPs indicate that the six-coordinated species is a hemichrome in which the distal E7 Histidine is coordinated to the heme Fe and blocks irreversibly the ligand binding site. Although the Mb-MRPs complex is a six-coordinated species, the 1608 cm-1 FTIR band characteristic of a hemichrome was not observed.

  5. Quantitative structure activity relationship studies on the flavonoid mediated inhibition of multidrug resistance proteins 1 and 2

    NARCIS (Netherlands)

    Zanden, J.J. van; Wortelboer, H.M.; Bijlsma, S.; Punt, A.; Usta, M.; Bladeren, P.J.V.; Rietjens, I.M.C.M.; Cnubben, N.H.P.

    2005-01-01

    In the present study, the effects of a large series of flavonoids on multidrug resistance proteins (MRPs) were studied in MRP1 and MRP2 transfected MDCKII cells. The results were used to define the structural requirements of flavonoids necessary for potent inhibition of MRP1- and MRP2-mediated

  6. Hepatocyte SLAMF3 reduced specifically the multidrugs resistance protein MRP-1 and increases HCC cells sensitization to anti-cancer drugs.

    Science.gov (United States)

    Fouquet, Grégory; Debuysscher, Véronique; Ouled-Haddou, Hakim; Eugenio, Mélanie Simoes; Demey, Baptiste; Singh, Amrathlal Rabbind; Ossart, Christèle; Al Bagami, Mohammed; Regimbeau, Jean-Marc; Nguyen-Khac, Eric; Naassila, Mickael; Marcq, Ingrid; Bouhlal, Hicham

    2016-05-31

    Multidrug resistance MDR proteins (MRPs) are members of the C family of a group of proteins named ATP binding cassette (ABC) transporters. MRPs can transport drugs including anticancer drugs, nucleoside analogs, antimetabolites and tyrosine kinase inhibitors. Drugs used in HCC therapy, such as tyrosine kinase inhibitor sorafenib, are substrates of uptake and/or efflux transporters. Variable expression of MRPs at the plasma membrane of tumor cells may contribute to drug resistance and subsequent clinical response. Recently, we reported that the hepatocyte SLAMF3 expression (Signaling Lymphocytic Activation Molecule Family member 3) was reduced in tumor cells from hepatocellular carcinoma (HCC) compared to its high expression in adjacent tissues. In the present study, we make a strong correlation between induced SLAMF3 overexpression and the specific loss of MRP-1 expression and its functionalities as a drugs resistance transporter. No changes were observed on expression of ABCG2 and MDR. More importantly, we highlight a strong inverse correlation between MRP-1 and SLAMF3 expression in patients with HCC. We propose that the SLAMF3 overexpression in cancerous cells could represent a potential therapeutic strategy to improve the drugs sensibility of resistant cells and thus control the therapeutic failure in HCC patients.

  7. Upregulation of Mrps18a in breast cancer identified by selecting phage antibody libraries on breast tissue sections

    DEFF Research Database (Denmark)

    Sørensen, Karen Marie Juul; Meldgaard, Theresa; Melchjorsen, Connie Jenning

    2017-01-01

    BACKGROUND: One of the hallmarks of cancer is an altered energy metabolism, and here, mitochondria play a central role. Previous studies have indicated that some mitochondrial ribosomal proteins change their expression patterns upon transformation. METHOD: In this study, we have used the selection...... of recombinant antibody libraries displayed on the surface of filamentous bacteriophage as a proteomics discovery tool for the identification of breast cancer biomarkers. A small subpopulation of breast cells expressing both cytokeratin 19 and cytokeratin 14 was targeted using a novel selection procedure....... RESULTS: We identified the mitochondrial ribosomal protein s18a (Mrps18a) as a protein which is upregulated in breast cancer. However, Mrps18a was not homogeneously upregulated in all cancer cells, suggesting the existence of sub-populations within the tumor. The upregulation was not confined...

  8. Expression and activity of multidrug resistance protein 1 in a murine thymoma cell line

    Science.gov (United States)

    Echevarria-Lima, Juliana; Kyle-Cezar, Fernanda; Leite, Daniela F P; Capella, Luiz; Capella, Márcia A M; Rumjanek, Vivian M

    2005-01-01

    Multidrug resistance proteins [MRPs and P-glycoprotein (Pgp)] are members of the family of ATP-binding cassette (ABC) transport proteins, originally described as being involved in the resistance against anti-cancer agents in tumour cells. These proteins act as ATP-dependent efflux pumps and have now been described in normal cells where they exert physiological roles. The aim of this work was to investigate the expression and activity of MRP and Pgp in the thymoma cell line, EL4. It was observed that EL4 cells expressed mRNA for MRP1, but not for MRP2, MRP3 or Pgp. The activity of ABC transport proteins was evaluated by using the efflux of the fluorescent probes carboxy-2′-7′-dichlorofluorescein diacetate (CFDA) and rhodamine 123 (Rho 123). EL4 cells did not retain CFDA intracellularly, and MRP inhibitors (probenecid, indomethacin and MK 571) decreased MRP1 activity in a concentration-dependent manner. As expected, EL4 cells accumulated Rho 123, and the presence of cyclosporin A and verapamil did not modify this accumulation. Most importantly, when EL4 cells were incubated in the presence of the MRP1 inhibitors indomethacin and MK 571 for 6 days, they started to express CD4 and CD8 molecules on their surface, producing double-positive cells and CD8 single-positive cells. Our results suggest that MRP activity is important for the maintenance of the undifferentiated state in this cell type. This finding might have implications in the physiological process of normal thymocyte maturation. PMID:15804283

  9. Expression of multidrug resistance proteins in retinoblastoma

    Directory of Open Access Journals (Sweden)

    Swati Shukla

    2017-11-01

    Full Text Available AIM: To elucidate the mechanism of multidrug resistance in retinoblastoma, and to acquire more insights into in vivo drug resistance. METHODS: Three anticancer drug resistant Y79 human RB cells were generated against vincristine, etoposide or carboplatin, which are used for conventional chemotherapy in RB. Primary cultures from enucleated eyes after chemotherapy (PCNC were also prepared. Their chemosensitivity to chemotherapeutic agents (vincristine, etoposide and carboplatin were measured using MTT assay. Western blot analysis was performed to evaluate the expression of p53, Bcl-2 and various multidrug resistant proteins in retinoblastoma cells. RESULTS: Following exposure to chemotherapeutic drugs, PCNC showed less sensitivity to drugs. No significant changes observed in the p53 expression, whereas Bcl-2 expression was found to be increased in the drug resistant cells as well as in PCNC. Increased expression of P-glycoprotein (P-gp was observed in drug resistant Y79 cells; however there was no significant change in the expression of P-gp found between primary cultures of primarily enucleated eyes and PCNC. Multidrug resistance protein 1 (Mrp-1 expression was found to be elevated in the drug resistant Y79 cells as well as in PCNC. No significant change in the expression of lung resistance associated protein (Lrp was observed in the drug resistant Y79 cells as well as in PCNC. CONCLUSION: Our results suggest that multidrug resistant proteins are intrinsically present in retinoblastoma which causes treatment failure in managing retinoblastoma with chemotherapy.

  10. Expression of multidrug resistance proteins in retinoblastoma.

    Science.gov (United States)

    Shukla, Swati; Srivastava, Arpna; Kumar, Sunil; Singh, Usha; Goswami, Sandeep; Chawla, Bhavna; Bajaj, Mandeep Singh; Kashyap, Seema; Kaur, Jasbir

    2017-01-01

    To elucidate the mechanism of multidrug resistance in retinoblastoma, and to acquire more insights into in vivo drug resistance. Three anticancer drug resistant Y79 human RB cells were generated against vincristine, etoposide or carboplatin, which are used for conventional chemotherapy in RB. Primary cultures from enucleated eyes after chemotherapy (PCNC) were also prepared. Their chemosensitivity to chemotherapeutic agents (vincristine, etoposide and carboplatin) were measured using MTT assay. Western blot analysis was performed to evaluate the expression of p53, Bcl-2 and various multidrug resistant proteins in retinoblastoma cells. Following exposure to chemotherapeutic drugs, PCNC showed less sensitivity to drugs. No significant changes observed in the p53 expression, whereas Bcl-2 expression was found to be increased in the drug resistant cells as well as in PCNC. Increased expression of P-glycoprotein (P-gp) was observed in drug resistant Y79 cells; however there was no significant change in the expression of P-gp found between primary cultures of primarily enucleated eyes and PCNC. Multidrug resistance protein 1 (Mrp-1) expression was found to be elevated in the drug resistant Y79 cells as well as in PCNC. No significant change in the expression of lung resistance associated protein (Lrp) was observed in the drug resistant Y79 cells as well as in PCNC. Our results suggest that multidrug resistant proteins are intrinsically present in retinoblastoma which causes treatment failure in managing retinoblastoma with chemotherapy.

  11. Expression of multidrug resistance proteins in retinoblastoma

    Science.gov (United States)

    Shukla, Swati; Srivastava, Arpna; Kumar, Sunil; Singh, Usha; Goswami, Sandeep; Chawla, Bhavna; Bajaj, Mandeep Singh; Kashyap, Seema; Kaur, Jasbir

    2017-01-01

    AIM To elucidate the mechanism of multidrug resistance in retinoblastoma, and to acquire more insights into in vivo drug resistance. METHODS Three anticancer drug resistant Y79 human RB cells were generated against vincristine, etoposide or carboplatin, which are used for conventional chemotherapy in RB. Primary cultures from enucleated eyes after chemotherapy (PCNC) were also prepared. Their chemosensitivity to chemotherapeutic agents (vincristine, etoposide and carboplatin) were measured using MTT assay. Western blot analysis was performed to evaluate the expression of p53, Bcl-2 and various multidrug resistant proteins in retinoblastoma cells. RESULTS Following exposure to chemotherapeutic drugs, PCNC showed less sensitivity to drugs. No significant changes observed in the p53 expression, whereas Bcl-2 expression was found to be increased in the drug resistant cells as well as in PCNC. Increased expression of P-glycoprotein (P-gp) was observed in drug resistant Y79 cells; however there was no significant change in the expression of P-gp found between primary cultures of primarily enucleated eyes and PCNC. Multidrug resistance protein 1 (Mrp-1) expression was found to be elevated in the drug resistant Y79 cells as well as in PCNC. No significant change in the expression of lung resistance associated protein (Lrp) was observed in the drug resistant Y79 cells as well as in PCNC. CONCLUSION Our results suggest that multidrug resistant proteins are intrinsically present in retinoblastoma which causes treatment failure in managing retinoblastoma with chemotherapy. PMID:29181307

  12. Evidence that the 5p12 Variant rs10941679 Confers Susceptibility to Estrogen-Receptor-Positive Breast Cancer through FGF10 and MRPS30 Regulation

    DEFF Research Database (Denmark)

    Ghoussaini, Maya; French, Juliet D; Michailidou, Kyriaki

    2016-01-01

    expression of FGF10 and MRPS30. Functional assays demonstrated that SNP rs10941679 maps to an enhancer element that physically interacts with the FGF10 and MRPS30 promoter regions in breast cancer cell lines. FGF10 is an oncogene that binds to FGFR2 and is overexpressed in ∼10% of human breast cancers......, whereas MRPS30 plays a key role in apoptosis. These data suggest that the strongest signal of association at 5p12 is mediated through coordinated activation of FGF10 and MRPS30, two candidate genes for breast cancer pathogenesis....

  13. Conserved allosteric hot spots in the transmembrane domains of cystic fibrosis transmembrane conductance regulator (CFTR) channels and multidrug resistance protein (MRP) pumps.

    Science.gov (United States)

    Wei, Shipeng; Roessler, Bryan C; Chauvet, Sylvain; Guo, Jingyu; Hartman, John L; Kirk, Kevin L

    2014-07-18

    ATP-binding cassette (ABC) transporters are an ancient family of transmembrane proteins that utilize ATPase activity to move substrates across cell membranes. The ABCC subfamily of the ABC transporters includes active drug exporters (the multidrug resistance proteins (MRPs)) and a unique ATP-gated ion channel (cystic fibrosis transmembrane conductance regulator (CFTR)). The CFTR channel shares gating principles with conventional ligand-gated ion channels, but the allosteric network that couples ATP binding at its nucleotide binding domains (NBDs) with conformational changes in its transmembrane helices (TMs) is poorly defined. It is also unclear whether the mechanisms that govern CFTR gating are conserved with the thermodynamically distinct MRPs. Here we report a new class of gain of function (GOF) mutation of a conserved proline at the base of the pore-lining TM6. Multiple substitutions of this proline promoted ATP-free CFTR activity and activation by the weak agonist, 5'-adenylyl-β,γ-imidodiphosphate (AMP-PNP). TM6 proline mutations exhibited additive GOF effects when combined with a previously reported GOF mutation located in an outer collar of TMs that surrounds the pore-lining TMs. Each TM substitution allosterically rescued the ATP sensitivity of CFTR gating when introduced into an NBD mutant with defective ATP binding. Both classes of GOF mutations also rescued defective drug export by a yeast MRP (Yor1p) with ATP binding defects in its NBDs. We conclude that the conserved TM6 proline helps set the energy barrier to both CFTR channel opening and MRP-mediated drug efflux and that CFTR channels and MRP pumps utilize similar allosteric mechanisms for coupling conformational changes in their translocation pathways to ATP binding at their NBDs. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  14. DIRProt: a computational approach for discriminating insecticide resistant proteins from non-resistant proteins.

    Science.gov (United States)

    Meher, Prabina Kumar; Sahu, Tanmaya Kumar; Banchariya, Anjali; Rao, Atmakuri Ramakrishna

    2017-03-24

    Insecticide resistance is a major challenge for the control program of insect pests in the fields of crop protection, human and animal health etc. Resistance to different insecticides is conferred by the proteins encoded from certain class of genes of the insects. To distinguish the insecticide resistant proteins from non-resistant proteins, no computational tool is available till date. Thus, development of such a computational tool will be helpful in predicting the insecticide resistant proteins, which can be targeted for developing appropriate insecticides. Five different sets of feature viz., amino acid composition (AAC), di-peptide composition (DPC), pseudo amino acid composition (PAAC), composition-transition-distribution (CTD) and auto-correlation function (ACF) were used to map the protein sequences into numeric feature vectors. The encoded numeric vectors were then used as input in support vector machine (SVM) for classification of insecticide resistant and non-resistant proteins. Higher accuracies were obtained under RBF kernel than that of other kernels. Further, accuracies were observed to be higher for DPC feature set as compared to others. The proposed approach achieved an overall accuracy of >90% in discriminating resistant from non-resistant proteins. Further, the two classes of resistant proteins i.e., detoxification-based and target-based were discriminated from non-resistant proteins with >95% accuracy. Besides, >95% accuracy was also observed for discrimination of proteins involved in detoxification- and target-based resistance mechanisms. The proposed approach not only outperformed Blastp, PSI-Blast and Delta-Blast algorithms, but also achieved >92% accuracy while assessed using an independent dataset of 75 insecticide resistant proteins. This paper presents the first computational approach for discriminating the insecticide resistant proteins from non-resistant proteins. Based on the proposed approach, an online prediction server DIRProt has

  15. Drug efflux proteins in multidrug resistant bacteria

    NARCIS (Netherlands)

    vanVeen, HW; Konings, WN

    Bacteria contain an array of transport proteins in their cytoplasmic membrane. Many of these proteins play an important role in conferring resistance to toxic compounds. The multidrug efflux systems encountered in prokaryotic cells are very similar to those observed in eukaryotic cells. Therefore, a

  16. Tyrosine and aurora kinase inhibitors diminish transport function of multidrug resistance-associated protein (MRP 4 and breast cancer resistance protein (BCRP

    Directory of Open Access Journals (Sweden)

    Rhiannon N. Hardwick

    2016-12-01

    Full Text Available Tyrosine and aurora kinases are important effectors in signal transduction pathways that are often involved in aberrant cancer cell growth. Tyrosine (TKI and aurora (AKI kinase inhibitors are anti-cancer agents specifically designed to target such signaling pathways through TKI/AKI binding to the ATP-binding pocket of kinases thereby leading to diminished kinase activity. Some TKIs have been identified as inhibitors of ATP-binding cassette (ABC transporters such as P-glycoprotein and breast cancer resistance protein (BCRP, which are commonly upregulated in malignant cells. TKI/AKIs have been investigated as ABC transporter inhibitors in order to facilitate the accumulation of concomitantly administered chemo-therapeutics within cancer cells. However, ABC transporters are prominently expressed in the liver and other eliminating organs, and their inhibition has been linked to intracellular accumulation of drugs, altered disposition, and toxicity. The potential for TKIs/AKIs to inhibit other important hepatic efflux transporters, particularly multidrug resistance-associated proteins (MRPs, remains unknown. The aim of the current study was to compare the inhibitory potency of 20 selected TKI/AKIs against MRP4 and BCRP through the use of inverted membrane vesicle assays. Relative IC50 values were estimated by determining TKI/AKI inhibition of MRP4-mediated [3H]-dehydroepiandrosterone sulfate uptake and BCRP-mediated [3H]-estrone sulfate uptake. To provide insight to the clinical relevance of TKI/AKI inhibition of ABC efflux transporters, the ratio of the steady-state maximum total plasma concentration (Css to the IC50 for each compound was calculated with Css/IC50 ratio >0.1 deemed potentially clinically relevant. Such analysis identified several potentially clinically relevant inhibitors of MRP4: alisertib, danusertib, erlotinib, lapatinib, neratinib, nilotinib, pazopanib, sorafenib, and tozasertib. The potentially clinically relevant inhibition of

  17. Bipolar resistive switching in different plant and animal proteins

    KAUST Repository

    Bag, A.; Hota, Mrinal Kanti; Mallik, Sandipan B.; Maì ti, Chinmay Kumar

    2014-01-01

    We report bipolar resistive switching phenomena observed in different types of plant and animal proteins. Using protein as the switching medium, resistive switching devices have been fabricated with conducting indium tin oxide (ITO) and Al as bottom and top electrodes, respectively. A clockwise bipolar resistive switching phenomenon is observed in all proteins. It is shown that the resistive switching phenomena originate from the local redox process in the protein and the ion exchange from the top electrode/protein interface.

  18. Bipolar resistive switching in different plant and animal proteins

    KAUST Repository

    Bag, A.

    2014-06-01

    We report bipolar resistive switching phenomena observed in different types of plant and animal proteins. Using protein as the switching medium, resistive switching devices have been fabricated with conducting indium tin oxide (ITO) and Al as bottom and top electrodes, respectively. A clockwise bipolar resistive switching phenomenon is observed in all proteins. It is shown that the resistive switching phenomena originate from the local redox process in the protein and the ion exchange from the top electrode/protein interface.

  19. Simple Coatings to Render Polystyrene Protein Resistant

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    Marcelle Hecker

    2018-02-01

    Full Text Available Non-specific protein adsorption is detrimental to the performance of many biomedical devices. Polystyrene is a commonly used material in devices and thin films. Simple reliable surface modification of polystyrene to render it protein resistant is desired in particular for device fabrication and orthogonal functionalisation schemes. This report details modifications carried out on a polystyrene surface to prevent protein adsorption. The trialed surfaces included Pluronic F127 and PLL-g-PEG, adsorbed on polystyrene, using a polydopamine-assisted approach. Quartz crystal microbalance with dissipation (QCM-D results showed only short-term anti-fouling success of the polystyrene surface modified with F127, and the subsequent failure of the polydopamine intermediary layer in improving its stability. In stark contrast, QCM-D analysis proved the success of the polydopamine assisted PLL-g-PEG coating in preventing bovine serum albumin adsorption. This modified surface is equally as protein-rejecting after 24 h in buffer, and thus a promising simple coating for long term protein rejection of polystyrene.

  20. Multidrug resistance-associated protein 4 is a bile transporter of Clonorchis sinensis simulated by in silico docking.

    Science.gov (United States)

    Dai, Fuhong; Yoo, Won Gi; Lee, Ji-Yun; Lu, Yanyan; Pak, Jhang Ho; Sohn, Woon-Mok; Hong, Sung-Jong

    2017-11-21

    Multidrug resistance-associated protein 4 (MRP4) is a member of the C subfamily of the ABC family of ATP-binding cassette (ABC) transporters. MRP4 regulates ATP-dependent efflux of various organic anionic substrates and bile acids out of cells. Since Clonorchis sinensis lives in host's bile duct, accumulation of bile juice can be toxic to the worm's tissues and cells. Therefore, C. sinensis needs bile transporters to reduce accumulation of bile acids within its body. We cloned MRP4 (CsMRP4) from C. sinensis and obtained a cDNA encoding an open reading frame of 1469 amino acids. Phylogenetic analysis revealed that CsMRP4 belonged to the MRP/SUR/CFTR subfamily. A tertiary structure of CsMRP4 was generated by homology modeling based on multiple structures of MRP1 and P-glycoprotein. CsMRP4 had two membrane-spanning domains (MSD1 & 2) and two nucleotide-binding domains (NBD1 & 2) as common structural folds. Docking simulation with nine bile acids showed that CsMRP4 transports bile acids through the inner cavity. Moreover, it was found that CsMRP4 mRNA was more abundant in the metacercariae than in the adults. Mouse immune serum, generated against the CsMRP4-NBD1 (24.9 kDa) fragment, localized CsMRP4 mainly in mesenchymal tissues and oral and ventral suckers of the metacercariae and the adults. Our findings shed new light on MRPs and their homologs and provide a platform for further structural and functional investigations on the bile transporters and parasites' survival.

  1. Multidrug resistance-associated protein 4 is a bile transporter of Clonorchis sinensis simulated by in silico docking

    Directory of Open Access Journals (Sweden)

    Fuhong Dai

    2017-11-01

    Full Text Available Abstract Background Multidrug resistance-associated protein 4 (MRP4 is a member of the C subfamily of the ABC family of ATP-binding cassette (ABC transporters. MRP4 regulates ATP-dependent efflux of various organic anionic substrates and bile acids out of cells. Since Clonorchis sinensis lives in host’s bile duct, accumulation of bile juice can be toxic to the worm’s tissues and cells. Therefore, C. sinensis needs bile transporters to reduce accumulation of bile acids within its body. Results We cloned MRP4 (CsMRP4 from C. sinensis and obtained a cDNA encoding an open reading frame of 1469 amino acids. Phylogenetic analysis revealed that CsMRP4 belonged to the MRP/SUR/CFTR subfamily. A tertiary structure of CsMRP4 was generated by homology modeling based on multiple structures of MRP1 and P-glycoprotein. CsMRP4 had two membrane-spanning domains (MSD1 & 2 and two nucleotide-binding domains (NBD1 & 2 as common structural folds. Docking simulation with nine bile acids showed that CsMRP4 transports bile acids through the inner cavity. Moreover, it was found that CsMRP4 mRNA was more abundant in the metacercariae than in the adults. Mouse immune serum, generated against the CsMRP4-NBD1 (24.9 kDa fragment, localized CsMRP4 mainly in mesenchymal tissues and oral and ventral suckers of the metacercariae and the adults. Conclusions Our findings shed new light on MRPs and their homologs and provide a platform for further structural and functional investigations on the bile transporters and parasites’ survival.

  2. High MRPS23 expression contributes to hepatocellular carcinoma proliferation and indicates poor survival outcomes.

    Science.gov (United States)

    Pu, Meng; Wang, Jianlin; Huang, Qike; Zhao, Ge; Xia, Congcong; Shang, Runze; Zhang, Zhuochao; Bian, Zhenyuan; Yang, Xishegn; Tao, Kaishan

    2017-07-01

    Hepatocellular carcinoma is one of the most prevalent neoplasms and the leading cause of cancer-related mortality worldwide. Mitochondrial ribosomal protein S23 is encoded by a nuclear gene and participates in mitochondrial protein translation. Mitochondrial ribosomal protein S23 overexpression has been found in many types of cancer. In this study, we explored mitochondrial ribosomal protein S23 expression in primary hepatocellular carcinoma tissues compared with matched adjacent non-tumoral liver tissues using mitochondrial ribosomal protein S23 messenger RNA and protein levels collected from public databases and clinical samples. Immunohistochemistry was performed to analyze the relationship between mitochondrial ribosomal protein S23 and various clinicopathological features. The results indicated that mitochondrial ribosomal protein S23 was significantly overexpressed in hepatocellular carcinoma. High mitochondrial ribosomal protein S23 expression was correlated with the tumor size and tumor-metastasis-node stage. Moreover, patients with high mitochondrial ribosomal protein S23 expression levels presented poorer survival rates. Mitochondrial ribosomal protein S23 was an independent prognostic factor for survival, especially at the early stage of hepatocellular carcinoma. In addition, the downregulation of mitochondrial ribosomal protein S23 decreased the proliferation of hepatocellular carcinoma in vitro and in vivo. In conclusion, we verified for the first time that mitochondrial ribosomal protein S23 expression was upregulated in hepatocellular carcinoma. High mitochondrial ribosomal protein S23 levels can predict poor clinical outcomes in hepatocellular carcinoma, and this protein plays a key role in tumor proliferation. Therefore, mitochondrial ribosomal protein S23 may be a potential therapeutic target for hepatocellular carcinoma.

  3. Receptor-like proteins involved in plant disease resistance

    NARCIS (Netherlands)

    Kruijt, M.; Kock, de M.J.D.; Wit, de P.J.G.M.

    2005-01-01

    Race-specific resistance in plants against microbial pathogens is governed by several distinct classes of resistance (R) genes. This review focuses on the class that consists of the plasma membrane-bound leucine-rich repeat proteins known as receptor-like proteins (RLPs). The first isolated

  4. Expression of the breast cancer resistance protein in breast cancer

    NARCIS (Netherlands)

    Faneyte, Ian F.; Kristel, Petra M. P.; Maliepaard, Marc; Scheffer, George L.; Scheper, Rik J.; Schellens, Jan H. M.; van de Vijver, Marc J.

    2002-01-01

    PURPOSE: The breast cancer resistance protein (BCRP) is involved in in vitro multidrug resistance and was first identified in the breast cancer cell line MCF7/AdrVp. The aim of this study was to investigate the role of BCRP in resistance of breast cancer to anthracycline treatment. EXPERIMENTAL

  5. Macrolide Resistance Mediated by a Bifidobacterium breve Membrane Protein

    OpenAIRE

    Margolles, Abelardo; Moreno, José Antonio; van Sinderen, Douwe; de los Reyes-Gavilán, Clara G.

    2005-01-01

    A gene coding for a hypothetical membrane protein from Bifidobacterium breve was expressed in Lactococcus lactis. Immunoblotting demonstrated that this protein is located in the membrane. Phenotypical changes in sensitivity towards 21 antibiotics were determined. The membrane protein-expressing cells showed higher levels of resistance to several macrolides.

  6. Protein function prediction involved on radio-resistant bacteria

    International Nuclear Information System (INIS)

    Mezhoud, Karim; Mankai, Houda; Sghaier, Haitham; Barkallah, Insaf

    2009-01-01

    Previously, we identified 58 proteins under positive selection in ionizing-radiation-resistant bacteria (IRRB) but absent in all ionizing-radiation-sensitive bacteria (IRSB). These are good reasons to believe these 58 proteins with their interactions with other proteins (interactomes) are a part of the answer to the question as to how IRRB resist to radiation, because our knowledge of interactomes of positively selected orphan proteins in IRRB might allow us to define cellular pathways important to ionizing-radiation resistance. Using the Database of Interacting Proteins and the PSIbase, we have predicted interactions of orthologs of the 58 proteins under positive selection in IRRB but absent in all IRSB. We used integrate experimental data sets with molecular interaction networks and protein structure prediction from databases. Among these, 18 proteins with their interactomes were identified in Deinococcus radiodurans R1. DNA checkpoint and repair, kinases pathways, energetic and nucleotide metabolisms were the important biological process that found. We predicted the interactomes of 58 proteins under positive selection in IRRB. It is hoped our data will provide new clues as to the cellular pathways that are important for ionizing-radiation resistance. We have identified news proteins involved on DNA management which were not previously mentioned. It is an important input in addition to protein that studied. It does still work to deepen our study on these new proteins

  7. Increased Prevalence of Activated Protein C Resistance During ...

    African Journals Online (AJOL)

    Background: Acquired resistance to protein C in pregnancy has been established as one of the factors associated with ..... diabetes, sickle cell disease, smoking, anti-phospholipid syndrome inherited thrombophilia, and previous history of.

  8. Neisseria meningitidis rifampicin resistant strains: analysis of protein differentially expressed

    Directory of Open Access Journals (Sweden)

    Schininà Maria

    2010-09-01

    Full Text Available Abstract Background Several mutations have been described as responsible for rifampicin resistance in Neisseria meningitidis. However, the intriguing question on why these strains are so rare remains open. The aim of this study was to investigate the protein content and to identify differential expression in specific proteins in two rifampicin resistant and one susceptible meningococci using two-dimensional electrophoresis (2-DE combined with mass spectrometry. Results In our experimental conditions, able to resolve soluble proteins with an isoelectric point between 4 and 7, twenty-three proteins have been found differentially expressed in the two resistant strains compared to the susceptible. Some of them, involved in the main metabolic pathways, showed an increased expression, mainly in the catabolism of pyruvate and in the tricarboxylic acid cycle. A decreased expression of proteins belonging to gene regulation and to those involved in the folding of polypeptides has also been observed. 2-DE analysis showed the presence of four proteins displaying a shift in their isoelectric point in both resistant strains, confirmed by the presence of amino acid changes in the sequence analysis, absent in the susceptible. Conclusions The analysis of differentially expressed proteins suggests that an intricate series of events occurs in N. meningitidis rifampicin resistant strains and the results here reported may be considered a starting point in understanding their decreased invasion capacity. In fact, they support the hypothesis that the presence of more than one protein differentially expressed, having a role in the metabolism of the meningococcus, influences its ability to infect and to spread in the population. Different reports have described and discussed how a drug resistant pathogen shows a high biological cost for survival and that may also explain why, for some pathogens, the rate of resistant organisms is relatively low considering the

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

    Science.gov (United States)

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

    2016-03-22

    Members of the ABC-F subfamily of ATP-binding cassette proteins mediate resistance to a broad array of clinically important antibiotic classes that target the ribosome of Gram-positive pathogens. The mechanism by which these proteins act has been a subject of long-standing controversy, with two competing hypotheses each having gained considerable support: antibiotic efflux versus ribosomal protection. Here, we report on studies employing a combination of bacteriological and biochemical techniques to unravel the mechanism of resistance of these proteins, and provide several lines of evidence that together offer clear support to the ribosomal protection hypothesis. Of particular note, we show that addition of purified ABC-F proteins to anin vitrotranslation assay prompts dose-dependent rescue of translation, and demonstrate that such proteins are capable of displacing antibiotic from the ribosomein vitro To our knowledge, these experiments constitute the first direct evidence that ABC-F proteins mediate antibiotic resistance through ribosomal protection.IMPORTANCEAntimicrobial resistance ranks among the greatest threats currently facing human health. Elucidation of the mechanisms by which microorganisms resist the effect of antibiotics is central to understanding the biology of this phenomenon and has the potential to inform the development of new drugs capable of blocking or circumventing resistance. Members of the ABC-F family, which includelsa(A),msr(A),optr(A), andvga(A), collectively yield resistance to a broader range of clinically significant antibiotic classes than any other family of resistance determinants, although their mechanism of action has been controversial since their discovery 25 years ago. Here we present the first direct evidence that proteins of the ABC-F family act to protect the bacterial ribosome from antibiotic-mediated inhibition. Copyright © 2016 Sharkey et al.

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

    Science.gov (United States)

    Bosse, John D; Dixon, Brian M

    2012-09-08

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

  11. Mitochondrial ribosomal protein S18-2 evokes chromosomal instability and transforms primary rat skin fibroblasts

    KAUST Repository

    Kashuba, Elena; Carbone, Ennio; Di Fabrizio, Enzo M.; Tirinato, Luca; Petruchek, Maria; Drummond, Catherine; Kovalevska, Larysa; Gurrapu, Sreeharsha; Mushtaq, Muhammad; Darekar, Suhas D.

    2015-01-01

    We have shown earlier that overexpression of the human mitochondrial ribosomal protein MRPS18-2 (S18-2) led to immortalization of primary rat embryonic fibroblasts. The derived cells expressed the embryonic stem cell markers, and cellular pathways

  12. Molecular and structural characteristics of multidrug resistance-associated protein 7 in Chinese liver fluke Clonorchis sinensis.

    Science.gov (United States)

    Dai, Fuhong; Yoo, Won Gi; Lee, Ji-Yun; Lu, Yanyan; Pak, Jhang Ho; Sohn, Woon-Mok; Hong, Sung-Jong

    2017-03-01

    Multidrug resistance-associated protein 7 (MRP7, ABCC10) is a C subfamily member of the ATP-binding cassette (ABC) superfamily. MRP7 is a lipophilic anion transporter that pumps endogenous and xenobiotic substrates from the cytoplasm to the extracellular milieu. Here, we cloned and characterized CsMRP7 as a novel ABC transporter from the Chinese liver fluke, Clonorchis sinensis. Full-length cDNA of CsMRP7 was 5174 nt, encoded 1636 amino acids (aa), and harbored a 147-bp 5'-untranslated region (5'-UTR) and 116-bp 3'-UTR. Phylogenetic analysis confirmed that CsMRP7 was closer to the ABCC subfamily than the ABCB subfamily. Tertiary structures of the N-terminal region (1-322 aa) and core region (323-1621 aa) of CsMRP7 were generated by homology modeling using glucagon receptor (PDB ID: 5ee7_A) and P-glycoprotein (PDB ID: 4f4c_A) as templates, respectively. CsMRP7 nucleotide-binding domain 2 (NBD2) was conserved more than NBD1, which was the sites of ATP binding and hydrolysis. Like typical long MRPs, CsMRP7 has an additional membrane-spanning domain 0 (MSD0) and cytoplasmic loop, along with a common structural fold consisting of MSD1-NBD1-MSD2-NBD2 as a single polypeptide assembly. MSD0, MSD1, and MSD2 consisted of TM1-7, TM8-13, and TM14-19, respectively. The CsMRP7 transcript was more abundant in the metacercariae than in the adult worms. Truncated NBD1 (39 kDa) and NBD2 (44 kDa) were produced in bacteria and mouse immune sera were raised. CsMRP7 was localized in the apical side of the intestinal epithelium, sperm in the testes and seminal receptacle, receptacle membrane, and mesenchymal tissue around intestine in the adult worm. These results provide molecular information and insights into structural and functional characteristics of CsMRP7 and homologs of flukes.

  13. Heterologously expressed bacterial and human multidrug resistance proteins confer cadmium resistance to Escherichia coli

    NARCIS (Netherlands)

    Achard-Joris, M; van Saparoea, HBV; Driessen, AJM; Bourdineaud, JP; Bourdineaud, Jean-Paul

    2005-01-01

    The human MDR1 gene is induced by cadmium exposure although no resistance to this metal is observed in human cells overexpressing hMDR1. To access the role of MDR proteins in cadmium resistance, human MDR1, Lactococcus lactis lmrA, and Oenococcus oeni omrA were expressed in an Escherichia coli tolC

  14. Multiple drug resistance protein (MDR-1, multidrug resistance-related protein (MRP and lung resistance protein (LRP gene expression in childhood acute lymphoblastic leukemia

    Directory of Open Access Journals (Sweden)

    Elvis Terci Valera

    Full Text Available CONTEXT: Despite the advances in the cure rate for acute lymphoblastic leukemia, approximately 25% of affected children suffer relapses. Expression of genes for the multiple drug resistance protein (MDR-1, multidrug resistance-related protein (MRP, and lung resistance protein (LRP may confer the phenotype of resistance to the treatment of neoplasias. OBJECTIVE: To analyze the expression of the MDR-1, MRP and LRP genes in children with a diagnosis of acute lymphoblastic leukemia via the semiquantitative reverse transcription polymerase chain reaction (RT-PCR, and to determine the correlation between expression and event-free survival and clinical and laboratory variables. DESIGN: A retrospective clinical study. SETTING: Laboratory of Pediatric Oncology, Department of Pediatrics, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Brazil. METHODS: Bone marrow aspirates from 30 children with a diagnosis of acute lymphoblastic leukemia were assessed for the expression of messenger RNA for the MDR-1, MRP and LRP genes by semi-quantitative RT-PCR. RESULTS: In the three groups studied, only the increased expression of LRP was related to worsened event-free survival (p = 0.005. The presence of the common acute lymphoblastic leukemia antigen (CALLA was correlated with increased LRP expression (p = 0.009 and increased risk of relapse or death (p = 0.05. The relative risk of relapse or death was six times higher among children with high LRP expression upon diagnosis (p = 0.05, as confirmed by multivariate analysis of the three genes studied (p = 0.035. DISCUSSION: Cell resistance to drugs is a determinant of the response to chemotherapy and its detection via RT-PCR may be of clinical importance. CONCLUSIONS: Evaluation of the expression of genes for resistance to antineoplastic drugs in childhood acute lymphoblastic leukemia upon diagnosis, and particularly the expression of the LRP gene, may be of clinical relevance, and should be the

  15. Breast cancer resistance protein is localized at the plasma membrane in mitoxantrone- and topotecan-resistant cell lines

    NARCIS (Netherlands)

    Scheffer, GL; Maliepaard, M; Pijnenborg, ACLM; van Gastelen, MA; Schroeijers, AB; Allen, JD; Ross, DD; van der Valk, P; Dalton, WS; Schellens, JHM; Scheper, RJ; de Jong, MC

    2000-01-01

    Tumor cells may display a multidrug resistant phenotype by overexpression of ATP-binding cassette transporters such as multidrug resistance (,MDR1) P-glycoprotein, multidrug resistance protein 1 (MRP1), and breast cancer resistance protein (BCRP). The presence of BCRP has thus far been reported

  16. Impact of neutrophil-secreted myeloid related proteins 8 and 14 (MRP 8/14) on leishmaniasis progression.

    Science.gov (United States)

    Contreras, Irazú; Shio, Marina T; Cesaro, Annabelle; Tessier, Philippe A; Olivier, Martin

    2013-01-01

    The myeloid-related proteins (MRPs) 8/14 are small proteins mainly produced by neutrophils, which have been reported to induce NO production in macrophages. On the other hand, Leishmania survives and multiplies within phagocytes by inactivating several of their microbicidal functions. Whereas MRPs are rapidly released during the innate immune response, their role in the regulation of Leishmaniasis is still unknown. In vitro experiments revealed that Leishmania infection alters MRP-induced signaling, leading to inhibition of macrophage functions (NO, TNF-α). In contrast, MRP-primed cells showed normal signaling activation and NO production in response to Leishmania infection. Using a murine air-pouch model, we observed that infection with L. major induced leukocyte recruitment and MRP secretion comparable to LPS-treated mice. Depletion of MRPs significantly reduced these inflammatory events and augmented both parasite load and footpad swelling during the first 8 weeks post-infection, as also observed in MRP KO mice. On the contrary, mouse treatment with recombinant MRPs (rMRPs) had the opposite effect. Collectively, our results suggest that rapid secretion of MRPs by neutrophils at the site of infection may protect uninfected macrophages and favor a more efficient innate inflammatory response against Leishmania infection. In summary, our study reveals the critical role played by MRPs in the regulation of Leishmania infection and how this pathogen can subvert its action.

  17. Retinol binding protein 4, obesity, and insulin resistance in adolescents

    Directory of Open Access Journals (Sweden)

    Ronaldi Noor

    2017-02-01

    Full Text Available Background Obesity is a global problem. Even in poor and developing countries, obesity has reached alarming levels. In childhood, obesity may lead to insulin resistance. Retinol binding protein (RBP4, secreted primarily by liver and adipose tissues, was recently proposed as a link between obesity and insulin resistance. The role of RBP4 in pediatric obesity and its relationship with insulin resistance have not been well elucidated. Objective To compare RBP4 levels in obese and lean adolescents and to assess for a relationship between RBP4 levels and insulin resistance. Method This cross-sectional study was conducted in three senior high schools in Padang, West Sumatera, Indonesia. Subjects were adolescents aged 14-18 years, who were obese or normal weight (n=56. We measured subjects’ body mass index (BMI and serum RBP4 concentrations. Insulin resistance was assessed using the homeostasis model assessment of insulin resistance (HOMA-IR index. Results Similar RBP4 levels were found in the obese and normoweight groups (P>0.05. Higher RBP4 levels were found in the insulin resistant compared to the non-insulin resistant group, but the difference was not significant (P > 0.05. Conclusion There is no significant difference in mean RBP4 levels in obese adolescents compared to normoweight adolescents. Nor are mean RBP4 levels significantly different between obese adolescents with and without insulin resistance.

  18. Epithelial membrane protein-1 is a biomarker of gefitinib resistance.

    Science.gov (United States)

    Jain, Anjali; Tindell, Charles A; Laux, Isett; Hunter, Jacob B; Curran, John; Galkin, Anna; Afar, Daniel E; Aronson, Nina; Shak, Steven; Natale, Ronald B; Agus, David B

    2005-08-16

    We describe a molecular resistance biomarker to gefitinib, epithelial membrane protein-1 (EMP-1). Gefitinib is a small-molecule inhibitor that competes for the ATP-binding site on EGF receptor (EGFR) and has been approved for patients with advanced lung cancers. Treatment with gefitinib has resulted in clinical benefit in patients, and, recently, heterozygous somatic mutations within the EGFR catalytic domain have been identified as a clinical correlate to objective response to gefitinib. However, clinical resistance to gefitinib limits the utility of this therapeutic to a fraction of patients, and objective clinical responses are rare. We aimed to assess the molecular phenotype and mechanism of in vivo gefitinib resistance in xenograft models and in patient samples. We generated in vivo gefitinib-resistance models in an adenocarcinoma xenograft model by serially passaging tumors in nude mice in presence of gefitinib until resistance was acquired. EMP-1 was identified as a surface biomarker whose expression correlated with acquisition of gefitinib resistance. EMP-1 expression was further correlated with lack of complete or partial response to gefitinib in lung cancer patient samples as well as clinical progression to secondary gefitinib resistance. EMP-1 expression and acquisition of gefitinib clinical resistance was independent of gefitinib-sensitizing EGFR somatic mutations. This report suggests the role of the adhesion molecule, EMP-1, as a biomarker of gefitinib clinical resistance, and further suggests a probable cross-talk between this molecule and the EGFR signaling pathway.

  19. Coexpression of multidrug resistance involve proteins: a flow cytometric analysis.

    Science.gov (United States)

    Boutonnat, J; Bonnefoix, T; Mousseau, M; Seigneurin, D; Ronot, X

    1998-01-01

    Cross resistance to multiple natural cytotoxic products represents a major obstacle in myeloblastic acute leukaemia (AML). Multidrug resistance (MDR) often involves overexpression of plasma membrane drug transporter P-glycoprotein (PGP) or the resistance associated protein (MRP). Recently, a protein overexpressed in a non-PGP MDR lung cancer cell line and termed lung resistance related protein (LRP) was identified. These proteins are known to be associated with a bad prognosis in AML. We have developed a triple indirect labelling analysed by flow cytometry to detect the coexpression of these proteins. Since no cell line expressing all three antigens is known, we mixed K562 cells (resistant to Adriblastine, PGP+, MRP-, LRP-) with GLC4 cells (resistant to Adriblastine, PGP-, MRP+, LRP+) to create a model system to test the method. The antibodies used were UIC2 for PGP, MRPm6 for MRP and LRP56 for LRP. They were revealed by Fab'2 coupled with Fluoresceine-isothiocyanate, Phycoerythrin or Tricolor with isotype specificity. Cells were fixed and permeabilized after PGP labelling because MRPm6 and LRP56 recognize intracellular epitopes. PGP and LRP were easily detected. MRP is expressed at relatively low levels and was more difficult to detect because in the triple labelling the non specific staining was higher than in a single labelling. Despite the increased background in the triple labelling we were able to detect coexpression of PGP, MRP, LRP by flow cytometry. This method appears to be very useful to detect coexpression of markers in AML. Such coexpression could modify the therapeutic approach with revertants.

  20. Food Processing: The Influence of the Maillard Reaction on Immunogenicity and Allergenicity of Food Proteins

    Science.gov (United States)

    Teodorowicz, Malgorzata; van Neerven, Joost

    2017-01-01

    The majority of foods that are consumed in our developed society have been processed. Processing promotes a non-enzymatic reaction between proteins and sugars, the Maillard reaction (MR). Maillard reaction products (MRPs) contribute to the taste, smell and color of many food products, and thus influence consumers’ choices. However, in recent years, MRPs have been linked to the increasing prevalence of diet- and inflammation-related non-communicable diseases including food allergy. Although during the last years a better understanding of immunogenicity of MRPs has been achieved, still only little is known about the structural/chemical characteristics predisposing MRPs to interact with antigen presenting cells (APCs). This report provides a comprehensive review of recent studies on the influence of the Maillard reaction on the immunogenicity and allergenicity of food proteins. PMID:28777346

  1. Food Processing: The Influence of the Maillard Reaction on Immunogenicity and Allergenicity of Food Proteins.

    Science.gov (United States)

    Teodorowicz, Malgorzata; van Neerven, Joost; Savelkoul, Huub

    2017-08-04

    The majority of foods that are consumed in our developed society have been processed. Processing promotes a non-enzymatic reaction between proteins and sugars, the Maillard reaction (MR). Maillard reaction products (MRPs) contribute to the taste, smell and color of many food products, and thus influence consumers' choices. However, in recent years, MRPs have been linked to the increasing prevalence of diet- and inflammation-related non-communicable diseases including food allergy. Although during the last years a better understanding of immunogenicity of MRPs has been achieved, still only little is known about the structural/chemical characteristics predisposing MRPs to interact with antigen presenting cells (APCs). This report provides a comprehensive review of recent studies on the influence of the Maillard reaction on the immunogenicity and allergenicity of food proteins.

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

    Science.gov (United States)

    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. Copyright © 2015 Elsevier Ltd. All rights reserved.

  3. Testing of disease-resistance of pokeweed antiviral protein gene ...

    African Journals Online (AJOL)

    Transformation of pokeweed antiviral protein gene (PAP) into plants was shown to improve plant resistance to several viruses or fungi pathogens with no much negative effect on plant growth. The non-virulent defective PAP inhibits only the virus but does not interfere with the host. A non-virulent defective PAP gene ...

  4. Evaluating the Role of Multidrug Resistance Protein 3 (MDR3) Inhibition in Predicting Drug-Induced Liver Injury Using 125 Pharmaceuticals.

    Science.gov (United States)

    Aleo, Michael D; Shah, Falgun; He, Kan; Bonin, Paul D; Rodrigues, A David

    2017-05-15

    The role of bile salt export protein (BSEP) inhibition in drug-induced liver injury (DILI) has been investigated widely, while inhibition of the canalicular multidrug resistant protein 3 (MDR3) has received less attention. This transporter plays a pivotal role in secretion of phospholipids into bile and functions coordinately with BSEP to mediate the formation of bile acid-containing biliary micelles. Therefore, inhibition of MDR3 in human hepatocytes was examined across 125 drugs (70 of Most-DILI-concern and 55 of No-DILI-concern). Of these tested, 41% of Most-DILI-concern and 47% of No-DILI-concern drugs had MDR3 IC 50 values of <50 μM. A better distinction across DILI classifications occurred when systemic exposure was considered where safety margins of 50-fold had low sensitivity (0.29), but high specificity (0.96). Analysis of physical chemical property space showed that basic compounds were twice as likely to be MDR3 inhibitors as acids, neutrals, and zwitterions and that inhibitors were more likely to have polar surface area (PSA) values of <100 Å 2 and cPFLogD values between 1.5 and 5. These descriptors, with different cutoffs, also highlighted a group of compounds that shared dual potency as MDR3 and BSEP inhibitors. Nine drugs classified as Most-DILI-concern compounds (four withdrawn, four boxed warning, and one liver injury warning in their approved label) had intrinsic potency features of <20 μM in both assays, thereby reinforcing the notion that multiple inhibitory mechanisms governing bile formation (bile acid and phospholipid efflux) may confer additional risk factors that play into more severe forms of DILI as shown by others for BSEP inhibitors combined with multidrug resistance-associated protein (MRP2, MRP3, MRP4) inhibitory properties. Avoiding physical property descriptors that highlight dual BSEP and MDR3 inhibition or testing drug candidates for inhibition of multiple efflux transporters (e.g., BSEP, MDR3, and MRPs) may be an effective

  5. Structural basis of protein oxidation resistance: a lysozyme study.

    Directory of Open Access Journals (Sweden)

    Marion Girod

    Full Text Available 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.

  6. An innovative strategy to clone positive modifier genes of defects caused by mtDNA mutations: MRPS18C as suppressor gene of m.3946G>A mutation in MT-ND1 gene.

    Science.gov (United States)

    Rodríguez-García, María Elena; Cotrina-Vinagre, Francisco Javier; Carnicero-Rodríguez, Patricia; Martínez-Azorín, Francisco

    2017-07-01

    We have developed a new functional complementation approach to clone modifier genes which overexpression is able to suppress the biochemical defects caused by mtDNA mutations (suppressor genes). This strategy consists in transferring human genes into respiratory chain-deficient fibroblasts, followed by a metabolic selection in a highly selective medium. We used a normalized expression cDNA library in an episomal vector (pREP4) to transfect the fibroblasts, and a medium with glutamine and devoid of any carbohydrate source to select metabolically. Growing the patient's fibroblasts in this selective medium, the deficient cells rapidly disappear unless they are rescued by the cDNA of a suppressor gene. The use of an episomal vector allows us to carry out several rounds of transfection/selection (cyclical phenotypic rescue) to enrich the rescue with true clones of suppressor genes. Using fibroblasts from a patient with epileptic encephalopathy with the m.3946G>A (p.E214K) mutation in the MT-ND1 gene, several candidate genes were identified and one of them was characterized functionally. Thus, overexpression of MRPS18C gene (that encode for bS18m protein) suppressed the molecular defects produced by this mtDNA mutation, recovering the complex I activity and reducing the ROS produced by this complex to normal levels. We suggest that modulation of bS18m expression may be an effective therapeutic strategy for the patients with this mutation.

  7. Using Resurrected Ancestral Proviral Proteins to Engineer Virus Resistance

    Directory of Open Access Journals (Sweden)

    Asunción Delgado

    2017-05-01

    Full Text Available Proviral factors are host proteins hijacked by viruses for processes essential for virus propagation such as cellular entry and replication. Pathogens and their hosts co-evolve. It follows that replacing a proviral factor with a functional ancestral form of the same protein could prevent viral propagation without fatally compromising organismal fitness. Here, we provide proof of concept of this notion. Thioredoxins serve as general oxidoreductases in all known cells. We report that several laboratory resurrections of Precambrian thioredoxins display substantial levels of functionality within Escherichia coli. Unlike E. coli thioredoxin, however, these ancestral thioredoxins are not efficiently recruited by the bacteriophage T7 for its replisome and therefore prevent phage propagation in E. coli. These results suggest an approach to the engineering of virus resistance. Diseases caused by viruses may have a devastating effect in agriculture. We discuss how the suggested approach could be applied to the engineering of plant virus resistance.

  8. Prevalence of Resistence to Activated Protein C (Apc-Resistance in Blood Donors in Kosovo

    Directory of Open Access Journals (Sweden)

    Ymer Mekaj

    2009-11-01

    Full Text Available AbstractOne of the most frequent hereditary causes of thrombophilia is, without a doubt, resistance to Activated Protein C (APC-resistance, which is a consequence of point mutation in gene coding for coagulation Factor V (Factor V Leiden in 90-95% of cases.The aim of this paper was to determine prevalence of APC-resistance in a group of healthy blood donors. The size of the group is quite representative of Kosovo Albanians.A total of 944 blood donors were examined (537 males and 407 females, for whom APC-resistance was determined by functional methods of coagulation using the kit ACTICLOT® Protein C Resistance. Method is based on the test of APTT determined twice: first in the presence and second in the absence of activated Protein C (APC. The ratio of these two values constitutes is called Activated Protein C - Sensitivity Ratio (APC-SR.From 944 examined donors, pathologic values of APC-SR (1,3-1,9 were found in 32 persons (3,4% of the total number. The distribution among sexes was 3,35% (18/537 in male and 3,43% (14/407 in female subjects. The mean values of APC-SR (1,64 in male and 1,71 in female subjects were not significantly different (P = 0,22.Based on these results, we conclude that the prevalence of APC resistance in Albanian population of Kosovo is within the lower limit of prevalence in general population in different countries of European countries, which, according to some authors ranges is from 3 to 7%.

  9. Protein resistance of dextran and dextran-PEG copolymer films

    Science.gov (United States)

    Kozak, Darby; Chen, Annie; Bax, Jacinda; Trau, Matt

    2011-01-01

    The protein resistance of dextran and dextran-poly(ethylene glycol) (PEG) copolymer films was examined on an organosilica particle-based assay support. Comb-branched dextran-PEG copolymer films were synthesized in a two step process using the organosilica particle as a solid synthetic support. Particles modified with increasing amounts (0.1-1.2 mg m−2) of three molecular weights (10 000, 66 900, 400 000 g mol−1) of dextran were found to form relatively poor protein-resistant films compared to dextran-PEG copolymers and previously studied PEG films. The efficacy of the antifouling polymer films was found to be dependent on the grafted amount and its composition, with PEG layers being the most efficient, followed by dextran-PEG copolymers, and dextran alone being the least efficient. Immunoglobulin gamma (IgG) adsorption decreased from ~ 5 to 0.5 mg m−2 with increasing amounts of grafted dextran, but bovine serum albumin (BSA) adsorption increased above monolayer coverage (to ~2 mg m−2) indicating ternary adsorption of the smaller protein within the dextran layer. PMID:21614699

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

    Science.gov (United States)

    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

  11. The expression and significance of P-glycoprotein, lung resistance protein and multidrug resistance-associated protein in gastric cancer

    Directory of Open Access Journals (Sweden)

    Li Yan

    2009-11-01

    Full Text Available Abstract Background To detect the expression of multidrug resistance molecules P-glycoprotein (P-gp, Lung resistnce protein (LRP and Multidrug resistance-associated protein (MRP and analyze the relationship between them and the clinico-pathological features. Methods The expressions of P-gp, LRP and MRP in formalin-fixed paraffin-embedded tissue sections from 59 gastric cancer patients were determined by a labbelled Streptavidin-Peroxidase (SP immunohistochemical technique, and the results were analyzed in correlation with clinicopathological data. None of these patients received chemotherapy prior to surgery. Results The positive rates of P-gp, LRP, MRP were 86.4%, 84.7% and 27.1%, respectively. The difference between the positive rate of P-gp and MRP was significant statistically, as well as the difference between the expression of MRP and LRP. No significant difference was observed between P-gp and LRP, but the positively correlation between the expression of P-gp and LRP had been found. No significant correlation between the expression of P-gp, LRP, MRP and the grade of differentiation were observed. The expression of P-gp was correlated with clinical stages positively (r = 0.742, but the difference with the expression of P-gp in different stages was not significant. Conclusion The expressions of P-gp, LRP and MRP in patients with gastric cancer without prior chemotherapy are high, indicating that innate drug resistance may exist in gastric cancer.

  12. Using Resurrected Ancestral Proviral Proteins to Engineer Virus Resistance.

    Science.gov (United States)

    Delgado, Asunción; Arco, Rocio; Ibarra-Molero, Beatriz; Sanchez-Ruiz, Jose M

    2017-05-09

    Proviral factors are host proteins hijacked by viruses for processes essential for virus propagation such as cellular entry and replication. Pathogens and their hosts co-evolve. It follows that replacing a proviral factor with a functional ancestral form of the same protein could prevent viral propagation without fatally compromising organismal fitness. Here, we provide proof of concept of this notion. Thioredoxins serve as general oxidoreductases in all known cells. We report that several laboratory resurrections of Precambrian thioredoxins display substantial levels of functionality within Escherichia coli. Unlike E. coli thioredoxin, however, these ancestral thioredoxins are not efficiently recruited by the bacteriophage T7 for its replisome and therefore prevent phage propagation in E. coli. These results suggest an approach to the engineering of virus resistance. Diseases caused by viruses may have a devastating effect in agriculture. We discuss how the suggested approach could be applied to the engineering of plant virus resistance. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

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

    NARCIS (Netherlands)

    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

  14. Role of breast cancer resistance protein in the bioavailability and fetal penetration of topotecan

    NARCIS (Netherlands)

    Jonker, JW; Smit, JW; Brinkhuis, RF; Maliepaard, M; Beijnen, JH; Schellens, JHM; Schinkel, AH

    2000-01-01

    Background and Methods: Breast cancer resistance protein (BCRP/MXR/ABCP) is a multidrug-resistance protein that is a member of the adenosine triphosphate-binding cassette family of drug transporters. BCRP can render tumor cells resistant to the anticancer drugs topotecan, mitoxantrone, doxorubicin,

  15. Natural coagulation inhibitors and active protein c resistance in preeclampsia

    Directory of Open Access Journals (Sweden)

    Cengiz Demir

    2010-01-01

    Full Text Available INTRODUCTION: The etiology of preeclampsia is not fully established. A few studies have shown a relationship between natural coagulation inhibitors and preeclampsia. OBJECTIVES: The purpose of this study was to investigate the status of natural coagulation inhibitors and active protein C resistance (APC-R in preeclampsia. PATIENTS AND METHODS: We studied 70 women with preeclampsia recruited consecutively and 70 healthy pregnant and 70 nonpregnant women as controls. Plasma protein C (PC, free protein S (fPS, antithrombin III (ATIII and APC-R were evaluated. RESULTS: ATIII values were found to be significantly lower in preeclamptic patients than in the control groups (p< 0.001. Nevertheless, there was no significant difference between the healthy pregnant and nonpregnant women groups (p=0.141. The fPS values of the preeclamptic and healthy pregnant groups were lower than that of the nonpregnant group (p< 0.001, and the fPS value of the preeclamptic pregnant women was lower than that of healthy pregnant women (p<0.001. The PC value of the preeclamptic pregnant women was lower than that of the control groups (p< 0.001. The PC value of the healthy pregnant women was lower than that of the nonpregnant women (p< 0.001. The mean APC activity values were lower in the preeclamptic patients than that of the control groups (p< 0.001, p< 0.001. The APC-R positivity rates of the preeclamptic groups were higher than that of the control groups (p<0.001. CONCLUSIONS: This study demonstrated that ATIII, fPS, PC values and APC resistance were lower and APC-R positivity was higher in preeclamptic women than in normal pregnant and nonpregnant women.

  16. Protease-resistant prions selectively decrease Shadoo protein.

    Directory of Open Access Journals (Sweden)

    Joel C Watts

    2011-11-01

    Full Text Available 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.

  17. Extracellular proteins: Novel key components of metal resistance in cyanobacteria?

    Directory of Open Access Journals (Sweden)

    Joaquin eGiner-Lamia

    2016-06-01

    Full Text Available 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 towards 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.

  18. Positive muscle protein net balance and differential regulation of atrogene expression after resistance exercise and milk protein supplementation

    DEFF Research Database (Denmark)

    Reitelseder, Søren; Agergaard, Jakob; Doessing, Simon

    2014-01-01

    Purpose Resistance exercise and amino acid availability are positive regulators of muscle protein net balance (NB). However, anabolic responses to resistance exercise and protein supplementation deserve further elucidation. The purpose was to compare intakes of whey, caseinate (both: 0.30 g/kg lean...... body mass), or a non-caloric control after heavy resistance exercise on protein turnover and mRNA expressions of forkhead homeobox type O (FOXO) isoforms, muscle RING finger 1 (MuRF1), and Atrogin1 in young healthy males. Methods Protein turnover was determined by stable isotope-labeled leucine...

  19. Mitonuclear protein imbalance as a conserved longevity mechanism

    NARCIS (Netherlands)

    Houtkooper, Riekelt H.; Mouchiroud, Laurent; Ryu, Dongryeol; Moullan, Norman; Katsyuba, Elena; Knott, Graham; Williams, Robert W.; Auwerx, Johan

    2013-01-01

    Longevity is regulated by a network of closely linked metabolic systems. We used a combination of mouse population genetics and RNA interference in Caenorhabditis elegans to identify mitochondrial ribosomal protein S5 (Mrps5) and other mitochondrial ribosomal proteins as metabolic and longevity

  20. Retinol-Binding Protein 4 and Insulin Resistance in Polycystic Ovary Syndrome

    OpenAIRE

    Hutchison, Samantha K.; Harrison, Cheryce; Stepto, Nigel; Meyer, Caroline; Teede, Helena J.

    2008-01-01

    OBJECTIVE?Polycystic ovary syndrome (PCOS) is an insulin-resistant state with insulin resistance being an established therapeutic target; however, measurement of insulin resistance remains challenging. We aimed to 1) determine serum retinol-binding protein 4 (RBP4) levels (purported to reflect insulin resistance) in women with PCOS and control subjects, 2) examine the relationship of RBP4 to conventional markers of insulin resistance, and 3) examine RBP4 changes with interventions modulating ...

  1. Resistance training reduces whole-body protein turnover and improves net protein retention in untrained young males.

    Science.gov (United States)

    Hartman, Joseph W; Moore, Daniel R; Phillips, Stuart M

    2006-10-01

    It is thought that resistance exercise results in an increased need for dietary protein; however, data also exists to support the opposite conclusion. The purpose of this study was to determine the impact of resistance exercise training on protein metabolism in novices with the hypothesis that resistance training would reduce protein turnover and improve whole-body protein retention. Healthy males (n = 8, 22 +/- 1 y, BMI = 25.3 +/- 1.8 kg.m(-2)) participated in a progressive whole-body split routine resistance-training program 5d/week for 12 weeks. Before (PRE) and after (POST) the training, oral [15N]-glycine ingestion was used to assess nitrogen flux (Q), protein synthesis (PS), protein breakdown (PB), and net protein balance (NPB = PS-PB). Macronutrient intake was controlled over a 5d period PRE and POST, while estimates of protein turnover and urinary nitrogen balance (N(bal) = N(in) - urine N(out)) were conducted. Bench press and leg press increased 40% and 50%, respectively (p training-induced increases in both NPB (PRE = 0.22 +/- 0.13 g.kg(-1).d(-1); POST = 0.54 +/- 0.08 g.kg(-1).d(-1)) and urinary nitrogen balance (PRE = 2.8 +/- 1.7 g N.d(-1); POST = 6.5 +/- 0.9 g N.d(-1)) were observed. A program of resistance training that induced significant muscle hypertrophy resulted in reductions of both whole-body PS and PB, but an improved NPB, which favoured the accretion of skeletal muscle protein. Urinary nitrogen balance increased after training. The reduction in PS and PB and a higher NPB in combination with an increased nitrogen balance after training suggest that dietary requirements for protein in novice resistance-trained athletes are not higher, but lower, after resistance training.

  2. Role of multidrug resistance protein (MRP) in glutathione S-conjugate transport in mammalian cells

    NARCIS (Netherlands)

    Müller, M.; de Vries, E. G.; Jansen, P. L.

    1996-01-01

    The human multidrug resistance protein (MRP), a 190-kDa member of the ABC-protein superfamily, is an ATP-dependent glutathione S-conjugate carrier (GS-X pump) and is present in membranes of many, if not all, cells. Overexpression of MRP in tumor cells contributes to resistance to natural product

  3. Role of multidrug resistance protein (MRP) in glutathione S-conjugate transport in mammalian cells

    NARCIS (Netherlands)

    Muller, M; deVries, EGE; Jansen, PLM

    1996-01-01

    The human multidrug resistance protein (MRP), a 190-kDa member of the ABC-protein superfamily, is an ATP-dependent glutathione S-conjugate carrier (GS-X pump) and is present in membranes of many, if not all, cells, Overexpression of MRP in tumor cells contributes to resistance to natural product

  4. The overexpression of MRP4 is related to multidrug resistance in osteosarcoma cells

    Directory of Open Access Journals (Sweden)

    Zhonghui He

    2015-01-01

    Full Text Available Doxorubicin (Adriamycin, ADM is an antimitotic drug used in the treatment of a wide range of malignant tumors, including acute leukemia, lymphoma, osteosarcoma, breast cancer, and lung cancer. Multidrug resistance-associated proteins (MRPs are members of a superfamily of ATP-binding cassette (ABC transporters, which can transport various molecules across extra- and intra-cellular membranes. The aim of this study was to investigate whether there was a correlation between MRP4 and primary ADM resistance in osteosarcoma cells. In this paper, we chose the human osteosarcoma cell line MG63, ADM resistant cell line MG63/DOX, and the patient′s primary cell GSF-0686. We checked the ADM sensitivity and cytotoxicity of all the three cells by cell proliferation assay. The intracellular drug concentrations were measured by using LC-MS/MS. We also examined MRP4 gene expression by RT-PCR and Western Blot. We found that the intracellular ADM concentration of the parent osteosarcoma cell line MG63 was higher than the ADM resistant osteosarcoma MG63/DOX cell line or the GSF-0686 cell after ADM treatment (P < 0.05. In addition, MRP4 mRNA and protein levels in ADM resistant osteosarcoma cells were higher than in MG63 cell (P < 0.05. Taking together, this work suggests that overexpression of MRP4 may confer ADM resistance in osteosarcoma cells.

  5. Effect of protein/essential amino acids and resistance training on skeletal muscle hypertrophy: A case for whey protein

    Directory of Open Access Journals (Sweden)

    Stout Jeffrey R

    2010-06-01

    Full Text Available Abstract Regardless of age or gender, resistance training or provision of adequate amounts of dietary protein (PRO or essential amino acids (EAA can increase muscle protein synthesis (MPS in healthy adults. Combined PRO or EAA ingestion proximal to resistance training, however, can augment the post-exercise MPS response and has been shown to elicit a greater anabolic effect than exercise plus carbohydrate. Unfortunately, chronic/adaptive response data comparing the effects of different protein sources is limited. A growing body of evidence does, however, suggest that dairy PRO, and whey in particular may: 1 stimulate the greatest rise in MPS, 2 result in greater muscle cross-sectional area when combined with chronic resistance training, and 3 at least in younger individuals, enhance exercise recovery. Therefore, this review will focus on whey protein supplementation and its effects on skeletal muscle mass when combined with heavy resistance training.

  6. The Maillard reaction of a shrimp by-product protein hydrolysate: chemical changes and inhibiting effects of reactive oxygen species in human HepG2 cells.

    Science.gov (United States)

    Zha, Fengchao; Wei, Binbin; Chen, Shengjun; Dong, Shiyuan; Zeng, Mingyong; Liu, Zunying

    2015-06-01

    Recently, much attention has been given to improving the antioxidant activity of protein hydrolysates via the Maillard reaction, but little is known about the cellular antioxidant activity of Maillard reaction products (MRPs) from protein hydrolysates. We first investigated chemical characterization and the cellular antioxidant activity of MRPs in a shrimp (Litopenaeus vannamei) by-product protein hydrolysate (SBH)-glucose system at 110 °C for up to 10 h of heating. Solutions of SBH and glucose were also heated alone as controls. The Maillard reaction greatly resulted in the increase of hydroxymethylfurfural (HMF) and browning intensity, high molecular weight fraction, and reduction of the total amino acid in SBH with the heating time, which correlated well with the free radical scavenging activity of MRPs. MRPs had stronger inhibiting effects on oxidative stress of human HepG2 cells than the original SBH, and its cellular antioxidant activity strongly correlated with free radical scavenging activity, but less affected by the browning intensity and HMF level. The caramelization of glucose partially affected the HMF level and free radical scavenging activity of MRPs, but it was not related to the cellular antioxidant activity. The cellular antioxidant activity of MRPs for 5 h of heating time appeared to reach a maximum level, which was mainly due to carbonyl ammonia condensation reaction. In conclusion, the Maillard reaction is a potential method to increase the cellular antioxidant activity of a shrimp by-product protein hydrolysate, but the higher HMF levels and the lower amino acid content in MRPs should also be considered.

  7. Structure, function and subcellular localization of the potato Resistance protein Rx1

    NARCIS (Netherlands)

    Slootweg, E.J.

    2009-01-01

    Resistance proteins are part of the plant’s immune system and mediate a defence response upon recognizing their cognate pathogens. They are thought to be present in the cell as part of a larger protein complex. The modular architecture of R proteins suggests that they form a scaffold for various

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

    DEFF Research Database (Denmark)

    Esmarck, B.; Andersen, J.L.; Olsen, S.

    2001-01-01

    1. Age-associated loss of skeletal muscle mass and strength can partly be counteracted by resistance training, causing a net synthesis of muscular proteins. Protein synthesis is influenced synergistically by postexercise amino acid supplementation, but the importance of the timing of protein intake...

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

    Science.gov (United States)

    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. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. Functional evidence of multidrug resistance transporters (MDR in rodent olfactory epithelium.

    Directory of Open Access Journals (Sweden)

    Adrien Molinas

    Full Text Available P-glycoprotein (Pgp and multidrug resistance-associated protein (MRP1 are membrane transporter proteins which function as efflux pumps at cell membranes and are considered to exert a protective function against the entry of xenobiotics. While evidence for Pgp and MRP transporter activity is reported for olfactory tissue, their possible interaction and participation in the olfactory response has not been investigated.Functional activity of putative MDR transporters was assessed by means of the fluorometric calcein acetoxymethyl ester (calcein-AM accumulation assay on acute rat and mouse olfactory tissue slices. Calcein-AM uptake was measured as fluorescence intensity changes in the presence of Pgp or MRP specific inhibitors. Epifluorescence microscopy measured time course analysis in the olfactory epithelium revealed significant inhibitor-dependent calcein uptake in the presence of each of the selected inhibitors. Furthermore, intracellular calcein accumulation in olfactory receptor neurons was also significantly increased in the presence of either one of the Pgp or MRP inhibitors. The presence of Pgp or MRP1 encoding genes in the olfactory mucosa of rat and mouse was confirmed by RT-PCR with appropriate pairs of species-specific primers. Both transporters were expressed in both newborn and adult olfactory mucosa of both species. To assess a possible involvement of MDR transporters in the olfactory response, we examined the electrophysiological response to odorants in the presence of the selected MDR inhibitors by recording electroolfactograms (EOG. In both animal species, MRPs inhibitors induced a marked reduction of the EOG magnitude, while Pgp inhibitors had only a minor or no measurable effect.The findings suggest that both Pgp and MRP transporters are functional in the olfactory mucosa and in olfactory receptor neurons. Pgp and MRPs may be cellular constituents of olfactory receptor neurons and represent potential mechanisms for modulation

  11. Maillard reaction products of rice protein hydrolysates with mono-, oligo- and polysaccharides

    Science.gov (United States)

    Rice protein, a byproduct of rice syrup production, is abundant but, its lack of functionality prevents its wide use as a food ingredient. Maillard reaction products of (MRPs) hydrolysates from the limited hydrolysis of rice protein (LHRP) and various mono-, oligo- and polysaccharides were evaluat...

  12. Whey Protein Supplementation Enhances Whole Body Protein Metabolism and Performance Recovery after Resistance Exercise: A Double-Blind Crossover Study.

    Science.gov (United States)

    West, Daniel W D; Abou Sawan, Sidney; Mazzulla, Michael; Williamson, Eric; Moore, Daniel R

    2017-07-11

    No study has concurrently measured changes in free-living whole body protein metabolism and exercise performance during recovery from an acute bout of resistance exercise. We aimed to determine if whey protein ingestion enhances whole body net protein balance and recovery of exercise performance during overnight (10 h) and 24 h recovery after whole body resistance exercise in trained men. In a double-blind crossover design, 12 trained men (76 ± 8 kg, 24 ± 4 years old, 14% ± 5% body fat; means ± standard deviation (SD)) performed resistance exercise in the evening prior to consuming either 25 g of whey protein (PRO; MuscleTech 100% Whey) or an energy-matched placebo (CHO) immediately post-exercise (0 h), and again the following morning (~10 h of recovery). A third randomized trial, completed by the same participants, involving no exercise and no supplement served as a rested control trial (Rest). Participants ingested [ 15 N]glycine to determine whole body protein kinetics and net protein balance over 10 and 24 h of recovery. Performance was assessed pre-exercise and at 0, 10, and 24 h of recovery using a battery of tests. Net protein balance tended to improve in PRO ( P = 0.064; effect size (ES) = 0.61, PRO vs. CHO) during overnight recovery. Over 24 h, net balance was enhanced in PRO ( P = 0.036) but not in CHO ( P = 0.84; ES = 0.69, PRO vs. CHO), which was mediated primarily by a reduction in protein breakdown (PRO protein supplementation improved MVC (ES = 0.76), REP (ES = 0.44), and peak power (ES = 0.55). In conclusion, whey protein supplementation enhances whole body anabolism, and may improve acute recovery of exercise performance after a strenuous bout of resistance exercise.

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

    Directory of Open Access Journals (Sweden)

    Wilson Jacob

    2006-06-01

    Full Text Available Abstract 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.

  14. Configurable Resistive Switching between Memory and Threshold Characteristics for Protein-Based Devices

    KAUST Repository

    Wang, Hong; Du, Yuanmin; Li, Yingtao; Zhu, Bowen; Leow, Wan Ru; Li, Yuangang; Pan, Jisheng; Wu, Tao; Chen, Xiaodong

    2015-01-01

    The employ of natural biomaterials as the basic building blocks of electronic devices is of growing interest for biocompatible and green electronics. Here, resistive switching (RS) devices based on naturally silk protein with configurable

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

    OpenAIRE

    Rigalli, Juan Pablo; Ciriaci, Nadia; Arias, Agostina; Ceballos, Mar?a Paula; Villanueva, Silvina Stella Maris; Luquita, Marcelo Gabriel; Mottino, Aldo Domingo; Ghanem, Carolina In?s; Catania, Viviana Alicia; Ruiz, Mar?a 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 t...

  16. Polyglycerol coatings of glass vials for protein resistance.

    Science.gov (United States)

    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. Copyright © 2013 Elsevier B.V. All rights reserved.

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

    NARCIS (Netherlands)

    A. Umar (Arzu); J.W.M. Martens (John); J.A. Foekens (John); L. Paša-Tolić (Ljiljana); H. Kang; A.M. Timmermans (Mieke); M.P. Look (Maxime); M.E. Meijer van Gelder (Marion); N. Jaitly (Navdeep); M.A. den Bakker (Michael)

    2009-01-01

    textabstractTamoxifen-resistance is a major cause of death in patients with recurrent breast cancer. Current clinical parameters can correctly predict therapy response in only half of the treated patients. Identification of proteins that associate with tamoxifen-resistance is a first step towards

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

    DEFF Research Database (Denmark)

    Farup, Jean; Rahbek, S K; Vendelbo, M H

    2014-01-01

    In a comparative study, we investigated the effects of maximal eccentric or concentric resistance training combined with whey protein or placebo on muscle and tendon hypertrophy. 22 subjects were allocated into either a high-leucine whey protein hydrolysate + carbohydrate group (WHD) or a carbohy......In a comparative study, we investigated the effects of maximal eccentric or concentric resistance training combined with whey protein or placebo on muscle and tendon hypertrophy. 22 subjects were allocated into either a high-leucine whey protein hydrolysate + carbohydrate group (WHD...... or contraction mode effects. In conclusion, high-leucine whey protein hydrolysate augments muscle and tendon hypertrophy following 12 weeks of resistance training – irrespective of contraction mode....

  19. High dietary protein intake, reducing or eliciting insulin resistance?

    NARCIS (Netherlands)

    Rietman, A.; Schwarz, J.; Tome, D.; Kok, F.J.; Mensink, M.R.

    2014-01-01

    Dietary proteins have an insulinotropic effect and thus promote insulin secretion, which indeed leads to enhanced glucose clearance from the blood. In the long term, however, a high dietary protein intake is associated with an increased risk of type 2 diabetes. Moreover, branched-chain amino acids

  20. Identification of protein expression alterations in gefitinib-resistant human lung adenocarcinoma: PCNT and mPR play key roles in the development of gefitinib-associated resistance

    Energy Technology Data Exchange (ETDEWEB)

    Lin, Chi-Chen [Institute of Biomedical Science, National Chung-Hsing University, Taichung, Taiwan (China); Institute of Biomedical Science, and Rong Hsing Research Center for Translational Medicine, National Chung Hsing University, Taiwan (China); Department of Medical Research and Education, Taichung Veterans General Hospital, Taichung, Taiwan (China); Division of Chest Medicine, Department of Internal Medicine, Changhua Christian Hospital, Changhua, Taiwan (China); Chen, Jing-Ting [Department of Medical Science and Institute of Bioinformatics and Structural Biology, National Tsing Hua University, Hsinchu, Taiwan (China); Lin, Meng-Wei [Department of Medical Science and Institute of Bioinformatics and Structural Biology, National Tsing Hua University, Hsinchu, Taiwan (China); Department of Applied Science, National Hsinchu University of Education, Hsinchu, Taiwan (China); Chan, Chia-Hao [Department of Obstetrics and Gynecology, Hsinchu Mackay Memorial Hospital, Hsinchu 30071, Taiwan (China); Wen, Yueh-Feng [Department of Medical Science and Institute of Bioinformatics and Structural Biology, National Tsing Hua University, Hsinchu, Taiwan (China); Department of Internal Medicine, National Taiwan University Hospital Hsinchu Branch, Hsinchu, Taiwan (China); Wu, Shin-Bei [Department of Applied Science, National Hsinchu University of Education, Hsinchu, Taiwan (China); Chung, Ting-Wen [Department of Medical Science and Institute of Bioinformatics and Structural Biology, National Tsing Hua University, Hsinchu, Taiwan (China); Lyu, Kevin W. [Lutheran Medical Center, Brooklyn, NY (United States); Global Scholars Program, St. George' s University/Northumbria University, Newcastle upon Tyne (United Kingdom); Chou, Hsiu-Chuan, E-mail: chouhc@mail.nhcue.edu.tw [Department of Applied Science, National Hsinchu University of Education, Hsinchu, Taiwan (China); and others

    2015-11-01

    Gefitinib is the first-line chemotherapeutic drug for treating non-small cell lung cancer (NSCLC), which comprises nearly 85% of all lung cancer cases worldwide. However, most patients eventually develop drug resistance after 12–18 months of treatment. Hence, investigating the drug resistance mechanism and resistance-associated biomarkers is necessary. Two lung adenocarcinoma cell lines, PC9 and gefitinib-resistant PC9/Gef, were established for examining resistance mechanisms and identifying potential therapeutic targets. Two-dimensional differential gel electrophoresis and matrix-assisted laser desorption ionization time-of-flight mass spectrometry were used for examining global protein expression changes between PC9 and PC9/Gef. The results revealed that 164 identified proteins were associated with the formation of gefitinib resistance in PC9 cells. Additional studies using RNA interference showed that progesterone receptor membrane component 1 and pericentrin proteins have major roles in gefitinib resistance. In conclusion, the proteomic approach enabled identifying of numerous proteins involved in gefitinib resistance. The results provide useful diagnostic markers and therapeutic candidates for treating gefitinib-resistant NSCLC. - Highlights: • 164 proteins associated with gefitinib resistance were identified through proteomic analysis. • In this study, a lung adenocarcinoma and its gefitinib resistant partner were established. • mPR and PCNT proteins have evidenced to play important roles in gefitinib resistance.

  1. Identification of protein expression alterations in gefitinib-resistant human lung adenocarcinoma: PCNT and mPR play key roles in the development of gefitinib-associated resistance

    International Nuclear Information System (INIS)

    Lin, Chi-Chen; Chen, Jing-Ting; Lin, Meng-Wei; Chan, Chia-Hao; Wen, Yueh-Feng; Wu, Shin-Bei; Chung, Ting-Wen; Lyu, Kevin W.; Chou, Hsiu-Chuan

    2015-01-01

    Gefitinib is the first-line chemotherapeutic drug for treating non-small cell lung cancer (NSCLC), which comprises nearly 85% of all lung cancer cases worldwide. However, most patients eventually develop drug resistance after 12–18 months of treatment. Hence, investigating the drug resistance mechanism and resistance-associated biomarkers is necessary. Two lung adenocarcinoma cell lines, PC9 and gefitinib-resistant PC9/Gef, were established for examining resistance mechanisms and identifying potential therapeutic targets. Two-dimensional differential gel electrophoresis and matrix-assisted laser desorption ionization time-of-flight mass spectrometry were used for examining global protein expression changes between PC9 and PC9/Gef. The results revealed that 164 identified proteins were associated with the formation of gefitinib resistance in PC9 cells. Additional studies using RNA interference showed that progesterone receptor membrane component 1 and pericentrin proteins have major roles in gefitinib resistance. In conclusion, the proteomic approach enabled identifying of numerous proteins involved in gefitinib resistance. The results provide useful diagnostic markers and therapeutic candidates for treating gefitinib-resistant NSCLC. - Highlights: • 164 proteins associated with gefitinib resistance were identified through proteomic analysis. • In this study, a lung adenocarcinoma and its gefitinib resistant partner were established. • mPR and PCNT proteins have evidenced to play important roles in gefitinib resistance.

  2. Fluoroquinolone resistance protein NorA of Staphylococcus aureus is a multidrug efflux transporter.

    OpenAIRE

    Neyfakh, A A; Borsch, C M; Kaatz, G W

    1993-01-01

    The gene of the Staphylococcus aureus fluoroquinolone efflux transporter protein NorA confers resistance to a number of structurally dissimilar drugs, not just to fluoroquinolones, when it is expressed in Bacillus subtilis. NorA provides B. subtilis with resistance to the same drugs and to a similar extent as the B. subtilis multidrug transporter protein Bmr does. NorA and Bmr share 44% sequence similarity. Both the NorA- and Bmr-conferred resistances can be completely reversed by reserpine.

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

    Directory of Open Access Journals (Sweden)

    Phillips Stuart M

    2011-10-01

    Full Text Available Abstract 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.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  5. TIMP-1 increases expression and phosphorylation of proteins associated with drug resistance in breast cancer cells

    DEFF Research Database (Denmark)

    Hekmat, Omid; Munk, Stephanie; Fogh, Louise

    2013-01-01

    may explain the resistance phenotype to topoisomerase inhibitors that was observed in cells with high TIMP-1 levels. Pathway analysis showed an enrichment of proteins from functional categories such as apoptosis, cell cycle, DNA repair, transcription factors, drug targets and proteins associated......Tissue inhibitor of metalloproteinase 1 (TIMP-1) is a protein with a potential biological role in drug resistance. To elucidate the unknown molecular mechanisms underlying the association between high TIMP-1 levels and increased chemotherapy resistance, we employed SILAC-based quantitative mass...... spectrometry to analyze global proteome and phosphoproteome differences of MCF-7 breast cancer cells expressing high or low levels of TIMP-1. In TIMP-1 high expressing cells, 312 proteins and 452 phosphorylation sites were up-regulated. Among these were the cancer drug targets topoisomerase 1, 2A and 2B, which...

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

    NARCIS (Netherlands)

    Nielen, van M.; Feskens, E.J.M.; Rietman, A.; Siebelink, E.; Mensink, M.R.

    2014-01-01

    Increasing protein intake and soy consumption appear to be promising approaches to prevent metabolic syndrome (MetS). However, the effect of soy consumption on insulin resistance, glucose homeostasis, and other characteristics of MetS is not frequently studied in humans. We aimed to investigate the

  7. Co-induction of glucose regulated proteins and adriamycin resistance in Chinese hamster cells

    International Nuclear Information System (INIS)

    Shen, J.; Hughes, C.; Cai, J.; Bartels, C.; Gessner, T.; Subjeck, J.

    1987-01-01

    Glucose deprivation, anoxia, calcium ionophore A23187 or 2-deoxyglucose all inducers of glucose regulated proteins (grps), also lead to a significant induction of resistance to the drug adriamycin. In the case of anoxia, A23187 and 2-deoxyglucose, the induction of resistance correlates with both the application of the inducing stress and the induction of grps. In the case of glucose deprivation, the onset of resistance correlates with the onset of glucose deprivation and precedes grp induction. Removal of each grp including condition results in the rapid disappearance of this resistance in a manner which correlates with the repression of the grps. This drug resistance can be induced in confluent cells or in actively proliferating cells, although the effect is greater in the more sensitive proliferating cells. Induction of heat shock proteins (hsps) does not appear to lead to any major change in adriamycin resistance. Grp induced cells retain less adriamycin than do controls with the greatest reduction occurring during anoxia, which is also the strongest inducer of grps and resistance. The authors propose that the application of a grp inducing stress leads to a concurrent induction in drug resistance, possibly via the translocation of grps in the cell. Finally, they also observed that adriamycin itself can induce both hsps and grps. It is possible that adriamycin exposure may correspondingly induce auto-resistance

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

    Science.gov (United States)

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

    2012-01-13

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

  9. Whey Protein Supplementation Enhances Whole Body Protein Metabolism and Performance Recovery after Resistance Exercise: A Double-Blind Crossover Study

    Directory of Open Access Journals (Sweden)

    Daniel W. D. West

    2017-07-01

    Full Text Available No study has concurrently measured changes in free-living whole body protein metabolism and exercise performance during recovery from an acute bout of resistance exercise. We aimed to determine if whey protein ingestion enhances whole body net protein balance and recovery of exercise performance during overnight (10 h and 24 h recovery after whole body resistance exercise in trained men. In a double-blind crossover design, 12 trained men (76 ± 8 kg, 24 ± 4 years old, 14% ± 5% body fat; means ± standard deviation (SD performed resistance exercise in the evening prior to consuming either 25 g of whey protein (PRO; MuscleTech 100% Whey or an energy-matched placebo (CHO immediately post-exercise (0 h, and again the following morning (~10 h of recovery. A third randomized trial, completed by the same participants, involving no exercise and no supplement served as a rested control trial (Rest. Participants ingested [15N]glycine to determine whole body protein kinetics and net protein balance over 10 and 24 h of recovery. Performance was assessed pre-exercise and at 0, 10, and 24 h of recovery using a battery of tests. Net protein balance tended to improve in PRO (P = 0.064; effect size (ES = 0.61, PRO vs. CHO during overnight recovery. Over 24 h, net balance was enhanced in PRO (P = 0.036 but not in CHO (P = 0.84; ES = 0.69, PRO vs. CHO, which was mediated primarily by a reduction in protein breakdown (PRO < CHO; P < 0.01. Exercise decreased repetitions to failure (REP, maximal strength (MVC, peak and mean power, and countermovement jump performance (CMJ at 0 h (all P < 0.05 vs. Pre. At 10 h, there were small-to-moderate effects for enhanced recovery of the MVC (ES = 0.56, mean power (ES = 0.49, and CMJ variables (ES: 0.27–0.49 in PRO. At 24 h, protein supplementation improved MVC (ES = 0.76, REP (ES = 0.44, and peak power (ES = 0.55. In conclusion, whey protein supplementation enhances whole body anabolism, and may improve acute recovery of

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

    Science.gov (United States)

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

    2018-04-01

    The sugarcane borer, Diatraea saccharalis (F.), is a major maize borer pest and a target of transgenic maize expressing Bacillus thuringiensis (Bt) proteins in South America and the mid-southern region of the United States. Evolution of resistance in target pest populations is a great threat to the long-term efficacy of Bt crops. In this study, we compared the genetic basis of resistance to Cry1Ab protein in 3 resistant colonies of sugarcane borer established from field populations in Louisiana, USA. Responses of larvae to the Cry1Ab protein for the parental and 10 other cross colonies were assayed in a diet-incorporated bioassay. All 3 resistant colonies were highly resistant to the Cry1Ab protein with a resistance ratio of >555.6 fold. No maternal effect or sex linkage was evident for the resistance in the 3 colonies; and the resistance was functionally nonrecessive at the Cry1Ab concentrations of ≤ 3.16 μg/g, but it became recessive at ≥10 μg/g. In an interstrain complementation test for allelism, the F 1 progeny from crosses between any 2 of the 3 resistant colonies exhibited the similar resistance levels as their parental colonies, indicating that the 3 colonies most likely shared a locus of Cry1Ab resistance. Results generated from this study should provide useful information in developing effective strategies for managing Bt resistance in the insect. © 2016 Institute of Zoology, Chinese Academy of Sciences.

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

    International Nuclear Information System (INIS)

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

    2011-01-01

    The pentapeptide repeat protein AlbG, provides self-resistance to the nonribosomally encoded hybrid polyketide-peptide termed albicidin. Analysis of the AlbG three-dimensional structure and the sequences of other pentapeptide repeat proteins that confer resistance to topiosomerase poisons suggests they have a similar dimer interface which may be critical to their interaction with topoisomerases. 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

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

    Science.gov (United States)

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

    2011-04-01

    Acquired resistance of mammalian cells to heavy metals is closely relevant to enhanced expression of several multidrug resistance-associated proteins (MRP), but it remains unclear whether MRP proteins confer resistance to heavy metals in zebrafish. In this study, we obtained zebrafish (Danio rerio) fibroblast-like ZF4 cells with resistance to toxic heavy metals after chronic cadmium exposure and selection for 6months. These cadmium-resistant cells (ZF4-Cd) were maintained in 5μM cadmium and displayed cross-resistance to cadmium, mercury, arsenite and arsenate. ZF4-Cd cells remained the resistance to heavy metals after protracted culture in cadmium-free medium. In comparison with ZF4-WT cells, ZF4-Cd cells exhibited accelerated rate of cadmium excretion, enhanced activity of MRP-like transport, elevated expression of abcc2, abcc4 and mt2 genes, and increased content of cellular GSH. Inhibition of MRP-like transport activity, GSH biosynthesis and GST activity significantly attenuated the resistance of ZF4-Cd cells to heavy metals. The results indicate that some of MRP transporters are involved in the efflux of heavy metals conjugated with cellular GSH and thus play crucial roles in heavy metal detoxification of zebrafish cells. Copyright © 2010 Elsevier Inc. All rights reserved.

  13. Analysis of the protein profiles of the antibiotic- resistant Salmonella ...

    African Journals Online (AJOL)

    Owner

    2005-05-18

    May 18, 2005 ... ice-cold 100% acetone and air-dried. The dried whole cell proteins and other samples (flagillin, CFUS) for 2DE were digested (100oC,. 5 min) in 4 µl of 10% SDS and dissolved in 100 µl of urea sample buffer containing 8 M urea, 4% Triton X-100, 20 mM dithiothreitol,. 2% ampholyte (pH 3.5~10) and traces ...

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

    Science.gov (United States)

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

    2015-07-22

    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.

  15. Supplementary Material for: Mycobacterium tuberculosis whole genome sequencing and protein structure modelling provides insights into anti-tuberculosis drug resistance

    KAUST Repository

    Phelan, Jody; Coll, Francesc; McNerney, Ruth; Ascher, David; Pires, Douglas; Furnham, Nick; Coeck, Nele; Hill-Cawthorne, Grant; Nair, Mridul; Mallard, Kim; Ramsay, Andrew; Campino, Susana; Hibberd, Martin; Pain, Arnab; Rigouts, Leen; Clark, Taane

    2016-01-01

    Abstract Background Combating the spread of drug resistant tuberculosis is a global health priority. Whole genome association studies are being applied to identify genetic determinants of resistance to anti-tuberculosis drugs. Protein structure

  16. The effects of whey protein with or without carbohydrates on resistance training adaptations

    OpenAIRE

    Hulmi, Juha; Laakso, Mia; Mero, Antti; Häkkinen, Keijo; Ahtiainen, Juha; Peltonen, Heikki

    2015-01-01

    Background: Nutrition intake in the context of a resistance training (RT) bout may affect body composition and muscle strength. However, the individual and combined effects of whey protein and carbohydrates on long-term resistance training adaptations are poorly understood. Methods: A four-week preparatory RT period was conducted in previously untrained males to standardize the training background of the subjects. Thereafter, the subjects were randomized into three groups: 30 g of...

  17. The Role of ABC Proteins in Drug Resistant Breast Cancer Cells

    Science.gov (United States)

    2008-04-01

    called the Plasmodium falciparum Chloroquine Transporter (PfCRT). While PfCRT is known to be the main molecular determinant of chloroquine resistance...proteins (such as human P-glycoprotein) and labeled PfCRT with a photoaffinity drug analogue . A manuscript is currently in preparation detailing my results...directly responsible for drug response, the Plasmodium falciparum Chloroquine Resistance Transporter (PfCRT) (Fidock et al 2000). While not a member of

  18. Insulin resistance enhances the mitogen-activated protein kinase signaling pathway in ovarian granulosa cells.

    Directory of Open Access Journals (Sweden)

    Linghui Kong

    Full Text Available The ovary is the main regulator of female fertility. Granulosa cell dysfunction may be involved in various reproductive endocrine disorders. Here we investigated the effect of insulin resistance on the metabolism and function of ovarian granulosa cells, and dissected the functional status of the mitogen-activated protein kinase signaling pathway in these cells. Our data showed that dexamethasone-induced insulin resistance in mouse granulosa cells reduced insulin sensitivity, accompanied with an increase in phosphorylation of p44/42 mitogen-activated protein kinase. Furthermore, up-regulation of cytochrome P450 subfamily 17 and testosterone and down-regulation of progesterone were observed in insulin-resistant mouse granulosa cells. Inhibition of p44/42 mitogen-activated protein kinase after induction of insulin resistance in mouse granulosa cells decreased phosphorylation of p44/42 mitogen-activated protein kinase, downregulated cytochrome P450 subfamily 17 and lowered progesterone production. This insulin resistance cell model can successfully demonstrate certain mechanisms such as hyperandrogenism, which may inspire a new strategy for treating reproductive endocrine disorders by regulating cell signaling pathways.

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

    Science.gov (United States)

    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

  20. A nucleotide phosphatase activity in the nucleotide binding domain of an orphan resistance protein from rice.

    Science.gov (United States)

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

    2012-02-03

    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.

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

    Directory of Open Access Journals (Sweden)

    Lowery Lonnie M

    2009-01-01

    Full Text Available Abstract 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.

  2. Colistin resistance associated with outer membrane protein change in Klebsiella pneumoniae and Enterobacter asburiae.

    Science.gov (United States)

    Kádár, Béla; Kocsis, Béla; Tóth, Ákos; Kristóf, Katalin; Felső, Péter; Kocsis, Béla; Böddi, Katalin; Szabó, Dóra

    2017-06-01

    In this study, outer membrane proteins (OMPs) of colistin-resistant Klebsiella pneumoniae and Enterobacter asburiae were analyzed. One colistin-susceptible and three colistin-resistant K. pneumoniae sequence type 258 strains as well as one colistin-susceptible E. asburiae and its colistin-heteroresistant counterpart strain were involved in the study. OMP analysis of each strain was performed by microchip method. Matrix-assisted laser desorption ionization time of flight/mass spectrometry (MALDI-TOF/MS) investigation was carried out after separation of OMPs by two-dimensional gel electrophoresis and in-gel digestion. The MALDI-TOF/MS analysis of OMPs in the colistin-susceptible K. pneumoniae found 16 kDa proteins belonging to the LysM domain/BON superfamily, as well as DNA starvation proteins, whereas OmpX and OmpW were detected in the colistin-resistant counterpart strains. OmpC and OmpW were detected in the colistin-susceptible E. asburiae, whereas OmpA and OmpX were identified in the colistin-resistant counterpart. This study demonstrated that OMP differences were between colistin-susceptible and -resistant counterpart strains. The altered Gram-negative cell wall may contribute to acquired colistin resistance in Enterobacteriaceae.

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

    Directory of Open Access Journals (Sweden)

    Yang Yifan

    2012-06-01

    Full Text Available Abstract 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 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.

  4. Overexpression of protein kinase A - RIalpha reduces lipofection efficiency of cisplatin-resistant human tumor cells.

    Science.gov (United States)

    Son, K K; Rosenblatt, J

    2001-04-10

    Cisplatin-resistant variant A2780CP/vector cells were 4.0-5.3-fold more transfectable and 7.6-fold more resistant to cisplatin than their parent cisplatin-sensitive human ovarian carcinoma A2780/vector cells. Overexpression of cAMP-dependent protein kinase Type I regulatory alpha subunit (PKA-RIalpha) gene in A2780CP cells significantly reduced (maximum 47.0%) the transfection activity, with a slight reduction (maximum 27.3%) of cisplatin resistance, of A2780CP cells. However, RIalpha-overexpressing A2780CP (A2780CP/RIalpha) cells were still 2.5-to 3.0-fold more transfectable and 5.5-fold more resistant to cisplatin than A2780 cells. This results suggest that gene transfer efficiency is associated with cisplatin resistance, in part, through the PKA-mediated cAMP signal transduction pathway.

  5. The heat shock protein/chaperone network and multiple stress resistance

    KAUST Repository

    Jacob, Pierre

    2016-11-15

    Crop yield has been greatly enhanced during the last century. However, most elite cultivars are adapted to temperate climates and are not well suited to more stressful conditions. In the context of climate change, stress resistance is a major concern. To overcome these difficulties, scientists may help breeders by providing genetic markers associated with stress resistance. However, multi-stress resistance cannot be obtained from the simple addition of single stress resistance traits. In the field, stresses are unpredictable and several may occur at once. Consequently, the use of single stress resistance traits is often inadequate. Although it has been historically linked with the heat stress response, the heat shock protein (HSP)/chaperone network is a major component of multiple stress responses. Among the HSP/chaperone

  6. The heat shock protein/chaperone network and multiple stress resistance

    KAUST Repository

    Jacob, Pierre; Hirt, Heribert; Bendahmane, Abdelhafid

    2016-01-01

    Crop yield has been greatly enhanced during the last century. However, most elite cultivars are adapted to temperate climates and are not well suited to more stressful conditions. In the context of climate change, stress resistance is a major concern. To overcome these difficulties, scientists may help breeders by providing genetic markers associated with stress resistance. However, multi-stress resistance cannot be obtained from the simple addition of single stress resistance traits. In the field, stresses are unpredictable and several may occur at once. Consequently, the use of single stress resistance traits is often inadequate. Although it has been historically linked with the heat stress response, the heat shock protein (HSP)/chaperone network is a major component of multiple stress responses. Among the HSP/chaperone

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

    Science.gov (United States)

    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.

  8. Protein-protein interactions as a proxy to monitor conformational changes and activation states of the tomato resistance protein I-2

    NARCIS (Netherlands)

    Lukasik-Shreepaathy, E.; Vossen, J.H.; Tameling, W.I.L.; de Vroomen, M.J.; Cornelissen, B.J.C.; Takken, F.L.W.

    2012-01-01

    Plant resistance proteins (R) are involved in pathogen recognition and subsequent initiation of defence responses. Their activity is regulated by inter- and intramolecular interactions. In a yeast two-hybrid screen two clones (I2I-1 and I2I-2) specifically interacting with I-2, a Fusarium oxysporum

  9. Insulin resistance and protein energy metabolism in patients with advanced chronic kidney disease.

    Science.gov (United States)

    Siew, Edward D; Ikizler, Talat Alp

    2010-01-01

    Insulin resistance (IR), the reciprocal of insulin sensitivity is a known complication of advanced chronic kidney disease (CKD) and is associated with a number of metabolic derangements. The complex metabolic abnormalities observed in CKD such as vitamin D deficiency, obesity, metabolic acidosis, inflammation, and accumulation of "uremic toxins" are believed to contribute to the etiology of IR and acquired defects in the insulin-receptor signaling pathway in this patient population. Only a few investigations have explored the validity of commonly used assessment methods in comparison to gold standard hyperinsulinemic hyperglycemic clamp technique in CKD patients. An important consequence of insulin resistance is its role in the pathogenesis of protein energy wasting, a state of metabolic derangement characterized by loss of somatic and visceral protein stores not entirely accounted for by inadequate nutrient intake. In the general population, insulin resistance has been associated with accelerated protein catabolism. Among end-stage renal disease (ESRD) patients, enhanced muscle protein breakdown has been observed in patients with Type II diabetes compared to ESRD patients without diabetes. In the absence of diabetes mellitus (DM) or severe obesity, insulin resistance is detectable in dialysis patients and strongly associated with increased muscle protein breakdown, primarily mediated by the ubiquitin-proteasome pathway. Recent epidemiological data indicate a survival advantage and better nutritional status in insulin-free Type II DM patients treated with insulin sensitizer thiazolidinediones. Given the high prevalence of protein energy wasting in ESRD and its unequivocal association with adverse clinical outcomes, insulin resistance may represent an important modifiable target for intervention in the ESRD population.

  10. Evolution of protein bound Maillard reaction end-products and free Amadori compounds in low lactose milk in presence of fructosamine oxidase I.

    Science.gov (United States)

    Troise, Antonio Dario; Buonanno, Martina; Fiore, Alberto; Monti, Simona Maria; Fogliano, Vincenzo

    2016-12-01

    Thermal treatments and storage influence milk quality, particularly in low lactose milk as the higher concentration of reducing sugars can lead to the increased formation of the Maillard reaction products (MRPs). The control of the Amadori products (APs) formation is the key step to mitigate the Maillard reaction (MR) in milk. The use of fructosamine oxidases, (Faox) provided promising results. In this paper, the effects of Faox I were evaluated by monitoring the concentration of free and bound MRPs in low lactose milk during shelf life. Results showed that the enzyme reduced the formation of protein-bound MRPs down to 79% after six days at 37°C. Faox I lowered the glycation of almost all the free amino acids resulting effective on basic and polar amino acids. Data here reported corroborate previous findings on the potentiality of Faox enzymes in controlling the early stage of the MR in foods. Copyright © 2016 Elsevier Ltd. All rights reserved.

  11. Coat protein-mediated resistance against an Indian isolate of the ...

    Indian Academy of Sciences (India)

    Coat protein (CP)-mediated resistance against an Indian isolate of the Cucumber mosaic virus (CMV) subgroup IB was demonstrated in transgenic lines of Nicotiana benthamiana through Agrobacterium tumefaciens-mediated transformation. Out of the fourteen independently transformed lines developed, two lines were ...

  12. Ghrelin- and GH-induced insulin resistance: no association with retinol-binding protein-4

    DEFF Research Database (Denmark)

    Vestergaard, Esben Thyssen; Krag, Morten B; Poulsen, Morten M

    2013-01-01

    Supraphysiological levels of ghrelin and GH induce insulin resistance. Serum levels of retinol-binding protein-4 (RBP4) correlate inversely with insulin sensitivity in patients with type 2 diabetes. We aimed to determine whether ghrelin and GH affect RBP4 levels in human subjects....

  13. C-reactive protein, insulin resistance and risk of cardiovascular disease: a population-based study

    DEFF Research Database (Denmark)

    Hansen, T.W.; Olsen, M.H.; Rasmussen, S.

    2008-01-01

    BACKGROUND: C-reactive protein (CRP), a marker of inflammation, and insulin resistance (IR), a metabolic disorder, are closely related. CRP and IR have both been identified as significant risk factors of cardiovascular disease (CVD) after adjustment for conventional CVD risk factors...

  14. Association of ERCC1 protein expression to platinum resistance in epithelial ovarian cancer

    DEFF Research Database (Denmark)

    Dahl Steffensen, Karina; Waldstrøm, Marianne; Jakobsen, Anders

    was to investigate if immunohistochemical expression of ERCC1 protein was associated with resistance to standard combination carboplatin and paclitaxel chemotherapy in newly diagnosed ovarian cancer patients. Methods: Formalin-fixed, paraffin-embedded tissue sections from 101 patients with newly diagnosed ovarian...

  15. Analysis of the protein profiles of the antibiotic-resistant Salmonella ...

    African Journals Online (AJOL)

    The emergent Salmonella typhimurium definitive phage type (DT) 104 is of particular global concern due to its frequent isolation and multiple antibiotic resistances. There is thus a need to know the kind of proteins expressed by S. typhimurium DT104 so as to provide a basis for developing an intervention. This study ...

  16. Protein source in a high-protein diet modulates reductions in insulin resistance and hepatic steatosis in fa/fa Zucker rats.

    Science.gov (United States)

    Wojcik, Jennifer L; Devassy, Jessay G; Wu, Yinghong; Zahradka, Peter; Taylor, Carla G; Aukema, Harold M

    2016-01-01

    High-protein diets are being promoted to reduce insulin resistance and hepatic steatosis in metabolic syndrome. Therefore, the effect of protein source in high-protein diets on reducing insulin resistance and hepatic steatosis was examined. Fa/fa Zucker rats were provided normal-protein (15% of energy) casein, high-protein (35% of energy) casein, high-protein soy, or high-protein mixed diets with animal and plant proteins. The high-protein mixed diet reduced area under the curve for insulin during glucose tolerance testing, fasting serum insulin and free fatty acid concentrations, homeostatic model assessment index, insulin to glucose ratio, and pancreatic islet cell area. The high-protein mixed and the high-protein soy diets reduced hepatic lipid concentrations, liver to body weight ratio, and hepatic steatosis rating. These improvements were observed despite no differences in body weight, feed intake, or adiposity among high-protein diet groups. The high-protein casein diet had minimal benefits. A high-protein mixed diet was the most effective for modulating reductions in insulin resistance and hepatic steatosis independent of weight loss, indicating that the source of protein within a high-protein diet is critical for the management of these metabolic syndrome parameters. © 2015 The Obesity Society.

  17. Effects of aging and insulin resistant states on protein anabolic responses in older adults.

    Science.gov (United States)

    Morais, Jose A; Jacob, Kathryn Wright; Chevalier, Stéphanie

    2018-07-15

    Insulin is the principal postprandial anabolic hormone and resistance to its action could contribute to sarcopenia. We developed different types of hyperinsulinemic clamp protocols to measure simultaneously glucose and protein metabolism in insulin resistant states (older adults, obesity, diabetes, etc.). To define effects of healthy aging in response to insulin, we employed the hyperinsulinemic, euglycemic and isoaminoacidemic (HYPER-1) clamp. The net whole-body anabolic (protein balance) response to hyperinsulinemia was lower in the elderly vs young (p = 0.007) and was highly correlated with the clamp glucose rate of disposal (r = 0.671, p anabolism compared with young ones. As most of the anabolism occurs during feeding, we studied the fed-state metabolic responses with aging using the hyperinsulinemic, hyperglycemic and hyperaminoacidemic clamp, including muscle biopsies. Older women showed comparable whole-body protein anabolic responses and stimulation of mixed-muscle protein synthesis by feeding to the young. The responses of skeletal muscle insulin signaling through the Akt-mTORC1 pathway were also unaltered, and therefore consistent with muscle protein synthesis results. Given that type 2 diabetes infers insulin resistance of protein metabolism with aging, we studied 10 healthy, 8 obese, and 8 obese type 2 diabetic elderly women using the HYPER-1 clamp. When compared to the group of young lean women to define the effects of obesity and diabetes with aging, whole-body change in net protein balance with hyperinsulinemia was similarly blunted in obese and diabetic older women. However, only elderly obese women with diabetes had lower net balance than lean older women. We conclude that with usual aging, the blunted whole-body anabolic response in elderly subjects is mediated by the failure of insulin to stimulate protein synthesis to the same extent as in the young, especially in men. This blunted response can be overcome at the whole-body and muscle

  18. The heat-shock protein/chaperone network and multiple stress resistance.

    Science.gov (United States)

    Jacob, Pierre; Hirt, Heribert; Bendahmane, Abdelhafid

    2017-04-01

    Crop yield has been greatly enhanced during the last century. However, most elite cultivars are adapted to temperate climates and are not well suited to more stressful conditions. In the context of climate change, stress resistance is a major concern. To overcome these difficulties, scientists may help breeders by providing genetic markers associated with stress resistance. However, multistress resistance cannot be obtained from the simple addition of single stress resistance traits. In the field, stresses are unpredictable and several may occur at once. Consequently, the use of single stress resistance traits is often inadequate. Although it has been historically linked with the heat stress response, the heat-shock protein (HSP)/chaperone network is a major component of multiple stress responses. Among the HSP/chaperone 'client proteins', many are primary metabolism enzymes and signal transduction components with essential roles for the proper functioning of a cell. HSPs/chaperones are controlled by the action of diverse heat-shock factors, which are recruited under stress conditions. In this review, we give an overview of the regulation of the HSP/chaperone network with a focus on Arabidopsis thaliana. We illustrate the role of HSPs/chaperones in regulating diverse signalling pathways and discuss several basic principles that should be considered for engineering multiple stress resistance in crops through the HSP/chaperone network. © 2016 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

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

    Science.gov (United States)

    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. Pea proteins oral supplementation promotes muscle thickness gains during resistance training: a double-blind, randomized, Placebo-controlled clinical trial vs. Whey protein

    OpenAIRE

    Babault, Nicolas; Pa?zis, Christos; Deley, Ga?lle; Gu?rin-Deremaux, Laetitia; Saniez, Marie-H?l?ne; Lefranc-Millot, Catherine; Allaert, Fran?ois A

    2015-01-01

    Background The effects of protein supplementation on muscle thickness and strength seem largely dependent on its composition. The current study aimed at comparing the impact of an oral supplementation with vegetable Pea protein (NUTRALYS?) vs. Whey protein and Placebo on biceps brachii muscle thickness and strength after a 12-week resistance training program. Methods One hundred and sixty one males, aged 18 to 35?years were enrolled in the study and underwent 12?weeks of resistance training o...

  1. Bioinformatics and structural characterization of a hypothetical protein from Streptococcus mutans: implication of antibiotic resistance.

    Directory of Open Access Journals (Sweden)

    Jie Nan

    2009-10-01

    Full Text Available As an oral bacterial pathogen, Streptococcus mutans has been known as the aetiologic agent of human dental caries. Among a total of 1960 identified proteins within the genome of this organism, there are about 500 without any known functions. One of these proteins, SMU.440, has very few homologs in the current protein databases and it does not fall into any protein functional families. Phylogenetic studies showed that SMU.440 is related to a particular ecological niche and conserved specifically in some oral pathogens, due to lateral gene transfer. The co-occurrence of a MarR protein within the same operon among these oral pathogens suggests that SMU.440 may be associated with antibiotic resistance. The structure determination of SMU.440 revealed that it shares the same fold and a similar pocket as polyketide cyclases, which indicated that it is very likely to bind some polyketide-like molecules. From the interlinking structural and bioinformatics studies, we have concluded that SMU.440 could be involved in polyketide-like antibiotic resistance, providing a better understanding of this hypothetical protein. Besides, the combination of multiple methods in this study can be used as a general approach for functional studies of a protein with unknown function.

  2. Yersinia enterocolitica serum resistance proteins YadA and ail bind the complement regulator C4b-binding protein.

    Directory of Open Access Journals (Sweden)

    Vesa Kirjavainen

    Full Text Available Many pathogens are equipped with factors providing resistance against the bactericidal action of complement. Yersinia enterocolitica, a Gram-negative enteric pathogen with invasive properties, efficiently resists the deleterious action of human complement. The major Y. enterocolitica serum resistance determinants include outer membrane proteins YadA and Ail. Lipopolysaccharide (LPS O-antigen (O-ag and outer core (OC do not contribute directly to complement resistance. The aim of this study was to analyze a possible mechanism whereby Y. enterocolitica could inhibit the antibody-mediated classical pathway of complement activation. We show that Y. enterocolitica serotypes O:3, O:8, and O:9 bind C4b-binding protein (C4bp, an inhibitor of both the classical and lectin pathways of complement. To identify the C4bp receptors on Y. enterocolitica serotype O:3 surface, a set of mutants expressing YadA, Ail, O-ag, and OC in different combinations was tested for the ability to bind C4bp. The studies showed that both YadA and Ail acted as C4bp receptors. Ail-mediated C4bp binding, however, was blocked by the O-ag and OC, and could be observed only with mutants lacking these LPS structures. C4bp bound to Y. enterocolitica was functionally active and participated in the factor I-mediated degradation of C4b. These findings show that Y. enterocolitica uses two proteins, YadA and Ail, to bind C4bp. Binding of C4bp could help Y. enterocolitica to evade complement-mediated clearance in the human host.

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

    Science.gov (United States)

    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. Copyright © 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

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

    Science.gov (United States)

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

    2011-11-01

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

  5. Bactobolin resistance is conferred by mutations in the L2 ribosomal protein.

    Science.gov (United States)

    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

    2012-12-18

    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). Currently available antibiotics target surprisingly few cellular functions, and there is a need to identify novel antibiotic targets. We have been interested in the Burkholderia thailandensis bactobolins, and we sought to learn about the target of bactobolin activity by mapping spontaneous resistance mutations in the bactobolin-sensitive Bacillus subtilis. Our results indicate that the bactobolin target is the 50S ribosome-associated L2 protein or a region of the ribosome affected by L2. Bactobolin-resistant mutants are not resistant to other known ribosome inhibitors. Our evidence indicates that bactobolins

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

    Directory of Open Access Journals (Sweden)

    Lv Y

    2015-07-01

    Full Text Available Yingqian Lv, Shan Zhao, Jinzhu Han, Likang Zheng, Zixin Yang, Li Zhao Department of Oncology, The Second Hospital, Hebei Medical University, Shijiazhuang, Hebei Province, People’s Republic of China Abstract: 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. Keywords: hypoxia, hypoxia-inducible factor-1α, multidrug resistance associated protein, transcriptional regulation, chemotherapy tolerance

  7. Attenuation of iron-binding proteins in ARPE-19 cells reduces their resistance to oxidative stress.

    Science.gov (United States)

    Karlsson, Markus; Kurz, Tino

    2016-09-01

    Oxidative stress-related damage to retinal pigment epithelial (RPE) cells is an important feature in the development of age-related macular degeneration. Iron-catalysed intralysosomal production of hydroxyl radicals is considered a major pathogenic factor, leading to lipofuscin formation with ensuing depressed cellular autophagic capacity, lysosomal membrane permeabilization and apoptosis. Previously, we have shown that cultured immortalized human RPE (ARPE-19) cells are extremely resistant to exposure to bolus doses of hydrogen peroxide and contain considerable amounts of the iron-binding proteins metallothionein (MT), heat-shock protein 70 (HSP70) and ferritin (FT). According to previous findings, autophagy of these proteins depresses lysosomal redox-active iron. The aim of this study was to investigate whether up- or downregulation of these proteins would affect the resistance of ARPE-19 cells to oxidative stress. The sensitivity of ARPE-19 cells to H2 O2 exposure was tested following upregulation of MT, HSP70 and/or FT by pretreatment with ZnSO4 , heat shock or FeCl3 , as well as siRNA-mediated downregulation of the same proteins. Upregulation of MT, HSP70 and FT did not improve survival following exposure to H2 O2 . This was interpreted as existence of an already maximal protection. Combined siRNA-mediated attenuation of both FT chains (H and L), or simultaneous downregulation of all three proteins, made the cells significantly more susceptible to oxidative stress confirming the importance of iron-binding proteins. The findings support our hypothesis that the oxidative stress resistance exhibited by RPE cells may be explained by a high autophagic influx of iron-binding proteins that would keep levels of redox-active lysosomal iron low. © 2016 Acta Ophthalmologica Scandinavica Foundation. Published by John Wiley & Sons Ltd.

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

    Science.gov (United States)

    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

  9. P-glycoprotein and multidrug resistance protein activities in relation to treatment outcome in acute myeloid leukemia

    NARCIS (Netherlands)

    de Vries, EGE; van Putten, WLJ; Verdonck, LF; Ossenkoppele, GJ; Verhoef, GEG; Vellenga, E

    Despite treatment with intensive chemotherapy, a considerable number of patients with acute myeloid leukemia (AML) die from their disease due to the occurrence of resistance. Overexpression of the transporter proteins P-glycoprotein (P-gp) and multidrug resistance protein (MRP) 1 has been identified

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

    Directory of Open Access Journals (Sweden)

    Li Ling

    2010-11-01

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

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

    Directory of Open Access Journals (Sweden)

    Juliana Ide Aoki

    2016-09-01

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

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

    Science.gov (United States)

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

    2016-09-01

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

  13. Recruitment of a penicillin-binding protein gene from Neisseria flavescens during the emergence of penicillin resistance in Neisseria meningitidis

    OpenAIRE

    SPRATT, BG; ZHANG, QY; JONES, DM; HUTCHISON, A; BRANNIGAN, JA; DOWSON, CG

    1989-01-01

    Non-beta-lactamase-producing, penicillin-resistant strains of Neisseria meningitidis produce altered forms of penicillin-binding protein 2 that have decreased affinity for penicillin. The sequence of the penicillin-binding protein 2 gene (penA) from a penicillin-resistant strain of N. meningitidis was compared to the sequence of the same gene from penicillin-sensitive strains and from penicillin-sensitive and penicillin-resistant strains of Neisseria gonorrhoeae. The penA genes from penicilli...

  14. Identification of Adenyl Cyclase Activity in a Disease Resistance Protein in Arabidopsis thaliana

    KAUST Repository

    Hussein, Rana

    2012-11-01

    Cyclic nucleotide, cAMP, is an important signaling molecule in animals and plants. However, in plants the enzymes that synthesize this second messenger, adenyl cyclases (ACs), remain elusive. Given the physiological importance of cAMP in signaling, particularly in response to biotic and abiotic stresses, it is thus important to identify and characterize ACs in higher plants. Using computational approaches, a disease resistance protein from Arabidopsis thaliana, At3g04220 was found to have an AC catalytic center motif. In an attempt to prove that this candidate has adenyl cyclases activity in vitro, the coding sequence of the putative AC catalytic domain of this protein was cloned and expressed in E. coli and the recombinant protein was purified. The nucleotide cyclase activity of the recombinant protein was examined using cyclic nucleotide enzyme immunoassays. In parallel, the expression of At3g04220 was measured in leaves under three different stress conditions in order to determine under which conditions the disease resistance protein could function. Results show that the purified recombinant protein has Mn2+ dependent AC activity in vitro, and the expression analysis supports a role for At3g04220 and cAMP in plant defense.

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

    Directory of Open Access Journals (Sweden)

    Leddy John J

    2009-03-01

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

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

    Directory of Open Access Journals (Sweden)

    Sinning Wayne E

    2005-08-01

    Full Text Available Abstract 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

  17. Major vault protein/lung resistance-related protein (MVP/LRP) expression in nervous system tumors.

    Science.gov (United States)

    Sasaki, Tsutomu; Hankins, Gerald R; Helm, Gregory A

    2002-01-01

    Lung resistance-related protein (LRP) was identified as the major vault protein (MVP), the main component of multimeric vault particles. It functions as a transport-associated protein that can be associated with multidrug resistance. In previous studies, expression of MVP/LRP has been documented in tumors of various types. In general, good correlations have been reported for expression of MVP/LRP and decreased sensitivity to chemotherapy and poor prognosis. MVP/LRP expression has been documented in glioblastomas, but its expression in nervous system tumors in general has not been well characterized. Immunohistochemistry using anti-human MVP/LRP antibody (LRP-56) was performed on formalin-fixed, paraffin-embedded archival tissue from 69 primary central nervous system tumors. Expression of MVP/LRP was observed in 81.2% (56/69) of primary nervous system tumors, including astrocytomas (11/13), oligodendrogliomas (1/2), oligoastrocytomas (5/5), ependymoma (1/1), meningiomas (35/45), schwannomas (2/2), and neurofibroma (1/1). Various degrees and distributions of immunoreactivity to MVP/ LRP were observed. Neither the presence nor the degree of immunoreactivity to MVP/LRP showed any correlation with either tumor grade or the presence of brain invasion.

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

    Directory of Open Access Journals (Sweden)

    Elena eIlina

    2013-07-01

    Full Text Available 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 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 ribosomal protein S5 to the recipient N. gonorrhoeae strains was analyzed. The relatively high rate of transformation (ca. 10-5 CFUs indicates the possibility of spread of spectinonycin resistance within gonococcal population due to the horizontal gene transfer.

  19. Stress proteins and phytohormones: their role in formation of plant resistance

    International Nuclear Information System (INIS)

    Kosakivska, I.V.

    2005-01-01

    Full text: Using the disc-electrophoresis methods, we have studied protein biosynthesis of different plants, including 11 species of Orchidaceae, some other tropical and subtropical plants, 9 different fruit plants, and 4 cultivars of Triticum aestivum L. under stresses factors such as high and low temperature, clinostating, radioactive irradiation and osmotic shock. Specific and unspecific reactions of plants protein system on stresses were found. De novo synthesis of 35 and 45 kD polypeptides were observed in total and mitochondrial proteins fractions after heat-shock and radioactive irradiation. This suggests that mitochondries participate in formation of plant resistance. Intensive synthesis of ABA revealed as the universal reaction of all studied plants on action of different kinds of stresses. Specific changes in balance of phytohormones were found under different stresses. We observed the correlation between endogenous ABA, IAA and cytokinin level and plant resistance. We also found the interaction between the process of biosynthesis of proteins and phytohormone balance, as well as their direct participation in formation of plant resistance. (author)

  20. Stimulation of muscle protein synthesis by whey and caseinate ingestion after resistance exercise in elderly individuals

    DEFF Research Database (Denmark)

    Dideriksen, K J; Reitelseder, S; Petersen, S G

    2011-01-01

    Sarcopenia is a well-known phenomenon in elderly individuals and resistance exercise together with sufficient amino acid (AA) availability has proved to be a counteractive implement. However, the source of AA and supplement timing require further investigation. The objective was to compare muscle...... protein synthesis (MPS) to intakes of whey and caseinate after heavy resistance exercise in healthy elderly individuals, and, furthermore, to compare the timing effect of caseinate intake. Twenty-four elderly men and women (mean ± SEM; 68 ± 1 years) were randomized to one of four groups: caseinate intake...

  1. Systems biology analysis of mitogen activated protein kinase inhibitor resistance in malignant melanoma.

    Science.gov (United States)

    Zecena, Helma; Tveit, Daniel; Wang, Zi; Farhat, Ahmed; Panchal, Parvita; Liu, Jing; Singh, Simar J; Sanghera, Amandeep; Bainiwal, Ajay; Teo, Shuan Y; Meyskens, Frank L; Liu-Smith, Feng; Filipp, Fabian V

    2018-04-04

    Kinase inhibition in the mitogen activated protein kinase (MAPK) pathway is a standard therapy for cancer patients with activating BRAF mutations. However, the anti-tumorigenic effect and clinical benefit are only transient, and tumors are prone to treatment resistance and relapse. To elucidate mechanistic insights into drug resistance, we have established an in vitro cellular model of MAPK inhibitor resistance in malignant melanoma. The cellular model evolved in response to clinical dosage of the BRAF inhibitor, vemurafenib, PLX4032. We conducted transcriptomic expression profiling using RNA-Seq and RT-qPCR arrays. Pathways of melanogenesis, MAPK signaling, cell cycle, and metabolism were significantly enriched among the set of differentially expressed genes of vemurafenib-resistant cells vs control. The underlying mechanism of treatment resistance and pathway rewiring was uncovered to be based on non-genomic adaptation and validated in two distinct melanoma models, SK-MEL-28 and A375. Both cell lines have activating BRAF mutations and display metastatic potential. Downregulation of dual specific phosphatases, tumor suppressors, and negative MAPK regulators reengages mitogenic signaling. Upregulation of growth factors, cytokines, and cognate receptors triggers signaling pathways circumventing BRAF blockage. Further, changes in amino acid and one-carbon metabolism support cellular proliferation despite MAPK inhibitor treatment. In addition, treatment-resistant cells upregulate pigmentation and melanogenesis, pathways which partially overlap with MAPK signaling. Upstream regulator analysis discovered significant perturbation in oncogenic forkhead box and hypoxia inducible factor family transcription factors. The established cellular models offer mechanistic insight into cellular changes and therapeutic targets under inhibitor resistance in malignant melanoma. At a systems biology level, the MAPK pathway undergoes major rewiring while acquiring inhibitor resistance

  2. Biomimetic Membranes for Multi-Redox Center Proteins

    Directory of Open Access Journals (Sweden)

    Renate L. C. Naumann

    2016-03-01

    Full Text Available His-tag technology was applied for biosensing purposes involving multi-redox center proteins (MRPs. An overview is presented on various surfaces ranging from flat to spherical and modified with linker molecules with nitrile-tri-acetic acid (NTA terminal groups to bind his-tagged proteins in a strict orientation. The bound proteins are submitted to in situ dialysis in the presence of lipid micelles to form a so-called protein-tethered bilayer lipid membrane (ptBLM. MRPs, such as the cytochrome c oxidase (CcO from R. sphaeroides and P. denitrificans, as well as photosynthetic reactions centers (RCs from R. sphaeroides, were thus investigated. Electrochemical and surface-sensitive optical techniques, such as surface plasmon resonance, surface plasmon-enhanced fluorescence, surface-enhanced infrared absorption spectroscopy (SEIRAS and surface-enhanced resonance Raman spectroscopy (SERRS, were employed in the case of the ptBLM structure on flat surfaces. Spherical particles ranging from µm size agarose gel beads to nm size nanoparticles modified in a similar fashion were called proteo-lipobeads (PLBs. The particles were investigated by laser-scanning confocal fluorescence microscopy (LSM and UV/Vis spectroscopy. Electron and proton transfer through the proteins were demonstrated to take place, which was strongly affected by the membrane potential. MRPs can thus be used for biosensing purposes under quasi-physiological conditions.

  3. Resistance Training and Co-supplementation with Creatine and Protein in Older Subjects with Frailty.

    Science.gov (United States)

    Collins, J; Longhurst, G; Roschel, H; Gualano, B

    2016-01-01

    Studies assessing the effects co-supplementation with creatine and protein, along with resistance training, in older individuals with frailty are lacking. This is an exploratory trial from the Pro-Elderly study ("Protein Intake and Resistance Training in Aging") aimed at gathering knowledge on the feasibility, safety, and efficacy of co-supplementation with creatine and protein supplementation, combined with resistance training, in older individuals with frailty. A 14-week, double-blind, randomized, parallel-group, placebo controlled exploratory trial. The subjects were randomly assigned to whey protein and creatine co-supplementation (WHEY+CR) or whey protein supplementation (WHEY) group. All subjects undertook a supervised exercise training program and were assessed at baseline and after 14 weeks. Muscle function, body composition, blood parameters, and self-reported adverse events were assessed. No interaction effects (between-group differences) were observed for any dependent variables (p > 0.05 for all). However, there were main time-effects in handgrip (WHEY+CR = 26.65 ± 31.29; WHEY = 13.84 ± 14.93 Kg; p = 0.0005), timed-up-and-go (WHEY+CR = -11.20 ± 9.37; WHEY = -17.76 ± 21.74 sec; p = 0.006), and timed-stands test (WHEY+CR = 47.50 ± 35.54; WHEY = 46.87 ± 24.23 reps; p = 0.0001), suggesting that WHEY+CR and WHEY were similarly effective in improving muscle function. All of the subjects showed improvements in at least two of the three functional tests, regardless of their treatments. Body composition and blood parameters were not changed (p > 0.05). No severe adverse effects were observed. Co-supplementation with creatine and whey protein was well-tolerable and free of adverse events in older subjects with frailty undertaking resistance training. Creatine supplementation did not augment the adaptive effects of resistance training along with whey protein on body composition or muscle function in this population. Clinicaltrials.gov: NCT01890382.

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

    Directory of Open Access Journals (Sweden)

    Jinlong Guo

    2015-01-01

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

  5. Resistance to the peptidyl transferase inhibitor tiamulin caused by mutation of ribosomal protein l3.

    Science.gov (United States)

    Bøsling, Jacob; Poulsen, Susan M; Vester, Birte; Long, Katherine S

    2003-09-01

    The antibiotic tiamulin targets the 50S subunit of the bacterial ribosome and interacts at the peptidyl transferase center. Tiamulin-resistant Escherichia coli mutants were isolated in order to elucidate mechanisms of resistance to the drug. No mutations in the rRNA were selected as resistance determinants using a strain expressing only a plasmid-encoded rRNA operon. Selection in a strain with all seven chromosomal rRNA operons yielded a mutant with an A445G mutation in the gene coding for ribosomal protein L3, resulting in an Asn149Asp alteration. Complementation experiments and sequencing of transductants demonstrate that the mutation is responsible for the resistance phenotype. Chemical footprinting experiments show a reduced binding of tiamulin to mutant ribosomes. It is inferred that the L3 mutation, which points into the peptidyl transferase cleft, causes tiamulin resistance by alteration of the drug-binding site. This is the first report of a mechanism of resistance to tiamulin unveiled in molecular detail.

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

    Science.gov (United States)

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

    2014-11-01

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

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

    Science.gov (United States)

    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. Copyright © 2016 Elsevier B.V. All

  8. Metabolic responses to high protein diet in Korean elite bodybuilders with high-intensity resistance exercise

    Directory of Open Access Journals (Sweden)

    Choue Ryowon

    2011-07-01

    Full Text Available Abstract Background High protein diet has been known to cause metabolic acidosis, which is manifested by increased urinary excretion of nitrogen and calcium. Bodybuilders habitually consumed excessive dietary protein over the amounts recommended for them to promote muscle mass accretion. This study investigated the metabolic response to high protein consumption in the elite bodybuilders. Methods Eight elite Korean bodybuilders within the age from 18 to 25, mean age 21.5 ± 2.6. For data collection, anthropometry, blood and urinary analysis, and dietary assessment were conducted. Results They consumed large amounts of protein (4.3 ± 1.2 g/kg BW/day and calories (5,621.7 ± 1,354.7 kcal/day, as well as more than the recommended amounts of vitamins and minerals, including potassium and calcium. Serum creatinine (1.3 ± 0.1 mg/dl and potassium (5.9 ± 0.8 mmol/L, and urinary urea nitrogen (24.7 ± 9.5 mg/dl and creatinine (2.3 ± 0.7 mg/dl were observed to be higher than the normal reference ranges. Urinary calcium (0.3 ± 0.1 mg/dl, and phosphorus (1.3 ± 0.4 mg/dl were on the border of upper limit of the reference range and the urine pH was in normal range. Conclusions Increased urinary excretion of urea nitrogen and creatinine might be due to the high rates of protein metabolism that follow high protein intake and muscle turnover. The obvious evidence of metabolic acidosis in response to high protein diet in the subjects with high potassium intake and intensive resistance exercise were not shown in this study results. However, this study implied that resistance exercise with adequate mineral supplementation, such as potassium and calcium, could reduce or offset the negative effects of protein-generated metabolic changes. This study provides preliminary information of metabolic response to high protein intake in bodybuilders who engaged in high-intensity resistance exercise. Further studies will be needed to determine the effects of the intensity

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

    Directory of Open Access Journals (Sweden)

    Rong Di

    2015-03-01

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

  10. Multidrug resistance-associated protein-1 (MRP1 genetic variants, MRP1 protein levels and severity of COPD

    Directory of Open Access Journals (Sweden)

    Rutgers Bea

    2010-05-01

    Full Text Available Abstract Background Multidrug resistance-associated protein-1 (MRP1 protects against oxidative stress and toxic compounds generated by cigarette smoking, which is the main risk factor for chronic obstructive pulmonary disease (COPD. We have previously shown that single nucleotide polymorphisms (SNPs in MRP1 significantly associate with level of FEV1 in two independent population based cohorts. The aim of our study was to assess the associations of MRP1 SNPs with FEV1 level, MRP1 protein levels and inflammatory markers in bronchial biopsies and sputum of COPD patients. Methods Five SNPs (rs212093, rs4148382, rs504348, rs4781699, rs35621 in MRP1 were genotyped in 110 COPD patients. The effects of MRP1 SNPs were analyzed using linear regression models. Results One SNP, rs212093 was significantly associated with a higher FEV1 level and less airway wall inflammation. Another SNP, rs4148382 was significantly associated with a lower FEV1 level, higher number of inflammatory cells in induced sputum and with a higher MRP1 protein level in bronchial biopsies. Conclusions This is the first study linking MRP1 SNPs with lung function and inflammatory markers in COPD patients, suggesting a role of MRP1 SNPs in the severity of COPD in addition to their association with MRP1 protein level in bronchial biopsies.

  11. Dual regulatory roles of the extended N terminus for activation of the tomato MI-1.2 resistance protein

    NARCIS (Netherlands)

    Lukasik-Shreepaathy, E.; Slootweg, E.; Richter, H.; Goverse, A.; Cornelissen, B.J.C.; Takken, F.L.W.

    2012-01-01

    Plant resistance (R) proteins mediate race-specific immunity and initiate host defenses that are often accompanied by a localized cell-death response. Most R proteins belong to the nucleotide binding-leucine-rich repeat (NB-LRR) protein family, as they carry a central NB-ARC domain fused to an LRR

  12. Macrophage replication screen identifies a novel Francisella hydroperoxide resistance protein involved in virulence.

    Directory of Open Access Journals (Sweden)

    Anna C Llewellyn

    Full Text Available 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

  13. Stimulation of muscle protein synthesis by whey and caseinate ingestion after resistance exercise in elderly individuals

    DEFF Research Database (Denmark)

    Dideriksen, K J; Reitelseder, S; Petersen, S G

    2011-01-01

    Sarcopenia is a well-known phenomenon in elderly individuals and resistance exercise together with sufficient amino acid (AA) availability has proved to be a counteractive implement. However, the source of AA and supplement timing require further investigation. The objective was to compare muscle...... protein synthesis (MPS) to intakes of whey and caseinate after heavy resistance exercise in healthy elderly individuals, and, furthermore, to compare the timing effect of caseinate intake. Twenty-four elderly men and women (mean ± SEM; 68 ± 1 years) were randomized to one of four groups: caseinate intake...... and caseinate feeding immediately after heavy resistance exercise in elderly individuals, and MPS is similar with caseinate ingestion before and after exercise....

  14. Whey protein consumption after resistance exercise reduces energy intake at a post-exercise meal.

    Science.gov (United States)

    Monteyne, Alistair; Martin, Alex; Jackson, Liam; Corrigan, Nick; Stringer, Ellen; Newey, Jack; Rumbold, Penny L S; Stevenson, Emma J; James, Lewis J

    2018-03-01

    Protein consumption after resistance exercise potentiates muscle protein synthesis, but its effects on subsequent appetite in this context are unknown. This study examined appetite and energy intake following consumption of protein- and carbohydrate-containing drinks after resistance exercise. After familiarisation, 15 resistance training males (age 21 ± 1 years, body mass 78.0 ± 11.9 kg, stature 1.78 ± 0.07 m) completed two randomised, double-blind trials, consisting of lower-body resistance exercise, followed by consumption of a whey protein (PRO 23.9 ± 3.6 g protein) or dextrose (CHO 26.5 ± 3.8 g carbohydrate) drink in the 5 min post-exercise. An ad libitum meal was served 60 min later, with subjective appetite measured throughout. Drinks were flavoured and matched for energy content and volume. The PRO drink provided 0.3 g/kg body mass protein. Ad libitum energy intake (PRO 3742 ± 994 kJ; CHO 4172 ± 1132 kJ; P = 0.007) and mean eating rate (PRO 339 ± 102 kJ/min; CHO 405 ± 154 kJ/min; P = 0.009) were lower during PRO. The change in eating rate was associated with the change in energy intake (R = 0.661, P = 0.007). No interaction effects were observed for subjective measures of appetite. The PRO drink was perceived as creamier and thicker, and less pleasant, sweet and refreshing (P consumption after resistance exercise reduces subsequent energy intake, and this might be partially mediated by a reduced eating rate. Whilst this reduced energy intake is unlikely to impair hypertrophy, it may be of value in supporting an energy deficit for weight loss.

  15. Selection and characterization of resistance to the Vip3Aa20 protein from Bacillus thuringiensis in Spodoptera frugiperda.

    Science.gov (United States)

    Bernardi, Oderlei; Bernardi, Daniel; Horikoshi, Renato J; Okuma, Daniela M; Miraldo, Leonardo L; Fatoretto, Julio; Medeiros, Fernanda Cl; Burd, Tony; Omoto, Celso

    2016-09-01

    Spodoptera frugiperda is one the main target pests of maize events expressing Vip3Aa20 protein from Bacillus thuringiensis (Bt) in Brazil. In this study, we selected a resistant strain of S. frugiperda on Bt maize expressing Vip3Aa20 protein and characterized the inheritance and fitness costs of the resistance. The resistance ratio of the Vip3Aa20-resistant strain of S. frugiperda was >3200-fold. Neonates of the Vip3Aa20-resistant strain were able to survive and emerge as fertile adults on Vip3Aa20 maize, while larvae from susceptible and heterozygous strains did not survive. The inheritance of Vip3Aa20 resistance was autosomal recessive and monogenic. Life history studies to investigate fitness cost revealed an 11% reduction in the survival rate until adult stage and a ∼50% lower reproductive rate of the Vip3Aa20-resistant strain compared with susceptible and heterozygous strains. This is the first characterization of S. frugiperda resistance to Vip3Aa protein. Our results provide useful information for resistance management programs designed to prevent or delay resistance evolution to Vip3Aa proteins in S. frugiperda. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

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

    International Nuclear Information System (INIS)

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

    2008-01-01

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

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

    Science.gov (United States)

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

    2014-10-01

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

  18. XA23 is an executor R protein and confers broad-spectrum disease resistance in rice.

    Science.gov (United States)

    Wang, Chunlian; Zhang, Xiaoping; Fan, Yinglun; Gao, Ying; Zhu, Qinlong; Zheng, Chongke; Qin, Tengfei; Li, Yanqiang; Che, Jinying; Zhang, Mingwei; Yang, Bing; Liu, Yaoguang; Zhao, Kaijun

    2014-11-09

    The majority of plant disease resistance (R) genes encode proteins that share common structural features. However, the transcription activator-like effector (TALE) associated executor type R genes show no considerable sequence homology to any known R genes. We adopted a map-based cloning approach and TALE-based technology to isolate and characterize Xa23, a new executor R gene derived from the wild rice (Oryza rufipogon) that confers an extremely broad spectrum of resistance to bacterial blight caused by Xanthomonas oryzae pv. oryzae (Xoo). Xa23 encodes a 113-amino acid protein that shares 50% identity to the known executor R protein XA10. The predicted transmembrane helices in XA23 also overlap with those of XA10. Unlike Xa10, however, Xa23 transcription is specifically activated by AvrXa23, a TALE present in all examined Xoo field isolates. Moreover, the susceptible xa23 allele has an identical open reading frame of Xa23, but differs in promoter region by lacking the TALE binding-element (EBE) for AvrXa23. XA23 can trigger strong hypersensitive response in rice, tobacco and tomato. Our results provide the first evidence that plant genomes have an executor R gene family in which members execute their function and spectrum of disease resistance by recognizing the cognate TALEs in pathogen. © The Author 2014. Published by the Molecular Plant Shanghai Editorial Office in association with Oxford University Press on behalf of CSPB and IPPE, SIBS, CAS.

  19. The anti-apoptotic BAG3 protein is involved in BRAF inhibitor resistance in melanoma cells.

    Science.gov (United States)

    Guerriero, Luana; Palmieri, Giuseppe; De Marco, Margot; Cossu, Antonio; Remondelli, Paolo; Capunzo, Mario; Turco, Maria Caterina; Rosati, Alessandra

    2017-10-06

    BAG3 protein, a member of BAG family of co-chaperones, has a pro-survival role in several tumour types. BAG3 anti-apoptotic properties rely on its characteristic to bind several intracellular partners, thereby modulating crucial events such as apoptosis, differentiation, cell motility, and autophagy. In human melanomas, BAG3 positivity is correlated with the aggressiveness of the tumour cells and can sustain IKK-γ levels, allowing a sustained activation of NF-κB. Furthermore, BAG3 is able to modulate BRAFV600E levels and activity in thyroid carcinomas. BRAFV600E is the most frequent mutation detected in malignant melanomas and is targeted by Vemurafenib, a specific inhibitor found to be effective in the treatment of advanced melanoma. However, patients with BRAF-mutated melanoma may result insensitive ab initio or, mostly, develop acquired resistance to the treatment with this molecule. Here we show that BAG3 down-modulation interferes with BRAF levels in melanoma cells and sensitizes them to Vemurafenib treatment. Furthermore, the down-modulation of BAG3 protein in an in vitro model of acquired resistance to Vemurafenib can induce sensitization to the BRAFV600E specific inhibition by interfering with BRAF pathway through reduction of ERK phosphorylation, but also on parallel survival pathways. Future studies on BAG3 molecular interactions with key proteins responsible of acquired BRAF inhibitor resistance may represent a promising field for novel multi-drugs treatment design.

  20. Functional analysis of P-glycoprotein and multidrug resistance associated protein related multidrug resistance in AML-blasts.

    Science.gov (United States)

    Brügger, D; Herbart, H; Gekeler, V; Seitz, G; Liu, C; Klingebiel, T; Orlikowsky, T; Einsele, H; Denzlinger, C; Bader, P; Niethammer, D; Beck, J F

    1999-05-01

    Despite the high effectiveness of various P-glycoprotein (P-gp) modulating substances in vitro their clinical value e.g. for combination treatment of acute myelogenous leukemias (AML) remains still unclear. This might be explainable by recent findings that other factors than P-gp (e.g. the multidrug resistance associated protein (MRP)) may also be involved in clinical occurring drug resistance. To study P-gp and MRP mediated MDR in AML blasts from patients with relapses at the functional level we measured rhodamine 123 (RHO) efflux in combination with a P-gp specific (SDZ PSC 833) or a MRP specific (MK571) modulator, respectively. Furthermore, direct antineoplastic drug action was monitored by determination of damaged cell fraction of a blast population using flow cytometry. We generally found strongly modulated RHO efflux by SDZ PSC 833 but slight RHO-efflux modulation by MK571 in blasts from relapsed states of AML expressing MDR1 or MRP mRNA at various levels. We could not demonstrate, though, significant PSC 833 or MK571 mediated modulation of the cytotoxic effects of etoposide. The results point to the possibility that combination of etoposide and a modulator might not improve responses to chemotherapy by targeting P-gp or MRP exclusively.

  1. MDM2 Antagonist Nutlin-3a Reverses Mitoxantrone Resistance by Inhibiting Breast Cancer Resistance Protein Mediated Drug Transport

    Science.gov (United States)

    Zhang, Fan; Throm, Stacy L.; Murley, Laura L.; Miller, Laura A.; Zatechka, D. Steven; Guy, R. Kiplin; Kennedy, Rachel; Stewart, Clinton F.

    2011-01-01

    Breast cancer resistance protein (BCRP; ABCG2), a clinical marker for identifying the side population (SP) cancer stem cell subgroup, affects intestinal absorption, brain penetration, hepatobiliary excretion, and multidrug resistance of many anti-cancer drugs. Nutlin-3a is currently under pre-clinical investigation in a variety of solid tumor and leukemia models as a p53 reactivation agent, and has been recently demonstrated to also have p53 independent actions in cancer cells. In the present study, we first report that nutlin-3a can inhibit the efflux function of BCRP. We observed that although the nutlin-3a IC50 did not differ between BCRP over-expressing and vector control cells, nutlin-3a treatment significantly potentiated the cells to treatment with the BCRP substrate mitoxantrone. Combination index calculations suggested synergism between nutlin-3a and mitoxantrone in cell lines over-expressing BCRP. Upon further investigation, it was confirmed that nutlin-3a increased the intracellular accumulation of BCRP substrates such as mitoxantrone and Hoechst 33342 in cells expressing functional BCRP without altering the expression level or localization of BCRP. Interestingly, nutlin-3b, considered virtually “inactive” in disrupting the MDM2/p53 interaction, reversed Hoechst 33342 efflux with the same potency as nutlin-3a. Intracellular accumulation and bi-directional transport studies using MDCKII cells suggested that nutlin-3a is not a substrate of BCRP. Additionally, an ATPase assay using Sf9 insect cell membranes over-expressing wild-type BCRP indicated that nutlin-3a inhibits BCRP ATPase activity in a dose-dependent fashion. In conclusion, our studies demonstrate that nutlin-3a inhibits BCRP efflux function, which consequently reverses BCRP-related drug resistance. PMID:21459080

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

    Science.gov (United States)

    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. Published by Elsevier B.V.

  3. Production of low-affinity penicillin-binding protein by low- and high-resistance groups of methicillin-resistant Staphylococcus aureus.

    Science.gov (United States)

    Murakami, K; Nomura, K; Doi, M; Yoshida, T

    1987-01-01

    Methicillin- and cephem-resistant Staphylococcus aureus (137 strains) for which the cefazolin MICs are at least 25 micrograms/ml could be classified into low-resistance (83% of strains) and high-resistance (the remaining 17%) groups by the MIC of flomoxef (6315-S), a 1-oxacephalosporin. The MICs were less than 6.3 micrograms/ml and more than 12.5 micrograms/ml in the low- and high-resistance groups, respectively. All strains produced penicillin-binding protein 2' (PBP 2'), which has been associated with methicillin resistance and which has very low affinity for beta-lactam antibiotics. Production of PBP 2' was regulated differently in low- and high-resistance strains. With penicillinase-producing strains of the low-resistance group, cefazolin, cefamandole, and cefmetazole induced PBP 2' production about 5-fold, while flomoxef induced production 2.4-fold or less. In contrast, penicillinase-negative variants of low-resistance strains produced PBP 2' constitutively in large amounts and induction did not occur. With high-resistance strains, flomoxef induced PBP 2' to an extent similar to that of cefazolin in both penicillinase-producing and -negative strains, except for one strain in which the induction did not occur. The amount of PBP 2' induced by beta-lactam antibiotics in penicillinase-producing strains of the low-resistance group correlated well with resistance to each antibiotic. Large amounts of PBP 2' in penicillinase-negative variants of the low-resistance group did not raise the MICs of beta-lactam compounds, although these strains were more resistant when challenged with flomoxef for 2 h. Different regulation of PBP 2' production was demonstrated in the high- and low-resistance groups, and factor(s) other than PBP 2' were suggested to be involved in the methicillin resistance of high-resistance strains. Images PMID:3499861

  4. Mechanosensitive molecular networks involved in transducing resistance exercise-signals into muscle protein accretion

    Directory of Open Access Journals (Sweden)

    Emil Rindom

    2016-11-01

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

  5. Cross-resistance to purified Bt proteins, Bt corn and Bt cotton in a Cry2Ab2-corn resistant strain of Spodoptera frugiperda.

    Science.gov (United States)

    Yang, Fei; Kerns, David L; Head, Graham P; Price, Paula; Huang, Fangneng

    2017-12-01

    Gene-pyramiding by combining two or more dissimilar Bacillus thuringiensis (Bt) proteins into a crop has been used to delay insect resistance. The durability of gene-pyramiding can be reduced by cross-resistance. Fall armyworm, Spodoptera frugiperda, is a major target pest of the Cry2Ab2 protein used in pyramided Bt corn and cotton. Here, we provide the first experimental evaluation of cross-resistance in S. frugiperda selected with Cry2Ab2 corn to multiple Bt sources including purified Bt proteins, Bt corn and Bt cotton. Concentration - response bioassays showed that resistance ratios for Cry2Ab2-resistant (RR) relative to Cry2Ab2-susceptible (SS) S. frugiperda were -1.4 for Cry1F, 1.2 for Cry1A.105, >26.7 for Cry2Ab2, >10.0 for Cry2Ae and -1.1 for Vip3A. Larvae of Cry2Ab2-heterozygous (RS), SS and RR S. frugiperda were all susceptible to Bt corn and Bt cotton containing Cry1 (Cry1F or Cry1A.105) and/or Vip3A proteins. Pyramided Bt cotton containing Cry1Ac + Cry2Ab2 or Cry1Ab + Cry2Ae were also effective against SS and RS, but not RR. These findings suggest that Cry2Ab2-corn-selected S. frugiperda is not cross-resistant to Cry1F, Cry1A.105 or Vip3A protein, or corn and cotton plants containing these Bt proteins, but it can cause strong cross-resistance to Cry2Ae and Bt crops expressing similar Bt proteins. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

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

    Directory of Open Access Journals (Sweden)

    John E McLaughlin

    Full Text Available 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

  7. Autophagy is induced by resistance exercise in young men but unfolded protein response is induced regardless of age.

    Science.gov (United States)

    Hentilä, Jaakko; Ahtiainen, Juha P; Paulsen, Gøran; Raastad, Truls; Häkkinen, Keijo; Mero, Antti A; Hulmi, Juha J

    2018-04-02

    Autophagy and unfolded protein response (UPR) appear to be important for skeletal muscle homeostasis and may be altered by exercise. Our aim was to investigate the effects of resistance exercise and training on indicators of UPR and autophagy in healthy untrained young men (n = 12, 27 ± 4 years) and older men (n = 8, 61 ± 6 years) as well as in resistance-trained individuals (n = 15, 25 ± 5 years). Indicators of autophagy and UPR were investigated from the muscle biopsies after a single resistance exercise bout and after 21 weeks of resistance training. Lipidated LC3II as an indicator of autophagosome content increased at 48 hours post resistance exercise (P resistance-training period (P resistance exercise in untrained young and older men (P resistance-training period regardless of age. UPR was unchanged within the first few hours after the resistance exercise bout regardless of the training status. Changes in autophagy and UPR ER indicators did not correlate with a resistance-training-induced increase in muscle strength and size. Autophagosome content is increased by resistance training in young previously untrained men, but this response may be blunted by aging. However, unfolded protein response is induced by an unaccustomed resistance exercise bout in a delayed manner regardless of age. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

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

    Science.gov (United States)

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

    2010-08-01

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

  9. Free and Protein-Bound Maillard Reaction Products in Beer: Method Development and a Survey of Different Beer Types.

    Science.gov (United States)

    Hellwig, Michael; Witte, Sophia; Henle, Thomas

    2016-09-28

    The Maillard reaction is important for beer color and flavor, but little is known about the occurrence of individual glycated amino acids in beer. Therefore, seven Maillard reaction products (MRPs), namely, fructosyllysine, maltulosyllysine, pyrraline, formyline, maltosine, MG-H1, and argpyrimidine, were synthesized and quantitated in different types of beer (Pilsner, dark, bock, wheat, and nonalcoholic beers) by HPLC-ESI-MS/MS in the multiple reaction monitoring mode through application of the standard addition method. Free MRPs were analyzed directly. A high molecular weight fraction was isolated by dialysis and hydrolyzed enzymatically prior to analysis. Maltulosyllysine was quantitated for the first time in food. The most important free MRPs in beer are fructosyllysine (6.8-27.0 mg/L) and maltulosyllysine (3.7-21.8 mg/L). Beer contains comparatively high amounts of late-stage free MRPs such as pyrraline (0.2-1.6 mg/L) and MG-H1 (0.3-2.5 mg/L). Minor amounts of formyline (4-230 μg/L), maltosine (6-56 μg/L), and argpyrimidine (0.1-4.1 μg/L) were quantitated. Maltulosyllysine was the most significant protein-bound MRP, but both maltulosyllysine and fructosyllysine represent only 15-60% of the total protein-bound lysine-derived Amadori products. Differences in the patterns of protein-bound and free individual MRPs and the ratios between them were identified, which indicate differences in their chemical, biochemical, and microbiological stabilities during the brewing process.

  10. The effects of whey protein with or without carbohydrates on resistance training adaptations.

    Science.gov (United States)

    Hulmi, Juha J; Laakso, Mia; Mero, Antti A; Häkkinen, Keijo; Ahtiainen, Juha P; Peltonen, Heikki

    2015-01-01

    Nutrition intake in the context of a resistance training (RT) bout may affect body composition and muscle strength. However, the individual and combined effects of whey protein and carbohydrates on long-term resistance training adaptations are poorly understood. A four-week preparatory RT period was conducted in previously untrained males to standardize the training background of the subjects. Thereafter, the subjects were randomized into three groups: 30 g of whey proteins (n = 22), isocaloric carbohydrates (maltodextrin, n = 21), or protein + carbohydrates (n = 25). Within these groups, the subjects were further randomized into two whole-body 12-week RT regimens aiming either for muscle hypertrophy and maximal strength or muscle strength, hypertrophy and power. The post-exercise drink was always ingested immediately after the exercise bout, 2-3 times per week depending on the training period. Body composition (by DXA), quadriceps femoris muscle cross-sectional area (by panoramic ultrasound), maximal strength (by dynamic and isometric leg press) and serum lipids as basic markers of cardiovascular health, were analysed before and after the intervention. Twelve-week RT led to increased fat-free mass, muscle size and strength independent of post-exercise nutrient intake (P carbohydrate group independent of the type of RT (P carbohydrate group (P carbohydrates or combination of proteins and carbohydrates did not have a major effect on muscle size or strength when ingested two to three times a week. However, whey proteins may increase abdominal fat loss and relative fat-free mass adaptations in response to resistance training when compared to fast-acting carbohydrates.

  11. The effect of resistance training combined with timed ingestion of protein on muscle fiber size and muscle strength

    DEFF Research Database (Denmark)

    Andersen, L.L.; Tufekovic, G.; Zebis, M.K.

    2005-01-01

    of resistance training combined with timed ingestion of isoenergetic protein vs carbohydrate supplementation on muscle fiber hypertrophy and mechanical muscle performance. Supplementation was administered before and immediately after each training bout and, in addition, in the morning on nontraining days...

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

    Science.gov (United States)

    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

  13. Localization and activity of multidrug resistance protein 1 in the secretory pathway of Leishmania parasites.

    Science.gov (United States)

    Dodge, Matthew A; Waller, Ross F; Chow, Larry M C; Zaman, Muhammad M; Cotton, Leanne M; McConville, Malcolm J; Wirth, Dyann F

    2004-03-01

    Upregulation of the multidrug resistance protein 1 (LeMDR1) in the protozoan parasite, Leishmania enriettii, confers resistance to hydrophobic drugs such as vinblastine, but increases the sensitivity of these parasites to the mitochondrial drug, rhodamine 123. In order to investigate the mechanism of action of LeMDR1, the subcellular localization of green fluorescent protein (GFP)-tagged versions of LeMDR1 and the fate of the traceable-fluorescent LeMDR1 substrate calcein AM were examined in both Leishmania mexicana and L. enriettii LeMDR1 -/- and overexpressing cell lines. The LeMDR1-GFP chimera was localized by fluorescence microscopy to a number of secretory and endocytic compartments, including the Golgi apparatus, endoplasmic reticulum (ER) and a multivesicular tubule (MVT)-lysosome. Pulse-chase labelling experiments with calcein AM suggested that the Golgi and ER pools, but not the MVT-lysosome pool, of LeMDR1 were active in pumping calcein AM out of the cell. Cells labelled with calcein AM under conditions that slow vesicular transport (low temperature and stationary growth) inhibited export and resulted in the accumulation of fluorescent calcein in both the Golgi and the mitochondria. We propose that LeMDR1 substrates are pumped into secretory compartments and exported from the parasite by exocytosis. Accumulation of MDR substrates in the ER can result in alternative transport to the mitochondrion, explaining the reciprocal sensitivity of drug-resistant Leishmania to vinblastine and rhodamine 123.

  14. Effects of high-protein diet containing isolated whey protein in rats submitted to resistance training of aquatic jumps.

    Science.gov (United States)

    Avila, Eudes Thiago Pereira; da Rosa Lima, Thiago; Tibana, Ramires Alsamir; de Almeida, Paula Caroline; Fraga, Géssica Alves; de Souza Sena, Mariana; Corona, Luiz Felipe Petusk; Navalta, James Wilfred; Rezaei, Sajjad; Ghayomzadeh, Morteza; Damazo, Amílcar Sabino; Prestes, Jonato; Voltarelli, Fabrício Azevedo

    2018-02-13

    Isolated whey protein (IWP) can decrease body fat compared with other protein sources. The present study verified the effects of high protein diet (HD) containing IWP on several parameters of rats subjected to resistance training (RT). Thirty-two male Wistar rats (60 days of age) were separated into four groups (n = 8/group): sedentary normoproteic (IWP 14%; SN); sedentary hyperproteic (IWP 35%; SH); trained normoproteic (IWP 14%; TN), and trained hyperproteic (WPI 35%; TH). Relative tissue/organ weight (g): perirenal and retroperitoneal adipose tissues were lower in SH and TH compared with SN (no difference to TN); omental and subcutaneous adipose tissues were higher in SN compared with SH. Epididymal adipose tissue was higher in SN compared with other groups. Heart weight was higher in TH compared with TN and SN, but not SH; kidney and liver higher in TH and SH compared with SN and TN; gastrocnemius lower in SN compared with other groups; soleus higher in SH in relation to other groups. The triglycerides levels (mg/dL) was reduced in the TH groups compared with SH, TN, and SN. There were no changes both in the concentrations of adiponectin and leptin and in the protein expression of GLUT-4 and p70 s6k . HD containing WPI improved body composition, increased the weight of the heart, kidneys, liver and gastrocnemius and soleus muscles; however, this diet maintained the normal histomorphology of muscle and liver and, when associated with RT, reduced the serum levels of triglycerides. Copyright © 2018 Elsevier Inc. All rights reserved.

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

    Science.gov (United States)

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

    2017-03-01

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

  16. Effects of whey protein supplement in the elderly submitted to resistance training: systematic review and meta-analysis.

    Science.gov (United States)

    Colonetti, Tamy; Grande, Antonio Jose; Milton, Karen; Foster, Charlie; Alexandre, Maria Cecilia Manenti; Uggioni, Maria Laura Rodrigues; Rosa, Maria Inês da

    2017-05-01

    We performed a systematic review to map the evidence and analyze the effect of whey protein supplementation in the elderly submitted to resistance training. A comprehensive search on Medline, LILACS, EMBASE, and the Cochrane Library for relevant publications was conducted until August 2015. The terms used in the search were: "Resistance training"; "Whey protein"; "Elderly". A total of 632 studies were screened. Five studies were included composing a sample of 391 patients. The supplement whey protein was associated with higher total protein ingestion 9.40 (95% CI: 4.03-14.78), and with an average change in plasma leucine concentration. The supplementation was also associated with increased mixed muscle protein synthesis 1.26 (95% CI: 0.46-2.07) compared to the control group. We observed an increase in total protein intake, resulting in increased concentration of leucine and mixed muscle protein fractional synthesis rate.

  17. Comparison of protein profiles of beech bark disease-resistant or beech bark disease-susceptible American beech

    Science.gov (United States)

    Mary E. Mason; Marek Krasowski; Judy Loo; Jennifer. Koch

    2011-01-01

    Proteomic analysis of beech bark proteins from trees resistant and susceptible to beech bark disease (BBD) was conducted. Sixteen trees from eight geographically isolated stands, 10 resistant (healthy) and 6 susceptible (diseased/infested) trees, were studied. The genetic complexity of the sample unit, the sampling across a wide geographic area, and the complexity of...

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

    Directory of Open Access Journals (Sweden)

    Heran Ma

    Full Text Available 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.

  19. P-body proteins regulate transcriptional rewiring to promote DNA replication stress resistance.

    Science.gov (United States)

    Loll-Krippleber, Raphael; Brown, Grant W

    2017-09-15

    mRNA-processing (P-) bodies are cytoplasmic granules that form in eukaryotic cells in response to numerous stresses to serve as sites of degradation and storage of mRNAs. Functional P-bodies are critical for the DNA replication stress response in yeast, yet the repertoire of P-body targets and the mechanisms by which P-bodies promote replication stress resistance are unknown. In this study we identify the complete complement of mRNA targets of P-bodies during replication stress induced by hydroxyurea treatment. The key P-body protein Lsm1 controls the abundance of HHT1, ACF4, ARL3, TMA16, RRS1 and YOX1 mRNAs to prevent their toxic accumulation during replication stress. Accumulation of YOX1 mRNA causes aberrant downregulation of a network of genes critical for DNA replication stress resistance and leads to toxic acetaldehyde accumulation. Our data reveal the scope and the targets of regulation by P-body proteins during the DNA replication stress response.P-bodies form in response to stress and act as sites of mRNA storage and degradation. Here the authors identify the mRNA targets of P-bodies during DNA replication stress, and show that P-body proteins act to prevent toxic accumulation of these target transcripts.

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

    Science.gov (United States)

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

    2014-10-01

    In a comparative study, we investigated the effects of maximal eccentric or concentric resistance training combined with whey protein or placebo on muscle and tendon hypertrophy. 22 subjects were allocated into either a high-leucine whey protein hydrolysate + carbohydrate group (WHD) or a carbohydrate group (PLA). Subjects completed 12 weeks maximal knee extensor training with one leg using eccentric contractions and the other using concentric contractions. Before and after training cross-sectional area (CSA) of m. quadriceps and patellar tendon CSA was quantified with magnetic resonance imaging and a isometric strength test was used to assess maximal voluntary contraction (MVC) and rate of force development (RFD). Quadriceps CSA increased by 7.3 ± 1.0% (P tendon CSA increased by 14.9 ± 3.1% (P effect of contraction mode. MVC and RFD increased by 15.6 ± 3.5% (P effects. In conclusion, high-leucine whey protein hydrolysate augments muscle and tendon hypertrophy following 12 weeks of resistance training - irrespective of contraction mode. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  1. Configurable Resistive Switching between Memory and Threshold Characteristics for Protein-Based Devices

    KAUST Repository

    Wang, Hong

    2015-05-01

    The employ of natural biomaterials as the basic building blocks of electronic devices is of growing interest for biocompatible and green electronics. Here, resistive switching (RS) devices based on naturally silk protein with configurable functionality are demonstrated. The RS type of the devices can be effectively and exactly controlled by controlling the compliance current in the set process. Memory RS can be triggered by a higher compliance current, while threshold RS can be triggered by a lower compliance current. Furthermore, two types of memory devices, working in random access and WORM modes, can be achieved with the RS effect. The results suggest that silk protein possesses the potential for sustainable electronics and data storage. In addition, this finding would provide important guidelines for the performance optimization of biomaterials based memory devices and the study of the underlying mechanism behind the RS effect arising from biomaterials. Resistive switching (RS) devices with configurable functionality based on protein are successfully achieved. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

    Energy Technology Data Exchange (ETDEWEB)

    Rusen, L. [National Institute for Lasers, Plasma and Radiation Physics, 409 Atomistilor Street, PO Box MG-16, 077125, Magurele, Bucharest (Romania); Mustaciosu, C. [Horia Hulubei National Institute of Physics and Nuclear Engineering - IFIN HH, Magurele, Bucharest (Romania); Mitu, B.; Filipescu, M.; Dinescu, M. [National Institute for Lasers, Plasma and Radiation Physics, 409 Atomistilor Street, PO Box MG-16, 077125, Magurele, Bucharest (Romania); Dinca, V., E-mail: dinali@nipne.ro [National Institute for Lasers, Plasma and Radiation Physics, 409 Atomistilor Street, PO Box MG-16, 077125, Magurele, Bucharest (Romania)

    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(ε-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{sup −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.

  3. ISOELECTRIC FOCUSING OF MEMBRANE PROTEINS OF PROBIOTIC B. COAGULANS AND ITS BACTERIOPHAGE RESISTANT MUTANTS

    Directory of Open Access Journals (Sweden)

    Kavita Rajesh Pandey

    2016-09-01

    Full Text Available Bacteriophages are the most notorious type of infection in the probiotic and dairy fermentations. Two phage resistant mutants viz. B. co PIII and B. co MIII (B. coagulans mutants PIII and MIII obtained in previous studies (Dubey and Vakil, 2010, were further characterized for their protein profile in comparison with the parental probiotic strain –B. coagulans. The cell lysates were subjected to ultra-centrifugation and the purified membrane fractions were resolved using 2D gel electrophoresis. The Isoelectric focussing showed 187, 202 and 154 protein spots for the parental strain, mutant B. co PIII and mutant B. co MIII, respectively. Ten and 18 protein spots were missing as compared to parent for mutants B.co PIII and B.co MIII whereas there were 21 and 14 new spots noticed for these two mutants. Eight membrane proteins present only in the phage sensitive parental culture could be tentatively identified by comparison with the complete proteome of B. coagulans by use of UniprotKB and then CELLO database It is quite likely that some of these identified membrane proteins may be also functioning as receptors for phage adsorption followed by entry of nucleic acid into the phage sensitive host cell.

  4. Melatonin Promotes Apoptosis of Oxaliplatin-resistant Colorectal Cancer Cells Through Inhibition of Cellular Prion Protein.

    Science.gov (United States)

    Lee, Jun Hee; Yoon, Yeo Min; Han, Yong-Seok; Yun, Chul Won; Lee, Sang Hun

    2018-04-01

    Drug resistance restricts the efficacy of chemotherapy in colorectal cancer. However, the detailed molecular mechanism of drug resistance in colorectal cancer cells remains unclear. The level of cellular prion protein (PrP C ) in oxaliplatin-resistant colorectal cancer (SNU-C5/Oxal-R) cells was assessed. PrP C level in SNU-C5/Oxal-R cells was significantly increased compared to that in wild-type (SNU-C5) cells. Superoxide dismutase and catalase activities were higher in SNU-C5/Oxal-R cells than in SNU-C5 cells. Treatment of SNU-C5/Oxal-R cells with oxaliplatin and melatonin reduced PrP C expression, while suppressing antioxidant enzyme activity and increasing superoxide anion generation. In SNU-C5/Oxal-R cells, endoplasmic reticulum stress and apoptosis were significantly increased following co-treatment with oxaliplatin and melatonin compared to treatment with oxaliplatin alone. Co-treatment with oxaliplatin and melatonin increased endoplasmic reticulum stress in and apoptosis of SNU-C5/Oxal-R cells through inhibition of PrP C , suggesting that PrP C could be a key molecule in oxaliplatin resistance of colorectal cancer cells. Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

  5. The role of cytoskeleton and adhesion proteins in the resistance to photodynamic therapy. Possible therapeutic interventions.

    Science.gov (United States)

    Di Venosa, Gabriela; Perotti, Christian; Batlle, Alcira; Casas, Adriana

    2015-08-01

    It is known that Photodynamic Therapy (PDT) induces changes in the cytoskeleton, the cell shape, and the adhesion properties of tumour cells. In addition, these targets have also been demonstrated to be involved in the development of PDT resistance. The reversal of PDT resistance by manipulating the cell adhesion process to substrata has been out of reach. Even though the existence of cell adhesion-mediated PDT resistance has not been reported so far, it cannot be ruled out. In addition to its impact on the apoptotic response to photodamage, the cytoskeleton alterations are thought to be associated with the processes of metastasis and invasion after PDT. In this review, we will address the impact of photodamage on the microfilament and microtubule cytoskeleton components and its regulators on PDT-treated cells as well as on cell adhesion. We will also summarise the impact of PDT on the surviving and resistant cells and their metastatic potential. Possible strategies aimed at taking advantage of the changes induced by PDT on actin, tubulin and cell adhesion proteins by targeting these molecules will also be discussed.

  6. Penicillin-resistant viridans streptococci have obtained altered penicillin-binding protein genes from penicillin-resistant strains of Streptococcus pneumoniae.

    OpenAIRE

    Dowson, C G; Hutchison, A; Woodford, N; Johnson, A P; George, R C; Spratt, B G

    1990-01-01

    Penicillin-resistant strains of Streptococcus pneumoniae possess altered forms of penicillin-binding proteins (PBPs) with decreased affinity for penicillin. The PBP2B genes of these strains have a mosaic structure, consisting of regions that are very similar to those in penicillin-sensitive strains, alternating with regions that are highly diverged. Penicillin-resistant strains of viridans groups streptococci (e.g., S. sanguis and S. oralis) that produce altered PBPs have also been reported. ...

  7. Leucine supplementation improves acquired growth hormone resistance in rats with protein-energy malnutrition.

    Science.gov (United States)

    Gao, Xuejin; Tian, Feng; Wang, Xinying; Zhao, Jie; Wan, Xiao; Zhang, Li; Wu, Chao; Li, Ning; Li, Jieshou

    2015-01-01

    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. 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 the MC-L and MC-H groups than in the MC

  8. Application of protein typing in molecular epidemiological investigation of nosocomial infection outbreak of aminoglycoside-resistant Pseudomonas aeruginosa.

    Science.gov (United States)

    Song, Min; Tang, Min; Ding, Yinghuan; Wu, Zecai; Xiang, Chengyu; Yang, Kui; Zhang, Zhang; Li, Baolin; Deng, Zhenghua; Liu, Jinbo

    2017-12-16

    Pseudomonas aeruginosan has emerged as an important pathogen elated to serious infections and nosocomial outbreaks worldwide. This study was conducted to understand the prevalence of aminoglycoside (AMG)-resistant P. aeruginosa in our hospital and to provide a scientific basis for control measures against nosocomial infections. Eighty-two strains of P. aeruginosa were isolated from clinical departments and divided into AMG-resistant strains and AMG-sensitive strains based on susceptibility test results. AMG-resistant strains were typed by drug resistance gene typing (DRGT) and protein typing. Five kinds of aminoglycoside-modifying enzyme (AME) genes were detected in the AMG-resistant group. AMG-resistant P. aeruginosa strains were classified into three types and six subtypes by DRGT. Four protein peaks, namely, 9900.02, 7600.04, 9101.25 and 10,372.87 Da, were significantly and differentially expressed between the two groups. AMG-resistant P. aeruginosa strains were also categorised into three types and six subtypes at the distance level of 10 by protein typing. AMG-resistant P. aeruginosa was cloned spread in our hospital; the timely implementation of nosocomial infection prevention and control strategies were needed in preventing outbreaks and epidemic of AMG-resistant P. aeruginosa. SELDI-TOF MS technology can be used for bacterial typing, which provides a new method of clinical epidemiological survey and nosocomial infection control.

  9. Functional markers based molecular characterization and cloning of resistance gene analogs encoding NBS-LRR disease resistance proteins in finger millet (Eleusine coracana).

    Science.gov (United States)

    Panwar, Preety; Jha, Anand Kumar; Pandey, P K; Gupta, Arun K; Kumar, Anil

    2011-06-01

    Magnaporthe grisea, the blast fungus is one of the main pathological threats to finger millet crop worldwide. A systematic search for the blast resistance gene analogs was carried out, using functional molecular markers. Three-fourths of the recognition-dependent disease resistance genes (R-genes) identified in plants encodes nucleotide binding site (NBS) leucine-rich repeat (LRR) proteins. NBS-LRR homologs have only been isolated on a limited scale from Eleusine coracana. Genomic DNA sequences sharing homology with NBS region of resistance gene analogs were isolated and characterized from resistant genotypes of finger millet using PCR based approach with primers designed from conserved regions of NBS domain. Attempts were made to identify molecular markers linked to the resistance gene and to differentiate the resistant bulk from the susceptible bulk. A total of 9 NBS-LRR and 11 EST-SSR markers generated 75.6 and 73.5% polymorphism respectively amongst 73 finger millet genotypes. NBS-5, NBS-9, NBS-3 and EST-SSR-04 markers showed a clear polymorphism which differentiated resistant genotypes from susceptible genotypes. By comparing the banding pattern of different resistant and susceptible genotypes, five DNA amplifications of NBS and EST-SSR primers (NBS-05(504,) NBS-09(711), NBS-07(688), NBS-03(509) and EST-SSR-04(241)) were identified as markers for the blast resistance in resistant genotypes. Principal coordinate plot and UPGMA analysis formed similar groups of the genotypes and placed most of the resistant genotypes together showing a high level of genetic relatedness and the susceptible genotypes were placed in different groups on the basis of differential disease score. Our results provided a clue for the cloning of finger millet blast resistance gene analogs which not only facilitate the process of plant breeding but also molecular characterization of blast resistance gene analogs from Eleusine coracana.

  10. Dynamic proteome profiling of individual proteins in human skeletal muscle after a high-fat diet and resistance exercise.

    Science.gov (United States)

    Camera, Donny M; Burniston, Jatin G; Pogson, Mark A; Smiles, William J; Hawley, John A

    2017-12-01

    It is generally accepted that muscle adaptation to resistance exercise (REX) training is underpinned by contraction-induced, increased rates of protein synthesis and dietary protein availability. By using dynamic proteome profiling (DPP), we investigated the contribution of both synthesis and breakdown to changes in abundance on a protein-by-protein basis in human skeletal muscle. Age-matched, overweight males consumed 9 d of a high-fat, low-carbohydrate diet during which time they either undertook 3 sessions of REX or performed no exercise. Precursor enrichment and the rate of incorporation of deuterium oxide into newly synthesized muscle proteins were determined by mass spectrometry. Ninety proteins were included in the DPP, with 28 proteins exhibiting significant responses to REX. The most common pattern of response was an increase in turnover, followed by an increase in abundance with no detectable increase in protein synthesis. Here, we provide novel evidence that demonstrates that the contribution of synthesis and breakdown to changes in protein abundance induced by REX differ on a protein-by-protein basis. We also highlight the importance of the degradation of individual muscle proteins after exercise in human skeletal muscle.-Camera, D. M., Burniston, J. G., Pogson, M. A., Smiles, W. J., Hawley, J. A. Dynamic proteome profiling of individual proteins in human skeletal muscle after a high-fat diet and resistance exercise. © FASEB.

  11. Branched-Chain Amino Acid Ingestion Stimulates Muscle Myofibrillar Protein Synthesis following Resistance Exercise in Humans

    Directory of Open Access Journals (Sweden)

    Sarah R. Jackman

    2017-06-01

    Full Text Available The ingestion of intact protein or essential amino acids (EAA stimulates mechanistic target of rapamycin complex-1 (mTORC1 signaling and muscle protein synthesis (MPS following resistance exercise. The purpose of this study was to investigate the response of myofibrillar-MPS to ingestion of branched-chain amino acids (BCAAs only (i.e., without concurrent ingestion of other EAA, intact protein, or other macronutrients following resistance exercise in humans. Ten young (20.1 ± 1.3 years, resistance-trained men completed two trials, ingesting either 5.6 g BCAA or a placebo (PLA drink immediately after resistance exercise. Myofibrillar-MPS was measured during exercise recovery with a primed, constant infusion of L-[ring13C6] phenylalanine and collection of muscle biopsies pre and 4 h-post drink ingestion. Blood samples were collected at time-points before and after drink ingestion. Western blotting was used to measure the phosphorylation status of mTORC1 signaling proteins in biopsies collected pre, 1-, and 4 h-post drink. The percentage increase from baseline in plasma leucine (300 ± 96%, isoleucine (300 ± 88%, and valine (144 ± 59% concentrations peaked 0.5 h-post drink in BCAA. A greater phosphorylation status of S6K1Thr389 (P = 0.017 and PRAS40 (P = 0.037 was observed in BCAA than PLA at 1 h-post drink ingestion. Myofibrillar-MPS was 22% higher (P = 0.012 in BCAA (0.110 ± 0.009%/h than PLA (0.090 ± 0.006%/h. Phenylalanine Ra was ~6% lower in BCAA (18.00 ± 4.31 μmol·kgBM−1 than PLA (21.75 ± 4.89 μmol·kgBM−1; P = 0.028 after drink ingestion. We conclude that ingesting BCAAs alone increases the post-exercise stimulation of myofibrillar-MPS and phosphorylation status mTORC1 signaling.

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

    Science.gov (United States)

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

    2016-09-28

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

  13. Unique Pattern of Protein-Bound Maillard Reaction Products in Manuka (Leptospermum scoparium) Honey.

    Science.gov (United States)

    Hellwig, Michael; Rückriemen, Jana; Sandner, Daniel; Henle, Thomas

    2017-05-03

    As a unique feature, honey from the New Zealand manuka tree (Leptospermum scoparium) contains substantial amounts of dihydroxyacetone (DHA) and methylglyoxal (MGO). Although MGO is a reactive intermediate in the Maillard reaction, very little is known about reactions of MGO with honey proteins. We hypothesized that the abundance of MGO should result in a particular pattern of protein-bound Maillard reaction products (MRPs) in manuka honey. A protein-rich high-molecular-weight fraction was isolated from 12 manuka and 8 non-manuka honeys and hydrolyzed enzymatically. By HPLC-MS/MS, 8 MRPs, namely, N-ε-fructosyllysine, N-ε-maltulosyllysine, carboxymethyllysine, carboxyethyllysine (CEL), pyrraline, formyline, maltosine, and methylglyoxal-derived hydroimidazolone 1 (MG-H1), were quantitated. Compared to non-manuka honeys, the manuka honeys were characterized by high concentrations of CEL and MG-H1, whereas the formation of N-ε-fructosyllysine was suppressed, indicating concurrence reactions of glucose and MGO at the ε-amino group of protein-bound lysine. Up to 31% of the lysine and 8% of the arginine residues, respectively, in the manuka honey protein can be modified to CEL and MG-H1, respectively. CEL and MG-H1 concentrations correlated strongly with the MGO concentration of the honeys. Manuka honey possesses a special pattern of protein-bound MRPs, which might be used to prove the reliability of labeled MGO levels in honeys and possibly enable the detection of fraudulent MGO or DHA addition to honey.

  14. Resistant starch and protein intake enhances fat oxidation and feelings of fullness in lean and overweight/obese women

    DEFF Research Database (Denmark)

    Gentile, Christopher L; Ward, Emery; Holst, Jens Juul

    2015-01-01

    and overweight/obese women. METHODS: Women of varying levels of adiposity consumed one of four pancake test meals in a single-blind, randomized crossover design: 1) waxy maize (control) starch (WMS); 2) waxy maize starch and whey protein (WMS+WP); 3) resistant starch (RS); or 4) RS and whey protein (RS...

  15. Scrapie susceptibility-linked polymorphisms modulate the in vitro conversion of sheep prion protein to protease-resistant forms

    NARCIS (Netherlands)

    Bossers, A.; Belt, P.B.G.M.; Raymond, G.J.; Caughey, B.; Vries, de R.; Smits, M.

    1997-01-01

    Prion diseases are natural transmissible neurodegenerative disorders in humans and animals. They are characterized by the accumulation of a protease-resistant scrapie-associated prion protein (PrPSc) of the host-encoded cellular prion protein (PrPC) mainly in the central nervous system.

  16. Impaired renal secretion of substrates for the multidrug resistance protein 2 in mutant transport-deficient (TR-) rats.

    NARCIS (Netherlands)

    Masereeuw, R.; Notenboom, S.; Smeets, P.H.E.; Wouterse, A.C.; Russel, F.G.M.

    2003-01-01

    Previous studies with mutant transport-deficient rats (TR(-)), in which the multidrug resistance protein 2 (Mrp2) is lacking, have emphasized the importance of this transport protein in the biliary excretion of a wide variety of glutathione conjugates, glucuronides, and other organic anions. Mrp2 is

  17. Protein-protein association and cellular localization of four essential gene products encoded by tellurite resistance-conferring cluster "ter" from pathogenic Escherichia coli.

    Science.gov (United States)

    Valkovicova, Lenka; Vavrova, Silvia Minarikova; Mravec, Jozef; Grones, Jozef; Turna, Jan

    2013-12-01

    Gene cluster "ter" conferring high tellurite resistance has been identified in various pathogenic bacteria including Escherichia coli O157:H7. However, the precise mechanism as well as the molecular function of the respective gene products is unclear. Here we describe protein-protein association and localization analyses of four essential Ter proteins encoded by minimal resistance-conferring fragment (terBCDE) by means of recombinant expression. By using a two-plasmid complementation system we show that the overproduced single Ter proteins are not able to mediate tellurite resistance, but all Ter members play an irreplaceable role within the cluster. We identified several types of homotypic and heterotypic protein-protein associations among the Ter proteins by in vitro and in vivo pull-down assays and determined their cellular localization by cytosol/membrane fractionation. Our results strongly suggest that Ter proteins function involves their mutual association, which probably happens at the interface of the inner plasma membrane and the cytosol.

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

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    Jinxing Song

    2016-02-01

    Full Text Available 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.

  19. Effects of protein kinase C activators on phorbol ester-sensitive and -resistant EL4 thymoma cells.

    Science.gov (United States)

    Sansbury, H M; Wisehart-Johnson, A E; Qi, C; Fulwood, S; Meier, K E

    1997-09-01

    Phorbol ester-sensitive EL4 murine thymoma cells respond to phorbol 12-myristate 13-acetate with activation of ERK mitogen-activated protein kinases, synthesis of interleukin-2, and death, whereas phorbol ester-resistant variants of this cell line do not exhibit these responses. Additional aspects of the resistant phenotype were examined, using a newly-established resistant cell line. Phorbol ester induced morphological changes, ERK activation, calcium-dependent activation of the c-Jun N-terminal kinase (JNK), interleukin-2 synthesis, and growth inhibition in sensitive but not resistant cells. A series of protein kinase C activators caused membrane translocation of protein kinase C's (PKCs) alpha, eta, and theta in both cell lines. While PKC eta was expressed at higher levels in sensitive than in resistant cells, overexpression of PKC eta did not restore phorbol ester-induced ERK activation to resistant cells. In sensitive cells, PKC activators had similar effects on cell viability and ERK activation, but differed in their abilities to induce JNK activation and interleukin-2 synthesis. PD 098059, an inhibitor of the mitogen activated protein (MAP)/ERK kinase kinase MEK, partially inhibited ERK activation and completely blocked phorbol ester-induced cell death in sensitive cells. Thus MEK and/or ERK activation, but not JNK activation or interleukin-2 synthesis, appears to be required for phorbol ester-induced toxicity. Alterations in phorbol ester response pathways, rather than altered expression of PKC isoforms, appear to confer phorbol ester resistance to EL4 cells.

  20. Human Skeletal Muscle Stem Cells in Adaptations to Exercise; Effects of Resistance Exercise Contraction Mode and Protein Supplementation

    DEFF Research Database (Denmark)

    Farup, Jean

    2014-01-01

    the effect of contraction mode specific resistance training and protein supplementation on whole muscle and tendon hypertrophy. Quadriceps muscle and patellar tendon cross-sectional area (CSA) was quantified using magnetic resonance imaging pre and post 12 weeks of eccentric (Ecc) or concentric (Conc...... concentric resistance training and ingestion of protein influence myocellular adaptations, with special emphasis on muscle stem cell adaptations, during both acute and prolonged resistance exercise in human skeletal muscle. Paper I. Whey protein supplementation accelerates satellite cell proliferation during...... recovery from eccentric exercise In paper I, we evaluated the effect of a single bout of unaccustomed eccentric exercise on fiber type specific SC content by immunohistochemistry. Subjects received either hydrolysed whey protein (Whey) or iso-caloric carbohydrate (Placebo) in the days post eccentric...

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

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    Isabella Massimi

    2015-01-01

    Full Text Available 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.

  2. Detection and characterisation of multi-drug resistance protein 1 (MRP-1) in human mitochondria.

    Science.gov (United States)

    Roundhill, E A; Burchill, S A

    2012-03-13

    Overexpression of plasma membrane multi-drug resistance protein 1 (MRP-1) can lead to multidrug resistance. In this study, we describe for the first time the expression of mitochondrial MRP-1 in untreated human normal and cancer cells and tissues. MRP-1 expression and subcellular localisation in normal and cancer cells and tissues was examined by differential centrifugation and western blotting, and immunofluorescence microscopy. Viable mitochondria were isolated and MRP-1 efflux activity measured using the calcein-AM functional assay. MRP-1 expression was increased using retroviral infection and specific overexpression confirmed by RNA array. Cell viability was determined by trypan blue exclusion and annexin V-propidium iodide labelling of cells. MRP-1 was detected in the mitochondria of cancer and normal cells and tissues. The efflux activity of mitochondrial MRP-1 was more efficient (55-64%) than that of plasma membrane MRP-1 (11-22%; PMRP-1 expression resulted in a preferential increase in mitochondrial MRP-1, suggesting selective targeting to this organelle. Treatment with a non-lethal concentration of doxorubicin (0.85 nM, 8 h) increased mitochondrial and plasma membrane MRP-1, increasing resistance to MRP-1 substrates. For the first time, we have identified MRP-1 with efflux activity in human mitochondria. Mitochondrial MRP-1 may be an exciting new therapeutic target where historically MRP-1 inhibitor strategies have limited clinical success.

  3. Association between insulin resistance and c-reactive protein among Peruvian adults

    Directory of Open Access Journals (Sweden)

    Gelaye Bizu

    2010-05-01

    Full Text Available Abstract Objective Insulin resistance (IR, a reduced physiological response of peripheral tissues to the action of insulin, is one of the major causes of type 2 diabetes. We sought to evaluate the relationship between serum C-reactive protein (CRP, a marker of systemic inflammation, and prevalence of IR among Peruvian adults. Methods This population based study of 1,525 individuals (569 men and 956 women; mean age 39 years old was conducted among residents in Lima and Callao, Peru. Fasting plasma glucose, insulin, and CRP concentrations were measured using standard approaches. Insulin resistance was assessed using the homeostasis model (HOMA-IR. Categories of CRP were defined by the following tertiles: 2.53 mg/l. Logistic regression procedures were employed to estimate odds ratios (OR and 95% confidence intervals (CI. Results Elevated CRP were significantly associated with increased mean fasting insulin and mean HOMA-IR concentrations (p 2.53 mg/l (upper tertile had a 2.18-fold increased risk of IR (OR = 2.18 95% CI 1.51-3.16 as compared with those in the lowest tertile ( Conclusion Our observations among Peruvians suggest that chronic systemic inflammation, as evidenced by elevated CRP, may be of etiologic importance in insulin resistance and diabetes.

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

    Science.gov (United States)

    Nyström, Sofie; Hammarström, Per

    2015-05-11

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

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

    Directory of Open Access Journals (Sweden)

    Melisa Edith Gantner

    2013-01-01

    Full Text Available 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.

  6. Loss of regulator of G protein signaling 5 exacerbates obesity, hepatic steatosis, inflammation and insulin resistance.

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    Wei Deng

    Full Text Available BACKGROUND: The effect of regulator of G protein signaling 5 (RGS5 on cardiac hypertrophy, atherosclerosis and angiogenesis has been well demonstrated, but the role in the development of obesity and insulin resistance remains completely unknown. We determined the effect of RGS5 deficiency on obesity, hepatic steatosis, inflammation and insulin resistance in mice fed either a normal-chow diet (NC or a high-fat diet (HF. METHODOLOGY/PRINCIPAL FINDINGS: Male, 8-week-old RGS5 knockout (KO and littermate control mice were fed an NC or an HF for 24 weeks and were phenotyped accordingly. RGS5 KO mice exhibited increased obesity, fat mass and ectopic lipid deposition in the liver compared with littermate control mice, regardless of diet. When fed an HF, RGS5 KO mice had a markedly exacerbated metabolic dysfunction and inflammatory state in the blood serum. Meanwhile, macrophage recruitment and inflammation were increased and these increases were associated with the significant activation of JNK, IκBα and NF-κBp65 in the adipose tissue, liver and skeletal muscle of RGS5 KO mice fed an HF relative to control mice. These exacerbated metabolic dysfunction and inflammation are accompanied with decreased systemic insulin sensitivity in the adipose tissue, liver and skeletal muscle of RGS5 KO mice, reflected by weakened Akt/GSK3β phosphorylation. CONCLUSIONS/SIGNIFICANCE: Our data suggest that loss of RGS5 exacerbates HF-induced obesity, hepatic steatosis, inflammation and insulin resistance.

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

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    Shiori Sekine

    2016-03-01

    Full Text Available 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.

  9. Anti-Restriction Protein, KlcAHS, Promotes Dissemination of Carbapenem Resistance

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    Xiaofei Jiang

    2017-05-01

    Full Text Available Carbapenemase-producing Klebsiella pneumoniae (KPC has emerged and spread throughout the world. A retrospective analysis was performed on carbapenem-resistant K. pneumoniae isolated at our teaching hospital during the period 2009–2010, when the initial outbreak occurred. To determine the mechanism(s that underlies the increased infectivity exhibited by KPC, Multilocus Sequence Typing (MLST was conducted. A series of plasmids was also extracted, sequenced and analyzed. Concurrently, the complete sequences of blaKPC−2-harboring plasmids deposited in GenBank were summarized and aligned. The blaKPC−2 and KlcAHS genes in the carbapenem-resistant K. pneumoniae isolates were examined. E. coli strains, carrying different Type I Restriction and Modification (RM systems, were selected to study the interaction between RM systems, anti-RM systems and horizontal gene transfer (HGT. The ST11 clone predominated among 102 carbapenem-resistant K. pneumoniae isolates, all harbored the blaKPC−2 gene; 98% contained the KlcAHS gene. KlcAHS was one of the core genes in the backbone region of most blaKPC−2 carrying plasmids. Type I RM systems in the host bacteria reduced the rate of pHS10842 plasmid transformation by 30- to 40-fold. Presence of the anti-restriction protein, KlcAHS, on the other hand, increased transformation efficiency by 3- to 6-fold. These results indicate that RM systems can significantly restrict HGT. In contrast, KlcAHS can disrupt the RM systems and promote HGT by transformation. These findings suggest that the anti-restriction protein, KlcAHS, represents a novel mechanism that facilitates the increased transfer of blaKPC-2 and KlcAHS-carrying plasmids among K. pneumoniae strains.

  10. The 1p-encoded protein stathmin and resistance of malignant gliomas to nitrosoureas.

    Science.gov (United States)

    Ngo, Teri-T B; Peng, Tien; Liang, Xing-Jie; Akeju, Oluwaseun; Pastorino, Sandra; Zhang, Wei; Kotliarov, Yuri; Zenklusen, Jean C; Fine, Howard A; Maric, Dragan; Wen, Patrick Y; De Girolami, Umberto; Black, Peter McL; Wu, Wells W; Shen, Rong-Fong; Jeffries, Neal O; Kang, Dong-Won; Park, John K

    2007-04-18

    Malignant gliomas are generally resistant to all conventional therapies. Notable exceptions are anaplastic oligodendrogliomas with loss of heterozygosity on chromosome 1p (1p+/-). Patients with 1p+/- anaplastic oligodendroglioma frequently respond to procarbazine, 1-(2-chloroethyl)-3-cyclohexyl-l-nitrosourea, and vincristine. Because the underlying biologic basis for this clinical finding is unclear, we evaluated differentially expressed 1p-encoded proteins in 1p+/- and 1p+/+ malignant glioma cell lines and then examined whether their expression was associated with outcome of patients with anaplastic oligodendroglioma. We used a comparative proteomic screen of A172 (1p+/-) and U251 (1p+/+) malignant glioma cell lines to identify differentially expressed 1p-encoded proteins, including stathmin, a microtubule-associated protein. 1p+/- and 1p+/+ anaplastic oligodendroglioma specimens from 24 patients were assessed for stathmin expression by immunohistochemistry. The relationship between stathmin expression and clinical outcome was assessed with Kaplan-Meier analyses. RNA inhibition and cDNA transfection experiments tested effects of stathmin under- and overexpression, respectively, on the in vitro and in vivo resistance of malignant glioma cells to treatment with nitrosourea. For in vivo resistance studies, 36 mice with intracranial and 16 mice with subcutaneous xenograft tumor implants were used (one tumor per mouse). Flow cytometry was used for cell cycle analysis. Immunoblotting was used to assess protein expression. All statistical tests were two-sided. Decreased stathmin expression in tumors was statistically significantly associated with loss of heterozygosity in 1p (Pnitrosourea-treated mice carrying xenograft tumors. Median survival of mice with stathmin+/- tumors was 95 days (95% CI = 68.7 to 121.3 days) and that of mice with stathmin+/+ tumors was 64 days (95% CI = 58.2 to 69.8 days) (difference = 31 days, 95% CI = 4.1 to 57.9 days; PNitrosoureas induced

  11. Comparative genomics of Fusarium oxysporum f. sp. melonis reveals the secreted protein recognized by the Fom-2 resistance gene in melon

    NARCIS (Netherlands)

    Schmidt, S.M.; Lukasiewicz, J.; Farrer, R.; van Dam, P.; Bertoldo, C.; Rep, M.

    Development of resistant crops is the most effective way to control plant diseases to safeguard food and feed production. Disease resistance is commonly based on resistance genes, which generally mediate the recognition of small proteins secreted by invading pathogens. These proteins secreted by

  12. Decreased UV light resistance of spores of Bacillus subtilis strains deficient in pyrimidine dimer repair and small, acid-soluble spore proteins

    International Nuclear Information System (INIS)

    Setlow, B.; Setlow, P.

    1988-01-01

    Loss of small, acid-soluble spore protein alpha reduced spore UV resistance 30- to 50-fold in Bacillus subtilis strains deficient in pyrimidine dimer repair, but gave only a 5- to 8-fold reduction in UV resistance in repair-proficient strains. However, both repair-proficient and -deficient spores lacking this protein had identical heat and gamma-radiation resistance

  13. Involvement of p38 mitogen-activated protein kinase in acquired gemcitabine-resistant human urothelial carcinoma sublines

    Directory of Open Access Journals (Sweden)

    Yu-Ting Kao

    2014-07-01

    Full Text Available Resistance to chemotherapeutic drugs is one of the major challenges in the treatment of cancer. A better understanding of how resistance arises and what molecular alterations correlate with resistance is the key to developing novel effective therapeutic strategies. To investigate the underlying mechanisms of gemcitabine (Gem resistance and provide possible therapeutic options, three Gem-resistant urothelial carcinoma sublines were established (NG0.6, NG0.8, and NG1.0. These cells were cross-resistant to arabinofuranosyl cytidine and cisplatin, but sensitive to 5-fluorouracil. The resistant cells expressed lower values of [hENT1 × dCK/RRM1 × RRM2] mRNA ratio. Two adenosine triphosphate-binding cassette proteins ABCD1 as well as multidrug resistance protein 1 were elevated. Moreover, cyclin D1, cyclin-dependent kinases 2 and 4 were upregulated, whereas extracellular signal-regulated kinase 1/2 and p38 mitogen-activated protein kinase (MAPK activity were repressed significantly. Administration of p38 MAPK inhibitor significantly reduced the Gem sensitivity in NTUB1 cells, whereas that of an extracellular signal-regulated kinase MAPK inhibitor did not. Furthermore, the Gem-resistant sublines also exhibited higher migration ability. Forced expression of p38 MAPK impaired the cell migration activity and augmented Gem sensitivity in NG1.0 cells. Taken together, these results demonstrate that complex mechanisms were merged in acquiring Gem resistance and provide information that can be important for developing therapeutic targets for treating Gem-resistant tumors.

  14. Bodipy-FL-Verapamil: A Fluorescent Probe for the Study of Multidrug Resistance Proteins

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    Anna Rosati

    2004-01-01

    Full Text Available Most of the substances used as fluorescent probes to study drug transport and the effect of efflux blockers in multidrug resistant cells have many drawbacks, such as toxicity, unspecific background, accumulation in mitochondria. New fluorescent compounds, among which Bodipy‐FL‐verapamil (BV, have been therefore proposed as more useful tools. The uptake of BV has been evaluated by cytofluorimetry and fluorescence microscopy using cell lines that overexpress P‐glycoprotein (P388/ADR and LLC‐PK1/ADR or MRP (multidrug resistance‐related protein (PANC‐1 and clinical specimens from patients. The effect of specific inhibitors for P‐glycoprotein (verapamil and vinblastine or MRP (MK571 and probenecid has been also studied. BV intracellular concentrations were significantly lower in the two P‐glycoprotein overexpressing cell lines in comparison with the parental lines. In addition, verapamil and vinblastine increased the intracellular concentrations of the dye; MK571 and probenecid, two MRP inhibitors, increased BV levels in PANC‐1 cells, that express this protein. These findings were confirmed in clinical specimens from patients. Fluorescence microscopy revealed a faint fluorescence emission in P‐glycoprotein or MRP expressing cell lines; however, treatment with specific inhibitors significantly increased the fluorescence. BV is a useful tool for studying multidrug resistance proteins with different techniques such as cytofluorimetry and fluorescence microscopy, but does not discriminate between P‐glycoprotein and MRP. In comparison with other classic fluorescent probes, the assay with this dye is extremely rapid, simple, not toxic for cells, devoid of fluorescent background, and can be useful in the clinical settings.

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

    Science.gov (United States)

    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.

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

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    Isabelle Bleiziffer

    2017-01-01

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

  17. Reduced expression of Jak-1 and Tyk-2 proteins leads to interferon resistance in Hepatitis C virus replicon

    Directory of Open Access Journals (Sweden)

    Luftig Ronald

    2007-09-01

    Full Text Available Abstract Background Alpha interferon in combination with ribavirin is the standard therapy for hepatitis C virus infection. Unfortunately, a significant number of patients fail to eradicate their infection with this regimen. The mechanisms of IFN-resistance are unclear. The aim of this study was to determine the contribution of host cell factors to the mechanisms of interferon resistance using replicon cell lines. Results HCV replicons with high and low activation of the IFN-promoter were cultured for a prolonged period of time in the presence of interferon-alpha (IFN-alpha2b. Stable replicon cell lines with resistant phenotype were isolated and characterized by their ability to continue viral replication in the presence of IFN-alpha. Interferon resistant cell colonies developed only in replicons having lower activation of the IFN promoter and no resistant colonies arose from replicons that exhibit higher activation of the IFN promoter. Individual cell clones were isolated and nine IFN resistant cell lines were established. HCV RNA and protein levels in these cells were not altered by IFN- alpha2b. Reduced signaling and IFN-resistant phenotype was found in all Huh-7 cell lines even after eliminating HCV, suggesting that cellular factors are involved. Resistant phenotype in the replicons is not due to lack of interferon receptor expression. All the cell lines show defect in the JAK-STAT signaling and phosphorylation of STAT 1 and STAT 2 proteins were strongly inhibited due to reduced expression of Tyk2 and Jak-1 protein. Conclusion This in vitro study provides evidence that altered expression of the Jak-Stat signaling proteins can cause IFN resistance using HCV replicon cell clones.

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

    Science.gov (United States)

    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. © 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

  19. Comparative sequence analysis of acid sensitive/resistance proteins in Escherichia coli and Shigella flexneri

    Science.gov (United States)

    Manikandan, Selvaraj; Balaji, Seetharaaman; Kumar, Anil; Kumar, Rita

    2007-01-01

    The molecular basis for the survival of bacteria under extreme conditions in which growth is inhibited is a question of great current interest. A preliminary study was carried out to determine residue pattern conservation among the antiporters of enteric bacteria, responsible for extreme acid sensitivity especially in Escherichia coli and Shigella flexneri. Here we found the molecular evidence that proved the relationship between E. coli and S. flexneri. Multiple sequence alignment of the gadC coded acid sensitive antiporter showed many conserved residue patterns at regular intervals at the N-terminal region. It was observed that as the alignment approaches towards the C-terminal, the number of conserved residues decreases, indicating that the N-terminal region of this protein has much active role when compared to the carboxyl terminal. The motif, FHLVFFLLLGG, is well conserved within the entire gadC coded protein at the amino terminal. The motif is also partially conserved among other antiporters (which are not coded by gadC) but involved in acid sensitive/resistance mechanism. Phylogenetic cluster analysis proves the relationship of Escherichia coli and Shigella flexneri. The gadC coded proteins are converged as a clade and diverged from other antiporters belongs to the amino acid-polyamine-organocation (APC) superfamily. PMID:21670792

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

    International Nuclear Information System (INIS)

    McBride, Corrin E.; Machamer, Carolyn E.

    2010-01-01

    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.

  1. Effect of administration of high-protein diet in rats submitted to resistance training.

    Science.gov (United States)

    da Rosa Lima, Thiago; Ávila, Eudes Thiago Pereira; Fraga, Géssica Alves; de Souza Sena, Mariana; de Souza Dias, Arlyson Batista; de Almeida, Paula Caroline; Dos Santos Trombeta, Joice Cristina; Junior, Roberto Carlos Vieira; Damazo, Amílcar Sabino; Navalta, James Wilfred; Prestes, Jonato; Voltarelli, Fabrício Azevedo

    2018-04-01

    Although there is limited evidence regarding the pathophysiological effects of a high-protein diet (HD), it is believed that this type of diet could overload the body and cause damage to the organs directly involved with protein metabolism and excretion. The aim of this study was to verify the effects of HD on biochemical and morphological parameters of rats that completed a resistance training protocol (RT; aquatic jump) for 8 weeks. Thirty-two adult male Wistar rats were divided into four groups (n = 8 for each group): sedentary normal protein diet (SN-14%), sedentary high-protein diet (SH-35%), trained normal protein diet (TN-14%), and trained high-protein diet (TH-35%). Biochemical, tissue, and morphological measurements were made. Kidney (1.91 ± 0.34) and liver weights (12.88 ± 1.42) were higher in the SH. Soleus muscle weight was higher in the SH (0.22 ± 0.03) when compared to all groups. Blood glucose (123.2 ± 1.8), triglycerides (128.5 ± 44.0), and HDL cholesterol levels (65.7 ± 20.9) were also higher in the SH compared with the other experimental groups. Exercise reduced urea levels in the trained groups TN and TH (31.0 ± 4.1 and 36.8 ± 6.6), respectively. Creatinine levels were lower in TH and SH groups (0.68 ± 0.12; 0.54 ± 0.19), respectively. HD negatively altered renal morphology in SH, but when associated with RT, the apparent damage was partially reversed. In addition, the aquatic jump protocol reversed the damage to the gastrocnemius muscle caused by the HD. A high-protein diet promoted negative metabolic and morphological changes, while RT was effective in reversing these deleterious effects.

  2. No Difference Between the Effects of Supplementing With Soy Protein Versus Animal Protein on Gains in Muscle Mass and Strength in Response to Resistance Exercise.

    Science.gov (United States)

    Messina, Mark; Lynch, Heidi; Dickinson, Jared M; Reed, Katharine E

    2018-05-03

    Much attention has been given to determining the influence of total protein intake and protein source on gains in lean body mass (LBM) and strength in response to resistance exercise training (RET). Acute studies indicate that whey protein, likely related to its higher leucine content, stimulates muscle protein synthesis (MPS) to a greater extent than proteins such as soy and casein. Less clear is the extent to which the type of protein supplemented impacts strength and LBM in longer term studies (≥6 weeks). Therefore, a meta-analysis was conducted to compare the effect of supplementation with soy protein to animal protein supplementation on strength and LBM in response to RET. Nine studies involving 266 participants suitable for inclusion in the meta-analysis were identified. Five studies compared whey with soy protein and four compared soy protein with other proteins (beef, milk or dairy protein). Meta-analysis showed that supplementing RET with whey or soy protein resulted in significant increases in strength but found no difference between groups (bench press Chi 2 = 0.02, p=0.90; squat Chi 2 =0.22, p =0.64). There was no significant effect of whey or soy alone (n=5) on LBM change, and no differences between groups (Chi 2 =0.00, p=0.96). Strength and LBM both increased significantly in the 'other protein' and the soy groups (n=9), but there were no between group differences (bench Chi 2 =0.02, p=0.88; squat Chi 2 =0.78, p=0.38 and LBM Chi 2 =0.06, p=0.80). The results of this meta-analysis indicate that soy protein supplementation produces similar gains in strength and LBM in response to RET as whey protein.

  3. Role of G-protein-coupled receptor-related genes in insecticide resistance of the mosquito, Culex quinquefasciatus.

    Science.gov (United States)

    Li, Ting; Liu, Lena; Zhang, Lee; Liu, Nannan

    2014-09-29

    G-protein-coupled receptors regulate signal transduction pathways and play diverse and pivotal roles in the physiology of insects, however, the precise function of GPCRs in insecticide resistance remains unclear. Using quantitative RT-PCR and functional genomic methods, we, for the first time, explored the function of GPCRs and GPCR-related genes in insecticide resistance of mosquitoes, Culex quinquefasciatus. A comparison of the expression of 115 GPCR-related genes at a whole genome level between resistant and susceptible Culex mosquitoes identified one and three GPCR-related genes that were up-regulated in highly resistant Culex mosquito strains, HAmCq(G8) and MAmCq(G6), respectively. To characterize the function of these up-regulated GPCR-related genes in resistance, the up-regulated GPCR-related genes were knockdown in HAmCq(G8) and MAmCq(G6) using RNAi technique. Knockdown of these four GPCR-related genes not only decreased resistance of the mosquitoes to permethrin but also repressed the expression of four insecticide resistance-related P450 genes, suggesting the role of GPCR-related genes in resistance is involved in the regulation of resistance P450 gene expression. This results help in understanding of molecular regulation of resistance development in Cx. quinquefasciatus.

  4. Effects of Insect Protein Supplementation during Resistance Training on Changes in Muscle Mass and Strength in Young Men

    Directory of Open Access Journals (Sweden)

    Mathias T. Vangsoe

    2018-03-01

    Full Text Available During prolonged resistance training, protein supplementation is known to promote morphological changes; however, no previous training studies have tested the effect of insect protein isolate in a human trial. The aim of this study was to investigate the potential effect of insect protein as a dietary supplement to increase muscle hypertrophy and strength gains during prolonged resistance training in young men. Eighteen healthy young men performed resistance training four day/week for eight weeks. Subjects were block randomized into two groups consuming either an insect protein isolate or isocaloric carbohydrate supplementation within 1 h after training and pre-sleep on training days. Strength and body composition were measured before and after intervention to detect adaptions to the resistance training. Three-day weighed dietary records were completed before and during intervention. Fat- and bone- free mass (FBFM improved significantly in both groups (Mean (95% confidence interval (CI, control group (Con: (2.5 kg (1.5, 3.5 p < 0.01, protein group (Pro: (2.7 kg (1.6, 3.8 p < 0.01 from pre- to post-. Leg and bench press one repetition maximum (1 RM improved by Con: (42.0 kg (32.0, 52.0 p < 0.01 and (13.8 kg (10.3, 17.2 p < 0.01, Pro: (36.6 kg (27.3, 45.8 p < 0.01 and (8.1 kg (4.5, 11.8 p < 0.01, respectively. No significant differences in body composition and muscle strength improvements were found between groups. In young healthy men, insect protein supplementation did not improve adaptations to eight weeks of resistance training in comparison to carbohydrate supplementation. A high habitual protein intake in both Con and Pro may partly explain our observation of no superior effect of insect protein supplementation.

  5. C-reactive protein levels and treatment resistance in schizophrenia - A Danish population-based cohort study

    DEFF Research Database (Denmark)

    Horsdal, Henriette Thisted; Wimberley, Theresa; Benros, Michael Eriksen

    2017-01-01

    -time schizophrenia diagnosis and a baseline C-reactive protein measurement (a commonly available marker of systemic inflammation) from 2000 to 2012. We defined treatment resistance as the earliest observed instance of either clozapine initiation or hospital admission due to schizophrenia after having received......OBJECTIVE: Schizophrenia is associated with increased levels of inflammatory markers. However, it remains unclear whether inflammatory markers are associated with treatment-resistant schizophrenia. METHODS: We conducted a population-based follow-up study among individuals with a first...... (4.0 vs. 3.1 mg/L, p = .13) was observed among the 52 (13.3%) treatment-resistant individuals. Increased levels of C-reactive protein (above 3 mg/L) at baseline were not associated with treatment resistance (adjusted hazard ratio = 0.99, 95% confidence interval [0.56, 1.73]). CONCLUSIONS: C...

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

    Science.gov (United States)

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

    2009-07-01

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

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

    Science.gov (United States)

    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

  8. Localization of multidrug resistance-associated protein 2 in the nonpigmented ciliary epithelium of the eye.

    Science.gov (United States)

    Pelis, Ryan M; Shahidullah, Mohammad; Ghosh, Sikha; Coca-Prados, Miguel; Wright, Stephen H; Delamere, Nicholas A

    2009-05-01

    The nonpigmented epithelium (NPE) of the ciliary body represents an important component of the blood-aqueous barrier of the eye. Many therapeutic drugs penetrate poorly across the NPE into the aqueous humor of the eye interior. Several of these therapeutic drugs, such as methotrexate, vincristine, and etoposide, are substrates of the multidrug resistance-associated protein 2 (MRP2). Abundant MRP2 protein was detected by Western blot in homogenates of human ciliary body and freshly dissected porcine NPE. In cultured porcine NPE, the intracellular accumulation of the MRP2 substrates calcein (1.8-fold), 5-(and-6)-carboxy-2',7'-dichlorofluorescein (22.1-fold), and doxorubicin (1.9-fold) was significantly increased in the presence of 50 microM MK571 ((E)-3-[[[3-[2-(7-chloro-2-quinolinyl)-ethenyl]phenyl]-[[3-dimethylamino)-3-oxopropyl]thio]methyl]thio]-propanoic acid), an MRP inhibitor. In addition, the intracellular accumulation of the MRP2 substrate glutathione methylfluorescein was increased by 50 microM MK571 (4.3-fold), 500 microM indomethacin (2.6-fold), and 50 microM cyclosporin A (2.1-fold) but not by 500 microM sulfinpyrazone. These data are consistent with MRP2-mediated transport activity in cultured NPE, and MRP2 mRNA (reverse transcriptase-polymerase chain reaction) and protein (Western blot) were detected in the cultured cells. Immunolocalization studies in native human and porcine eyes showed MRP2 protein at the apical interface of the NPE and pigmented cell layers. Close examination of MRP2 immunoreactivity supported the conclusion that MRP2 is localized in the apical membrane of the NPE. MRP2 at the apical membrane of NPE cells may be involved in protecting intraocular tissues from exposure to potentially harmful toxins.

  9. Initial infection of roots and leaves reveals different resistance phenotypes associated with coat protein gene-mediated resistance to Potato mop-top virus.

    Science.gov (United States)

    Germundsson, Anna; Sandgren, Maria; Barker, Hugh; Savenkov, Eugene I; Valkonen, Jari P T

    2002-05-01

    Resistance to the pomovirus Potato mop-top virus (PMTV) was studied in potato (Solanum tuberosum cv. Saturna) and Nicotiana benthamiana transformed with the coat protein (CP) gene of PMTV. The incidence of PMTV infections was reduced in tubers of the CP-transgenic potatoes grown in the field in soil infested with the viruliferous vector, Spongospora subterranea. However, in those tubers that were infected, all three virus RNAs were detected and virus titres were high. The CP-transgenic N. benthamiana plants were inoculated with PMTV using two methods. Following mechanical inoculation of leaves, no RNA 3 (the CP-encoding RNA homologous to the transgene) was detected in leaves, but in some plants low amounts of RNA 3 were detected in roots; RNA 2 was readily detected in leaves and roots of several plants. Inoculation of roots using viruliferous S. subterranea resulted in infection of roots in all plants and the three PMTV RNAs were detected. However, no systemic movement of PMTV from roots to the above-ground parts was observed, indicating a novel expression of resistance. These data indicate that the CP gene-mediated resistance to PMTV specifically restricts accumulation of PMTV RNA 3, and is more effective in leaves than roots. Furthermore, expression of resistance is different depending on whether leaves or roots are inoculated. Data do not exclude the possibility that both a protein-mediated and an RNA-mediated resistance mechanism are involved.

  10. Plasmid-Mediated Resistance to Thrombin-Induced Platelet Microbicidal Protein in Staphylococci: Role of the qacA Locus

    OpenAIRE

    Kupferwasser, Leon Iri; Skurray, Ronald A.; Brown, Melissa H.; Firth, Neville; Yeaman, Michael R.; Bayer, Arnold S.

    1999-01-01

    Thrombin-induced platelet microbicidal protein 1 (tPMP-1) is a small, cationic peptide released from rabbit platelets following thrombin stimulation. In vitro resistance to this peptide among strains of Staphylococcus aureus correlates with the survival advantage of such strains at sites of endothelial damage in humans as well as in experimental endovascular infections. The mechanisms involved in the phenotypic resistance of S. aureus to tPMP-1 are not fully delineated. The plasmid-encoded st...

  11. C-reactive protein, insulin resistance and risk of cardiovascular disease: a population-based study

    DEFF Research Database (Denmark)

    Jeppesen, Jørgen; Hansen, Tine Willum; Olsen, Michael H

    2008-01-01

    were recorded at baseline. CRP was determined by a high-sensitivity assay, and IR was determined by the homoeostasis model assessment (HOMA-IR) method. RESULTS: Over a median follow-up of 9.4 years, the incidence of the prespecified CV event, defined as the composite event of CV death, nonfatal...... and HOMA-IR, the hazard ratio (95% confidence interval) of a CV event was 1.33 (1.14-1.55; PHOMA-IR level. CONCLUSION......BACKGROUND: C-reactive protein (CRP), a marker of inflammation, and insulin resistance (IR), a metabolic disorder, are closely related. CRP and IR have both been identified as significant risk factors of cardiovascular disease (CVD) after adjustment for conventional CVD risk factors...

  12. Coexistence of protease sensitive and resistant prion protein in 129VV homozygous sporadic Creutzfeldt–Jakob disease: a case report

    Directory of Open Access Journals (Sweden)

    Rodríguez-Martínez Ana B

    2012-10-01

    Full Text Available Abstract Introduction The coexistence of different molecular types of classical protease-resistant prion protein in the same individual have been described, however, the simultaneous finding of these with the recently described protease-sensitive variant or variably protease-sensitive prionopathy has, to the best of our knowledge, not yet been reported. Case presentation A 74-year-old Caucasian woman showed a sporadic Creutzfeldt–Jakob disease clinical phenotype with reactive depression, followed by cognitive impairment, akinetic-rigid Parkinsonism with pseudobulbar syndrome and gait impairment with motor apraxia, visuospatial disorientation, and evident frontal dysfunction features such as grasping, palmomental reflex and brisk perioral reflexes. She died at age 77. Neuropathological findings showed: spongiform change in the patient’s cerebral cortex, striatum, thalamus and molecular layer of the cerebellum with proteinase K-sensitive synaptic-like, dot-like or target-like prion protein deposition in the cortex, thalamus and striatum; proteinase K-resistant prion protein in the same regions; and elongated plaque-like proteinase K-resistant prion protein in the molecular layer of the cerebellum. Molecular analysis of prion protein after proteinase K digestion revealed decreased signal intensity in immunoblot, a ladder-like protein pattern, and a 71% reduction of PrPSc signal relative to non-digested material. Her cerebellum showed a 2A prion protein type largely resistant to proteinase K. Genotype of polymorphism at codon 129 was valine homozygous. Conclusion Molecular typing of prion protein along with clinical and neuropathological data revealed, to the best of our knowledge, the first case of the coexistence of different protease-sensitive prion proteins in the same patient in a rare case that did not fulfill the current clinical diagnostic criteria for either probable or possible sporadic Creutzfeldt–Jakob disease. This highlights the

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

    Directory of Open Access Journals (Sweden)

    Sylvie Cornelie

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

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

    Science.gov (United States)

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

    2007-01-01

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

  15. Identification of distinct specificity determinants in resistance protein Cf-4 allows construction of a Cf-9 mutant that confers recognition of avirulence protein AVR4

    NARCIS (Netherlands)

    Hoorn, Van der R.A.L.; Roth, R.; Wit, De P.J.G.M.

    2001-01-01

    The tomato resistance genes Cf-4 and Cf-9 confer specific, hypersensitive response-associated recognition of Cladosporium carrying the avirulence genes Avr4 and Avr9, respectively. Cf-4 and Cf-9 encode type I transmembrane proteins with extracellular leucine-rich repeats (LRRs). Compared with Cf-9,

  16. Pronounced energy restriction with elevated protein intake results in no change in proteolysis and reductions in skeletal muscle protein synthesis that are mitigated by resistance exercise.

    Science.gov (United States)

    Hector, Amy J; McGlory, Chris; Damas, Felipe; Mazara, Nicole; Baker, Steven K; Phillips, Stuart M

    2018-01-01

    Preservation of lean body mass (LBM) may be important during dietary energy restriction (ER) and requires equal rates of muscle protein synthesis (MPS) and muscle protein breakdown (MPB). Currently, the relative contribution of MPS and MPB to the loss of LBM during ER in humans is unknown. We aimed to determine the impact of dietary protein intake and resistance exercise on MPS and MPB during a controlled short-term energy deficit. Adult men (body mass index, 28.6 ± 0.6 kg/m 2 ; age 22 ± 1 yr) underwent 10 d of 40%-reduced energy intake while performing unilateral resistance exercise and consuming lower protein (1.2 g/kg/d, n = 12) or higher protein (2.4 g/kg/d, n = 12). Pre- and postintervention testing included dual-energy X-ray absorptiometry, primed constant infusion of ring -[ 13 C 6 ]phenylalanine, and 15 [N]phenylalanine to measure acute postabsorptive MPS and MPB; D 2 O to measure integrated MPS; and gene and protein expression. There was a decrease in acute MPS after ER (higher protein, 0.059 ± 0.006 to 0.051 ± 0.009%/h; lower protein, 0.061 ± 0.005 to 0.045 ± 0.006%/h; P resistance exercise (higher protein, 0.067 ± 0.01%/h; lower protein, 0.061 ± 0.006%/h), and integrated MPS followed a similar pattern. There was no change in MPB (energy balance, 0.080 ± 0.01%/hr; ER rested legs, 0.078 ± 0.008%/hr; ER exercised legs, 0.079 ± 0.006%/hr). We conclude that a reduction in MPS is the main mechanism that underpins LBM loss early in ER in adult men.-Hector, A. J., McGlory, C., Damas, F., Mazara, N., Baker, S. K., Phillips, S. M. Pronounced energy restriction with elevated protein intake results in no change in proteolysis and reductions in skeletal muscle protein synthesis that are mitigated by resistance exercise. © FASEB.

  17. A secretory protein of necrotrophic fungus Sclerotinia sclerotiorum that suppresses host resistance.

    Directory of Open Access Journals (Sweden)

    Wenjun Zhu

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

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

    Science.gov (United States)

    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. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.

  19. Overcoming chemotherapy drug resistance by targeting inhibitors of apoptosis proteins (IAPs).

    Science.gov (United States)

    Rathore, Rama; McCallum, Jennifer E; Varghese, Elizabeth; Florea, Ana-Maria; Büsselberg, Dietrich

    2017-07-01

    Inhibitors of apoptosis (IAPs) are a family of proteins that play a significant role in the control of programmed cell death (PCD). PCD is essential to maintain healthy cell turnover within tissue but also to fight disease or infection. Uninhibited, IAPs can suppress apoptosis and promote cell cycle progression. Therefore, it is unsurprising that cancer cells demonstrate significantly elevated expression levels of IAPs, resulting in improved cell survival, enhanced tumor growth and subsequent metastasis. Therapies to target IAPs in cancer has garnered substantial scientific interest and as resistance to anti-cancer agents becomes more prevalent, targeting IAPs has become an increasingly attractive strategy to re-sensitize cancer cells to chemotherapies, antibody based-therapies and TRAIL therapy. Antagonism strategies to modulate the actions of XIAP, cIAP1/2 and survivin are the central focus of current research and this review highlights advances within this field with particular emphasis upon the development and specificity of second mitochondria-derived activator of caspase (SMAC) mimetics (synthetic analogs of endogenously expressed inhibitors of IAPs SMAC/DIABLO). While we highlight the potential of SMAC mimetics as effective single agent or combinatory therapies to treat cancer we also discuss the likely clinical implications of resistance to SMAC mimetic therapy, occasionally observed in cancer cell lines.

  20. The Escherichia coli BtuE protein functions as a resistance determinant against reactive oxygen species.

    Directory of Open Access Journals (Sweden)

    Felipe A Arenas

    2011-01-01

    Full Text Available This work shows that the recently described Escherichia coli BtuE peroxidase protects the bacterium against oxidative stress that is generated by tellurite and by other reactive oxygen species elicitors (ROS. Cells lacking btuE (ΔbtuE displayed higher sensitivity to K(2TeO(3 and other oxidative stress-generating agents than did the isogenic, parental, wild-type strain. They also exhibited increased levels of cytoplasmic reactive oxygen species, oxidized proteins, thiobarbituric acid reactive substances, and lipoperoxides. E. coli ΔbtuE that was exposed to tellurite or H(2O(2 did not show growth changes relative to wild type cells either in aerobic or anaerobic conditions. Nevertheless, the elimination of btuE from cells deficient in catalases/peroxidases (Hpx(- resulted in impaired growth and resistance to these toxicants only in aerobic conditions, suggesting that BtuE is involved in the defense against oxidative damage. Genetic complementation of E. coli ΔbtuE restored toxicant resistance to levels exhibited by the wild type strain. As expected, btuE overexpression resulted in decreased amounts of oxidative damage products as well as in lower transcriptional levels of the oxidative stress-induced genes ibpA, soxS and katG.

  1. Influence of multidrug resistance and drug transport proteins on chemotherapy drug metabolism.

    Science.gov (United States)

    Joyce, Helena; McCann, Andrew; Clynes, Martin; Larkin, Annemarie

    2015-05-01

    Chemotherapy involving the use of anticancer drugs remains an important strategy in the overall management of patients with metastatic cancer. Acquisition of multidrug resistance remains a major impediment to successful chemotherapy. Drug transporters in cell membranes and intracellular drug metabolizing enzymes contribute to the resistance phenotype and determine the pharmacokinetics of anticancer drugs in the body. ATP-binding cassette (ABC) transporters mediate the transport of endogenous metabolites and xenobiotics including cytotoxic drugs out of cells. Solute carrier (SLC) transporters mediate the influx of cytotoxic drugs into cells. This review focuses on the substrate interaction of these transporters, on their biology and what role they play together with drug metabolizing enzymes in eliminating therapeutic drugs from cells. The majority of anticancer drugs are substrates for the ABC transporter and SLC transporter families. Together, these proteins have the ability to control the influx and the efflux of structurally unrelated chemotherapeutic drugs, thereby modulating the intracellular drug concentration. These interactions have important clinical implications for chemotherapy because ultimately they determine therapeutic efficacy, disease progression/relapse and the success or failure of patient treatment.

  2. Alpha-tocopherol transfer protein disruption confers resistance to malarial infection in mice

    Directory of Open Access Journals (Sweden)

    Takeya Motohiro

    2010-04-01

    Full Text Available Abstract Background Various factors impact the severity of malaria, including the nutritional status of the host. Vitamin E, an intra and extracellular anti-oxidant, is one such nutrient whose absence was shown previously to negatively affect Plasmodium development. However, mechanisms of this Plasmodium inhibition, in addition to means by which to exploit this finding as a therapeutic strategy, remain unclear. Methods α-TTP knockout mice were infected with Plasmodium berghei NK65 or Plasmodium yoelii XL-17, parasitaemia, survival rate were monitored. In one part of the experiments mice were fed with a supplemented diet of vitamin E and then infected. In addition, parasite DNA damage was monitored by means of comet assay and 8-OHdG test. Moreover, infected mice were treated with chloroquine and parasitaemia and survival rate were monitored. Results Inhibition of α-tocopherol transfer protein (α-TTP, a determinant of vitamin E concentration in circulation, confers resistance to malarial infection as a result of oxidative damage to the parasites. Furthermore, in combination with the anti-malarial drug chloroquine results were even more dramatic. Conclusion Considering that these knockout mice lack observable negative impacts typical of vitamin E deficiency, these results suggest that inhibition of α-TTP activity in the liver may be a useful strategy in the prevention and treatment of malaria infection. Moreover, a combined strategy of α-TTP inhibition and chloroquine treatment might be effective against drug resistant parasites.

  3. Enhanced disease resistance and drought tolerance in transgenic rice plants overexpressing protein elicitors from Magnaporthe oryzae.

    Science.gov (United States)

    Wang, Zhenzhen; Han, Qiang; Zi, Qian; Lv, Shun; Qiu, Dewen; Zeng, Hongmei

    2017-01-01

    Exogenous application of the protein elicitors MoHrip1 and MoHrip2, which were isolated from the pathogenic fungus Magnaporthe oryzae (M. oryzae), was previously shown to induce a hypersensitive response in tobacco and to enhance resistance to rice blast. In this work, we successfully transformed rice with the mohrip1 and mohrip2 genes separately. The MoHrip1 and MoHrip2 transgenic rice plants displayed higher resistance to rice blast and stronger tolerance to drought stress than wild-type (WT) rice and the vector-control pCXUN rice. The expression of salicylic acid (SA)- and abscisic acid (ABA)-related genes was also increased, suggesting that these two elicitors may trigger SA signaling to protect the rice from damage during pathogen infection and regulate the ABA content to increase drought tolerance in transgenic rice. Trypan blue staining indicated that expressing MoHrip1 and MoHrip2 in rice plants inhibited hyphal growth of the rice blast fungus. Relative water content (RWC), water usage efficiency (WUE) and water loss rate (WLR) were measured to confirm the high capacity for water retention in transgenic rice. The MoHrip1 and MoHrip2 transgenic rice also exhibited enhanced agronomic traits such as increased plant height and tiller number.

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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. © 2015 Société Française de Pharmacologie et de Thérapeutique.

  6. The SNARE protein SNAP23 and the SNARE-interacting protein Munc18c in human skeletal muscle are implicated in insulin resistance/type 2 diabetes

    DEFF Research Database (Denmark)

    Boström, Pontus; Andersson, Linda; Vind, Birgitte

    2010-01-01

    /cytosolic compartment in the patients with the type 2 diabetes. Expression of the SNARE-interacting protein Munc18c was higher in skeletal muscle from patients with type 2 diabetes. Studies in L6 cells showed that Munc18c promoted the expression of SNAP23. CONCLUSIONS: We have translated our previous in vitro results......OBJECTIVE: Our previous studies suggest that the SNARE protein synaptosomal-associated protein of 23 kDa (SNAP23) is involved in the link between increased lipid levels and insulin resistance in cardiomyocytes. The objective was to determine whether SNAP23 may also be involved in the known...... association between lipid accumulation in skeletal muscle and insulin resistance/type 2 diabetes in humans, as well as to identify a potential regulator of SNAP23. RESEARCH DESIGN AND METHODS: We analyzed skeletal muscle biopsies from patients with type 2 diabetes and healthy, insulin-sensitive control...

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

    Science.gov (United States)

    Isshiki, Marina; Umezawa, Kazuo; Tamura, Hiroomi

    2011-01-01

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

  8. The synthesis and protein resistance of amphiphilic PDMS-b-(PDMS-g-cysteine) copolymers

    Science.gov (United States)

    Lei, Yufeng; Lin, Yaling; Zhang, Anqiang

    2017-10-01

    Zwitterionic polymers have been used to cope with nonspecific protein adsorption and bio-fouling problems for a wide range of materials, including biomedical devices, marine coatings and membrane separation. However, direct surface modification with highly water-soluble zwitterionic polymers is rather difficult due to their poor attachment to hydrophobic solid surfaces. In this work, we utilize the hydrophobic interaction to anchor zwitterionic polysiloxanes grafted with cysteine onto surfaces by adding an hydrophobic block of polydimethylsiloxanes, referred as PDMS-b-(PDMS-g-Cys)s. The synthesis involves only three steps of reactions, and the structures of each product were characterized using GPC, FT-IR and 1H NMR. The adsorption and protein resistance of PDMS-b-(PDMS-g-Cys)s on a gold surface are investigated with QCM-D. The results show that the hydrophobic interaction moieties of the additional PDMS blocks help the hydrophilic cysteine-grafted blocks stably attach and then function on the sensor. These findings suggest that the addition of hydrophobic moieties provides an effective approach to construct anti-fouling interfaces with zwitterionic polymers in aqueous solution.

  9. Histone acetylation and CREB binding protein are required for neuronal resistance against ischemic injury.

    Directory of Open Access Journals (Sweden)

    Ferah Yildirim

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

  10. Plasma phospholipid transfer protein activity is related to insulin resistance : impaired acute lowering by insulin in obese Type II diabetic patients

    NARCIS (Netherlands)

    Riemens, SC; van Tol, A; Sluiter, WJ; Dullaart, RPF

    Cholesteryl ester transfer protein (CETP) and phospholipid transfer protein (PLTP) have important functions in high density lipoprotein (HDL) metabolism. We determined the association of plasma CETP and PLTP activities (measured with exogenous' substrate assays) with insulin resistance, plasma

  11. Plasminogen stimulates propagation of protease-resistant prion protein in vitro.

    Science.gov (United States)

    Mays, Charles E; Ryou, Chongsuk

    2010-12-01

    To clarify the role of plasminogen as a cofactor for prion propagation, we conducted functional assays using a cell-free prion protein (PrP) conversion assay termed protein misfolding cyclic amplification (PMCA) and prion-infected cell lines. Here, we report that plasminogen stimulates propagation of the protease-resistant scrapie PrP (PrP(Sc)). Compared to control PMCA conducted without plasminogen, addition of plasminogen in PMCA using wild-type brain material significantly increased PrP conversion, with an EC(50) = ∼56 nM. PrP conversion in PMCA was substantially less efficient with plasminogen-deficient brain material than with wild-type material. The activity stimulating PrP conversion was specific for plasminogen and conserved in its kringle domains. Such activity was abrogated by modification of plasminogen structure and interference of PrP-plasminogen interaction. Kinetic analysis of PrP(Sc) generation demonstrated that the presence of plasminogen in PMCA enhanced the PrP(Sc) production rate to ∼0.97 U/μl/h and reduced turnover time to ∼1 h compared to those (∼0.4 U/μl/h and ∼2.5 h) obtained without supplementation. Furthermore, as observed in PMCA, plasminogen and kringles promoted PrP(Sc) propagation in ScN2a and Elk 21(+) cells. Our results demonstrate that plasminogen functions in stimulating conversion processes and represents the first cellular protein cofactor that enhances the hypothetical mechanism of prion propagation.

  12. Detection of First-Line Drug Resistance Mutations and Drug-Protein Interaction Dynamics from Tuberculosis Patients in South India.

    Science.gov (United States)

    Nachappa, Somanna Ajjamada; Neelambike, Sumana M; Amruthavalli, Chokkanna; Ramachandra, Nallur B

    2018-05-01

    Diagnosis of drug-resistant tuberculosis predominantly relies on culture-based drug susceptibility testing, which take weeks to produce a result and a more time-efficient alternative method is multiplex allele-specific PCR (MAS-PCR). Also, understanding the role of mutations in causing resistance helps better drug designing. To evaluate the ability of MAS-PCR in the detection of drug resistance and to understand the mechanism of interaction of drugs with mutant proteins in Mycobacterium tuberculosis. Detection of drug-resistant mutations using MAS-PCR and validation through DNA sequencing. MAS-PCR targeted five loci on three genes, katG 315 and inhA -15 for the drug isoniazid (INH), and rpoB 516, 526, and 531 for rifampicin (RIF). Furthermore, the sequence data were analyzed to study the effect on interaction of the anti-TB drug molecule with the target protein using in silico docking. We identified drug-resistant mutations in 8 out of 114 isolates with 2 of them as multidrug-resistant TB using MAS-PCR. DNA sequencing confirmed only six of these, recording a sensitivity of 85.7% and specificity of 99.3% for MAS-PCR. Molecular docking showed estimated free energy of binding (ΔG) being higher for RIF binding with RpoB S531L mutant. Codon 315 in KatG does not directly interact with INH but blocks the drug access to active site. We propose DNA sequencing-based drug resistance detection for TB, which is more accurate than MAS-PCR. Understanding the action of resistant mutations in disrupting the normal drug-protein interaction aids in designing effective drug alternatives.

  13. RACK1 downregulates levels of the pro-apoptotic protein Fem1b in apoptosis-resistant colon cancer cells.

    Science.gov (United States)

    Subauste, M Cecilia; Ventura-Holman, Tereza; Du, Liqin; Subauste, Jose S; Chan, Shing-Leng; Yu, Victor C; Maher, Joseph F

    2009-12-01

    Evasion of apoptosis plays an important role in colon cancer progression. Following loss of the Apc tumor suppressor gene in mice, the gene encoding Fem1b is upregulated early in neoplastic intestinal epithelium. Fem1b is a pro-apoptotic protein that interacts with Fas, TNFR1 and Apaf-1, and increased expression of Fem1b induces apoptosis of cancer cells. Fem1b is a homolog of FEM-1, a protein in Caenorhabditis elegans that is negatively regulated by ubiquitination and proteasomal degradation. To study Fem1b regulation in colon cancer progression, we used apoptotis-sensitive SW480 cells, derived from a primary colon cancer, and their isogenic, apoptosis-resistant counterparts SW620 cells, derived from a subsequent metastatic lesion in the same patient. Treatment with proteasome inhibitor increased Fem1b protein levels in SW620 cells, but not in SW480 cells. In SW620 cells we found that endogenous Fem1b co-immunoprecipitates in complexes with RACK1, a protein known to mediate ubiquitination and proteasomal degradation of other pro-apoptotic proteins and to be upregulated in colon cancer. Full-length Fem1b, or the N-terminal region of Fem1b, associated with RACK1 when co-expressed in HEK293T cells, and RACK1 stimulated ubiquitination of Fem1b. RACK1 overexpression in SW620 cells led to downregulation of Fem1b protein levels. Conversely, downregulation of RACK1 led to upregulation of Fem1b protein levels, associated with induction of apoptosis, and this apoptosis was inhibited by blocking Fem1b protein upregulation. In conclusion, RACK1 downregulates levels of the pro-apoptotic protein Fem1b in metastatic, apoptosis-resistant colon cancer cells, which may promote apoptosis-resistance during progression of colon cancer.

  14. Absence of association between major vault protein (MVP) gene polymorphisms and drug resistance in Chinese Han patients with partial epilepsy.

    Science.gov (United States)

    Zhou, Luo; Zhang, Mengqi; Long, Hongyu; Long, Lili; Xie, Yuanyuan; Liu, Zhaoqian; Kang, Jin; Chen, Qihua; Feng, Li; Xiao, Bo

    2015-11-15

    Drug resistance in epilepsy is common despite many antiepileptic drugs (AEDs) available for treatment. The development of drug resistant epilepsy may be a result of multiple factors. Several previous studies reported that the major vault protein (MVP) was significantly increased in epileptogenic brain tissues resected from patients with partial-onset seizures, indicating the possible involvement of MVP in drug resistance. In this article, we aimed to identify the association between single nucleotide polymorphisms (SNPs) of MVP gene and drug resistance of partial epilepsy in a Chinese Han population. A total of 510 patients with partial-onset seizures and 206 healthy controls were recruited. Among the patients, 222 were drug resistant and 288 were responsive. The selection of tagging SNPs was based on the Hapmap database and Haploview software and the genotyping was conducted on the Sequenom MassARRAY iPLEX platform. For the selected loci rs12149746, rs9938630 and rs4788186 in the MVP gene, there was no significant difference in allele or genotype distribution between the drug resistant and responsive groups, or between all of the patients and healthy controls. Linkage disequilibrium between any two loci was detected but there was no significant difference in haplotype frequency between the drug resistant and responsive groups. Our results suggest that MVP genetic polymorphisms and haplotypes may not be associated with drug resistance of partial epilepsy in the Chinese Han population. Copyright © 2015 Elsevier B.V. All rights reserved.

  15. Major vault protein (MVP) gene polymorphisms and drug resistance in mesial temporal lobe epilepsy with hippocampal sclerosis.

    Science.gov (United States)

    Balan, Shabeesh; Radhab, Saradalekshmi Koramannil; Radha, Koramannil; Sathyan, Sanish; Vijai, Joseph; Banerjee, Moinak; Radhakrishnan, Kurupath

    2013-09-10

    The human major vault protein (MVP) has been implicated in the development of drug resistance in cancer cells. Over expression of MVP has also been reported in brain tissue samples from antiepileptic drug (AED)-resistant human focal epilepsies. To investigate the relationship between single nucleotide polymorphisms (SNPs) involving the MVP gene and AED-resistance, we compared the distribution of three SNPs in the MVP gene, rs4788187, rs3815824 and rs3815823, among 220 patients with mesial temporal lobe epilepsy with hippocampal sclerosis (MTLE-HS) (prototype of AED-resistant epilepsy syndrome), 201 patients with juvenile myoclonic epilepsy (JME) (prototype of AED-responsive epilepsy syndrome) and 213 ethnically matched non-epilepsy controls. All the patients and controls were residents of the South Indian state of Kerala for more than three generations. We did not find any significant difference in allele and genotypic frequencies of the studied SNPs between AED-resistant and AED-responsive cohorts, and between AED-resistant and AED-responsive cohorts independently and pooled together when compared with the controls. We conclude that rs4788187, rs3815824, rs3815823 variants of the MVP gene are associated neither with predisposition for epilepsy nor with AED-resistance in the population that we have studied. Our results suggest the need for further research into the link between MVP and AED-resistance. Copyright © 2013 Elsevier B.V. All rights reserved.

  16. Pea proteins oral supplementation promotes muscle thickness gains during resistance training: a double-blind, randomized, Placebo-controlled clinical trial vs. Whey protein.

    Science.gov (United States)

    Babault, Nicolas; Païzis, Christos; Deley, Gaëlle; Guérin-Deremaux, Laetitia; Saniez, Marie-Hélène; Lefranc-Millot, Catherine; Allaert, François A

    2015-01-01

    The effects of protein supplementation on muscle thickness and strength seem largely dependent on its composition. The current study aimed at comparing the impact of an oral supplementation with vegetable Pea protein (NUTRALYS®) vs. Whey protein and Placebo on biceps brachii muscle thickness and strength after a 12-week resistance training program. One hundred and sixty one males, aged 18 to 35 years were enrolled in the study and underwent 12 weeks of resistance training on upper limb muscles. According to randomization, they were included in the Pea protein (n = 53), Whey protein (n = 54) or Placebo (n = 54) group. All had to take 25 g of the proteins or placebo twice a day during the 12-week training period. Tests were performed on biceps muscles at inclusion (D0), mid (D42) and post training (D84). Muscle thickness was evaluated using ultrasonography, and strength was measured on an isokinetic dynamometer. Results showed a significant time effect for biceps brachii muscle thickness (P Pea, Whey and Placebo, respectively; P Pea group as compared to Placebo whereas there was no difference between Whey and the two other conditions. Muscle strength also increased with time with no statistical difference between groups. In addition to an appropriate training, the supplementation with pea protein promoted a greater increase of muscle thickness as compared to Placebo and especially for people starting or returning to a muscular strengthening. Since no difference was obtained between the two protein groups, vegetable pea proteins could be used as an alternative to Whey-based dietary products. The present trial has been registered at ClinicalTrials.gov (NCT02128516).

  17. The stress protein BAG3 stabilizes Mcl-1 protein and promotes survival of cancer cells and resistance to antagonist ABT-737.

    Science.gov (United States)

    Boiani, Mariana; Daniel, Cristina; Liu, Xueyuan; Hogarty, Michael D; Marnett, Lawrence J

    2013-03-08

    Members of the Bcl-2 family of proteins are important inhibitors of apoptosis in human cancer and are targets for novel anticancer agents such as the Bcl-2 antagonists, ABT-263 (Navitoclax), and its analog ABT-737. Unlike Bcl-2, Mcl-1 is not antagonized by ABT-263 or ABT-737 and is considered to be a major factor in resistance. Also, Mcl-1 exhibits differential regulation when compared with other Bcl-2 family members and is a target for anticancer drug discovery. Here, we demonstrate that BAG3, an Hsp70 co-chaperone, protects Mcl-1 from proteasomal degradation, thereby promoting its antiapoptotic activity. Using neuroblastoma cell lines, with a defined Bcl-2 family dependence, we found that BAG3 expression correlated with Mcl-1 dependence and ABT-737 resistance. RNA silencing of BAG3 led to a marked reduction in Mcl-1 protein levels and overcame ABT-737 resistance in Mcl-1-dependent cells. In ABT-737-resistant cells, Mcl-1 co-immunoprecipitated with BAG3, and loss of Mcl-1 after BAG3 silencing was prevented by proteasome inhibition. BAG3 and Mcl-1 were co-expressed in a panel of diverse cancer cell lines resistant to ABT-737. Silencing BAG3 reduced Mcl-1 protein levels and overcame ABT-737 resistance in several of the cell lines, including triple-negative breast cancer (MDA-MB231) and androgen receptor-negative prostate cancer (PC3) cells. These studies identify BAG3-mediated Mcl-1 stabilization as a potential target for cancer drug discovery.

  18. The Stress Protein BAG3 Stabilizes Mcl-1 Protein and Promotes Survival of Cancer Cells and Resistance to Antagonist ABT-737*

    Science.gov (United States)

    Boiani, Mariana; Daniel, Cristina; Liu, Xueyuan; Hogarty, Michael D.; Marnett, Lawrence J.

    2013-01-01

    Members of the Bcl-2 family of proteins are important inhibitors of apoptosis in human cancer and are targets for novel anticancer agents such as the Bcl-2 antagonists, ABT-263 (Navitoclax), and its analog ABT-737. Unlike Bcl-2, Mcl-1 is not antagonized by ABT-263 or ABT-737 and is considered to be a major factor in resistance. Also, Mcl-1 exhibits differential regulation when compared with other Bcl-2 family members and is a target for anticancer drug discovery. Here, we demonstrate that BAG3, an Hsp70 co-chaperone, protects Mcl-1 from proteasomal degradation, thereby promoting its antiapoptotic activity. Using neuroblastoma cell lines, with a defined Bcl-2 family dependence, we found that BAG3 expression correlated with Mcl-1 dependence and ABT-737 resistance. RNA silencing of BAG3 led to a marked reduction in Mcl-1 protein levels and overcame ABT-737 resistance in Mcl-1-dependent cells. In ABT-737-resistant cells, Mcl-1 co-immunoprecipitated with BAG3, and loss of Mcl-1 after BAG3 silencing was prevented by proteasome inhibition. BAG3 and Mcl-1 were co-expressed in a panel of diverse cancer cell lines resistant to ABT-737. Silencing BAG3 reduced Mcl-1 protein levels and overcame ABT-737 resistance in several of the cell lines, including triple-negative breast cancer (MDA-MB231) and androgen receptor-negative prostate cancer (PC3) cells. These studies identify BAG3-mediated Mcl-1 stabilization as a potential target for cancer drug discovery. PMID:23341456

  19. Tomato Cf resistance proteins mediate recognition of cognate homologous effectors from fungi pathogenic on diots and monocots

    NARCIS (Netherlands)

    Stergiopoulos, I.; Burg, van den H.A.; Ökmen, B.; Beenen, H.G.; Liere, van S.; Kema, G.H.J.; Wit, de P.J.G.M.

    2010-01-01

    Most fungal effectors characterized so far are species-specific and facilitate virulence on a particular host plant. During infection of its host tomato, Cladosporium fulvum secretes effectors that function as virulence factors in the absence of cognate Cf resistance proteins and induce

  20. Zonal down-regulation and redistribution of the multidrug resistance protein 2 during bile duct ligation in rat liver

    NARCIS (Netherlands)

    Paulusma, C. C.; Kothe, M. J.; Bakker, C. T.; Bosma, P. J.; van Bokhoven, I.; van Marle, J.; Bolder, U.; Tytgat, G. N.; Oude Elferink, R. P.

    2000-01-01

    We have studied regulation of the multidrug resistance protein 2 (mrp2) during bile duct ligation (BDL) in the rat. In hepatocytes isolated after 16, 48, and 72 hours of BDL, mrp2-mediated dinitrophenyl-glutathione (DNP-GS) transport was decreased to 65%, 33%, and 33% of control values,

  1. The breast cancer resistance protein transporter ABCG2 is expressed in the human kidney proximal tubule apical membrane.

    NARCIS (Netherlands)

    Huls, M.; Brown, C.D.; Windass, A.S.; Sayer, R.; Heuvel, J.J.M.W. van den; Heemskerk, S.; Russel, F.G.M.; Masereeuw, R.

    2008-01-01

    The Breast Cancer Resistance Protein (BCRP/ABCG2) is a transporter restricting absorption and enhancing excretion of many compounds including anticancer drugs. This transporter is highly expressed in many tissues; however, in human kidney, only the mRNA was found in contrast to the mouse kidney,

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

    Science.gov (United States)

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

    2013-09-13

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

  3. Plasma phospholipid transfer protein activity is independently determined by obesity and insulin resistance in non-diabetic subjects

    NARCIS (Netherlands)

    de Vries, Rindert; Kappelle, Paul J. W. H.; Dallinga-Thie, Geesje M.; Dullaart, Robin P. F.

    2011-01-01

    Phospholipid transfer protein (PLTP) is an emerging cardio-metabolic risk factor which is intricately involved in lipoprotein metabolism. Elevated plasma PLTP activity levels are reported in obesity and diabetes mellitus, but the relative contributions of obesity and insulin resistance to plasma

  4. Plasma phospholipid transfer protein activity is independently determined by obesity and insulin resistance in non-diabetic subjects

    NARCIS (Netherlands)

    de Vries, Rindert; Kappelle, Paul J.W.H.; Dallinga-Thie, Geesje M.; Dullaart, Robin P. F.

    Background: Phospholipid transfer protein (PLTP) is an emerging cardio-metabolic risk factor which is intricately involved in lipoprotein metabolism. Elevated plasma PLTP activity levels are reported in obesity and diabetes mellitus, but the relative contributions of obesity and insulin resistance

  5. Low-dose oral contraceptives and acquired resistance to activated protein C: a randomised cross-over study

    NARCIS (Netherlands)

    Rosing, J.; Middeldorp, S.; Curvers, J.; Christella, M.; Thomassen, L. G.; Nicolaes, G. A.; Meijers, J. C.; Bouma, B. N.; Büller, H. R.; Prins, M. H.; Tans, G.

    1999-01-01

    BACKGROUND: We have reported previously that, compared with use of second-generation oral contraceptives, the use of third-generation oral contraceptives is associated with increased resistance to the anticoagulant action of activated protein C (APC). Owing to the cross-sectional design of that

  6. Decreased PARP and procaspase-2 protein levels are associated with cellular drug resistance in childhood acute lymphoblastic leukemia

    NARCIS (Netherlands)

    A. Holleman (Amy); M.L. den Boer (Monique); K.M. Kazemier (Karin); H.B. Beverloo (Berna); A.R.M. von Bergh (Anne); G.E. Janka-Schaub (Gritta); R. Pieters (Rob)

    2005-01-01

    textabstractDrug resistance in childhood acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) is associated with impaired ability to induce apoptosis. To elucidate causes of apoptotic defects, we studied the protein expression of Apaf-1, procaspases-2, -3, -6, -7,

  7. Plasmid-mediated resistance to thrombin-induced platelet microbicidal protein in staphylococci: role of the qacA locus.

    Science.gov (United States)

    Kupferwasser, L I; Skurray, R A; Brown, M H; Firth, N; Yeaman, M R; Bayer, A S

    1999-10-01

    Thrombin-induced platelet microbicidal protein 1 (tPMP-1) is a small, cationic peptide released from rabbit platelets following thrombin stimulation. In vitro resistance to this peptide among strains of Staphylococcus aureus correlates with the survival advantage of such strains at sites of endothelial damage in humans as well as in experimental endovascular infections. The mechanisms involved in the phenotypic resistance of S. aureus to tPMP-1 are not fully delineated. The plasmid-encoded staphylococcal gene qacA mediates multidrug resistance to multiple organic cations via a proton motive force-dependent efflux pump. We studied whether the qacA gene might also confer resistance to cationic tPMP-1. Staphylococcal plasmids encoding qacA were found to confer resistance to tPMP-1 in an otherwise susceptible parental strain. Deletions which removed the region containing the qacA gene in the S. aureus multiresistance plasmid pSK1 abolished tPMP-1 resistance. Resistance to tPMP-1 in the qacA-bearing strains was inoculum independent but peptide concentration dependent, with the level of resistance decreasing at higher peptide concentrations for a given inoculum. There was no apparent cross-resistance in qacA-bearing strains to other endogenous cationic antimicrobial peptides which are structurally distinct from tPMP-1, including human neutrophil defensin 1, protamine, or the staphylococcal lantibiotics pep5 and nisin. These data demonstrate that the staphylococcal multidrug resistance gene qacA also mediates in vitro resistance to cationic tPMP-1.

  8. Soy-dairy protein blend and whey protein ingestion after resistance exercise increases amino acid transport and transporter expression in human skeletal muscle

    Science.gov (United States)

    Reidy, P. T.; Walker, D. K.; Dickinson, J. M.; Gundermann, D. M.; Drummond, M. J.; Timmerman, K. L.; Cope, M. B.; Mukherjea, R.; Jennings, K.; Volpi, E.

    2014-01-01

    Increasing amino acid availability (via infusion or ingestion) at rest or postexercise enhances amino acid transport into human skeletal muscle. It is unknown whether alterations in amino acid availability, from ingesting different dietary proteins, can enhance amino acid transport rates and amino acid transporter (AAT) mRNA expression. We hypothesized that the prolonged hyperaminoacidemia from ingesting a blend of proteins with different digestion rates postexercise would enhance amino acid transport into muscle and AAT expression compared with the ingestion of a rapidly digested protein. In a double-blind, randomized clinical trial, we studied 16 young adults at rest and after acute resistance exercise coupled with postexercise (1 h) ingestion of either a (soy-dairy) protein blend or whey protein. Phenylalanine net balance and transport rate into skeletal muscle were measured using stable isotopic methods in combination with femoral arteriovenous blood sampling and muscle biopsies obtained at rest and 3 and 5 h postexercise. Phenylalanine transport into muscle and mRNA expression of select AATs [system L amino acid transporter 1/solute-linked carrier (SLC) 7A5, CD98/SLC3A2, system A amino acid transporter 2/SLC38A2, proton-assisted amino acid transporter 1/SLC36A1, cationic amino acid transporter 1/SLC7A1] increased to a similar extent in both groups (P protein blend resulted in a prolonged and positive net phenylalanine balance during postexercise recovery compared with whey protein (P protein synthesis increased similarly between groups. We conclude that, while both protein sources enhanced postexercise AAT expression, transport into muscle, and myofibrillar protein synthesis, postexercise ingestion of a protein blend results in a slightly prolonged net amino acid balance across the leg compared with whey protein. PMID:24699854

  9. Protein cross-linking, peroxidase and beta-1,3-endoglucanase involved in resistance of pea against Orobanche crenata.

    Science.gov (United States)

    Pérez-de-Luque, Alejandro; González-Verdejo, Clara I; Lozano, M Dolores; Dita, Miguel A; Cubero, José I; González-Melendi, Pablo; Risueño, María C; Rubiales, Diego

    2006-01-01

    Root holoparasitic angiosperms, like Orobanche spp, completely lack chlorophyll and totally depend on their host for their supply of nutrients. O. crenata is a severe constraint to the cultivation of legumes and breeding for resistance remains the most economical, feasible, and environmentally friendly method of control. Due to the lack of resistance in commercial pea cultivars, the use of wild relatives for breeding is necessary, and an understanding of the mechanisms underlying host resistance is needed in order to improve screening for resistance in breeding programmes. Compatible and incompatible interactions between O. crenata and pea have been studied using cytochemical procedures. The parasite was stopped in the host cortex before reaching the central cylinder, and accumulation of H2O2, peroxidases, and callose were detected in neighbouring cells. Protein cross-linking in the host cell walls appears as the mechanism of defence, halting penetration of the parasite. In situ hybridization studies have also shown that a peroxidase and a beta-glucanase are differently expressed in cells of the resistant host (Pf651) near the penetration point. The role of these proteins in the resistance to O. crenata is discussed.

  10. Altered protein expression in gestational diabetes mellitus placentas provides insight into insulin resistance and coagulation/fibrinolysis pathways.

    Directory of Open Access Journals (Sweden)

    Bin Liu

    Full Text Available OBJECTIVE: To investigate the placental proteome differences between pregnant women complicated with gestational diabetes mellitus (GDM and those with normal glucose tolerance (NGT. METHODS: We used two-dimensional electrophoresis (2DE to separate and compare placental protein levels from GDM and NGT groups. Differentially expressed proteins between the two groups were identified by MALDI-TOF/TOF mass spectrometry and further confirmed by Western blotting. The mRNA levels of related proteins were measured by realtime RT-PCR. Immunohistochemistry (IHC was performed to examine the cellular location of the proteins expressed in placenta villi. RESULTS: Twenty-one protein spots were differentially expressed between GDM and NGT placenta villi in the tested samples, fifteen of which were successfully identified by mass spectrometry. The molecular functions of these differentially expressed proteins include blood coagulation, signal transduction, anti-apoptosis, ATP binding, phospholipid binding, calcium ion binding, platelet activation, and tryptophan-tRNA ligase activity. Both protein and mRNA levels of Annexin A2, Annexin A5 and 14-3-3 protein ζ/δ were up-regulated, while the expression of the Ras-related protein Rap1A was down-regulated in the GDM placenta group. CONCLUSION: Placenta villi derived from GDM pregnant women exhibit significant proteome differences compared to those of NGT mothers. The identified differentially expressed proteins are mainly associated with the development of insulin resistance, transplacental transportation of glucose, hyperglucose-mediated coagulation and fibrinolysis disorders in the GDM placenta villi.

  11. Molecular evidence and functional expression of multidrug resistance associated protein (MRP) in rabbit corneal epithelial cells.

    Science.gov (United States)

    Karla, Pradeep K; Pal, Dananjay; Mitra, Ashim K

    2007-01-01

    Multidrug resistance associated protein (MRP) is a major family of efflux transporters involved in drug efflux leading to drug resistance. The objective of this study was to explore physical barriers for ocular drug absorption and to verify if the role of efflux transporters. MRP-2 is a major homologue of MRP family and found to express on the apical side of cell membrane. Cultured Rabbit Corneal Epithelial Cells (rCEC) were selected as an in vitro model for corneal epithelium. [14C]-erythromycin which is a proven substrate for MRP-2 was selected as a model drug for functional expression studies. MK-571, a known specific and potent inhibitor for MRP-2 was added to inhibit MRP mediated efflux. Membrane fraction of rCEC was used for western blot analysis. Polarized transport of [14C]-erythromycin was observed in rCEC and transport from B-->A was significantly high than from A-->B. Permeability's increased significantly from A-->B in the presence of MK-571 and ketoconozole. Uptake of [14C]-erythromycin in the presence of MK-571 was significantly higher than control in rCEC. RT-PCR analysis indicated a unique and distinct band at approximately 498 bp corresponding to MRP-2 in rCEC and MDCK11-MRP-2 cells. Immunoprecipitation followed by Western Blot analysis indicated a specific band at approximately 190 kDa in membrane fraction of rCEC and MDCK11-MRP-2 cells. For the first time we have demonstrated high expression of MRP-2 in rabbit corneal epithelium and its functional activity causing drug efflux. RT-PCR, immunoprecipitation followed by Western blot analysis further confirms the result.

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

    Directory of Open Access Journals (Sweden)

    Roberta Palorini

    2016-03-01

    Full Text Available Cancer cells often rely on glycolysis to obtain energy and support anabolic growth. Several studies showed that glycolytic cells are susceptible to cell death when subjected to low glucose availability or to lack of glucose. However, some cancer cells, including glycolytic ones, can efficiently acquire higher tolerance to glucose depletion, leading to their survival and aggressiveness. Although increased resistance to glucose starvation has been shown to be a consequence of signaling pathways and compensatory metabolic routes activation, the full repertoire of the underlying molecular alterations remain elusive. Using omics and computational analyses, we found that cyclic adenosine monophosphate-Protein Kinase A (cAMP-PKA axis activation is fundamental for cancer cell resistance to glucose starvation and anoikis. Notably, here we show that such a PKA-dependent survival is mediated by parallel activation of autophagy and glutamine utilization that in concert concur to attenuate the endoplasmic reticulum (ER stress and to sustain cell anabolism. Indeed, the inhibition of PKA-mediated autophagy or glutamine metabolism increased the level of cell death, suggesting that the induction of autophagy and metabolic rewiring by PKA is important for cancer cellular survival under glucose starvation. Importantly, both processes actively participate to cancer cell survival mediated by suspension-activated PKA as well. In addition we identify also a PKA/Src mechanism capable to protect cancer cells from anoikis. Our results reveal for the first time the role of the versatile PKA in cancer cells survival under chronic glucose starvation and anoikis and may be a novel potential target for cancer treatment.

  13. Supplementary Material for: Mycobacterium tuberculosis whole genome sequencing and protein structure modelling provides insights into anti-tuberculosis drug resistance

    KAUST Repository

    Phelan, Jody

    2016-01-01

    Abstract Background Combating the spread of drug resistant tuberculosis is a global health priority. Whole genome association studies are being applied to identify genetic determinants of resistance to anti-tuberculosis drugs. Protein structure and interaction modelling are used to understand the functional effects of putative mutations and provide insight into the molecular mechanisms leading to resistance. Methods To investigate the potential utility of these approaches, we analysed the genomes of 144 Mycobacterium tuberculosis clinical isolates from The Special Programme for Research and Training in Tropical Diseases (TDR) collection sourced from 20 countries in four continents. A genome-wide approach was applied to 127 isolates to identify polymorphisms associated with minimum inhibitory concentrations for first-line anti-tuberculosis drugs. In addition, the effect of identified candidate mutations on protein stability and interactions was assessed quantitatively with well-established computational methods. Results The analysis revealed that mutations in the genes rpoB (rifampicin), katG (isoniazid), inhA-promoter (isoniazid), rpsL (streptomycin) and embB (ethambutol) were responsible for the majority of resistance observed. A subset of the mutations identified in rpoB and katG were predicted to affect protein stability. Further, a strong direct correlation was observed between the minimum inhibitory concentration values and the distance of the mutated residues in the three-dimensional structures of rpoB and katG to their respective drugs binding sites. Conclusions Using the TDR resource, we demonstrate the usefulness of whole genome association and convergent evolution approaches to detect known and potentially novel mutations associated with drug resistance. Further, protein structural modelling could provide a means of predicting the impact of polymorphisms on drug efficacy in the absence of phenotypic data. These approaches could ultimately lead to novel

  14. Mycobacterium tuberculosis whole genome sequencing and protein structure modelling provides insights into anti-tuberculosis drug resistance

    KAUST Repository

    Phelan, Jody

    2016-03-23

    Background Combating the spread of drug resistant tuberculosis is a global health priority. Whole genome association studies are being applied to identify genetic determinants of resistance to anti-tuberculosis drugs. Protein structure and interaction modelling are used to understand the functional effects of putative mutations and provide insight into the molecular mechanisms leading to resistance. Methods To investigate the potential utility of these approaches, we analysed the genomes of 144 Mycobacterium tuberculosis clinical isolates from The Special Programme for Research and Training in Tropical Diseases (TDR) collection sourced from 20 countries in four continents. A genome-wide approach was applied to 127 isolates to identify polymorphisms associated with minimum inhibitory concentrations for first-line anti-tuberculosis drugs. In addition, the effect of identified candidate mutations on protein stability and interactions was assessed quantitatively with well-established computational methods. Results The analysis revealed that mutations in the genes rpoB (rifampicin), katG (isoniazid), inhA-promoter (isoniazid), rpsL (streptomycin) and embB (ethambutol) were responsible for the majority of resistance observed. A subset of the mutations identified in rpoB and katG were predicted to affect protein stability. Further, a strong direct correlation was observed between the minimum inhibitory concentration values and the distance of the mutated residues in the three-dimensional structures of rpoB and katG to their respective drugs binding sites. Conclusions Using the TDR resource, we demonstrate the usefulness of whole genome association and convergent evolution approaches to detect known and potentially novel mutations associated with drug resistance. Further, protein structural modelling could provide a means of predicting the impact of polymorphisms on drug efficacy in the absence of phenotypic data. These approaches could ultimately lead to novel resistance

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

    Science.gov (United States)

    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.

  16. Determination of proteins induced in response to jasmonic acid and salicylic acid in resistant and susceptible cultivars of tomato.

    Science.gov (United States)

    Afroz, Amber; Khan, Muhammad Rashid; Komatsu, Setsuko

    2010-07-01

    Jasmonic acid (JA) and salicylic acid (SA) are signaling molecules that play key roles in the regulation of metabolic processes, reproduction, and defense against pathogens. The proteomics approach was used to identify proteins that are induced by JA and SA in the tomato cultivars Roma and Pant Bahr, which are susceptible and resistant to bacterial wilt, respectively. Threonine deaminase and leucine amino peptidase were upregulated, and ribulose-1,5-bisphosphate carboxylase/oxygenase small chain was downregulated by time-course application of JA. Translationally controlled tumor protein was upregulated by time-course application of SA. Protein disulfide isomerase was upregulated by application of either JA or SA. Proteins related to defense, energy, and protein destination/storage are suspected to be responsible for the susceptibility or resistance of the cultivars. Furthermore, in Roma, iron ABC transporter was upregulated by JA and down-regulated by SA. Iron ABC transporter plays a part in the signal transduction of both JA and SA in cultivars of tomato that are resistant to bacterial wilt.

  17. vPARP Adjusts MVP Expression in Drug-resistant Cell Lines in Conjunction with MDR Proteins.

    Science.gov (United States)

    Wojtowicz, Karolina; Januchowski, Radoslaw; Nowicki, Michal; Zabel, Maciej

    2017-06-01

    The definition of vault (ribonucleoprotein particles) function remains highly complex. Vaults may cooperate with multidrug resistance (MDR) proteins, supporting their role in drug resistance. This topic is the main theme of this publication. The cell viability was determined by an MTT assay. The protein expression was detected by western blot analysis. The proteins were knocked-down using siRNA. No major vault protein (MVP) in the LoVo/Dx and W1PR cell lines after tunicamycin treatment was shown. In W1PR cells with knocked-down MVP, a statistically significant decrease in cell viability was noted. In LoVo/Dx, W1TR and A2780TR cells were vault poly-ADP-ribose polymerase (vPARP) was knockdown, a decrease in cell viability was shown. Also, MVP silencing induced an increase in glycoprotein P (Pgp) expression in LoVo/Dx cells. MVP is important for the drug resistance of cancer cells, but it probably requires the presence of vPARP for full activation. Some correlations between MDR proteins and vaults exist. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

  18. Improving protein resistance of {alpha}-Al{sub 2}O{sub 3} membranes by modification with POEGMA brushes

    Energy Technology Data Exchange (ETDEWEB)

    He Huating [State Key Laboratory of Materials-Oriented Chemical Engineering, College of Chemistry and Chemical Engineering, Nanjing University of Technology, Nanjing 210009 (China); Jing Wenheng, E-mail: jingwenheng@yahoo.com.cn [State Key Laboratory of Materials-Oriented Chemical Engineering, College of Chemistry and Chemical Engineering, Nanjing University of Technology, Nanjing 210009 (China); Xing Weihong; Fan Yiqun [State Key Laboratory of Materials-Oriented Chemical Engineering, College of Chemistry and Chemical Engineering, Nanjing University of Technology, Nanjing 210009 (China)

    2011-11-15

    A kind of protein-resistant ceramic membrane is prepared by grafting poly(oligo (ethylene glycol) methyl ether methacrylate) (POEGMA) brushes onto the surfaces and pore walls of {alpha}-Al{sub 2}O{sub 3} membrane (AM) by surface-initiated atom-transfer radical polymerization (SI-ATRP). Contact-angle, Fourier-transform infrared spectroscopy (FTIR), thermogravimetric analysis (TGA) and field-emission scanning electron microscopy (FESEM) were measured to confirm that the surfaces and pore walls of the ceramic porous membranes have been modified by the brushes with this method successfully. The protein interaction behavior with the POEGMA modified membranes (AM-POEGMA) was studied by the model protein of bovine serum albumin (BSA). A protein-resistant mechanism of AM-POEGMA was proposed to describe an interesting phenomenon discovered in the filtration experiment, in which the initial flux filtrating BSA solution is higher than the pure water flux. The fouling of AM-POEGMA was easier to remove than AM for the action of POEGMA brushes, indicated that the ceramic porous membranes modified with POEGMA brushes exhibit excellent protein resistance.

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

    DEFF Research Database (Denmark)

    Reitelseder, Søren; Agergaard, Jakob; Doessing, Simon

    2011-01-01

    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...

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

    Science.gov (United States)

    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.

  1. Mechanism of the pharmacokinetic interaction between methotrexate and benzimidazoles: potential role for breast cancer resistance protein in clinical drug-drug interactions

    NARCIS (Netherlands)

    Breedveld, Pauline; Zelcer, Noam; Pluim, Dick; Sönmezer, Ozgür; Tibben, Matthijs M.; Beijnen, Jos H.; Schinkel, Alfred H.; van Tellingen, Olaf; Borst, Piet; Schellens, Jan H. M.

    2004-01-01

    The antifolate drug methotrexate (MTX) is transported by breast cancer resistance protein (BCRP; ABCG2) and multidrug resistance-associated protein1-4 (MRP1-4; ABCC1-4). In cancer patients, coadministration of benzimidazoles and MTX can result in profound MTX-induced toxicity coinciding with an

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

    Science.gov (United States)

    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. Copyright © 2015 the American Physiological Society.

  3. R-Flurbiprofen Traps Prostaglandins within Cells by Inhibition of Multidrug Resistance-Associated Protein-4.

    Science.gov (United States)

    Wobst, Ivonne; Ebert, Lisa; Birod, Kerstin; Wegner, Marthe-Susanna; Hoffmann, Marika; Thomas, Dominique; Angioni, Carlo; Parnham, Michael J; Steinhilber, Dieter; Tegeder, Irmgard; Geisslinger, Gerd; Grösch, Sabine

    2016-12-30

    R -flurbiprofen is the non-COX-inhibiting enantiomer of flurbiprofen and is not converted to S -flurbiprofen in human cells. Nevertheless, it reduces extracellular prostaglandin E₂ (PGE₂) in cancer or immune cell cultures and human extracellular fluid. Here, we show that R -flurbiprofen acts through a dual mechanism: (i) it inhibits the translocation of cPLA 2α to the plasma membrane and thereby curtails the availability of arachidonic acid and (ii) R -flurbiprofen traps PGE₂ inside of the cells by inhibiting multidrug resistance-associated protein 4 (MRP4, ABCC4), which acts as an outward transporter for prostaglandins. Consequently, the effects of R -flurbiprofen were mimicked by RNAi-mediated knockdown of MRP4. Our data show a novel mechanism by which R -flurbiprofen reduces extracellular PGs at physiological concentrations, particularly in cancers with high levels of MRP4, but the mechanism may also contribute to its anti-inflammatory and immune-modulating properties and suggests that it reduces PGs in a site- and context-dependent manner.

  4. Classification of Breast Cancer Resistant Protein (BCRP) Inhibitors and Non-Inhibitors Using Machine Learning Approaches.

    Science.gov (United States)

    Belekar, Vilas; Lingineni, Karthik; Garg, Prabha

    2015-01-01

    The breast cancer resistant protein (BCRP) is an important transporter and its inhibitors play an important role in cancer treatment by improving the oral bioavailability as well as blood brain barrier (BBB) permeability of anticancer drugs. In this work, a computational model was developed to predict the compounds as BCRP inhibitors or non-inhibitors. Various machine learning approaches like, support vector machine (SVM), k-nearest neighbor (k-NN) and artificial neural network (ANN) were used to develop the models. The Matthews correlation coefficients (MCC) of developed models using ANN, k-NN and SVM are 0.67, 0.71 and 0.77, and prediction accuracies are 85.2%, 88.3% and 90.8% respectively. The developed models were tested with a test set of 99 compounds and further validated with external set of 98 compounds. Distribution plot analysis and various machine learning models were also developed based on druglikeness descriptors. Applicability domain is used to check the prediction reliability of the new molecules.

  5. Up-regulation of DNA-dependent protein kinase correlates with radiation resistance in oral squamous cell carcinoma

    International Nuclear Information System (INIS)

    Shintani, Satoru; Mihara, Mariko; Li, Chunnan; Nakahara Yuuji; Hino, Satoshi; Nakashiro, Koh-ichi; Hamakawa, Hiroyuki

    2003-01-01

    DNA-PK is a nuclear protein with serine/threonine kinase activity and forms a complex consisting of the DNA-PKcs and a heterodimer of Ku70 and Ku80 proteins. Recent laboratory experiments have demonstrated that the DNA-PK complex formation is one of the major pathways by which mammalian cells respond to DNA double-strand breaks induced by ionizing radiation. In this study, we evaluated the relationship between expression levels of DNA-PKcs, Ku70 and Ku80 proteins and radiation sensitivity in oral squamous cell carcinoma (OSCC) cell lines and in OSCC patients treated with preoperative radiation therapy. The OSCC cell lines greatly differed in their response to irradiation, as assessed by a standard colony formation assay. However, the expression levels of the DNA-PK complex proteins were all similar, and there was no association between the magnitude of their expression and the tumor radiation sensitivity. Expression of DNA-PK complex proteins increased after radiation treatment, and the increased values correlated with the tumor radiation resistance. Expression of DNA-PKcs and Ku70 after irradiation was increased in the surviving cells of OSCC tissues irradiated preoperatively. These results suggest that up-regulation of DNA-PK complex protein, especially DNA-PKcs, after radiation treatment correlates to radiation resistance. DNA-PKcs might be a molecular target for a novel radiation sensitization therapy of OSCC. (author)

  6. F-Box Protein FBXO22 Mediates Polyubiquitination and Degradation of CD147 to Reverse Cisplatin Resistance of Tumor Cells.

    Science.gov (United States)

    Wu, Bo; Liu, Zhen-Yu; Cui, Jian; Yang, Xiang-Min; Jing, Lin; Zhou, Yang; Chen, Zhi-Nan; Jiang, Jian-Li

    2017-01-20

    Drug resistance remains a major clinical obstacle to successful treatment of cancer. As posttranslational modification is becoming widely recognized to affect the function of oncoproteins, targeting specific posttranslational protein modification provides an attractive strategy for anticancer drug development. CD147 is a transmembrane glycoprotein contributing to chemo-resistance of cancer cells in a variety of human malignancies. Ubiquitination is an important posttranslational modification mediating protein degradation. Degradation of oncoproteins, CD147 included, emerges as an attractive alternative for tumor inhibition. However, the ubiquitination of CD147 remains elusive. Here in this study, we found that deletion of the CD147 intracellular domain (CD147-ICD) prolonged the half-life of CD147 in HEK293T cells, and we identified that CD147-ICD interacts with FBXO22 using mass spectrometry and Western blot. Then, we demonstrated that FBXO22 mediates the polyubiquitination and degradation of CD147 by recognizing CD147-ICD. While knocking down of FBXO22 prolonged the half-life of CD147 in HEK293T cells, we found that FBXO22 regulates CD147 protein turnover in SMMC-7721, Huh-7 and A549 cells. Moreover, we found that the low level of FBXO22 contributes to the accumulation of CD147 and thereafter the cisplatin resistance of A549/DDP cells. To conclude, our study demonstrated that FBXO22 mediated the polyubiquitination and degradation of CD147 by interacting with CD147-ICD, and CD147 polyubiquitination by FBXO22 reversed cisplatin resistance of tumor cells.

  7. Ribosomal protein L3 mutations are associated with cfr-mediated linezolid resistance in clinical isolates of Staphylococcus cohnii.

    Science.gov (United States)

    Xu, Hongtao; Tian, Rui; Li, Yanming; Chen, Dongke; Liu, Yalin; Hu, Yunjian; Xiao, Fei

    2015-06-01

    From June, 2012 to November, 2013 five linezolid-resistant Staphylococcus cohnii isolates were identified in our hospital in Beijing, China. The investigation of the resistance mechanisms confirmed that the cfr-carrying plasmids were the main cause of linezolid resistance in those clinical isolates. Moreover, all the five isolates had ribosomal protein L3 mutations, which had different coordinate effect on cfr-mediated linezolid resistance directly through the substitution of serine 158 by phenylalanine or tyrosine in L3 protein. In this study, two types of plasmids (p432, p438) (Accession No. KM114207) were found, which share high sequence identity with previously reported cfr-carrying pRM01 and pMHZ plasmids originated from northern and southern China, showing wide regional dissemination in China. The stability of linezolid resistance was studied by passaging single colonies serially on antibiotic-free blood medium, which showed that the susceptible derivatives emerged until the passages 39-42 with the elimination of cfr-carrying plasmid. Thus the high stability of this plasmid may pose a risk for the transmission among patients or even cause an outbreak in clinical settings.

  8. Ultraviolet light and ozone stimulate accumulation of salicylic acid, pathogenesis-related proteins and virus resistance in tobacco

    International Nuclear Information System (INIS)

    Yalpani, N.; Enyedi, A.J.; León, J.; Raskin, I.

    1994-01-01

    In tobacco (Nicotiana tabacum L. cv. Xanthinc), salicylic acid (SA) levels increase in leaves inoculated by necrotizing pathogens and in healthy leaves located above the inoculated site. Systemic SA increase may trigger disease resistance and synthesis of pathogenesis-related proteins (PR proteins). Here we report that ultraviolet (UV)-C light or ozone induced biochemical responses similar to those induced by necrotizing pathogens. Exposure of leaves to UV-C light or ozone resulted in a transient ninefold increase in SA compared to controls. In addition, in UV-light-irradiated plants, SA increased nearly fourfold to 0.77 μg·g −1 fresh weight in leaves that were shielded from UV light. Increased SA levels were accompanied by accumulation of an SA conjugate and by an increase in the activity of benzoic acid 2-hydroxylase which catalyzes SA biosynthesis. In irradiated and in unirradiated leaves of plants treated with UV light, as well as in plants fumigated with ozone, PR proteins 1a and 1b accumulated. This was paralleled by the appearance of induced resistance to a subsequent challenge with tobacco mosaic virus. The results suggest that UV light, ozone fumigation and tobacco mosaic virus can activate a common signal-transduction pathway that leads to SA and PR-protein accumulation and increased disease resistance. (author)

  9. One-year effectiveness of two hypocaloric diets with different protein/carbohydrate ratios in weight loss and insulin resistance.

    Science.gov (United States)

    Calleja Fernández, A; Vidal Casariego, A; Cano Rodríguez, I; Ballesteros Pomar, Ma D

    2012-01-01

    The maintenance of weight loss may be influenced by the distribution of macronutrients in the diet and insulin sensitivity. The objective of the study was to evaluate the longterm effect of two hypocaloric diets with different protein/carbohydrate ratios in overweight and obese individuals either with insulin resistance (IR) or without insulin resistance (IS). Prospective, randomized, clinical intervention study. Forty patients were classified as IR/IS after a 75 g oral glucose tolerance test and then randomized to a diet with either 40% carbohydrate/30% protein/30% fat (diet A) or 55% carbohydrate/15% protein/30% fat (diet B). After one year of follow-up there was no difference in weight loss between diets A and B in each group, but the IS group maintained weight loss better than the IR group [-5.7 (3.9) vs. -0.6 (4.1); P = 0.04]. No differences were found in either Homeostasis Model Assessment (HOMA) or other metabolic glucose parameters except lower insulin at 120 minutes with diet A [21.40 (8.30) vs. 71.40 (17.11); P = 0.02]. The hypocaloric diets with different protein/carbohydrate ratios produced similar changes in weight. Insulin resistance may play a negative role in maintaining weight loss.

  10. Protein Expression Modifications in Phage-Resistant Mutants of Aeromonas salmonicida after AS-A Phage Treatment

    Directory of Open Access Journals (Sweden)

    Catarina Moreirinha

    2018-03-01

    Full Text Available The occurrence of infections by pathogenic bacteria is one of the main sources of financial loss for the aquaculture industry. This problem often cannot be solved with antibiotic treatment or vaccination. Phage therapy seems to be an alternative environmentally-friendly strategy to control infections. Recognizing the cellular modifications that bacteriophage therapy may cause to the host is essential in order to confirm microbial inactivation, while understanding the mechanisms that drive the development of phage-resistant strains. The aim of this work was to detect cellular modifications that occur after phage AS-A treatment in A. salmonicida, an important fish pathogen. Phage-resistant and susceptible cells were subjected to five successive streak-plating steps and analysed with infrared spectroscopy, a fast and powerful tool for cell study. The spectral differences of both populations were investigated and compared with a phage sensitivity profile, obtained through the spot test and efficiency of plating. Changes in protein associated peaks were found, and these results were corroborated by 1-D electrophoresis of intracellular proteins analysis and by phage sensitivity profiles. Phage AS-A treatment before the first streaking-plate step clearly affected the intracellular proteins expression levels of phage-resistant clones, altering the expression of distinct proteins during the subsequent five successive streak-plating steps, making these clones recover and be phenotypically more similar to the sensitive cells.

  11. The influence of acute resistance exercise on cyclooxygenase-1 and -2 activity and protein levels in human skeletal muscle.

    Science.gov (United States)

    Carroll, Chad C; O'Connor, Devin T; Steinmeyer, Robert; Del Mundo, Jonathon D; McMullan, David R; Whitt, Jamie A; Ramos, Jahir E; Gonzales, Rayna J

    2013-07-01

    This study evaluated the activity and content of cyclooxygenase (COX)-1 and -2 in response to acute resistance exercise (RE) in human skeletal muscle. Previous work suggests that COX-1, but not COX-2, is the primary COX isoform elevated with resistance exercise in human skeletal muscle. COX activity, however, has not been assessed after resistance exercise in humans. It was hypothesized that RE would increase COX-1 but not COX-2 activity. Muscle biopsies were taken from the vastus lateralis of nine young men (25 ± 1 yr) at baseline (preexercise), 4, and 24 h after a single bout of knee extensor RE (three sets of 10 repetitions at 70% of maximum). Tissue lysate was assayed for COX-1 and COX-2 activity. COX-1 and COX-2 protein levels were measured via Western blot analysis. COX-1 activity increased at 4 h (P 0.05) with acute RE. In contrast, COX-2 protein levels were nearly 3-fold greater (P > 0.05) at 4 h and 5-fold greater (P = 0.06) at 24 h, compared with preexercise. In conclusion, COX-1 activity increases transiently with exercise independent of COX-1 protein levels. In contrast, both COX-2 activity and protein levels were elevated with exercise, and this elevation persisted to at least 24 h after RE.

  12. Induced resistance in plants and the role of pathogenesis-related proteins

    NARCIS (Netherlands)

    Loon, L.C. van

    1997-01-01

    The nature of induced resistance Resistance, according to Agrios (1988) is the ability of an organism to exclude or overcome, completely or in some degree, the effect of a pathogen or other damaging factor. Disease resistance in plants is manifested by limited symptoms, reflecting the

  13. Expression and activity of multidrug resistance proteins in mature endothelial cells and their precursors: A challenging correlation.

    Science.gov (United States)

    Krawczenko, Agnieszka; Bielawska-Pohl, Aleksandra; Wojtowicz, Karolina; Jura, Roksana; Paprocka, Maria; Wojdat, Elżbieta; Kozłowska, Urszula; Klimczak, Aleksandra; Grillon, Catherine; Kieda, Claudine; Duś, Danuta

    2017-01-01

    Active cellular transporters of harmful agents-multidrug resistance (mdr) proteins-are present in tumor, stem and endothelial cells, among others. While mdr proteins are broadly studied in tumor cells, their role in non-tumor cells and the significance of their action not connected with removal of harmful xenobiotics is less extensively documented. Proper assessment of mdr proteins expression is difficult. Mdr mRNA presence is most often evaluated but that does not necessarily correlate with the protein level. The protein expression itself is difficult to determine; usually cells with mdr overexpression are studied, not cells under physiological conditions, in which a low expression level of mdr protein is often insufficient for detection in vitro. Various methods are used to identify mdr mRNA and protein expression, together with functional tests demonstrating their biological drug transporting activities. Data comparing different methods of investigating expression of mdr mRNAs and their corresponding proteins are still scarce. In this article we present the results of a study concerning mdr mRNA and protein expression. Our goal was to search for the best method to investigate the expression level and functional activity of five selected mdr proteins-MDR1, BCRP, MRP1, MRP4 and MRP5-in established in vitro cell lines of human endothelial cells (ECs) and their progenitors. Endothelial cells demonstrated mdr presence at the mRNA level, which was not always confirmed at the protein level or in functional tests. Therefore, several different assays had to be applied for evaluation of mdr proteins expression and functions in endothelial cells. Among them functional tests seemed to be the most conclusive, although not very specific.

  14. A storage-protein marker associated with the suppressor of Pm8 for powdery mildew resistance in wheat.

    Science.gov (United States)

    Ren, S X; McIntosh, R A; Sharp, P J; The, T T

    1996-11-01

    A suppressor of resistance to powdery mildew conferred by Pm8 showed complete association with the presence of a storage-protein marker resolved by electrophoresis on SDS-PAGE gels. This marker was identified as the product of the gliadin allele Gli-A1a. The mildewresponse phenotypes of wheats possessing the 1BL.1RS translocation were completely predictable from electrophoretograms. The suppressor, designated SuPm8, was located on chromosome 1AS. It was specific in its suppression of Pm8, and did not affect the rye-derived resistance phenotypes of wheat lines with Pm17, also located in 1RS, or of lines with Pm7.

  15. The bacterial defensin resistance protein MprF consists of separable domains for lipid lysinylation and antimicrobial peptide repulsion.

    Directory of Open Access Journals (Sweden)

    Christoph M Ernst

    2009-11-01

    Full Text Available Many bacterial pathogens achieve resistance to defensin-like cationic antimicrobial peptides (CAMPs by the multiple peptide resistance factor (MprF protein. MprF plays a crucial role in Staphylococcus aureus virulence and it is involved in resistance to the CAMP-like antibiotic daptomycin. MprF is a large membrane protein that modifies the anionic phospholipid phosphatidylglycerol with l-lysine, thereby diminishing the bacterial affinity for CAMPs. Its widespread occurrence recommends MprF as a target for novel antimicrobials, although the mode of action of MprF has remained incompletely understood. We demonstrate that the hydrophilic C-terminal domain and six of the fourteen proposed trans-membrane segments of MprF are sufficient for full-level lysyl-phosphatidylglycerol (Lys-PG production and that several conserved amino acid positions in MprF are indispensable for Lys-PG production. Notably, Lys-PG production did not lead to efficient CAMP resistance and most of the Lys-PG remained in the inner leaflet of the cytoplasmic membrane when the large N-terminal hydrophobic domain of MprF was absent, indicating a crucial role of this protein part. The N-terminal domain alone did not confer CAMP resistance or repulsion of the cationic test protein cytochrome c. However, when the N-terminal domain was coexpressed with the Lys-PG synthase domain either in one protein or as two separate proteins, full-level CAMP resistance was achieved. Moreover, only coexpression of the two domains led to efficient Lys-PG translocation to the outer leaflet of the membrane and to full-level cytochrome c repulsion, indicating that the N-terminal domain facilitates the flipping of Lys-PG. Thus, MprF represents a new class of lipid-biosynthetic enzymes with two separable functional domains that synthesize Lys-PG and facilitate Lys-PG translocation. Our study unravels crucial details on the molecular basis of an important bacterial immune evasion mechanism and it may help

  16. Investigation the Response of Some Proteins That Involved in Cachexia Syndrome to Acute Resistance Exercise in Healthy Elderly People

    Directory of Open Access Journals (Sweden)

    Meysam Gholamali

    2015-01-01

    Full Text Available Objectives: The aim of this study was to investigate the response of plasma Myostatin and insulin growth factor like-1 (IGF-1, as two most important proteins that involved in Cachexia syndrome, to acute resistance exercise in healthy elderly people. Methods & Materials: Twelve healthy older men (Age=67±1.3 years, BMI=25±1.4 kg/m2 volunteered for participation in this study. 72 hours after the determination of muscular maximal strength (by 1-RM test, subjects participated in acute resistance exercises via 75% 1-RM. In this research, two blood samples were collected at before and immediately after the exercise from Antecubital vein. Plasma Myostatin and serum levels of IGF-1 were measured by ELISA methods. Paired T-Test used for statical analyses of research data. Significant level was set at P≤0.05. Results: The results of this study showed that plasma Myostatin significantly decreased in response to resistance exercise (P=0.0001. Also the serum levels of IGF-1 increased significantly in response to resistance exercise (P=0.0001. In turn, the results reveled that the IGF-1 to Myostatin ratio increased significantly in response to resistance exercise (P=0.001. Conclusion: The results of this study showed that resistance exercise through increases of IGF-1 and decreases of Myostatin causes increment of IGF-1 to Myostatin ratio. According to the results of this study it seems prescription of resistance exercise could positive changes in proteins that involved in Cachexia syndrome in elderly people. Presumably, through this way we can prevent from Cachexia and its many physiological and physical related dysfunctions in theses people. Although more study is needed to clear its mechanisms.

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

    Directory of Open Access Journals (Sweden)

    Wee Tek Tay

    2015-11-01

    Full Text Available 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

  18. Effect of resistance training and protein intake pattern on myofibrillar protein synthesis and proteome kinetics in older men in energy restriction.

    Science.gov (United States)

    Murphy, Caoileann H; Shankaran, Mahalakshmi; Churchward-Venne, Tyler A; Mitchell, Cameron J; Kolar, Nathan M; Burke, Louise M; Hawley, John A; Kassis, Amira; Karagounis, Leonidas G; Li, Kelvin; King, Chelsea; Hellerstein, Marc; Phillips, Stuart M

    2018-06-01

    Strategies to enhance the loss of fat while preserving muscle mass during energy restriction are of great importance to prevent sarcopenia in overweight older adults. We show for the first time that the integrated rate of synthesis of numerous individual contractile, cytosolic and mitochondrial skeletal muscle proteins was increased by resistance training (RT) and unaffected by dietary protein intake pattern during energy restriction in free-living, obese older men. We observed a correlation between the synthetic rates of skeletal muscle-derived proteins obtained in serum (creatine kinase M-type, carbonic anhydrase 3) and the synthetic rates of proteins obtained via muscle sampling; and that the synthesis rates of these proteins in serum revealed the stimulatory effects of RT. These results have ramifications for understanding the influence of RT on skeletal muscle and are consistent with the role of RT in maintaining muscle protein synthesis and potentially supporting muscle mass preservation during weight loss. We determined how the pattern of protein intake and resistance training (RT) influenced longer-term (2 weeks) integrated myofibrillar protein synthesis (MyoPS) during energy restriction (ER). MyoPS and proteome kinetics were measured during 2 weeks of ER alone and 2 weeks of ER plus RT (ER + RT) in overweight/obese older men. Participants were randomized to consume dietary protein in a balanced (BAL: 25% daily protein per meal × 4 meals) or skewed (SKEW: 7:17:72:4% daily protein per meal) pattern (n = 10 per group). Participants ingested deuterated water during the consecutive 2-week periods, and skeletal muscle biopsies and serum were obtained at the beginning and conclusion of ER and ER + RT. Bulk MyoPS (i.e. synthesis of the myofibrillar protein sub-fraction) and the synthetic rates of numerous individual skeletal muscle proteins were quantified. Bulk MyoPS was not affected by protein distribution during ER or ER + RT (ER: BAL = 1.24

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

    Science.gov (United States)

    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.

  20. Insulin resistance in Chinese patients with type 2 diabetes is associated with C-reactive protein independent of abdominal obesity

    Directory of Open Access Journals (Sweden)

    Feng Xiaocheng

    2010-12-01

    Full Text Available Abstract Background There is debate as to whether the association between C-reactive protein (CRP and insulin resistance is independent of body fatness, particularly central obesity. Therefore, the association among CRP, insulin resistance and obesity was analyzed in Chinese patients with type 2 diabetes. Methods The study included 520 Chinese patients diagnosed with type 2 diabetes with CRP levels not exceeding 10 mg/L. The degree of insulin resistance was determined with the homeostasis model assessment of insulin resistance (HOMA-IR. The CRP levels were categorized into quartiles from the lowest to the highest concentrations (Q1-Q4. Results Body mass index (BMI and waist circumference (WC were both higher in Q4, Q3 and Q2 than those in Q1. HOMA-IR was higher in Q2, Q3 and Q4 than that in Q1 (Q1 vs Q4, P Conclusion These findings showed that insulin resistance was associated with CRP levels independent of abdominal obesity in Chinese patients with type 2 diabetes, suggesting that abdominal obesity could only partly explain the link between subclinical inflammation and insulin resistance.

  1. Protein adsorption resistance of PVP-modified polyurethane film prepared by surface-initiated atom transfer radical polymerization

    Energy Technology Data Exchange (ETDEWEB)

    Yuan, Huihui; Qian, Bin; Zhang, Wei [Shanghai Key Laboratory of Functional Materials Chemistry and Research Center of Analysis and Test, East China University of Science and Technology, Shanghai 200237 (China); Lan, Minbo, E-mail: minbolan@ecust.edu.cn [Shanghai Key Laboratory of Functional Materials Chemistry and Research Center of Analysis and Test, East China University of Science and Technology, Shanghai 200237 (China); State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237 (China)

    2016-02-15

    Highlights: • Antifouling PVP brushes were successfully grafted on PU films by SI-ATRP. • The effect of polymerization time on surface property and topography was studied. • Hydrophilicity and protein fouling resistance of PVP–PU films were greatly promoted. • Competitive adsorption of three proteins on PVP–PU films was evaluated. - Abstract: 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/cm{sup 2}, 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.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  3. A High Protein Diet Has No Harmful Effects: A One-Year Crossover Study in Resistance-Trained Males

    Directory of Open Access Journals (Sweden)

    Jose Antonio

    2016-01-01

    Full Text Available The purpose of this investigation was to determine the effects of a high protein diet over a one-year period. Fourteen healthy resistance-trained men completed the study (mean ± SD; age 26.3±3.9 yr; height 178.5±8.4 cm; and average years of training 8.9±3.4 yr. In a randomized crossover design, subjects consumed their habitual or normal diet for 2 months and 4 months and alternated that with a higher protein diet (>3 g/kg/d for 2 months and 4 months. Thus, on average, each subject was on their normal diet for 6 months and a higher protein diet for 6 months. Body composition was assessed via the Bod Pod®. Each subject provided approximately 100–168 daily dietary self-reports. During the subjects’ normal eating phase, they consumed (mean ± SD 29.94±5.65 kcals/kg/day and 2.51±0.69 g/kg/day of protein. This significantly increased (p<0.05 during the high protein phase to 34.37±5.88 kcals/kg/day and 3.32±0.87 g/kg/day of protein. Our investigation discovered that, in resistance-trained men that consumed a high protein diet (~2.51–3.32 g/kg/d for one year, there were no harmful effects on measures of blood lipids as well as liver and kidney function. In addition, despite the total increase in energy intake during the high protein phase, subjects did not experience an increase in fat mass.

  4. Protein adsorption resistance of PVP-modified polyurethane film prepared by surface-initiated atom transfer radical polymerization

    International Nuclear Information System (INIS)

    Yuan, Huihui; Qian, Bin; Zhang, Wei; Lan, Minbo

    2016-01-01

    Highlights: • Antifouling PVP brushes were successfully grafted on PU films by SI-ATRP. • The effect of polymerization time on surface property and topography was studied. • Hydrophilicity and protein fouling resistance of PVP–PU films were greatly promoted. • Competitive adsorption of three proteins on PVP–PU films was evaluated. - Abstract: 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/cm"2, 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.

  5. Effect of manuka honey on the expression of universal stress protein A in meticillin-resistant Staphylococcus aureus.

    Science.gov (United States)

    Jenkins, Rowena; Burton, Neil; Cooper, Rose

    2011-04-01

    Staphylococcus aureus is an important pathogen that can cause many problems, from impetigo to endocarditis. With its continued resistance to multiple antibiotics, S. aureus remains a serious health threat. Honey has been used to eradicate meticillin-resistant S. aureus (MRSA) strains from wounds, but its mode of action is not yet understood. Proteomics provides a potent group of techniques that can be used to analyse differences in protein expression between untreated bacterial cells and those treated with inhibitory concentrations of manuka honey. In this study, two-dimensional (2D) electrophoresis was combined with matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) to determine the identities of proteins whose levels of expression were changed at least two-fold following treatment with manuka honey. Protein extracts were obtained from cells grown in tryptone soy broth (with or without manuka honey) by mechanical disruption and were separated on 2D polyacrylamide gels. A protein was isolated in gels prepared from untreated cell extract that was absent from gels made using honey-treated cell extract. Using MALDI-TOF MS, the protein was identified as universal stress protein A (UspA). Downregulation of this protein was confirmed by real-time polymerase chain reaction (PCR), which showed a 16-fold downregulation in honey-treated cells compared with untreated samples. This protein is involved in the stress stamina response and its downregulation could help to explain the inhibition of MRSA by manuka honey. Copyright © 2011 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.

  6. Anti-protein C antibodies are associated with resistance to endogenous protein C activation and a severe thrombotic phenotype in antiphospholipid syndrome.

    Science.gov (United States)

    Arachchillage, D R J; Efthymiou, M; Mackie, I J; Lawrie, A S; Machin, S J; Cohen, H

    2014-11-01

    Antiphospholipid antibodies may interfere with the anticoagulant activity of activated protein C (APC) to induce acquired APC resistance (APCr). To investigate the frequency and characteristics of APCr by using recombinant human APC (rhAPC) and endogenous protein C activation in antiphospholipid syndrome (APS). APCr was assessed in APS and non-APS venous thromboembolism (VTE) patients on warfarin and normal controls with rhAPC or Protac by thrombin generation. IgG anti-protein C and anti-protein S antibodies and avidity were assessed by ELISA. APS patients showed greater resistance to both rhAPC and Protac than non-APS patients and normal controls (median normalized endogenous thrombin potential inhibition): APS patients with rhAPC, 81.3% (95% confidence interval [CI] 75.2-88.3%; non-APS patients with rhAPC, 97.7% (95% CI 93.6-101.8%; APS patients with Protac, 66.0% (95% CI 59.5-72.6%); and non-APS patients with Protac, 80.7 (95% CI 74.2-87.2%). APS patients also had a higher frequency and higher levels of anti-protein C antibodies, with 60% (15/25) high-avidity antibodies. High-avidity anti-protein C antibodies were associated with greater APCr and with a severe thrombotic phenotype (defined as the development of recurrent VTE while patients were receiving therapeutic anticoagulation or both venous and arterial thrombosis). Twelve of 15 (80%) patients with high-avidity anti-protein C antibodies were classified as APS category I. Thrombotic APS patients showed greater APCr to both rhAPC and activation of endogenous protein C by Protac. High-avidity anti-protein C antibodies, associated with greater APCr, may provide a marker for a severe thrombotic phenotype in APS. However, in patients with category I APS, it remains to be established whether anti-protein C or anti-β2 -glycoprotein I antibodies are responsible for APCr. © 2014 International Society on Thrombosis and Haemostasis.

  7. Systemic acquired resistance in soybean is regulated by two proteins, Orthologous to Arabidopsis NPR1

    Directory of Open Access Journals (Sweden)

    Sandhu Devinder

    2009-08-01

    Full Text Available Abstract Background Systemic acquired resistance (SAR is induced in non-inoculated leaves following infection with certain pathogenic strains. SAR is effective against many pathogens. Salicylic acid (SA is a signaling molecule of the SAR pathway. The development of SAR is associated with the induction of pathogenesis related (PR genes. Arabidopsis non-expressor of PR1 (NPR1 is a regulatory gene of the SA signal pathway 123. SAR in soybean was first reported following infection with Colletotrichum trancatum that causes anthracnose disease. We investigated if SAR in soybean is regulated by a pathway, similar to the one characterized in Arabidopsis. Results Pathogenesis-related gene GmPR1 is induced following treatment of soybean plants with the SAR inducer, 2,6-dichloroisonicotinic acid (INA or infection with the oomycete pathogen, Phytophthora sojae. In P. sojae-infected plants, SAR was induced against the bacterial pathogen, Pseudomonas syringae pv. glycinea. Soybean GmNPR1-1 and GmNPR1-2 genes showed high identities to Arabidopsis NPR1. They showed similar expression patterns among the organs, studied in this investigation. GmNPR1-1 and GmNPR1-2 are the only soybean homologues of NPR1and are located in homoeologous regions. In GmNPR1-1 and GmNPR1-2 transformed Arabidopsis npr1-1 mutant plants, SAR markers: (i PR-1 was induced following INA treatment and (ii BGL2 following infection with Pseudomonas syringae pv. tomato (Pst, and SAR was induced following Pst infection. Of the five cysteine residues, Cys82, Cys150, Cys155, Cys160, and Cys216 involved in oligomer-monomer transition in NPR1, Cys216 in GmNPR1-1 and GmNPR1-2 proteins was substituted to Ser and Leu, respectively. Conclusion Complementation analyses in Arabidopsis npr1-1 mutants revealed that homoeologous GmNPR1-1 and GmNPR1-2 genes are orthologous to Arabidopsis NPR1. Therefore, SAR pathway in soybean is most likely regulated by GmNPR1 genes. Substitution of Cys216 residue, essential

  8. Systemic acquired resistance in soybean is regulated by two proteins, Orthologous to Arabidopsis NPR1.

    Science.gov (United States)

    Sandhu, Devinder; Tasma, I Made; Frasch, Ryan; Bhattacharyya, Madan K

    2009-08-05

    Systemic acquired resistance (SAR) is induced in non-inoculated leaves following infection with certain pathogenic strains. SAR is effective against many pathogens. Salicylic acid (SA) is a signaling molecule of the SAR pathway. The development of SAR is associated with the induction of pathogenesis related (PR) genes. Arabidopsis non-expressor of PR1 (NPR1) is a regulatory gene of the SA signal pathway 123. SAR in soybean was first reported following infection with Colletotrichum trancatum that causes anthracnose disease. We investigated if SAR in soybean is regulated by a pathway, similar to the one characterized in Arabidopsis. Pathogenesis-related gene GmPR1 is induced following treatment of soybean plants with the SAR inducer, 2,6-dichloroisonicotinic acid (INA) or infection with the oomycete pathogen, Phytophthora sojae. In P. sojae-infected plants, SAR was induced against the bacterial pathogen, Pseudomonas syringae pv. glycinea. Soybean GmNPR1-1 and GmNPR1-2 genes showed high identities to Arabidopsis NPR1. They showed similar expression patterns among the organs, studied in this investigation. GmNPR1-1 and GmNPR1-2 are the only soybean homologues of NPR1and are located in homoeologous regions. In GmNPR1-1 and GmNPR1-2 transformed Arabidopsis npr1-1 mutant plants, SAR markers: (i) PR-1 was induced following INA treatment and (ii) BGL2 following infection with Pseudomonas syringae pv. tomato (Pst), and SAR was induced following Pst infection. Of the five cysteine residues, Cys82, Cys150, Cys155, Cys160, and Cys216 involved in oligomer-monomer transition in NPR1, Cys216 in GmNPR1-1 and GmNPR1-2 proteins was substituted to Ser and Leu, respectively. Complementation analyses in Arabidopsis npr1-1 mutants revealed that homoeologous GmNPR1-1 and GmNPR1-2 genes are orthologous to Arabidopsis NPR1. Therefore, SAR pathway in soybean is most likely regulated by GmNPR1 genes. Substitution of Cys216 residue, essential for oligomer-monomer transition of Arabidopsis NPR1

  9. Heat-shock protein 40 is the key farnesylation target in meristem size control, abscisic acid signaling, and drought resistance.

    Science.gov (United States)

    Barghetti, Andrea; Sjögren, Lars; Floris, Maïna; Paredes, Esther Botterweg; Wenkel, Stephan; Brodersen, Peter

    2017-11-15

    Protein farnesylation is central to molecular cell biology. In plants, protein farnesyl transferase mutants are pleiotropic and exhibit defective meristem organization, hypersensitivity to the hormone abscisic acid, and increased drought resistance. The precise functions of protein farnesylation in plants remain incompletely understood because few relevant farnesylated targets have been identified. Here, we show that defective farnesylation of a single factor-heat-shock protein 40 (HSP40), encoded by the J2 and J3 genes-is sufficient to confer ABA hypersensitivity, drought resistance, late flowering, and enlarged meristems, indicating that altered function of chaperone client proteins underlies most farnesyl transferase mutant phenotypes. We also show that expression of an abiotic stress-related microRNA (miRNA) regulon controlled by the transcription factor SPL7 requires HSP40 farnesylation. Expression of a truncated SPL7 form mimicking its activated proteolysis fragment of the membrane-bound SPL7 precursor partially restores accumulation of SPL7-dependent miRNAs in farnesyl transferase mutants. These results implicate the pathway directing SPL7 activation from its membrane-bound precursor as an important target of farnesylated HSP40, consistent with our demonstration that HSP40 farnesylation facilitates its membrane association. The results also suggest that altered gene regulation via select miRNAs contributes to abiotic stress-related phenotypes of farnesyl transferase mutants. © 2017 Barghetti et al.; Published by Cold Spring Harbor Laboratory Press.

  10. Damage-recognition proteins as a potential indicator of DNA-damage-mediated sensitivity or resistance of human cells to ultraviolet radiation

    International Nuclear Information System (INIS)

    Chao, C.C.-K.

    1992-01-01

    The authors compared damage-recognition proteins in cells expressing different sensitivities to DNA damage. An increase in damage-recognition proteins and an enhancement of plasmid re-activation were detected in HeLa cells resistant to cisplatin and u.v. However, repair-defective cells derived from xeroderma-pigmentosum (a rare skin disease) patients did not express less cisplatin damage-recognition proteins than repair-competent cells, suggesting that damage-recognition-protein expression may not be related to DNA repair. By contrast, cells resistant to DNA damage consistently expressed high levels of u.v.-modified-DNA damage-recognition proteins. The results support the notion that u.v. damage-recognition proteins are different from those that bind to cisplatin. Findings also suggest that the damage-recognition proteins identified could be used as potential indicators of the sensitivity or resistance of cells to u.v. (author)

  11. Determination of coagulation inhibitor levels and resistance to activated protein C in patients undergoing gastric surgery for benign and malignant disorders

    DEFF Research Database (Denmark)

    Andersen, B S; Rahr, H B; Sørensen, J V

    1997-01-01

    The aim of the present study was to determine plasma levels of protein C antigen (PC:Ag) and activity (PC:Act), tissue factor pathway inhibitor (TFPI), protein S (PS), antithrombin (AT), heparin cofactor II (HCII), and resistance to activated protein C (APCR) before, during and after elective gas...

  12. Involvement of the Pleiotropic Drug Resistance Response, Protein Kinase C Signaling, and Altered Zinc Homeostasis in Resistance of Saccharomyces cerevisiae to Diclofenac ▿

    Science.gov (United States)

    van Leeuwen, Jolanda S.; Vermeulen, Nico P. E.; Vos, J. Chris

    2011-01-01

    Diclofenac is a widely used analgesic drug that can cause serious adverse drug reactions. We used Saccharomyces cerevisiae as a model eukaryote with which to elucidate the molecular mechanisms of diclofenac toxicity and resistance. Although most yeast cells died during the initial diclofenac treatment, some survived and started growing again. Microarray analysis of the adapted cells identified three major processes involved in diclofenac detoxification and tolerance. In particular, pleiotropic drug resistance (PDR) genes and genes under the control of Rlm1p, a transcription factor in the protein kinase C (PKC) pathway, were upregulated in diclofenac-adapted cells. We tested if these processes or pathways were directly involved in diclofenac toxicity or resistance. Of the pleiotropic drug resistance gene products, the multidrug transporter Pdr5p was crucially important for diclofenac tolerance. Furthermore, deletion of components of the cell wall stress-responsive PKC pathway increased diclofenac toxicity, whereas incubation of cells with the cell wall stressor calcofluor white before the addition of diclofenac decreased its toxicity. Also, diclofenac induced flocculation, which might trigger the cell wall alterations. Genes involved in ribosome biogenesis and rRNA processing were downregulated, as were zinc-responsive genes. Paradoxically, deletion of the zinc-responsive transcription factor Zap1p or addition of the zinc chelator 1,10-phenanthroline significantly increased diclofenac toxicity, establishing a regulatory role for zinc in diclofenac resistance. In conclusion, we have identified three new pathways involved in diclofenac tolerance in yeast, namely, Pdr5p as the main contributor to the PDR response, cell wall signaling via the PKC pathway, and zinc homeostasis, regulated by Zap1p. PMID:21724882

  13. Genipin-induced inhibition of uncoupling protein-2 sensitizes drug-resistant cancer cells to cytotoxic agents.

    Directory of Open Access Journals (Sweden)

    Ryan J Mailloux

    2010-10-01

    Full Text Available Uncoupling protein-2 (UCP2 is known to suppress mitochondrial reactive oxygen species (ROS production and is employed by drug-resistant cancer cells to mitigate oxidative stress. Using the drug-sensitive HL-60 cells and the drug-resistant MX2 subline as model systems, we show that genipin, a UCP2 inhibitor, sensitizes drug-resistant cells to cytotoxic agents. Increased MX2 cell death was observed upon co-treatment with genipin and different doses of menadione, doxorubicin, and epirubicin. DCFH-DA fluorimetry revealed that the increase in MX2 cell death was accompanied by enhanced cellular ROS levels. The drug-induced increase in ROS was linked to genipin-mediated inhibition of mitochondrial proton leak. State 4 and resting cellular respiratory rates were higher in the MX2 cells in comparison to the HL-60 cells, and the increased respiration was readily suppressed by genipin in the MX2 cells. UCP2 accounted for a remarkable 37% of the resting cellular oxygen consumption indicating that the MX2 cells are functionally reliant on this protein. Higher amounts of UCP2 protein were detected in the MX2 versus the HL-60 mitochondria. The observed effects of genipin were absent in the HL-60 cells pointing to the selectivity of this natural product for drug-resistant cells. The specificity of genipin for UCP2 was confirmed using CHO cells stably expressing UCP2 in which genipin induced an ∼22% decrease in state 4 respiration. These effects were absent in empty vector CHO cells expressing no UCP2. Thus, the chemical inhibition of UCP2 with genipin sensitizes multidrug-resistant cancer cells to cytotoxic agents.

  14. Expression of P-glycoprotein and multidrug resistance associated protein in Ehrlich ascites tumor cells after fractionated irradiation

    DEFF Research Database (Denmark)

    Nielsen, D; Maare, C; Eriksen, J

    2001-01-01

    PURPOSE: To characterize irradiated murine tumor cells with respect to drug resistance, drug kinetics, and ATPase activity, and to evaluate the possible role of P-glycoprotein (PGP) and murine multidrug resistance associated protein (Mrp1) in the drug-resistant phenotype of these cells. METHODS...... AND MATERIALS: Sensitive Ehrlich ascites tumor cells (EHR2) were in vitro exposed to fractionated irradiation (60 Gy). Western blot analysis was performed for determination of PGP and Mrp1, reverse transcriptase-polymerase chain reaction (RT-PCR) for determination of mdr1a + b mRNA, and semiquantitative RT......-PCR for Mrp1 mRNA. The clonogenic assay was applied to investigate sensitivity, whereas the steady-state drug accumulation of daunorubicin (DNR), 3H-vincristine (VCR), and 3H-etoposide (VP16) was measured by spectrofluorometry and scintillation counting, respectively. For determining of ATPase activity...

  15. Resistance of Sendai virus (HVJ) F-protein against gamma irradiation

    International Nuclear Information System (INIS)

    Hosokawa, Yasushi

    1980-01-01

    Sendai virus envelope proteins (hemagglutinin and F-protein), containing disulfide bonds in their molecules, were almost caused neither cleavage of protein molecules nor disappearance of antigenisitics by 10 5 R of γ-irradiation, but the liberated inner proteins (P, NP and M-protein) were severed into low molecules. By irradiation, hemagglutinin and F-protein of the virions were polymerized or aggregated together in such a way that the aggregate was not dissociated by SDS-2ME treatment on Slab gel electrophoresis, but in the liberated states hemagglutinin alone was aggregated and F-protein was separated with little loss of its anigenisity. From above results, the interaction between hemagglutinin and F-protein, and the utility of irradiation for isolation of F-protein were discussed. (author)

  16. Relationship of Triamine-Biocide Tolerance of Salmonella enterica Serovar Senftenberg to Antimicrobial Susceptibility, Serum Resistance and Outer Membrane Proteins.

    Science.gov (United States)

    Futoma-Kołoch, Bożena; Dudek, Bartłomiej; Kapczyńska, Katarzyna; Krzyżewska, Eva; Wańczyk, Martyna; Korzekwa, Kamila; Rybka, Jacek; Klausa, Elżbieta; Bugla-Płoskońska, Gabriela

    2017-07-11

    A new emerging phenomenon is the association between the incorrect use of biocides in the process of disinfection in farms and the emergence of cross-resistance in Salmonella populations. Adaptation of the microorganisms to the sub-inhibitory concentrations of the disinfectants is not clear, but may result in an increase of sensitivity or resistance to antibiotics, depending on the biocide used and the challenged Salmonella serovar. Exposure of five Salmonella enterica subsp. enterica serovar Senftenberg ( S. Senftenberg) strains to triamine-containing disinfectant did not result in variants with resistance to antibiotics, but has changed their susceptibility to normal human serum (NHS). Three biocide variants developed reduced sensitivity to NHS in comparison to the sensitive parental strains, while two isolates lost their resistance to serum. For S. Senftenberg, which exhibited the highest triamine tolerance (6 × MIC) and intrinsic sensitivity to 22.5% and 45% NHS, a downregulation of flagellin and enolase has been demonstrated, which might suggest a lower adhesion and virulence of the bacteria. This is the first report demonstrating the influence of biocide tolerance on NHS resistance. In conclusion, there was a potential in S. Senftenberg to adjust to the conditions, where the biocide containing triamine was present. However, the adaptation did not result in the increase of antibiotic resistance, but manifested in changes within outer membrane proteins' patterns. The strategy of bacterial membrane proteins' analysis provides an opportunity to adjust the ways of infection treatments, especially when it is connected to the life-threating bacteremia caused by Salmonella species.

  17. Protease resistance of infectious prions is suppressed by removal of a single atom in the cellular prion protein.

    Science.gov (United States)

    Leske, Henning; Hornemann, Simone; Herrmann, Uli Simon; Zhu, Caihong; Dametto, Paolo; Li, Bei; Laferriere, Florent; Polymenidou, Magdalini; Pelczar, Pawel; Reimann, Regina Rose; Schwarz, Petra; Rushing, Elisabeth Jane; Wüthrich, Kurt; Aguzzi, Adriano

    2017-01-01

    Resistance to proteolytic digestion has long been considered a defining trait of prions in tissues of organisms suffering from transmissible spongiform encephalopathies. Detection of proteinase K-resistant prion protein (PrPSc) still represents the diagnostic gold standard for prion diseases in humans, sheep and cattle. However, it has become increasingly apparent that the accumulation of PrPSc does not always accompany prion infections: high titers of prion infectivity can be reached also in the absence of protease resistant PrPSc. Here, we describe a structural basis for the phenomenon of protease-sensitive prion infectivity. We studied the effect on proteinase K (PK) resistance of the amino acid substitution Y169F, which removes a single oxygen atom from the β2-α2 loop of the cellular prion protein (PrPC). When infected with RML or the 263K strain of prions, transgenic mice lacking wild-type (wt) PrPC but expressing MoPrP169F generated prion infectivity at levels comparable to wt mice. The newly generated MoPrP169F prions were biologically indistinguishable from those recovered from prion-infected wt mice, and elicited similar pathologies in vivo. Surprisingly, MoPrP169F prions showed greatly reduced PK resistance and density gradient analyses showed a significant reduction in high-density aggregates. Passage of MoPrP169F prions into mice expressing wt MoPrP led to full recovery of protease resistance, indicating that no strain shift had taken place. We conclude that a subtle structural variation in the β2-α2 loop of PrPC affects the sensitivity of PrPSc to protease but does not impact prion replication and infectivity. With these findings a specific structural feature of PrPC can be linked to a physicochemical property of the corresponding PrPSc.

  18. Defended to the Nines: 25 Years of Resistance Gene Cloning Identifies Nine Mechanisms for R Protein Function[OPEN

    Science.gov (United States)

    2018-01-01

    Plants have many, highly variable resistance (R) gene loci, which provide resistance to a variety of pathogens. The first R gene to be cloned, maize (Zea mays) Hm1, was published over 25 years ago, and since then, many different R genes have been identified and isolated. The encoded proteins have provided clues to the diverse molecular mechanisms underlying immunity. Here, we present a meta-analysis of 314 cloned R genes. The majority of R genes encode cell surface or intracellular receptors, and we distinguish nine molecular mechanisms by which R proteins can elevate or trigger disease resistance: direct (1) or indirect (2) perception of pathogen-derived molecules on the cell surface by receptor-like proteins and receptor-like kinases; direct (3) or indirect (4) intracellular detection of pathogen-derived molecules by nucleotide binding, leucine-rich repeat receptors, or detection through integrated domains (5); perception of transcription activator-like effectors through activation of executor genes (6); and active (7), passive (8), or host reprogramming-mediated (9) loss of susceptibility. Although the molecular mechanisms underlying the functions of R genes are only understood for a small proportion of known R genes, a clearer understanding of mechanisms is emerging and will be crucial for rational engineering and deployment of novel R genes. PMID:29382771

  19. Defended to the Nines: 25 Years of Resistance Gene Cloning Identifies Nine Mechanisms for R Protein Function.

    Science.gov (United States)

    Kourelis, Jiorgos; van der Hoorn, Renier A L

    2018-02-01

    Plants have many, highly variable resistance ( R ) gene loci, which provide resistance to a variety of pathogens. The first R gene to be cloned, maize ( Zea mays ) Hm1 , was published over 25 years ago, and since then, many different R genes have been identified and isolated. The encoded proteins have provided clues to the diverse molecular mechanisms underlying immunity. Here, we present a meta-analysis of 314 cloned R genes. The majority of R genes encode cell surface or intracellular receptors, and we distinguish nine molecular mechanisms by which R proteins can elevate or trigger disease resistance: direct (1) or indirect (2) perception of pathogen-derived molecules on the cell surface by receptor-like proteins and receptor-like kinases; direct (3) or indirect (4) intracellular detection of pathogen-derived molecules by nucleotide binding, leucine-rich repeat receptors, or detection through integrated domains (5); perception of transcription activator-like effectors through activation of executor genes (6); and active (7), passive (8), or host reprogramming-mediated (9) loss of susceptibility. Although the molecular mechanisms underlying the functions of R genes are only understood for a small proportion of known R genes, a clearer understanding of mechanisms is emerging and will be crucial for rational engineering and deployment of novel R genes. © 2018 American Society of Plant Biologists. All rights reserved.

  20. Defects in mitochondrial fission protein dynamin-related protein 1 are linked to apoptotic resistance and autophagy in a lung cancer model.

    Directory of Open Access Journals (Sweden)

    Kelly Jean Thomas

    Full Text Available Evasion of apoptosis is implicated in almost all aspects of cancer progression, as well as treatment resistance. In this study, resistance to apoptosis was identified in tumorigenic lung epithelial (A549 cells as a consequence of defects in mitochondrial and autophagic function. Mitochondrial function is determined in part by mitochondrial morphology, a process regulated by mitochondrial dynamics whereby the joining of two mitochondria, fusion, inhibits apoptosis while fission, the division of a mitochondrion, initiates apoptosis. Mitochondrial morphology of A549 cells displayed an elongated phenotype-mimicking cells deficient in mitochondrial fission protein, Dynamin-related protein 1 (Drp1. A549 cells had impaired Drp1 mitochondrial recruitment and decreased Drp1-dependent fission. Cytochrome c release and caspase-3 and PARP cleavage were impaired both basally and with apoptotic stimuli in A549 cells. Increased mitochondrial mass was observed in A549 cells, suggesting defects in mitophagy (mitochondrial selective autophagy. A549 cells had decreased LC3-II lipidation and lysosomal inhibition suggesting defects in autophagy occur upstream of lysosomal degradation. Immunostaining indicated mitochondrial localized LC3 punctae in A549 cells increased after mitochondrial uncoupling or with a combination of mitochondrial depolarization and ectopic Drp1 expression. Increased inhibition of apoptosis in A549 cells is correlated with impeded mitochondrial fission and mitophagy. We suggest mitochondrial fission defects contribute to apoptotic resistance in A549 cells.

  1. Ageing has no effect on the regulation of the ubiquitin proteasome-related genes and proteins following resistance exercise

    Directory of Open Access Journals (Sweden)

    Renae Jane Stefanetti

    2014-01-01

    Full Text Available Skeletal muscle atrophy is a critical component of the ageing process. Age-related muscle wasting is due to disrupted muscle protein turnover, a process mediated in part by the ubiquitin proteasome pathway (UPP. Additionally, older subjects have been observed to have an attenuated anabolic response, at both the molecular and physiological levels, following a single-bout of resistance exercise (RE. We investigated the expression levels of the UPP-related genes and proteins involved in muscle protein degradation in 10 older (60-75 years versus 10 younger (18-30 years healthy male subjects at basal as well as 2 hours after a single-bout of RE. MURF1, atrogin-1 and FBXO40, their substrate targets PKM2, myogenin, MYOD, MHC and EIF3F as well as MURF1 and atrogin-1 transcriptional regulators FOXO1 and FOXO3 gene and/or protein expression levels were measured via real time PCR and western blotting, respectively. At basal, no age-related difference was observed in the gene/protein levels of atrogin-1, MURF1, myogenin, MYOD and FOXO1/3. However, a decrease in FBXO40 mRNA and protein levels was observed in older subjects, while PKM2 protein was increased in older subjects. In response to RE, MURF1, atrogin-1 and FBXO40 mRNA were upregulated in both the younger and older subjects, with changes observed in protein levels. In conclusion, UPP-related gene/protein expression is comparably regulated in healthy young and old male subjects at basal and following RE. These findings suggest that UPP signalling plays a limited role in the process of age-related muscle wasting. Future studies are required to investigate additional proteolytic mechanisms in conjunction with skeletal muscle protein breakdown measurements following RE in older versus younger subjects.

  2. Reverting doxorubicin resistance in colon cancer by targeting a key signaling protein, steroid receptor coactivator.

    Science.gov (United States)

    Xiong, Sang; Xiao, Gong-Wei

    2018-04-01

    Although there have been notable improvements in treatments against cancer, further research is required. In colon cancer, nearly all patients eventually experience drug resistance and stop responding to the approved drugs, making treatment difficult. Steroid receptor coactivator (SRC) is an oncogenic nuclear receptor coactivator that serves an important role in drug resistance. The present study generated a doxorubicin-resistant colon cancer cell line, in which the upregulation/activation of SRC was responsible for drug resistance, which in turn activated AKT. Overexpression of receptor tyrosine kinase-like epidermal growth factor receptor and insulin-like growth factor 1 receptor also induced SRC expression. It was observed that doxorubicin resistance in colon cancer also induced epithelial to mesenchymal transition, a decrease in expression of epithelial marker E-cadherin and an increase in the expression of mesenchymal markers, including N-cadherin and vimentin. Additionally, the present study indicated that SRC acts as a common signaling node, and inhibiting SRC in combination with doxorubicin treatment in doxorubicin-resistant cells aids in reversing the resistance. Thus, the present study suggests that activation of SRC is responsible for doxorubicin resistance in colon cancer. However, further research is required to understand the complete mechanism of how drug resistance occurs and how it may be tackled to treat patients.

  3. Analyzing pepsin degradation assay conditions used for allergenicity assessments to ensure that pepsin susceptible and pepsin resistant dietary proteins are distinguishable.

    Directory of Open Access Journals (Sweden)

    Rong Wang

    Full Text Available The susceptibility of a dietary protein to proteolytic degradation by digestive enzymes, such as gastric pepsin, provides information on the likelihood of systemic exposure to a structurally intact and biologically active macromolecule, thus informing on the safety of proteins for human and animal consumption. Therefore, the purpose of standardized in vitro degradation studies that are performed during protein safety assessments is to distinguish whether proteins of interest are susceptible or resistant to pepsin degradation via a study design that enables study-to-study comparison. Attempting to assess pepsin degradation under a wide-range of possible physiological conditions poses a problem because of the lack of robust and consistent data collected under a large-range of sub-optimal conditions, which undermines the needs to harmonize in vitro degradation conditions. This report systematically compares the effects of pH, incubation time, and pepsin-to-substrate protein ratio on the relative degradation of five dietary proteins: three pepsin susceptible proteins [ribulose 1,5-bisphosphate carboxylase-oxygenase (Rubisco, horseradish peroxidase (HRP, hemoglobin (Hb], and two pepsin resistant proteins [lipid transfer protein (LTP and soybean trypsin inhibitor (STI]. The results indicate that proteins susceptible to pepsin degradation are readily distinguishable from pepsin-resistant proteins when the reaction conditions are within the well-characterized optima for pepsin. The current standardized in vitro pepsin resistant assay with low pH and high pepsin-to-substrate ratio fits this purpose. Using non-optimal pH and/or pepsin-to-substrate protein ratios resulted in susceptible proteins no longer being reliably degraded by this stomach enzyme, which compromises the ability of this in vitro assay to distinguish between resistant and susceptible proteins and, therefore, no longer providing useful data to an overall weight-of-evidence approach to

  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

    Directory of Open Access Journals (Sweden)

    Charlene Desbonnet

    2016-04-01

    Full Text Available 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.

  5. Influence of source and quantity of protein on the development of immunity and resistance to African trypanosomiasis.

    Science.gov (United States)

    Norton, J D; Yang, S P; Diffley, P

    1986-01-01

    Although it is well documented that severe protein deprivation inhibits the development of the immune response and exacerbates certain infections, little has been done to study the effects of native diets on endemic diseases or immunity. Therefore, protein-restricted diets were formulated for mice to mimic the sources and amounts measured in human diets of the Batouri region of Cameroon, endemic for African trypanosomiasis. Weanling C57BL/6 female mice were fed a diet that contained 73% of the recommended daily allowance (RDA) of protein. The sources of protein were all plant (cornmeal), all animal (casein), or a ratio that reflected the native diet (2.2 parts plant to 1 part animal protein). Diets were isocaloric on a weight basis, equal in lipids, and adequate in vitamins and minerals. Control mice were fed laboratory chow or two times the RDA of animal protein (casein). Mice fed only cornmeal or the native diets consumed as much food but did not gain as much weight as mice fed only animal protein, indicating the poorer quality of protein in their diets. Upon infection with Trypanosoma brucei gambiense, however, significantly higher numbers of these mice controlled the first peak of parasitemia and survived the infection as compared with mice fed the other three diets. Since all mice developed patent infections and the parasite growth rate was unaffected by diet, innate immune factors were ruled out as the cause for the higher level of resistance to the parasite. To determine whether diet affected the development of the immune system, weanling mice were maintained on diets for 30 days before immunization with sheep erythrocytes or trinitrophenylated Ficoll. Mice fed only plant protein or native diets elicited higher direct plaque-forming-cell responses to both the T-cell-dependent and T-cell-independent antigens. Since variant-specific immunity which controls levels of African trypanosomes in the blood is a T-cell-independent humoral immunoglobulin M response

  6. Low-load high volume resistance exercise stimulates muscle protein synthesis more than high-load low volume resistance exercise in young men.

    Directory of Open Access Journals (Sweden)

    Nicholas A Burd

    Full Text Available 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.

  7. The TIR domain of TIR-NB-LRR resistance proteins is a signaling domain involved in cell death induction.

    Science.gov (United States)

    Swiderski, Michal R; Birker, Doris; Jones, Jonathan D G

    2009-02-01

    In plants, the TIR (toll interleukin 1 receptor) domain is found almost exclusively in nucleotide-binding (NB) leucine-rich repeat resistance proteins and their truncated homologs, and has been proposed to play a signaling role during resistance responses mediated by TIR containing R proteins. Transient expression in Nicotiana benthamiana leaves of "TIR + 80", the RPS4 truncation without the NB-ARC domain, leads to EDS1-, SGT1-, and HSP90-dependent cell death. Transgenic Arabidopsis plants expressing the RPS4 TIR+80 from either dexamethasone or estradiol-inducible promoters display inducer-dependent cell death. Cell death is also elicited by transient expression of similarly truncated constructs from two other R proteins, RPP1A and At4g19530, but is not elicited by similar constructs representing RPP2A and RPP2B proteins. Site-directed mutagenesis of the RPS4 TIR domain identified many loss-of-function mutations but also revealed several gain-of function substitutions. Lack of cell death induction by the E160A substitution suggests that amino acids outside of the TIR domain contribute to cell death signaling in addition to the TIR domain itself. This is consistent with previous observations that the TIR domain itself is insufficient to induce cell death upon transient expression.

  8. Combined Effect of the Cfr Methyltransferase and Ribosomal Protein L3 Mutations on Resistance to Ribosome-Targeting Antibiotics.

    Science.gov (United States)

    Pakula, Kevin K; Hansen, Lykke H; Vester, Birte

    2017-09-01

    Several groups of antibiotics inhibit bacterial growth by binding to bacterial ribosomes. Mutations in ribosomal protein L3 have been associated with resistance to linezolid and tiamulin, which both bind at the peptidyl transferase center in the ribosome. Resistance to these and other antibiotics also occurs through methylation of 23S rRNA at position A2503 by the methyltransferase Cfr. The mutations in L3 and the cfr gene have been found together in clinical isolates, raising the question of whether they have a combined effect on antibiotic resistance or growth. We transformed a plasmid-borne cfr gene into a uL3-depleted Escherichia coli strain containing either wild-type L3 or L3 with one of seven mutations, G147R, Q148F, N149S, N149D, N149R, Q150L, or T151P, expressed from plasmid-carried rplC genes. The L3 mutations are well tolerated, with small to moderate growth rate decreases. The presence of Cfr has a very minor influence on the growth rate. The resistance of the transformants to linezolid, tiamulin, florfenicol, and Synercid (a combination of quinupristin and dalfopristin [Q-D]) was measured by MIC assays. The resistance from Cfr was, in all cases, stronger than the effects of the L3 mutations, but various effects were obtained with the combinations of Cfr and L3 mutations ranging from a synergistic to an antagonistic effect. Linezolid and tiamulin susceptibility varied greatly among the L3 mutations, while no significant effects on florfenicol and Q-D susceptibility were seen. This study underscores the complex interplay between various resistance mechanisms and cross-resistance, even from antibiotics with overlapping binding sites. Copyright © 2017 American Society for Microbiology.

  9. A randomized trial of protein supplementation compared with extra fast food on the effects of resistance training to increase metabolism.

    Science.gov (United States)

    Hambre, David; Vergara, Marta; Lood, Yvonne; Bachrach-Lindström, Margareta; Lindström, Torbjörn; Nystrom, Fredrik H

    2012-10-01

    To prospectively evaluate the effects of resistance training combined with increased energy intake or protein-supplementation on lean body-mass, resting metabolic-rate (RMR) and cardiovascular risk factors. Twenty-four healthy males (aged 19-32 years) performed resistance exercise for 12 weeks aiming for at least 1 hour training-sessions 3 times a week. The participants were randomized to consume extra protein (33 g whey protein/day) or a meal of fast-food/day (1350 kcal, 41 g protein). Body-composition was measured with Dual-Energy X-ray Absorptiometry (DEXA) and RMR by indirect calorimetry. Fasting blood samples were drawn before and after the 3-month training period and after 12 months. The body weight increased from 75.1 ± 6.9 kg to 78.7 ± 7.2 kg (p < 0.0001), without differences between the groups. RMR increased from 1787 ± 143 kcal/24 h to 1954 ± 187 kcal/24 h (p < 0.0001, N = 24), which was more than expected from the increase in lean body-mass (increase from 59.7 ± 4.3 kg to 61.8 ± 4.1 kg p = 0.004). Fasting serum-insulin levels increased in the fast-food group compared with the extra-protein group (p = 0.03). ApoB increased from 0.691 ± 0.14 g/L to 0.768 ± 0.17 g/L, p = 0.004, in the fast-food group only. Long-term follow up after 12 months showed that RMR, body weight, total fat and lean body-masses did not differ from baseline (n = 19). Resistance training for 12 weeks increased RMR and lean body-mass similarly when based on either an increased energy-intake or protein supplement. However, the increase in RMR was higher than expected from the increase in lean body-mass. Thus resistance training could potentially decrease the risk of obesity by induction of increased RMR.

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

    Energy Technology Data Exchange (ETDEWEB)

    Tocchetti, Guillermo Nicolás [Instituto de Fisiología Experimental, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, CONICET, Suipacha 570, 2000 Rosario (Argentina); Rigalli, Juan Pablo [Instituto de Fisiología Experimental, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, CONICET, Suipacha 570, 2000 Rosario (Argentina); Department of Clinical Pharmacology and Pharmacoepidemiology, University of Heidelberg, Im Neuenheimer Feld 410, 69120 Heidelberg (Germany); Arana, Maite Rocío; Villanueva, Silvina Stella Maris [Instituto de Fisiología Experimental, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, CONICET, Suipacha 570, 2000 Rosario (Argentina); Mottino, Aldo Domingo, E-mail: amottino@unr.edu.ar [Instituto de Fisiología Experimental, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, CONICET, Suipacha 570, 2000 Rosario (Argentina)

    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. - Highlights: • Intestinal MRP2 (ABCC2) expression and activity can be regulated by xenobiotics. • PXR and CAR are major MRP2 modulators through a transcriptional mechanism. • Rifampicin

  11. Changes in Brain 14-3-3 Proteins in Response to Insulin Resistance Induced by a High Palatable Diet.

    Science.gov (United States)

    Bock, Hugo; Zimmer, Aline Rigon; Zimmer, Eduardo Rigon; de Souza, Diogo Onofre Gomes; Portela, Luis Valmor Cruz; Saraiva-Pereira, Maria Luiza

    2015-08-01

    The 14-3-3 protein family takes part in a wide range of cellular processes and is expressed in all eukaryotic organisms. In mammals, seven isoforms (β, ε, η, γ, τ, ζ, and σ) have been identified. 14-3-3 proteins are suggested to modulate the insulin-signaling cascade in the brain. The aim of this study was to investigate whether insulin resistance state induced by high palatable diet modulates expression of the 14-3-3 proteins in brain. Wistar male rats (n = 8) were divided into two experimental groups: insulin resistant (IR), induced by high palatable diet, and control (CO) group. Biochemical parameters (glucose tolerance test and plasma lipid profile) were evaluated after 130 days. Brain structures (cortex and hippocampus) were dissected for evaluation of messenger RNA (mRNA) and protein levels of different 14-3-3 proteins. Statistical analyses included Student t test and Pearson correlation. Significant decrease was observed in Ywhah and in Ywahq mRNA levels in the cortex of IR group, while no changes were observed in the hippocampus. Significant increase of θ isoform was observed in hippocampus IR group by immunodetection, while no differences were detected in the remaining isoforms. Inverse correlation was observed between blood glucose levels in cortex IR group and both Ywhah and Ywhaq mRNA levels. Protein levels of Creb and phosphatidylinositide 3-kinases (PI3K) showed to be increased in the hippocampus. These alterations may be due to a compensatory effect of impaired insulin signaling. We demonstrated differential expression of 14-3-3 isoforms throughout brain regions of rats with IR. As a whole, our results indicate that brain 14-3-3 levels are influenced by different diets.

  12. F-Box Protein FBXO22 Mediates Polyubiquitination and Degradation of CD147 to Reverse Cisplatin Resistance of Tumor Cells

    Directory of Open Access Journals (Sweden)

    Bo Wu

    2017-01-01

    Full Text Available Drug resistance remains a major clinical obstacle to successful treatment of cancer. As posttranslational modification is becoming widely recognized to affect the function of oncoproteins, targeting specific posttranslational protein modification provides an attractive strategy for anticancer drug development. CD147 is a transmembrane glycoprotein contributing to chemo-resistance of cancer cells in a variety of human malignancies. Ubiquitination is an important posttranslational modification mediating protein degradation. Degradation of oncoproteins, CD147 included, emerges as an attractive alternative for tumor inhibition. However, the ubiquitination of CD147 remains elusive. Here in this study, we found that deletion of the CD147 intracellular domain (CD147-ICD prolonged the half-life of CD147 in HEK293T cells, and we identified that CD147-ICD interacts with FBXO22 using mass spectrometry and Western blot. Then, we demonstrated that FBXO22 mediates the polyubiquitination and degradation of CD147 by recognizing CD147-ICD. While knocking down of FBXO22 prolonged the half-life of CD147 in HEK293T cells, we found that FBXO22 regulates CD147 protein turnover in SMMC-7721, Huh-7 and A549 cells. Moreover, we found that the low level of FBXO22 contributes to the accumulation of CD147 and thereafter the cisplatin resistance of A549/DDP cells. To conclude, our study demonstrated that FBXO22 mediated the polyubiquitination and degradation of CD147 by interacting with CD147-ICD, and CD147 polyubiquitination by FBXO22 reversed cisplatin resistance of tumor cells.

  13. Mutations of the Transporter Proteins GlpT and UhpT Confer Fosfomycin Resistance in Staphylococcus aureus

    Directory of Open Access Journals (Sweden)

    Su Xu

    2017-05-01

    Full Text Available With the increasing spread of methicillin-resistant Staphylococcus aureus worldwide, fosfomycin has begun to be used more often, either alone or in combination with other antibiotics, for treating methicillin-resistant S. aureus infections, resulting in the emergence of fosfomycin-resistant strains. Fosfomycin resistance is reported to be mediated by fosfomycin-modifying enzymes (FosA, FosB, FosC, and FosX and mutations of the target enzyme MurA or the membrane transporter proteins UhpT and GlpT. Our previous studies indicated that the fos genes might not the major fosfomycin resistance mechanism in S. aureus, whereas mutations of glpT and uhpT seemed to be more related to fosfomycin resistance. However, the precise role of these two genes in S. aureus fosfomycin resistance remains unclear. The aim of the present study was to investigate the role of glpT and uhpT in S. aureus fosfomycin resistance. Homologous recombination was used to knockout the uhpT and glpT genes in S. aureus Newman. Gene complementation was generated by the plasmid pRB473 carrying these two genes. The fosfomycin minimal inhibitory concentration (MIC of the strains was measured by the E-test to observe the influence of gene deletion on antibiotic susceptibility. In addition, growth curves were constructed to determine whether the mutations have a significant influence on bacterial growth. Deletion of uhpT, glpT, and both of them led to increased fosfomycin MIC 0.5 μg/ml to 32 μg/ml, 4 μg/ml, and >1024 μg/ml, respectively. By complementing uhpT and glpT into the deletion mutants, the fosfomycin MIC decreased from 32 to 0.5 μg/ml and from 4 to 0.25 μg/ml, respectively. Moreover, the transporter gene-deleted strains showed no obvious difference in growth curves compared to the parental strain. In summary, our study strongly suggests that mutations of uhpT and glpT lead to fosfomycin resistance in S. aureus, and that uhpT mutation may play a more important role. The high

  14. Downregulation of the proapoptotic protein MOAP-1 by the UBR5 ubiquitin ligase and its role in ovarian cancer resistance to cisplatin

    OpenAIRE

    Matsuura, K; Huang, N-J; Cocce, K; Zhang, L; Kornbluth, S

    2016-01-01

    Evasion of apoptosis allows many cancers to resist chemotherapy. Apoptosis is mediated by the serial activation of caspase family proteins. These proteases are often activated upon the release of cytochrome c from the mitochondria, which is promoted by the proapoptotic Bcl-2 family protein, Bax. This function of Bax is enhanced by the MOAP-1 (modulator of apoptosis protein 1) protein in response to DNA damage. Previously, we reported that MOAP-1 is targeted for ubiquitylation and degradation ...

  15. A single amino acid substitution in the core protein of West Nile virus increases resistance to acidotropic compounds.

    Directory of Open Access Journals (Sweden)

    Miguel A Martín-Acebes

    Full Text Available 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.

  16. A single amino acid substitution in the core protein of West Nile virus increases resistance to acidotropic compounds.

    Science.gov (United States)

    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.

  17. Expression of multidrug resistance-related protein (MRP-1), lung resistance-related protein (LRP) and topoisomerase-II (TOPO-II) in Wilms' tumor: immunohistochemical study using TMA methodology.

    Science.gov (United States)

    Fridman, Eduard; Skarda, Jozef; Pinthus, Jonatan H; Ramon, Jonathan; Mor, Yoran

    2008-06-01

    MRP-1, LRP and TOPO-II are all associated with protection of the cells from the adverse effects of various chemotherapeutics. The aim of this study was to measure the expression of these proteins in Wilms' tumor (WT). TMA block was constructed from 14 samples of WT's and from xenografts derived from them. Sections of the TMA were used for immunostaining against MRP-1, LRP and TOPO-IIa. All normal kidneys expressed MRP-1 but were either weakly or negatively stained for LRP and TOPO-IIa. In WT samples, MRP-1 was universally expressed, exclusively in the tubular component, while there was no expression of LRP and TOPO-IIa showed heterogeneous distribution. The xenografts varied in their MRP-1 and TOPO-IIa expression and exhibited weak/negative staining of LRP. This study shows that although all the proteins evaluated, had different expression patterns in the tumor samples, the most prominent changes in expression were found for MRP-1. The exact clinical implications of these changes in expression and their relevance to the resistance of these tumors to chemotherapy requires further investigation. The finding of different expression profiles for the multidrug resistance proteins in the original WT's and their xenografts suggests that the results of animal cancer models may be difficult to interpret.

  18. Resistance to the Peptidyl Transferase Inhibitor Tiamulin Caused by Mutation of Ribosomal Protein L3

    OpenAIRE

    Bøsling, Jacob; Poulsen, Susan M.; Vester, Birte; Long, Katherine S.

    2003-01-01

    The antibiotic tiamulin targets the 50S subunit of the bacterial ribosome and interacts at the peptidyl transferase center. Tiamulin-resistant Escherichia coli mutants were isolated in order to elucidate mechanisms of resistance to the drug. No mutations in the rRNA were selected as resistance determinants using a strain expressing only a plasmid-encoded rRNA operon. Selection in a strain with all seven chromosomal rRNA operons yielded a mutant with an A445G mutation in the gene coding for ri...

  19. Role of protein and amino acids in promoting lean mass accretion with resistance exercise and attenuating lean mass loss during energy deficit in humans.

    Science.gov (United States)

    Churchward-Venne, Tyler A; Murphy, Caoileann H; Longland, Thomas M; Phillips, Stuart M

    2013-08-01

    Amino acids are major nutrient regulators of muscle protein turnover. After protein ingestion, hyperaminoacidemia stimulates increased rates of skeletal muscle protein synthesis, suppresses muscle protein breakdown, and promotes net muscle protein accretion for several hours. These acute observations form the basis for strategized protein intake to promote lean mass accretion, or prevent lean mass loss over the long term. However, factors such as protein dose, protein source, and timing of intake are important in mediating the anabolic effects of amino acids on skeletal muscle and must be considered within the context of evaluating the reported efficacy of long-term studies investigating protein supplementation as part of a dietary strategy to promote lean mass accretion and/or prevent lean mass loss. Current research suggests that dietary protein supplementation can augment resistance exercise-mediated gains in skeletal muscle mass and strength and can preserve skeletal muscle mass during periods of diet-induced energy restriction. Perhaps less appreciated, protein supplementation can augment resistance training-mediated gains in skeletal muscle mass even in individuals habitually consuming 'adequate' (i.e., >0.8 g kg⁻¹ day⁻¹) protein. Additionally, overfeeding energy with moderate to high-protein intake (15-25 % protein or 1.8-3.0 g kg⁻¹ day⁻¹) is associated with lean, but not fat mass accretion, when compared to overfeeding energy with low protein intake (5 % protein or ~0.68 g kg⁻¹ day⁻¹). Amino acids represent primary nutrient regulators of skeletal muscle anabolism, capable of enhancing lean mass accretion with resistance exercise and attenuating the loss of lean mass during periods of energy deficit, although factors such as protein dose, protein source, and timing of intake are likely important in mediating these effects.

  20. EFFECT OF ADIPOSITY ON PLASMA-LIPID TRANSFER PROTEIN ACTIVITIES - A POSSIBLE LINK BETWEEN INSULIN-RESISTANCE AND HIGH-DENSITY-LIPOPROTEIN METABOLISM

    NARCIS (Netherlands)

    DULLAART, RPF; SLUITER, WJ; DIKKESCHEI, LD; HOOGENBERG, K; VANTOL, A

    The mechanisms responsible for the decreased high density lipoprotein (HDL) cholesterol levels associated with obesity and insulin resistance are not well understood. Lecithin: cholesterol acyltransferase (LCAT) and cholesterol ester transfer protein (CETP) are key factors in the esterification of

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

    International Nuclear Information System (INIS)

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

    2009-01-01

    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.

  2. Differentially expressed proteins in human MCF-7 breast cancer cells sensitive and resistant to paclitaxel

    Czech Academy of Sciences Publication Activity Database

    Pavlíková, N.; Bartoňová, I.; Balušíková, K.; Kopperová, D.; Halada, Petr; Kovář, J.

    2015-01-01

    Roč. 333, č. 1 (2015), s. 1-10 ISSN 0014-4827 Institutional support: RVO:61388971 Keywords : Breast cancer * Taxane resistance * 2-D electrophoresis Subject RIV: CE - Biochemistry Impact factor: 3.378, year: 2015

  3. Protein deficiency lowers resistance of Mormon crickets to the pathogenic fungus Beauveria bassiana.

    Science.gov (United States)

    Srygley, R B; Jaronski, S T

    Little is known about the effects of dietary macronutrients on the capacity of insects to ward off a fungal pathogen. Here we tested the hypothesis that Mormon crickets fed restricted protein diets have lower enzymatic assays of generalized immunity, slower rates of encapsulation of foreign bodies, and greater mortality from infection by Beauveria bassiana, a fungal pathogen. Beginning in the last nymphal instar, Mormon crickets were fed a high, intermediate, or low protein diet with correspondingly low, intermediate, or high carbohydrate proportions. After they eclosed to adult, we drew hemolymph, topically applied B. bassiana, maintained them on diet treatments, and measured mortality for 21 days. Mormon crickets fed high protein diets had higher prophenoloxidase titers, greater encapsulation response, and higher survivorship to Beauveria fungal infection than those on low protein diets. We replicated the study adding very high and very low protein diets to the treatments. A high protein diet increased phenoloxidase titers, and those fed the very high protein diet had more circulating prophenoloxidase. Mormon crickets fed the very low protein diet were the most susceptible to B. bassiana infection, but the more concentrated phenoloxidase and prophenoloxidase associated with the highest protein diets did not confer the greatest protection from the fungal pathogen as in the first replicate. We conclude that protein-restricted diets caused Mormon crickets to have lower phenoloxidase titers, slower encapsulation of foreign bodies, and greater mortality from B. bassiana infection than those fed high protein diets. These results support the nutrition-based dichotomy of migrating Mormon crickets, protein-deficient ones are more susceptible to pathogenic fungi whereas carbohydrate-deficient ones are more vulnerable to bacterial challenge. Published by Elsevier Ltd.

  4. Metabolic responses to high protein diet in Korean elite bodybuilders with high-intensity resistance exercise

    OpenAIRE

    Kim, Hyerang; Lee, Saningun; Choue, Ryowon

    2011-01-01

    Abstract Background High protein diet has been known to cause metabolic acidosis, which is manifested by increased urinary excretion of nitrogen and calcium. Bodybuilders habitually consumed excessive dietary protein over the amounts recommended for them to promote muscle mass accretion. This study investigated the metabolic response to high protein consumption in the elite bodybuilders. Methods Eight elite Korean bodybuilders within the age from 18 to 25, mean age 21.5 ± 2.6. For data collec...

  5. ERK mutations confer resistance to mitogen-activated protein kinase pathway inhibitors.

    Science.gov (United States)

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

    2014-12-01

    The use of targeted therapeutics directed against BRAF(V600)-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 BRAF(V600)-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. ©2014 American Association for Cancer Research.

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

    Energy Technology Data Exchange (ETDEWEB)

    Rudolf, Jeffrey D. [Scripps Research Inst., Jupiter, FL (United States); Bigelow, Lance [Argonne National Lab. (ANL), Argonne, IL (United States); Chang, Changsoo [Argonne National Lab. (ANL), Argonne, IL (United States); Cuff, Marianne E. [Argonne National Lab. (ANL), Argonne, IL (United States); Lohman, Jeremy R. [Scripps Research Inst., Jupiter, FL (United States); Chang, Chin-Yuan [Scripps Research Inst., Jupiter, FL (United States); Ma, Ming [Scripps Research Inst., Jupiter, FL (United States); Yang, Dong [Scripps Research Inst., Jupiter, FL (United States); Clancy, Shonda [Argonne National Lab. (ANL), Argonne, IL (United States); Babnigg, Gyorgy [Argonne National Lab. (ANL), Argonne, IL (United States); Joachimiak, Andrzej [Argonne National Lab. (ANL), Argonne, IL (United States); Phillips, George N. [Rice Univ., Houston, TX (United States); Shen, Ben [Scripps Research Inst., Jupiter, FL (United States)

    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. What a Difference Melphalan Makes! Differential Protein Expression in Melphalan Sensitive and Resistant Multiple Myeloma Cells Revealed by SILAC-based Quantitative Proteomics

    DEFF Research Database (Denmark)

    Zub, Kamila Anna; Sousa, Mirta M.L.; Young, Clifford

    have been suggested to contribute to drug resistance, including modulated drug transport and metabolism, enhanced DNA repair and decreased apoptotic signalling1,2,3. Major common determinants associated with melphalan resistance, however, remain elusive. To further identify potential protein biomarkers...

  8. The putative multidrug resistance protein MRP-7 inhibits methylmercury-associated animal toxicity and dopaminergic neurodegeneration in Caenorhabditis elegans.

    Science.gov (United States)

    VanDuyn, Natalia; Nass, Richard

    2014-03-01

    Parkinson's disease (PD) is the most prevalent neurodegenerative motor disorder worldwide, and results in the progressive loss of dopamine (DA) neurons in the substantia nigra pars compacta. Gene-environment interactions are believed to play a significant role in the vast majority of PD cases, yet the toxicants and the associated genes involved in the neuropathology are largely ill-defined. Recent epidemiological and biochemical evidence suggests that methylmercury (MeHg) may be an environmental toxicant that contributes to the development of PD. Here, we report that a gene coding for the putative multidrug resistance protein MRP-7 in Caenorhabditis elegans modulates whole animal and DA neuron sensitivity to MeHg. In this study, we demonstrate that genetic knockdown of MRP-7 results in a twofold increase in Hg levels and a dramatic increase in stress response proteins associated with the endoplasmic reticulum, golgi apparatus, and mitochondria, as well as an increase in MeHg-associated animal death. Chronic exposure to low concentrations of MeHg induces MRP-7 gene expression, while exposures in MRP-7 genetic knockdown animals results in a loss of DA neuron integrity without affecting whole animal viability. Furthermore, transgenic animals expressing a fluorescent reporter behind the endogenous MRP-7 promoter indicate that the transporter is expressed in DA neurons. These studies show for the first time that a multidrug resistance protein is expressed in DA neurons, and its expression inhibits MeHg-associated DA neuron pathology. © 2013 International Society for Neurochemistry.

  9. Acute post-exercise myofibrillar protein synthesis is not correlated with resistance training-induced muscle hypertrophy in young men.

    Science.gov (United States)

    Mitchell, Cameron J; Churchward-Venne, Tyler A; Parise, Gianni; Bellamy, Leeann; Baker, Steven K; Smith, Kenneth; Atherton, Philip J; Phillips, Stuart M

    2014-01-01

    Muscle hypertrophy following resistance training (RT) involves activation of myofibrillar protein synthesis (MPS) to expand the myofibrillar protein pool. The degree of hypertrophy following RT is, however, highly variable and thus we sought to determine the relationship between the acute activation of MPS and RT-induced hypertrophy. We measured MPS and signalling protein activation after the first session of resistance exercise (RE) in untrained men (n = 23) and then examined the relation between MPS with magnetic resonance image determined hypertrophy. To measure MPS, young men (24±1 yr; body mass index  = 26.4±0.9 kg•m²) underwent a primed constant infusion of L-[ring-¹³C₆] phenylalanine to measure MPS at rest, and acutely following their first bout of RE prior to 16 wk of RT. Rates of MPS were increased 235±38% (Pmuscle volume and acute rates of MPS measured over 1-3 h (r = 0.02), 3-6 h (r = 0.16) or the aggregate 1-6 h post-exercise period (r = 0.10). Hypertrophy after chronic RT was correlated (r = 0.42, P = 0.05) with phosphorylation of 4E-BP1(Thr37/46) at 1 hour post RE. We conclude that acute measures of MPS following an initial exposure to RE in novices are not correlated with muscle hypertrophy following chronic RT.

  10. Expression of hybrid fusion protein (Cry1Ac::ASAL) in transgenic rice plants imparts resistance against multiple insect pests.

    Science.gov (United States)

    Boddupally, Dayakar; Tamirisa, Srinath; Gundra, Sivakrishna Rao; Vudem, Dashavantha Reddy; Khareedu, Venkateswara Rao

    2018-05-31

    To evolve rice varieties resistant to different groups of insect pests a fusion gene, comprising DI and DII domains of Bt Cry1Ac and carbohydrate binding domain of garlic lectin (ASAL), was constructed. Transgenic rice lines were generated and evaluated to assess the efficacy of Cry1Ac::ASAL fusion protein against three major pests, viz., yellow stem borer (YSB), leaf folder (LF) and brown planthopper (BPH). Molecular analyses of transgenic plants revealed stable integration and expression of the fusion gene. In planta insect bioassays on transgenics disclosed enhanced levels of resistance compared to the control plants. High insect mortality of YSB, LF and BPH was observed on transgenics compared to that of control plants. Furthermore, honeydew assays revealed significant decreases in the feeding ability of BPH on transgenic plants as compared to the controls. Ligand blot analysis, using BPH insects fed on cry1Ac::asal transgenic rice plants, revealed a modified receptor protein-binding pattern owing to its ability to bind to additional receptors in insects. The overall results authenticate that Cry1Ac::ASAL protein is endowed with remarkable entomotoxic effects against major lepidopteran and hemipteran insects. As such, the fusion gene appears promising and can be introduced into various other crops to control multiple insect pests.

  11. Preparation of protein based surfactants from leather waste fleshings and their reutilization in leather as a water resisting agent

    International Nuclear Information System (INIS)

    Nawaz, H.; Nadeem, U.; Solangi, B.; Hany, O.E.

    2012-01-01

    Summary: Tanneries generate a huge amount of highly polluting solid and liquid wastes during leather processing at different stages such as fleshings, shavings, tanning, finishing etc. approximately, 250 kg of finished leather product is obtained from 1 ton of raw salted hide while other protein goes into wastes. leather fleshings are about 50-60% of the total solid waste generated in leather processing. three different surfactants have been prepared from soft wax, long chain fatty acid chlorides and leather waste protein isolated from alkaline hydrolysis of fleshings. products are milky in color and have been applied in goat leathers as a replacement of fat liquor and water resisting agent .the resulted crust leathers have been characterized for various physical parameters such as tensile strength, thickness, softness, tear strength, bursting load, water absorption etc, as per their standard test methods. leathers have also been evaluated for grain smoothness, fullness and feeling. leathers have shown satisfactory results as per international requirement specially for water resisting. thus a leather waste protein is converted into a useful product and reutilized in leather making. (author)

  12. Molecular and Functional Characterization of a Polygalacturonase-Inhibiting Protein from Cynanchum komarovii That Confers Fungal Resistance in Arabidopsis.

    Directory of Open Access Journals (Sweden)

    Nana Liu

    Full Text Available Compliance with ethical standards: This study did not involve human participants and animals, and the plant of interest is not an endangered species. Polygalacturonase-inhibiting proteins (PGIPs are leucine-rich repeat proteins that plants produce against polygalacturonase, a key virulence agent in pathogens. In this paper, we cloned and purified CkPGIP1, a gene product from Cynanchum komarovii that effectively inhibits polygalacturonases from Botrytis cinerea and Rhizoctonia solani. We found the expression of CkPGIP1 to be induced in response to salicylic acid, wounding, and infection with B. cinerea and R. solani. In addition, transgenic overexpression in Arabidopsis enhanced resistance against B. cinerea. Furthermore, CkPGIP1 obtained from transgenic Arabidopsis inhibited the activity of B. cinerea and R. solani polygalacturonases by 62.7-66.4% and 56.5-60.2%, respectively. Docking studies indicated that the protein interacts strongly with the B1-sheet at the N-terminus of the B. cinerea polygalacturonase, and with the C-terminus of the polygalacturonase from R. solani. This study highlights the significance of CkPGIP1 in plant disease resistance, and its possible application to manage fungal pathogens.

  13. Elicitin-induced distal systemic resistance in plants is mediated through the protein-protein interactions influenced by selected lysine residues

    Directory of Open Access Journals (Sweden)

    Hana eUhlíková

    2016-02-01

    Full Text Available Elicitins are a family of small proteins with sterol-binding activity that are secreted by Phytophthora and Pythium spp. classified as oomycete PAMPs. Although alfa- and beta-elicitins bind with the same affinity to one high affinity binding site on the plasma membrane, beta-elicitins (possessing 6-7 lysine residues are generally 50- to 100-fold more active at inducing distal HR and systemic resistance than the alfa-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 beta-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.

  14. Prunus domestica pathogenesis-related protein-5 activates the defense response pathway and enhances the resistance to fungal infection.

    Directory of Open Access Journals (Sweden)

    Ashraf El-kereamy

    Full Text Available Pathogenesis-related protein-5 (PR-5 has been implicated in plant disease resistance and its antifungal activity has been demonstrated in some fruit species. However, their roles, especially their interactions with the other defense responses in plant cells, are still not fully understood. In this study, we have cloned and characterized a new PR-5 cDNA named PdPR5-1 from the European plum (Prunus domestica. Expression of PdPR5-1 was studied in different cultivars varying in resistance to the brown rot disease caused by the necrotrophic fungus Monilinia fructicola. In addition transgenic Arabidopsis, ectopically expressing PdPR5-1 was used to study its role in other plant defense responses after fungal infection. We show that the resistant cultivars exhibited much higher levels of transcripts than the susceptible cultivars during fruit ripening. However, significant rise in the transcript levels after infection with M. fructicola was observed in the susceptible cultivars too. Transgenic Arabidopsis plants exhibited more resistance to Alternaria brassicicola. Further, there was a significant increase in the transcripts of genes involved in the phenylpropanoid biosynthesis pathway such as phenylalanine ammonia-lyase (PAL and phytoalexin (camalexin pathway leading to an increase in camalexin content after fungal infection. Our results show that PdPR5-1 gene, in addition to its anti-fungal properties, has a possible role in activating other defense pathways, including phytoalexin production.

  15. Analysis of the CYP51 gene and encoded protein in propiconazole-resistant isolates of Mycosphaerella fijiensis.

    Science.gov (United States)

    Cañas-Gutiérrez, Gloria P; Angarita-Velásquez, Mónica J; Restrepo-Flórez, Juan M; Rodríguez, Paola; Moreno, Claudia X; Arango, Rafael

    2009-08-01

    Mycosphaerella fijiensis Morelet causes black sigatoka, the most important disease in bananas and plantains. Disease control is mainly through the application of systemic fungicides, including sterol demethylation inhibitors (DMIs). Their intensive use has favoured the appearance of resistant strains. However, no studies have been published on the possible resistance mechanisms. In this work, the CYP51 gene was isolated and sequenced in 11 M. fijiensis strains that had shown different degrees of in vitro sensitivity to propiconazole, one of the most widely used DMI fungicides. Six mutations that could be related to the loss in sensitivity to this fungicide were found: Y136F, A313G, Y461D, Y463D, Y463H and Y463N. The mutations were analysed using a homology model of the protein that was constructed from the crystallographic structure of Mycobacterium tuberculosis (Zoff.) Lehmann & Neumann. Additionally, gene expression was determined in 13 M. fijiensis strains through quantitative analysis of products obtained by RT-PCR. Several changes in the sequence of the gene encoding sterol 14alpha-demethylase were found that have been described in other fungi as being correlated with resistance to azole fungicides. No correlation was found between gene expression and propiconazole resistance.

  16. Bone morphogenetic protein 4 is overexpressed in and promotes migration and invasion of drug-resistant cancer cells.

    Science.gov (United States)

    Zhou, Kairui; Shi, Xiaoli; Huo, Jinling; Liu, Weihua; Yang, Dongxiao; Yang, Tengjiao; Qin, Tiantian; Wang, Cong

    2017-08-01

    Drug resistance and metastasis significantly hinder chemotherapy and worsen prognoses in cancer. Bone morphogenetic protein 4 (BMP4) belongs to the TGF-β superfamily, has broad biological activities in cell proliferation and cartilage differentiation and is also able to induce migration and invasion. Herein, we investigated the role of BMP4 in the regulation of metastasis in paclitaxel-resistant human esophageal carcinoma EC109 cells (EC109/Taxol) and docetaxel-resistant human gastric cancer MGC803 cells (MGC/Doc). In these drug-resistant cell lines, we found the cell motility was enhanced and BMP4 was up-regulated relative to their respective parental cell lines. Consistent with in vitro assays, migration potential and BMP4 expression were increased in EC109/Taxol nude mice. Furthermore, to address whether BMP4 was required to enhance the metastatic in EC109/Taxol cells, the pharmacological inhibitor of BMP signaling dorsomorphin was used; meanwhile, we found that the migration and invasion abilities were inhibited. Moreover, the canonical Smad signaling pathway was investigated. Overall, our studies demonstrated that BMP4 participates in the regulation of invasion and migration by EC109/Taxol cells, and inhibition of BMP4 may be a novel strategy to interfere with metastasis in cancer therapy. Copyright © 2017 Elsevier B.V. All rights reserved.

  17. Bactericidal Specificity and Resistance Profile of Poly(Quaternary Ammonium) Polymers and Protein-Poly(Quaternary Ammonium) Conjugates.

    Science.gov (United States)

    Ji, Weihang; Koepsel, Richard R; Murata, Hironobu; Zadan, Sawyer; Campbell, Alan S; Russell, Alan J

    2017-08-14

    Antibacterial polymers are potentially powerful biocides that can destroy bacteria on contact. Debate in the literature has surrounded the mechanism of action of polymeric biocides and the propensity for bacteria to develop resistance to them. There has been particular interest in whether surfaces with covalently coupled polymeric biocides have the same mechanism of action and resistance profile as similar soluble polymeric biocides. We designed and synthesized a series of poly(quaternary ammonium) polymers, with tailorable molecular structures and architectures, to engineer their antibacterial specificity and their ability to delay the development of bacterial resistance. These linear poly(quaternary ammonium) homopolymers and block copolymers, generated using atom transfer radical polymerization, had structure-dependent antibacterial specificity toward Gram positive and negative bacterial species. When single block copolymers contained two polymer segments of differing antibacterial specificity, the polymer combined the specificities of its two components. Nanoparticulate human serum albumin-poly(quaternary ammonium) conjugates of these same polymers, synthesized via "grafting from" atom transfer radical polymerization, were strongly biocidal and also exhibited a marked decrease in the rate of bacterial resistance development relative to linear polymers. These protein-biocide conjugates mimicked the behavior of surface-presented polycationic biocides rather than their nonproteinaceous counterparts.

  18. Adipophilin protein expression in muscle - a possible protective role against insulin resistance

    NARCIS (Netherlands)

    Wilde, de J.; Smit, E.; Snepvangers, F.J.M.; Wit, de N.J.W.; Mohren, R.; Hulshof, M.F.M.; Mariman, E.C.M.

    2010-01-01

    Adipophilin is a 50 kDa protein that belongs to the PAT family (perilipin, adipophilin, TIP47, S3-12 and OXPAT), which comprises proteins involved in the coating of lipid droplets. Little is known about the functional role of adipophilin in muscle. Using the C2C12 cell line as a model, we

  19. Identification of Adenyl Cyclase Activity in a Disease Resistance Protein in Arabidopsis thaliana

    KAUST Repository

    Hussein, Rana

    2012-01-01

    center motif. In an attempt to prove that this candidate has adenyl cyclases activity in vitro, the coding sequence of the putative AC catalytic domain of this protein was cloned and expressed in E. coli and the recombinant protein was purified

  20. D-bifunctional protein deficiency associated with drug resistant infantile spasms

    NARCIS (Netherlands)

    Buoni, Sabrina; Zannolli, Raffaella; Waterham, Hans; Wanders, Ronald; Fois, Alberto

    2007-01-01

    Peroxisomal disorders appear with a frequency of about 1:5000 in newborns. Peroxisomal D-bifunctional protein (D-BP), encoded by the HSD17B4 gene (gene ID: 3294; locus tag: HGNC:5213, chromosome 5q2; official symbol: HSD17B4; name: hydroxysteroid (17-beta) dehydrogenase; gene type: protein coding)

  1. CopM is a novel copper-binding protein involved in copper resistance in Synechocystis sp. PCC 6803

    Science.gov (United States)

    Giner-Lamia, Joaquín; López-Maury, Luis; Florencio, Francisco J

    2015-01-01

    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

  2. Roles of circulating WNT-signaling proteins and WNT-inhibitors in human adiposity, insulin resistance, insulin secretion, and inflammation.

    Science.gov (United States)

    Almario, R U; Karakas, S E

    2015-02-01

    Wingless-type MMTV integration site family member (WNT) signaling and WNT-inhibitors have been implicated in regulation of adipogenesis, insulin resistance, pancreatic function, and inflammation. Our goal was to determine serum proteins involved in WNT signaling (WNT5 and WISP2) and WNT inhibition (SFRP4 and SFRP5) as they relate to obesity, serum adipokines, insulin resistance, insulin secretion, and inflammation in humans. Study population comprised 57 insulin resistant women with polycystic ovary syndrome (PCOS) and 27 reference women. In a cross-sectional study, blood samples were obtained at fasting, during oral, and frequently sampled intravenous glucose tolerance tests. Serum WNT5, WISP2, and SFRP4 concentrations did not differ between PCOS vs. reference women. Serum WNT5 correlated inversely with weight both in PCOS and reference women, and correlated directly with insulin response during oral glucose tolerance test in PCOS women. Serum WISP2 correlated directly with fatty acid binding protein 4. Serum SFRP5 did not differ between obese (n=32) vs. nonobese (n=25) PCOS women, but reference women had lower SFRP5 (pPCOS groups). Serum SFRP5 correlated inversely with IL-1β, TNF-α, cholesterol, and apoprotein B. These findings demonstrated that WNT5 correlated inversely with adiposity and directly with insulin response, and the WNT-inhibitor SFRP5 may be anti-inflammatory. Better understanding of the role of WNT signaling in obesity, insulin resistance, insulin secretion, lipoprotein metabolism, and inflammation is important for prevention and treatment of metabolic syndrome, diabetes and cardiovascular disease. © Georg Thieme Verlag KG Stuttgart · New York.

  3. Ursodeoxycholic acid pretreatment reduces oral bioavailability of the multiple drug resistance-associated protein 2 substrate baicalin in rats.

    Science.gov (United States)

    Wu, Tao; Li, Xi-Ping; Xu, Yan-Jiao; Du, Guang; Liu, Dong

    2013-11-01

    Baicalin is a major bioactive component of Scutellaria baicalensis and a substrate of multiple drug resistance-associated protein 2. Expression of multiple drug resistance-associated protein 2 is regulated by NF-E2-related factor 2. The aim of this study was to explore whether ursodeoxycholic acid, an NF-E2-related factor 2 activator, could influence the oral bioavailability of baicalin. A single dose of baicalin (200 mg/kg) was given orally to rats pretreated with ursodeoxycholic acid (75 mg/kg and 150 mg/kg, per day, intragastrically) or normal saline (per day, intragastrically) for six consecutive days. The plasma concentration of baicalin was measured with the HPLC method. The result indicated that the oral bioavailability of baicalin was significantly and dose-dependently reduced in rats pretreated with ursodeoxycholic acid. Compared with control rats, the mean area under concentration-time curve of baicalin was reduced from 13.25 ± 0.24 mg/L h to 7.62 ± 0.15 mg/L h and 4.97 ± 0.21 mg/L h, and the C(max) value was decreased from 1.31 ± 0.03 mg/L to 0.62 ± 0.05 mg/L and 0.36 ± 0.04 mg/L in rats pretreated with ursodeoxycholic acid at doses of 75 mg/kg and 150 mg/kg, respectively, for six consecutive days. Hence, ursodeoxycholic acid treatment reduced the oral bioavailability of baicalin in rats, probably due to the enhanced efflux of baicalin from the intestine and liver by multiple drug resistance-associated protein 2. Georg Thieme Verlag KG Stuttgart · New York.

  4. Moss Pathogenesis-Related-10 protein enhances resistance to Pythium irregulare in Physcomitrella patens and Arabidopsis thaliana

    Directory of Open Access Journals (Sweden)

    Alexandra eCastro

    2016-04-01

    Full Text Available Plants respond to pathogen infection by activating signaling pathways leading to the accumulation of proteins with diverse roles in defense. Here, we addressed the functional role of PpPR-10, a pathogenesis-related (PR-10 gene, of the moss Physcomitrella patens, in response to biotic stress. PpPR-10 belongs to a multigene family and encodes a protein twice the usual size of PR-10 proteins due to the presence of two Bet v1 domains. Moss PR-10 genes are differentially regulated during development and inoculation with the fungal pathogen Botrytis cinerea. Specifically, PpPR-10 transcript levels increase significantly by treatments with elicitors of Pectobacterium carotovorum subsp. carotovorum, spores of B. cinerea, and the defense hormone salicylic acid. To characterize the role of PpPR-10 in plant defense against pathogens, we conducted overexpression analysis in P. patens and in Arabidopsis thaliana. We demonstrate that constitutive expression of PpPR-10 in moss tissues increased resistance against the oomycete Pythium irregulare. PpPR-10 overexpressing moss plants developed less symptoms and decreased mycelium growth than wild type plants. In addition, PpPR-10 overexpressing plants constitutively produced cell wall depositions in protonemal tissue. Ectopic expression of PpPR-10 in Arabidopsis resulted in increased resistance against P. irregulare as well, evidenced by smaller lesions and less cellular damage compared to wild type plants. These results indicate that PpPR-10 is functionally active in the defense against the pathogen P. irregulare, in both P. patens and Arabidopsis, two evolutionary distant plants. Thus, P. patens can serve as an interesting source of genes to improve resistance against pathogen infection in flowering plants.

  5. Molecular cloning and characterization of novel Morus alba germin-like protein gene which encodes for a silkworm gut digestion-resistant antimicrobial protein.

    Directory of Open Access Journals (Sweden)

    Bharat Bhusan Patnaik

    Full Text Available Silkworm fecal matter is considered one of the richest sources of antimicrobial and antiviral protein (substances and such economically feasible and eco-friendly proteins acting as secondary metabolites from the insect system can be explored for their practical utility in conferring broad spectrum disease resistance against pathogenic microbial specimens.Silkworm fecal matter extracts prepared in 0.02 M phosphate buffer saline (pH 7.4, at a temperature of 60°C was subjected to 40% saturated ammonium sulphate precipitation and purified by gel-filtration chromatography (GFC. SDS-PAGE under denaturing conditions showed a single band at about 21.5 kDa. The peak fraction, thus obtained by GFC wastested for homogeneityusing C18reverse-phase high performance liquid chromatography (HPLC. The activity of the purified protein was tested against selected Gram +/- bacteria and phytopathogenic Fusarium species with concentration-dependent inhibitionrelationship. The purified bioactive protein was subjected to matrix-assisted laser desorption and ionization-time of flight mass spectrometry (MALDI-TOF-MS and N-terminal sequencing by Edman degradation towards its identification. The N-terminal first 18 amino acid sequence following the predicted signal peptide showed homology to plant germin-like proteins (Glp. In order to characterize the full-length gene sequence in detail, the partial cDNA was cloned and sequenced using degenerate primers, followed by 5'- and 3'-rapid amplification of cDNA ends (RACE-PCR. The full-length cDNA sequence composed of 630 bp encoding 209 amino acids and corresponded to germin-like proteins (Glps involved in plant development and defense.The study reports, characterization of novel Glpbelonging to subfamily 3 from M. alba by the purification of mature active protein from silkworm fecal matter. The N-terminal amino acid sequence of the purified protein was found similar to the deduced amino acid sequence (without the transit

  6. Molecular Cloning and Characterization of Novel Morus alba Germin-Like Protein Gene Which Encodes for a Silkworm Gut Digestion-Resistant Antimicrobial Protein

    Science.gov (United States)

    Patnaik, Bharat Bhusan; Kim, Dong Hyun; Oh, Seung Han; Song, Yong-Su; Chanh, Nguyen Dang Minh; Kim, Jong Sun; Jung, Woo-jin; Saha, Atul Kumar; Bindroo, Bharat Bhushan; Han, Yeon Soo

    2012-01-01

    Background Silkworm fecal matter is considered one of the richest sources of antimicrobial and antiviral protein (substances) and such economically feasible and eco-friendly proteins acting as secondary metabolites from the insect system can be explored for their practical utility in conferring broad spectrum disease resistance against pathogenic microbial specimens. Methodology/Principal Findings Silkworm fecal matter extracts prepared in 0.02 M phosphate buffer saline (pH 7.4), at a temperature of 60°C was subjected to 40% saturated ammonium sulphate precipitation and purified by gel-filtration chromatography (GFC). SDS-PAGE under denaturing conditions showed a single band at about 21.5 kDa. The peak fraction, thus obtained by GFC wastested for homogeneityusing C18reverse-phase high performance liquid chromatography (HPLC). The activity of the purified protein was tested against selected Gram +/− bacteria and phytopathogenic Fusarium species with concentration-dependent inhibitionrelationship. The purified bioactive protein was subjected to matrix-assisted laser desorption and ionization-time of flight mass spectrometry (MALDI-TOF-MS) and N-terminal sequencing by Edman degradation towards its identification. The N-terminal first 18 amino acid sequence following the predicted signal peptide showed homology to plant germin-like proteins (Glp). In order to characterize the full-length gene sequence in detail, the partial cDNA was cloned and sequenced using degenerate primers, followed by 5′- and 3′-rapid amplification of cDNA ends (RACE-PCR). The full-length cDNA sequence composed of 630 bp encoding 209 amino acids and corresponded to germin-like proteins (Glps) involved in plant development and defense. Conclusions/Significance The study reports, characterization of novel Glpbelonging to subfamily 3 from M. alba by the purification of mature active protein from silkworm fecal matter. The N-terminal amino acid sequence of the purified protein was found

  7. The Chloroplastic Protein THF1 Interacts with the Coiled-Coil Domain of the Disease Resistance Protein N′ and Regulates Light-Dependent Cell Death1[OPEN

    Science.gov (United States)

    Sekine, Ken-Taro; Wallon, Thérèse; Sugiwaka, Yuji; Kobayashi, Kappei

    2016-01-01

    One branch of plant immunity is mediated through nucleotide-binding/Leu-rich repeat (NB-LRR) family proteins that recognize specific effectors encoded by pathogens. Members of the I2-like family constitute a well-conserved subgroup of NB-LRRs from Solanaceae possessing a coiled-coil (CC) domain at their N termini. We show here that the CC domains of several I2-like proteins are able to induce a hypersensitive response (HR), a form of programmed cell death associated with disease resistance. Using yeast two-hybrid screens, we identified the chloroplastic protein Thylakoid Formation1 (THF1) as an interacting partner for several I2-like CC domains. Co-immunoprecipitations and bimolecular fluorescence complementation assays confirmed that THF1 and I2-like CC domains interact in planta and that these interactions take place in the cytosol. Several HR-inducing I2-like CC domains have a negative effect on the accumulation of THF1, suggesting that the latter is destabilized by active CC domains. To confirm this model, we investigated N′, which recognizes the coat protein of most Tobamoviruses, as a prototypical member of the I2-like family. Transient expression and gene silencing data indicated that THF1 functions as a negative regulator of cell death and that activation of full-length N′ results in the destabilization of THF1. Consistent with the known function of THF1 in maintaining chloroplast homeostasis, we show that the HR induced by N′ is light-dependent. Together, our results define, to our knowledge, novel molecular mechanisms linking light and chloroplasts to the induction of cell death by a subgroup of NB-LRR proteins. PMID:26951433

  8. The Chloroplastic Protein THF1 Interacts with the Coiled-Coil Domain of the Disease Resistance Protein N' and Regulates Light-Dependent Cell Death.

    Science.gov (United States)

    Hamel, Louis-Philippe; Sekine, Ken-Taro; Wallon, Thérèse; Sugiwaka, Yuji; Kobayashi, Kappei; Moffett, Peter

    2016-05-01

    One branch of plant immunity is mediated through nucleotide-binding/Leu-rich repeat (NB-LRR) family proteins that recognize specific effectors encoded by pathogens. Members of the I2-like family constitute a well-conserved subgroup of NB-LRRs from Solanaceae possessing a coiled-coil (CC) domain at their N termini. We show here that the CC domains of several I2-like proteins are able to induce a hypersensitive response (HR), a form of programmed cell death associated with disease resistance. Using yeast two-hybrid screens, we identified the chloroplastic protein Thylakoid Formation1 (THF1) as an interacting partner for several I2-like CC domains. Co-immunoprecipitations and bimolecular fluorescence complementation assays confirmed that THF1 and I2-like CC domains interact in planta and that these interactions take place in the cytosol. Several HR-inducing I2-like CC domains have a negative effect on the accumulation of THF1, suggesting that the latter is destabilized by active CC domains. To confirm this model, we investigated N', which recognizes the coat protein of most Tobamoviruses, as a prototypical member of the I2-like family. Transient expression and gene silencing data indicated that THF1 functions as a negative regulator of cell death and that activation of full-length N' results in the destabilization of THF1. Consistent with the known function of THF1 in maintaining chloroplast homeostasis, we show that the HR induced by N' is light-dependent. Together, our results define, to our knowledge, novel molecular mechanisms linking light and chloroplasts to the induction of cell death by a subgroup of NB-LRR proteins. © 2016 American Society of Plant Biologists. All Rights Reserved.

  9. Identification of multidrug resistance protein 1 (MRP1/ABCC1) as a molecular gate for cellular export of cobalamin

    DEFF Research Database (Denmark)

    Beedholm-Ebsen, Rasmus; van de Wetering, Koen; Hardlei, Tore

    2010-01-01

    transporters by cellular gene silencing showed a role in cellular Cbl efflux of the ATP-binding cassette (ABC)-drug transporter, ABCC1, alias multidrug resistance protein 1 (MRP1), which is present in the basolateral membrane of intestinal epithelium and in other cells. The ability of MRP1 to mediate ATP...... and kidney. In contrast, Cbl accumulates in the terminal part of the intestine of these mice, suggesting a functional malabsorption because of a lower epithelial basolateral Cbl efflux. The identification of this Cbl export mechanism now allows the delineation of a coherent pathway for Cbl trafficking from...

  10. High levels of X-linked Inhibitor-of-Apoptosis Protein (XIAP) are indicative of radio chemotherapy resistance in rectal cancer

    International Nuclear Information System (INIS)

    Flanagan, L.; Kehoe, J.; Fay, J.; Bacon, O.; Lindner, A.U.; Kay, E.W.; Deasy, J.; McNamara, D.A.; Prehn, J.H.M.

    2015-01-01

    The mainstay of treatment in rectal cancer is neoadjuvant radio chemotherapy prior to surgery, in an attempt to downstage the tumour, allowing for more complete removal during surgery. In 40 % of cases however, this neoadjuvant radio chemotherapy fails to achieve tumour regression, partly due insufficient apoptosis signaling. X-linked Inhibitor of Apoptosis Protein (XIAP) is an anti-apoptotic protein that has been reported to contribute to disease progression and chemotherapy resistance. We obtained rectal biopsy normal and matched tumour tissue from 29 rectal cancer patients with varying degrees of tumour regression, and using Western blot, examined anti-apoptotic XIAP and pro-apoptotic Smac protein levels in these tissues, with the aim to examine whether disturbed XIAP/Smac levels may be an indicator of neoadjuvant radio chemotherapy resistance. Expression of inhibitor of apoptosis proteins cIAP-1 and cIAP-2 was also examined. We found that levels of XIAP increased in accordance with the degree of radio chemotherapy resistance of the tissue. Levels of this protein were also significantly higher in tumour tissue, compared to matched normal tissue in highly resistant tissue. In contrast, Smac protein levels did not increase with radio chemotherapy resistance, and the protein was similarly expressed in normal and tumour tissue, indicating a shift in the balance of these proteins. Post treatment surgical resection tissue was available for 8 patients. When we compared matched tissue pre- and post- radio chemotherapy we found that XIAP levels increased significantly during treatment in both normal and tumour tissue, while Smac levels did not change. cIAP-1 and cIAP-2 levels were not differentially expressed in varying degrees of radio chemotherapy resistance, and neoadjuvant therapy did not alter expression of these proteins. These data indicate that disturbance of the XIAP/Smac balance may be a driver of radio chemotherapy resistance, and hence high levels of XIAP may

  11. Binding site alteration is responsible for field-isolated resistance to Bacillus thuringiensis Cry2A insecticidal proteins in two Helicoverpa species.

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    Silvia Caccia

    Full Text Available BACKGROUND: Evolution of resistance by target pests is the main threat to the long-term efficacy of crops expressing Bacillus thuringiensis (Bt insecticidal proteins. Cry2 proteins play a pivotal role in current Bt spray formulations and transgenic crops and they complement Cry1A proteins because of their different mode of action. Their presence is critical in the control of those lepidopteran species, such as Helicoverpa spp., which are not highly susceptible to Cry1A proteins. In Australia, a transgenic variety of cotton expressing Cry1Ac and Cry2Ab (Bollgard II comprises at least 80% of the total cotton area. Prior to the widespread adoption of Bollgard II, the frequency of alleles conferring resistance to Cry2Ab in field populations of Helicoverpa armigera and Helicoverpa punctigera was significantly higher than anticipated. Colonies established from survivors of F(2 screens against Cry2Ab are highly resistant to this toxin, but susceptible to Cry1Ac. METHODOLOGY/PRINCIPAL FINDINGS: Bioassays performed with surface-treated artificial diet on neonates of H. armigera and H. punctigera showed that Cry2Ab resistant insects were cross-resistant to Cry2Ae while susceptible to Cry1Ab. Binding analyses with (125I-labeled Cry2Ab were performed with brush border membrane vesicles from midguts of Cry2Ab susceptible and resistant insects. The results of the binding analyses correlated with bioassay data and demonstrated that resistant insects exhibited greatly reduced binding of Cry2Ab toxin to midgut receptors, whereas no change in (125I-labeled-Cry1Ac binding was detected. As previously demonstrated for H. armigera, Cry2Ab binding sites in H. punctigera were shown to be shared by Cry2Ae, which explains why an alteration of the shared binding site would lead to cross-resistance between the two Cry2A toxins. CONCLUSION/SIGNIFICANCE: This is the first time that a mechanism of resistance to the Cry2 class of insecticidal proteins has been reported

  12. A predicted protein interactome identifies conserved global networks and disease resistance subnetworks in maize.

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    Matt eGeisler

    2015-06-01

    Full Text Available Interactomes are genome-wide roadmaps of protein-protein interactions. They have been produced for humans, yeast, the fruit fly, and Arabidopsis thaliana and have become invaluable tools for generating and testing hypotheses. A predicted interactome for Zea mays (PiZeaM is presented here as an aid to the research community for this valuable crop species. PiZeaM was built using a proven method of interologs (interacting orthologs that were identified using both one-to-one and many-to-many orthology between genomes of maize and reference species. Where both maize orthologs occurred for an experimentally determined interaction in the reference species, we predicted a likely interaction in maize. A total of 49,026 unique interactions for 6,004 maize proteins were predicted. These interactions are enriched for processes that are evolutionarily conserved, but include many otherwise poorly annotated proteins in maize. The predicted maize interactions were further analyzed by comparing annotation of interacting proteins, including different layers of ontology. A map of pairwise gene co-expression was also generated and compared to predicted interactions. Two global subnetworks were constructed for highly conserved interactions. These subnetworks showed clear clustering of proteins by function. Another subnetwork was created for disease response using a bait and prey strategy to capture interacting partners for proteins that respond to other organisms. Closer examination of this subnetwork revealed the connectivity between biotic and abiotic hormone stress pathways. We believe PiZeaM will provide a useful tool for the prediction of protein function and analysis of pathways for Z. mays researchers and is presented in this paper as a reference tool for the exploration of protein interactions in maize.

  13. Silk Composite with a Fluoropolymer as a Water-Resistant Protein-Based Material

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    Keiji Numata

    2018-04-01

    Full Text Available Silk-based materials are water-sensitive and show different physical properties at different humidities and under wet/dry conditions. To overcome the water sensitivity of silk-based materials, we developed a silk composite material with a fluoropolymer. Blending and coating the silk protein-based materials, such as films and textiles, with the fluoropolymer enhanced the surface hydrophobicity, water vapor barrier properties, and size stability during shrinkage tests. This material design with a protein biopolymer and a fluoropolymer is expected to broaden the applicability of protein-based materials.

  14. Efficacy of whey protein supplementation on resistance exercise-induced changes in muscle strength, lean mass, and function in mobility-limited older adults

    Science.gov (United States)

    Whey protein supplementation may augment resistance exercise-induced increases in muscle strength and mass. Further studies are required to determine whether this effect extends to functionally compromised older adults. The objectives of the study were to compare the effects of whey protein concent...

  15. Protein supplementation increases muscle mass gain during prolonged resistance-type exercise training in frail elderly people: a randomized double-blind, placebo-controlled trial

    NARCIS (Netherlands)

    Tieland, C.A.B.; Dirks, M.L.; Zwaluw, van der N.L.; Verdijk, L.; Rest, van de O.; Groot, de C.P.G.M.; Loon, van L.C.

    2012-01-01

    Objectives Protein supplementation has been proposed as an effective dietary strategy to augment the skeletal muscle adaptive response to prolonged resistance-type exercise training in elderly people. Our objective was to assess the impact of protein supplementation on muscle mass, strength, and

  16. Serum liver fatty acid binding protein levels correlate positively with obesity and insulin resistance in Chinese young adults.

    Directory of Open Access Journals (Sweden)

    Juan Shi

    Full Text Available BACKGROUND: Liver fatty acid-binding protein (FABP1 plays an inconclusive role in adiposity. We investigated the association of serum FABP1 levels with obesity and insulin resistance in Chinese young people under 30 years old. METHODOLOGY AND PRINCIPAL FINDINGS: Cross-sectional analysis including 200 obese and 172 normal-weight subjects matched for age and sex, anthropometric measurements were performed and serum FABP1 and biochemical characteristics were measured. Insulin resistance was determined by homeostasis model assessment of insulin resistance (HOMA-IR and by the insulin sensitivity index (S(i derived from Bergman's minimal model. FABP1 levels in obese subjects were significantly higher than those in normal-weight subjects (p<0.001 and the significance remained after adjustment for age, gender, alanine and aspartate aminotransferases (p<0.001. Serum FABP1 levels were significantly correlated with many metabolic-related parameters, with BMI and triglycerides as the independent determinants. FABP1 levels remained an independent risk factor of insulin resistance assessed by binary S(i (OR = 1.868 per SD unit, 95% CI [1.035-3.373], p = 0.038 after adjustment for age, sex, BMI, waist circumference, systolic blood pressure, serum triacylglycerol, total cholesterol, HDL- and LDL-cholesterol,. FABP1 levels were also elevated with an increasing number of components of the metabolic syndrome (p for trend <0.001. Multiple regression modeling for the MetS and its components demonstrated that hypertriglyceridemia and low HDL-cholesterol were significantly correlated to serum FABP1 levels. CONCLUSIONS AND SIGNIFICANCE: Serum FABP1 correlates positively with obesity and insulin resistance in Chinese young adults. Our data supports the fact that FABP1 might be an important mediator participating in fatty acid metabolism and energy balance.

  17. Resistance to classical scrapie in experimentally challenged goats carrying mutation K222 of the prion protein gene

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    Acutis Pier Luigi

    2012-02-01

    Full Text Available Abstract Susceptibility of sheep to scrapie, a transmissible spongiform encephalopathy of small ruminants, is strongly influenced by polymorphisms of the prion protein gene (PRNP. Breeding programs have been implemented to increase scrapie resistance in sheep populations; though desirable, a similar approach has not yet been applied in goats. European studies have now suggested that several polymorphisms can modulate scrapie susceptibility in goats: in particular, PRNP variant K222 has been associated with resistance in case-control studies in Italy, France and Greece. In this study we investigated the resistance conferred by this variant using a natural Italian goat scrapie isolate to intracerebrally challenge five goats carrying genotype Q/Q 222 (wild type and five goats carrying genotype Q/K 222. By the end of the study, all five Q/Q 222 goats had died of scrapie after a mean incubation period of 19 months; one of the five Q/K 222 goats died after 24 months, while the other four were alive and apparently healthy up to the end of the study at 4.5 years post-challenge. All five of these animals were found to be scrapie negative. Statistical analysis showed that the probability of survival of the Q/K 222 goats versus the Q/Q 222 goats was significantly higher (p = 0.002. Our study shows that PRNP gene mutation K222 is strongly associated with resistance to classical scrapie also in experimental conditions, making it a potentially positive target for selection in the frame of breeding programs for resistance to classical scrapie in goats.

  18. SUMO-, MAPK- and resistance protein-signaling converge at transcription complexes that regulate plant innate immunity

    NARCIS (Netherlands)

    Burg, van den H.A.; Takken, F.L.W.

    2010-01-01

    Upon pathogen perception plant innate immune receptors activate various signaling pathways that trigger host defenses. PAMP-triggered defense signaling requires mitogen-activated protein kinase (MAPK) pathways, which modulate the activity of transcription factors through phosphorylation. Here, we

  19. SUMO-, MAPK-, and resistance protein-signaling converge at transcription complexes that regulate plant innate immunity

    NARCIS (Netherlands)

    van den Burg, H.A.; Takken, F.L.W.

    2010-01-01

    Upon pathogen perception plant innate immune receptors activate various signaling pathways that trigger host defenses. PAMP-triggered defense signaling requires mitogen-activated protein kinase (MAPK) pathways, which modulate the activity of transcription factors through phosphorylation. Here, we

  20. Eukaryotic resistance to fluoride toxicity mediated by a widespread family of fluoride export proteins

    OpenAIRE

    Li, Sanshu; Smith, Kathryn D.; Davis, Jared H.; Gordon, Patricia B.; Breaker, Ronald R.; Strobel, Scott A.

    2013-01-01

    Although fluoride is plentiful in the environment and is commonly used at high concentrations in oral hygiene products, little has been known about how biological systems overcome the toxic effects of this anion. We demonstrate that a protein called FEX in many fungi is essential for cell survival in the presence of high fluoride concentrations. The protein is required for the rapid expulsion of cytoplasmic fluoride, indicating that many eukaryotic species that carry FEX genes likely avoid fl...

  1. THE ROLE OF MULTIDRUG RESISTANCE ASSOCIATED PROTEIN (MRP) IN THE BLOOD-BRAIN BARRIER AND OPIOID ANALGESIA

    Science.gov (United States)

    Su, Wendy; Pasternak, Gavril W.

    2013-01-01

    The blood brain barrier protects the brain from circulating compounds and drugs. The ATP-binding cassette (ABC) transporter P-glycoprotein (Pgp) is involved with the barrier, both preventing the influx of agent from the blood into the brain and facilitating the efflux of compounds from the brain into the blood, raising the possibility of a similar role for other transporters. Multidrug resistance associated protein (MRP), a 190 kDa protein similar to Pgp is also ABC transport that has been implicated in the blood brain barrier. The current study explores its role in opioid action. Immunohistochemically, it is localized in the choroid plexus in ratsand can be selectively downregulated by antisense treatment at both the level of mRNA, as shown by RT-PCR, and protein, as demonstrated immunohistochemically. Behaviorally, downregulation of MRP significantly enhances the analgesic potency of systemic morphine in MRP knockout mice and in antisense-treated rats by lowering the blood brain barrier. Following intracerebroventricular administration, a number of compounds, including some opioids, are rapidly secreted from the brain into the blood where they contribute to the overall analgesic effects by activating peripheral systems. MRP plays a role in this efflux. Downregulating MRP expression leads to a corresponding decrease in the transport and a diminished analgesic response from opioids administered intracerebroventricularly. Thus, the transporter protein MRP plays a role in maintaining the blood-brain barrier and modulates the activity of opioids. PMID:23508590

  2. Immunization of Mice with Recombinant Brucella abortus Organic Hydroperoxide Resistance (Ohr) Protein Protects Against a Virulent Brucella abortus 544 Infection.

    Science.gov (United States)

    Hop, Huynh Tan; Reyes, Alisha Wehdnesday Bernardo; Simborio, Hannah Leah Tadeja; Arayan, Lauren Togonon; Min, Won Gi; Lee, Hu Jang; Lee, Jin Ju; Chang, Hong Hee; Kim, Suk

    2016-01-01

    In this study, the Brucella abortus ohr gene coding for an organic hydroperoxide resistance protein (Ohr) was cloned into a maltose fusion protein expression system (pMAL), inserted into Escherichia coli, and purified, and its immunogenicity was evaluated by western blot analysis using Brucella-positive mouse sera. The purified recombinant Ohr (rOhr) was treated with adjuvant and injected intraperitoneally into BALB/c mice. A protective immune response analysis revealed that rOhr induced a significant increase in both the IgG1 and IgG2a titers, and IgG2a reached a higher level than IgG1 after the second and third immunizations. Additionally, immunization with rOhr induced high production of IFN-γ as well as proinflammatory cytokines such as TNF, MCP-1, IL-12p70, and IL-6, but a lesser amount of IL-10, suggesting that rOhr predominantly elicited a cell-mediated immune response. In addition, immunization with rOhr caused a significantly higher degree of protection against a virulent B. abortus infection compared with a positive control group consisting of mice immunized with maltose-binding protein. These findings showed that B. abortus rOhr was able to induce both humoral and cell-mediated immunity in mice, which suggested that this recombinant protein could be a potential vaccine candidate for animal brucellosis.

  3. Acquired activated protein C resistance is associated with lupus anticoagulants and thrombotic events in pediatric patients with systemic lupus erythematosus.

    Science.gov (United States)

    Male, C; Mitchell, L; Julian, J; Vegh, P; Joshua, P; Adams, M; David, M; Andrew, M E

    2001-02-15

    Acquired activated protein C resistance (APCR) has been hypothesized as a possible mechanism by which antiphospholipid antibodies (APLAs) cause thrombotic events (TEs). However, available evidence for an association of acquired APCR with APLAs is limited. More importantly, an association of acquired APCR with TEs has not been demonstrated. The objective of the study was to determine, in pediatric patients with systemic lupus erythematosus (SLE), whether (1) acquired APCR is associated with the presence of APLAs, (2) APCR is associated with TEs, and (3) there is an interaction between APCR and APLAs in association with TEs. A cross-sectional cohort study of 59 consecutive, nonselected children with SLE was conducted. Primary clinical outcomes were symptomatic TEs, confirmed by objective radiographic tests. Laboratory testing included lupus anticoagulants (LAs), anticardiolipin antibodies (ACLAs), APC ratio, protein S, protein C, and factor V Leiden. The results revealed that TEs occurred in 10 (17%) of 59 patients. Acquired APCR was present in 18 (31%) of 58 patients. Acquired APCR was significantly associated with the presence of LAs but not ACLAs. Acquired APCR was also significantly associated with TEs. There was significant interaction between APCR and LAs in the association with TEs. Presence of both APCR and LAs was associated with the highest risk of a TE. Protein S and protein C concentrations were not associated with the presence of APLAs, APCR, or TEs. Presence of acquired APCR is a marker identifying LA-positive patients at high risk of TEs. Acquired APCR may reflect interference of LAs with the protein C pathway that may represent a mechanism of LA-associated TEs. (Blood. 2001;97:844-849)

  4. Ultrasensitive probing of the protein resistance of PEG surfaces by secondary ion mass spectrometry

    DEFF Research Database (Denmark)

    Kingshott, P.; McArthur, S.; Thissen, H.

    2002-01-01

    The highly sensitive surface analytical techniques X-ray photoelectron spectroscopy (XPS) and time-of-flight static secondary ion mass spectrometry (ToF-SIMS) were used to test the resistance of poly(ethylene glycol) (PEG) coatings towards adsorption of lysozyme (LYS) and fibronectin (FN). PEG co...

  5. A SNARE-protein has opposing functions in penetration resistance and defence signalling pathways

    DEFF Research Database (Denmark)

    Zhang, Ziguo; Feechan, Angela; Pedersen, Carsten

    2007-01-01

    Penetration resistance is often the first line of defence against fungal pathogens. Subsequently induced defences are mediated by the programmed cell death (PCD) reaction pathway and the salicylic acid (SA), jasmonic acid (JA) and ethylene (ET) signalling pathways. We previously demonstrated...

  6. Proteomic analysis of the maize rachis: potential roles of constitutive and induced proteins in resistance to Aspergillus flavus infection and aflatoxin accumulation.

    Science.gov (United States)

    Pechanova, Olga; Pechan, Tibor; Williams, W Paul; Luthe, Dawn S

    2011-01-01

    Infection of the maize (Zea mays L.) with aflatoxigenic fungus Aspergillus flavus and consequent contamination with carcinogenic aflatoxin is a persistent and serious agricultural problem causing disease and significant crop losses worldwide. The rachis (cob) is an important structure of maize ear that delivers essential nutrients to the developing kernels and A. flavus spreads through the rachis to infect kernels within the ear. Therefore, rachis plays an important role in fungal proliferation and subsequent kernel contamination. We used proteomic approaches and investigated the rachis tissue from aflatoxin accumulation resistant (Mp313E and Mp420) and susceptible (B73 and SC212m) maize inbred lines. First, we compared rachis proteins from resistant and susceptible inbred lines, which revealed that the young resistant rachis contains higher levels of abiotic stress-related proteins and proteins from phenylpropanoid metabolism, whereas susceptible young rachis contains pathogenesis-related proteins, which are generally inducible upon biotic stress. Second, we identified A. flavus-responsive proteins in rachis of both resistant and susceptible genotypes after 10- and 35-day infection. Differential expression of many stress/defense proteins during rachis juvenility, maturation and after A. flavus challenge demonstrates that resistant rachis relies on constitutive defenses, while susceptible rachis is more dependent on inducible defenses. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  7. Anti-cancer effect of HIV-1 viral protein R on doxorubicin resistant neuroblastoma.

    Directory of Open Access Journals (Sweden)

    Richard Y Zhao

    Full Text Available Several unique biological features of HIV-1 Vpr make it a potentially powerful agent for anti-cancer therapy. First, Vpr inhibits cell proliferation by induction of cell cycle G2 arrest. Second, it induces apoptosis through multiple mechanisms, which could be significant as it may be able to overcome apoptotic resistance exhibited by many cancerous cells, and, finally, Vpr selectively kills fast growing cells in a p53-independent manner. To demonstrate the potential utility of Vpr as an anti-cancer agent, we carried out proof-of-concept studies in vitro and in vivo. Results of our preliminary studies demonstrated that Vpr induces cell cycle G2 arrest and apoptosis in a variety of cancer types. Moreover, the same Vpr effects could also be detected in some cancer cells that are resistant to anti-cancer drugs such as doxorubicin (DOX. To further illustrate the potential value of Vpr in tumor growth inhibition, we adopted a DOX-resistant neuroblastoma model by injecting SK-N-SH cells into C57BL/6N and C57BL/6J-scid/scid mice. We hypothesized that Vpr is able to block cell proliferation and induce apoptosis regardless of the drug resistance status of the tumors. Indeed, production of Vpr via adenoviral delivery to neuroblastoma cells caused G2 arrest and apoptosis in both drug naïve and DOX-resistant cells. In addition, pre-infection or intratumoral injection of vpr-expressing adenoviral particles into neuroblastoma tumors in SCID mice markedly inhibited tumor growth. Therefore, Vpr could possibly be used as a supplemental viral therapeutic agent for selective inhibition of tumor growth in anti-cancer therapy especially when other therapies stop working.

  8. Expression of P-glycoprotein and multidrug resistance associated protein in Ehrlich ascites tumor cells after fractionated irradiation

    International Nuclear Information System (INIS)

    Nielsen, Dorte; Maare, Christian; Eriksen, Jens; Litman, Thomas; Skovsgaard, Torben

    2001-01-01

    Purpose: To characterize irradiated murine tumor cells with respect to drug resistance, drug kinetics, and ATPase activity, and to evaluate the possible role of P-glycoprotein (PGP) and murine multidrug resistance associated protein (Mrp1) in the drug-resistant phenotype of these cells. Methods and Materials: Sensitive Ehrlich ascites tumor cells (EHR2) were in vitro exposed to fractionated irradiation (60 Gy). Western blot analysis was performed for determination of PGP and Mrp1, reverse transcriptase-polymerase chain reaction (RT-PCR) for determination of mdr1a + b mRNA, and semiquantitative RT-PCR for Mrp1 mRNA. The clonogenic assay was applied to investigate sensitivity, whereas the steady-state drug accumulation of daunorubicin (DNR), 3 H-vincristine (VCR), and 3 H-etoposide (VP16) was measured by spectrofluorometry and scintillation counting, respectively. For determining of ATPase activity, the release of inorganic phosphate from ATP was quantified using a colorimetric method. Results: Compared with EHR2, the irradiated cell line EHR2/irr showed increased expression of PGP (threefold), Mrp1 (eightfold), and Mrp1 mRNA (sixfold), and a slight reduction of mdr1b mRNA, whereas mdr1a was present in EHR2 but could not be detected in EHR2/irr. EHR2/irr developed sixfold resistance to VP16, twofold resistance to vincristine, but remained sensitive to DNR. Addition of the PGP inhibitor, verapamil (VER) or depletion of glutathione by buthionine sulfoximine (BSO) partly reversed the resistance in EHR2/irr. In EHR2/irr, the steady-state accumulation of 3 H-VCR and 3 H-VP16 was significantly decreased as compared with EHR2, whereas the accumulation of DNR was unchanged. The ATPase activity of plasma membrane vesicles prepared from EHR2/irr cells was similar to that of wild-type EHR2 cells. The ATPase activity was neither stimulated by vinblastine nor VER. Conclusion: Irradiation induced a multidrug-resistant phenotype in sensitive tumor cells. This phenotype was

  9. Methamphetamine increases Prion Protein and induces dopamine-dependent expression of protease resistant PrPsc.

    Science.gov (United States)

    Ferrucci, M; Ryskalin, L; Biagioni, F; Gambardella, S; Busceti, C L; Falleni, A; Lazzeri, G; Fornai, F

    2017-07-01

    The cellular prion protein (PrPc) is physiologically expressed within selective brain areas of mammals. Alterations in the secondary structure of this protein lead to scrapie-like prion protein (PrPsc), which precipitates in the cell. PrPsc has been detected in infectious, inherited or sporadic neurodegenerative disorders. Prion protein metabolism is dependent on autophagy and ubiquitin proteasome. Despite not being fully elucidated, the physiological role of prion protein relates to chaperones which rescue cells under stressful conditions.Methamphetamine (METH) is a widely abused drug which produces oxidative stress in various brain areas causing mitochondrial alterations and protein misfolding. These effects produce a compensatory increase of chaperones while clogging cell clearing pathways. In the present study, we explored whether METH administration modifies the amount of PrPc. Since high levels of PrPc when the clearing systems are clogged may lead to its misfolding into PrPsc, we further tested whether METH exposure triggers the appearance of PrPsc. We analysed the effects of METH and dopamine administration in PC12 and striatal cells by using SDS-PAGE Coomassie blue, immune- histochemistry and immune-gold electron microscopy. To analyze whether METH administration produces PrPsc aggregates we used antibodies directed against PrP following exposure to proteinase K or sarkosyl which digest folded PrPc but misfolded PrPsc. We fond that METH triggers PrPsc aggregates in DA-containing cells while METH is not effective in primary striatal neurons which do not produce DA. In the latter cells exogenous DA is needed to trigger PrPsc accumulation similarly to what happens in DA containing cells under the effects of METH. The present findings, while fostering novel molecular mechanisms involving prion proteins, indicate that, cell pathology similar to prion disorders can be mimicked via a DA-dependent mechanism by a drug of abuse.

  10. Loss of Oca2 disrupts the unfolded protein response and increases resistance to endoplasmic reticulum stress in melanocytes.

    Science.gov (United States)

    Cheng, Tsing; Orlow, Seth J; Manga, Prashiela

    2013-11-01

    Accumulation of proteins in the endoplasmic reticulum (ER) typically induces stress and initiates the unfolded protein response (UPR) to facilitate recovery. If homeostasis is not restored, apoptosis is induced. However, adaptation to chronic UPR activation can increase resistance to subsequent acute ER stress. We therefore investigated adaptive mechanisms in Oculocutaneous albinism type 2 (Oca2)-null melanocytes where UPR signaling is arrested despite continued tyrosinase accumulation leading to resistance to the chemical ER stressor thapsigargin. Although thapsigargin triggers UPR activation, instead of Perk-mediated phosphorylation of eIF2α, in Oca2-null melanocytes, eIF2α was rapidly dephosphorylated upon treatment. Dephosphorylation was mediated by the Gadd34-PP1α phosphatase complex. Gadd34-complex inhibition blocked eIF2α dephosphorylation and significantly increased Oca2-null melanocyte sensitivity to thapsigargin. Thus, Oca2-null melanocytes adapt to acute ER stress by disruption of pro-apoptotic Perk signaling, which promotes cell survival. This is the first study to demonstrate rapid eIF2α dephosphorylation as an adaptive mechanism to ER stress. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  11. Incorporation of Ortho- and Meta-Tyrosine Into Cellular Proteins Leads to Erythropoietin-Resistance in an Erythroid Cell Line

    Directory of Open Access Journals (Sweden)

    Esztella Mikolás

    2014-04-01

    Full Text Available Background/Aims: Erythropoietin-resistance is an unsolved concern in the treatment of renal anaemia. We aimed to investigate the possible role of ortho- and meta-tyrosine - the hydroxyl free radical products of L-phenylalanine - in the development of erythropoietin-resistance. Methods: TF-1 erythroblast cell line was used. Cell concentration was determined on day 1; 2 and 3 by two independent observers simultaneously in Bürker cell counting chambers. Protein concentration was determined with colorimetric method. Para-, ortho- and meta-tyrosine levels were measured using reverse phase-HPLC with fluorescence detection. Using Western blot method activating phosphorylation of STAT5 and ERK1/2 were investigated. Results: We found a time- and concentration-dependent decrease of erythropoietin-induced proliferative activity in case of ortho- and meta-tyrosine treated TF-1 erythroblasts, compared to the para-tyrosine cultured cells. Decreased erythropoietin-response could be regained with a competitive dose of para-tyrosine. Proteins of erythroblasts treated by ortho- or meta-tyrosine had lower para-tyrosine and higher ortho- or meta-tyrosine content. Activating phosphorylation of ERK and STAT5 due to erythropoietin was practically prevented by ortho- or meta-tyrosine treatment. Conclusion: According to this study elevated ortho- and meta-tyrosine content of erythroblasts may lead to the dysfunction of intracellular signaling, resulting in erythropoietin-hyporesponsiveness.

  12. Heart type fatty acid binding protein response and subsequent development of atherosclerosis in insulin resistant polycystic ovary syndrome patients.

    Science.gov (United States)

    Cakir, Evrim; Ozbek, Mustafa; Sahin, Mustafa; Cakal, Erman; Gungunes, Askin; Ginis, Zeynep; Demirci, Taner; Delibasi, Tuncay

    2012-12-18

    Women with polycystic ovary syndrome (PCOS) have higher risk for cardiovascular disease (CVD). Heart type fatty acid binding protein (HFABP) has been found to be predictive for myocardial ischemia.Wet ested whether HFABP is the predictor for CVD in PCOS patients, who have an increased risk of cardiovascular disease. This was a prospective, cross sectional controlled study conducted in a training and research hospital.The study population consisted of 46 reproductive-age PCOS women and 28 control subjects. We evaluated anthropometric and metabolic parameters, carotid intima media thickness and HFABP levels in both PCOS patients and control group. Mean fasting insulin, homeostasis model assessment insulin resistance index (HOMA-IR), triglyceride, total cholesterol, low density lipoprotein cholesterol, free testosterone, total testosterone, carotid intima media thickness (CIMT) levels were significantly higher in PCOS patients. Although HFABP levels were higher in PCOS patients, the difference did not reach statistically significant in early age groups. After adjustment for age and body mass index, HFABP level was positive correlated with hsCRP, free testosterone levels, CIMT and HOMA-IR. Heart type free fatty acid binding protein appeared to have an important role in metabolic response and subsequent development of atherosclerosis in insulin resistant, hyperandrogenemic PCOS patients.

  13. Heart type fatty acid binding protein response and subsequent development of atherosclerosis in insulin resistant polycystic ovary syndrome patients

    Directory of Open Access Journals (Sweden)

    Cakir Evrim

    2012-12-01

    Full Text Available Abstract Background Women with polycystic ovary syndrome (PCOS have higher risk for cardiovascular disease (CVD. Heart type fatty acid binding protein (HFABP has been found to be predictive for myocardial ischemia.Wet ested whether HFABP is the predictor for CVD in PCOS patients, who have an increased risk of cardiovascular disease. Methods This was a prospective, cross sectional controlled study conducted in a training and research hospital.The study population consisted of 46 reproductive-age PCOS women and 28 control subjects. We evaluated anthropometric and metabolic parameters, carotid intima media thickness and HFABP levels in both PCOS patients and control group. Results Mean fasting insulin, homeostasis model assessment insulin resistance index (HOMA-IR, triglyceride, total cholesterol, low density lipoprotein cholesterol, free testosterone, total testosterone, carotid intima media thickness (CIMT levels were significantly higher in PCOS patients. Although HFABP levels were higher in PCOS patients, the difference did not reach statistically significant in early age groups. After adjustment for age and body mass index, HFABP level was positive correlated with hsCRP, free testosterone levels, CIMT and HOMA-IR. Conclusions Heart type free fatty acid binding protein appeared to have an important role in metabolic response and subsequent development of atherosclerosis in insulin resistant, hyperandrogenemic PCOS patients.

  14. Correlation of insulin resistance with serum C-reactive protein, adiponectin and leptin levels in patients with type 2 diabetes

    International Nuclear Information System (INIS)

    Duan Yangqiang; Wang Zuobing; Yu Hui

    2012-01-01

    Objective: To explore the relationship between serum C-reactive protein (CRP), adiponectin (APN), leptin (Leptin) levels, insulin resistance index (HOMA-IR) and type 2 diabetes mellitus (T2DM) disease susceptibility. Methods: The plasma leptin and insulin (FINS) levels in the DM patients were determined by RIA, and the serum ANP levels were determined by ELSIA. The CRP, conventional serum fasting plasma glucose (FPG) levels were determine by automatic biochemistry analyzer. The insulin resistance index (HOMA-IR, FPG x FINS/22.5) was calculated. The result was analyzed with normal healthy control group. Results: The serum CRP and leptin, HOMA-IR levels in T2DM group were significantly higher than that of in control group (P< 0.01), and the serum ANP was significantly lower than in control group (P<0.01). The HOMA-IR in T2DM was positively correlated with serum CRP (r= 0.36, P<0.05) and leptin(r= 0.39, P<0.05), and was negatively correlated with serum APN (r=0.32, P<0.05). Conclusion: The high serum CRP and leptin and low APN levels hyperlipidaemia might be factors for diabetes, and their metabolic disorders may be closely related with insulin resistance in patients with type 2 diabetes. (authors)

  15. Field-Evolved Resistance in Corn Earworm to Cry Proteins Expressed by Transgenic Sweet Corn

    Science.gov (United States)

    Dively, Galen P.; Finkenbinder, Chad

    2016-01-01

    Background Transgenic corn engineered with genes expressing insecticidal toxins from the bacterium Bacillus thuringiensis (Berliner) (Bt) are now a major tool in insect pest management. With its widespread use, insect resistance is a major threat to the sustainability of the Bt transgenic technology. For all Bt corn expressing Cry toxins, the high dose requirement for resistance management is not achieved for corn earworm, Helicoverpa zea (Boddie), which is more tolerant to the Bt toxins. Methodology/Major Findings We present field monitoring data using Cry1Ab (1996–2016) and Cry1A.105+Cry2Ab2 (2010–2016) expressing sweet corn hybrids as in-field screens to measure changes in field efficacy and Cry toxin susceptibility to H. zea. Larvae successfully damaged an increasing proportion of ears, consumed more kernel area, and reached later developmental stages (4th - 6th instars) in both types of Bt hybrids (Cry1Ab—event Bt11, and Cry1A.105+Cry2Ab2—event MON89034) since their commercial introduction. Yearly patterns of H. zea population abundance were unrelated to reductions in control efficacy. There was no evidence of field efficacy or tissue toxicity differences among different Cry1Ab hybrids that could contribute to the decline in control efficacy. Supportive data from laboratory bioassays demonstrate significant differences in weight gain and fitness characteristics between the Maryland H. zea strain and a susceptible strain. In bioassays with Cry1Ab expressing green leaf tissue, Maryland H. zea strain gained more weight than the susceptible strain at all concentrations tested. Fitness of the Maryland H. zea strain was significantly lower than that of the susceptible strain as indicated by lower hatch rate, longer time to adult eclosion, lower pupal weight, and reduced survival to adulthood. Conclusions/Significance After ruling out possible contributing factors, the rapid change in field efficacy in recent years and decreased susceptibility of H. zea to Bt

  16. Resistance to cyclosporin A derives from mutations in hepatitis C virus nonstructural proteins.

    Science.gov (United States)

    Arai, Masaaki; Tsukiyama-Kohara, Kyoko; Takagi, Asako; Tobita, Yoshimi; Inoue, Kazuaki; Kohara, Michinori

    2014-05-23

    Cyclosporine A (CsA) is an immunosuppressive drug that targets cyclophilins, cellular cofactors that regulate the immune system. Replication of hepatitis C virus (HCV) is suppressed by CsA, but the molecular basis of this suppression is still not fully understood. To investigate this suppression, we cultured HCV replicon cells (Con1, HCV genotype 1b, FLR-N cell) in the presence of CsA and obtained nine CsA-resistant FLR-N cell lines. We determined full-length HCV sequences for all nine clones, and chose two (clones #6 and #7) of the nine clones that have high replication activity in the presence of CsA for further analysis. Both clones showed two consensus mutations, one in NS3 (T1280V) and the other in NS5A (D2292E). Characterization of various mutants indicated that the D2292E mutation conferred resistance to high concentrations of CsA (up to 2 μM). In addition, the missense mutation T1280V contributed to the recovery of colony formation activity. The effects of these mutations are also evident in two established HCV replicon cell lines-HCV-RMT ([1], genotype 1a) and JFH1 (genotype 2a). Moreover, three other missense mutations in NS5A-D2303H, S2362G, and E2414K-enhanced the resistance to CsA conferred by D2292E; these double or all quadruple mutants could resist approximately 8- to 25-fold higher concentrations of CsA than could wild-type Con1. These four mutations, either as single or combinations, also made Con1 strain resistant to two other cyclophilin inhibitors, N-methyl-4-isoleucine-cyclosporin (NIM811) or Debio-025. Interestingly, the changes in IC50 values that resulted from each of these mutations were the lowest in the Debio-025-treated cells, indicating its highest resistant activity against the adaptive mutation. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

  17. Glucokinase regulatory protein genetic variant interacts with omega-3 PUFA to influence insulin resistance and inflammation in metabolic syndrome.

    Directory of Open Access Journals (Sweden)

    Pablo Perez-Martinez

    Full Text Available Glucokinase Regulatory Protein (GCKR plays a central role regulating both hepatic triglyceride and glucose metabolism. Fatty acids are key metabolic regulators, which interact with genetic factors and influence glucose metabolism and other metabolic traits. Omega-3 polyunsaturated fatty acids (n-3 PUFA have been of considerable interest, due to their potential to reduce metabolic syndrome (MetS risk.To examine whether genetic variability at the GCKR gene locus was associated with the degree of insulin resistance, plasma concentrations of C-reactive protein (CRP and n-3 PUFA in MetS subjects.Homeostasis model assessment of insulin resistance (HOMA-IR, HOMA-B, plasma concentrations of C-peptide, CRP, fatty acid composition and the GCKR rs1260326-P446L polymorphism, were determined in a cross-sectional analysis of 379 subjects with MetS participating in the LIPGENE dietary cohort.Among subjects with n-3 PUFA levels below the population median, carriers of the common C/C genotype had higher plasma concentrations of fasting insulin (P = 0.019, C-peptide (P = 0.004, HOMA-IR (P = 0.008 and CRP (P = 0.032 as compared with subjects carrying the minor T-allele (Leu446. In contrast, homozygous C/C carriers with n-3 PUFA levels above the median showed lower plasma concentrations of fasting insulin, peptide C, HOMA-IR and CRP, as compared with individuals with the T-allele.We have demonstrated a significant interaction between the GCKR rs1260326-P446L polymorphism and plasma n-3 PUFA levels modulating insulin resistance and inflammatory markers in MetS subjects. Further studies are needed to confirm this gene-diet interaction in the general population and whether targeted dietary recommendations can prevent MetS in genetically susceptible individuals.ClinicalTrials.gov NCT00429195.

  18. Technetium-99m-hexakis-2-methoxyisobutylisonitrile scintigraphy and multidrug resistance-related protein expression in human primary lung cancer

    International Nuclear Information System (INIS)

    Duan Xiaoyi; Wang Jiansheng; Liu Min; Guo Youmin

    2008-01-01

    The occurrence of multidrug resistance (MDR) is a major cause of resistance to chemotherapeutic agents in patients with lung cancer, in part owing to the overexpression of MDR-related proteins. Technetium-99m-hexakis-2-methoxyisobutylisonitrile ( 99m Tc-MIBI) has been shown to be a substrate for some MDR-related proteins. The aim of this study is to evaluate the role of 99m Tc-MIBI scintigraphy for functional imaging of MDR-related protein phenotypes. To determine the correlation between 99m Tc-MIBI scintigraphy and the expression level of P-glycoprotein (Pgp), multidrug-resistance protein (MRP), and glutathione-S-transferase Pi (GSTπ), 26 patients (17 men and 9 women, median age 57.5 years) with primary lung cancer were investigated. Following intravenous administration of 925 MBq 99m Tc-MIBI, single-photon emission computed tomography (SPECT) and computed tomography (CT) were performed at 15 min and 2 h. On the basis of the fused images, tumor to background (T/B) ratio of both early and delayed images, and washout rate (WR%) of 99m Tc-MIBI were calculated. The immunohistochemical staining of Pgp, MRP, and GSTπ was performed, and the expression level was semiquantitated using a pathoimage analysis system. The imaging results were compared with the status of Pgp, MRP, and GSTπ expression. The WR% of 99m Tc-MIBI showed a significant positive correlation with Pgp expression (r=0.560, P=0.003), as no correlation was observed between WR% and MRP or GSTπ (r=0.354, P=0.076; r=0.324, P=0.106). Neither early T/B nor delayed T/B correlated with the expression level of Pgp, MRP, and GSTπ. WR%, Pgp, and GSTπ expression showed significant differences between squamous cell carcinoma (group A) and adenocarcinoma (group B). There was no significant difference among Pgp, MRP, and GSTπ expression levels in any cases (P>0.05). Our data confirmed that 99m Tc-MIBI scintigraphy is useful for determining the MDR caused by Pgp in patients with primary lung cancer. (author)

  19. Reduction in mRNA and protein expression of a nicotinic acetylcholine receptor α8 subunit is associated with resistance to imidacloprid in the brown planthopper, Nilaparvata lugens.

    Science.gov (United States)

    Zhang, Yixi; Wang, Xin; Yang, Baojun; Hu, Yuanyuan; Huang, Lixin; Bass, Chris; Liu, Zewen

    2015-11-01

    Target-site resistance is commonly caused by qualitative changes in insecticide target-receptors and few studies have implicated quantitative changes in insecticide targets in resistance. Here we show that resistance to imidacloprid in a selected strain of Nilaparvata lugens is associated with a reduction in expression levels of the nicotinic acetylcholine receptor (nAChR) subunit Nlα8. Synergism bioassays of the selected strain suggested resistance was conferred, in part, by a target-site mechanism. Sequencing of N. lugens nAChR subunit genes identified no mutations associated with resistance, however, a decrease in mRNA and protein levels of Nlα8 was observed during selection. RNA interference knockdown of Nlα8 decreased the sensitivity of N. lugens to imidacloprid, demonstrating that a decrease in Nlα8 expression is sufficient to confer resistance in vivo. Radioligand binding assays revealed that the affinity of the high-affinity imidacloprid-binding site of native nAChRs was reduced by selection, and reducing the amount of Nlα8 cRNA injected into Xenopus oocytes significantly decreased imidacloprid potency on recombinant receptors. Taken together, these results provide strong evidence that a decrease in Nlα8 levels confers resistance to imidacloprid in N. lugens, and thus provides a rare example of target-site resistance associated with a quantitative rather than qualitative change. In insects, target-site mutations often cause high resistance to insecticides, such as neonicotinoids acting on nicotinic acetylcholine receptors (nAChRs). Here we found that a quantitative change in target-protein level, decrease in mRNA and protein levels of Nlα8, contributed importantly to imidacloprid resistance in Nilaparvata lugens. This finding provides a new target-site mechanism of insecticide resistance. © 2015 International Society for Neurochemistry.

  20. TARSyn: Tunable Antibiotic Resistance Devices Enabling Bacterial Synthetic Evolution and Protein Production

    DEFF Research Database (Denmark)

    Rennig, Maja; Martinez, Virginia; Mirzadeh, Kiavash

    2018-01-01

    Evolution can be harnessed to optimize synthetic biology designs. A prominent example is recombinant protein production-a dominating theme in biotechnology for more than three decades. Typically, a protein coding sequence (cds) is recombined with genetic elements, such as promoters, ribosome...... and allows expression levels in large clone libraries to be probed using a simple cell survival assay on the respective antibiotic. The power of the approach is demonstrated by substantially increasing production of two commercially interesting proteins, a Nanobody and an Affibody. The method is a simple......-level expression-an example of synthetic evolution. However, manual screening limits the ability to assay expression levels of all putative sequences in the libraries. Here we have solved this bottleneck by designing a collection of translational coupling devices based on a RNA secondary structure. Exchange...

  1. Differential in vivo gene expression of major Leptospira proteins in resistant or susceptible animal models.

    Science.gov (United States)

    Matsui, Mariko; Soupé, Marie-Estelle; Becam, Jérôme; Goarant, Cyrille

    2012-09-01

    Transcripts of Leptospira 16S rRNA, FlaB, LigB, LipL21, LipL32, LipL36, LipL41, and OmpL37 were quantified in the blood of susceptible (hamsters) and resistant (mice) animal models of leptospirosis. We first validated adequate reference genes and then evaluated expression patterns in vivo compared to in vitro cultures. LipL32 expression was downregulated in vivo and differentially regulated in resistant and susceptible animals. FlaB expression was also repressed in mice but not in hamsters. In contrast, LigB and OmpL37 were upregulated in vivo. Thus, we demonstrated that a virulent strain of Leptospira differentially adapts its gene expression in the blood of infected animals.

  2. The Impact of "Coat Protein-Mediated Virus Resistance" in Applied Plant Pathology and Basic Research.

    Science.gov (United States)

    Lindbo, John A; Falk, Bryce W

    2017-06-01

    Worldwide, plant viruses cause serious reductions in marketable crop yield and in some cases even plant death. In most cases, the most effective way to control virus diseases is through genetically controlled resistance. However, developing virus-resistant (VR) crops through traditional breeding can take many years, and in some cases is not even possible. Because of this, the demonstration of the first VR transgenic plants in 1985 generated much attention. This seminal report served as an inflection point for research in both basic and applied plant pathology, the results of which have dramatically changed both basic research and in a few cases, commercial crop production. The typical review article on this topic has focused on only basic or only applied research results stemming from this seminal discovery. This can make it difficult for the reader to appreciate the full impact of research on transgenic virus resistance, and the contributions from fundamental research that led to translational applications of this technology. In this review, we take a global view of this topic highlighting the significant changes to both basic and applied plant pathology research and commercial food production that have accumulated in the last 30 plus years. We present these milestones in the historical context of some of the scientific, economic, and environmental drivers for developing specific VR crops. The intent of this review is to provide a single document that adequately records the significant accomplishments of researchers in both basic and applied plant pathology research on this topic and how they relate to each other. We hope this review therefore serves as both an instructional tool for students new to the topic, as well as a source of conversation and discussion for how the technology of engineered virus resistance could be applied in the future.

  3. Tomato Cf resistance proteins mediate recognition of cognate homologous effectors from fungi pathogenic on dicots and monocots.

    Science.gov (United States)

    Stergiopoulos, Ioannis; van den Burg, Harrold A; Okmen, Bilal; Beenen, Henriek G; van Liere, Sabine; Kema, Gert H J; de Wit, Pierre J G M

    2010-04-20

    Most fungal effectors characterized so far are species-specific and facilitate virulence on a particular host plant. During infection of its host tomato, Cladosporium fulvum secretes effectors that function as virulence factors in the absence of cognate Cf resistance proteins and induce effector-triggered immunity in their presence. Here we show that homologs of the C. fulvum Avr4 and Ecp2 effectors are present in other pathogenic fungi of the Dothideomycete class, including Mycosphaerella fijiensis, the causal agent of black Sigatoka disease of banana. We demonstrate that the Avr4 homolog of M. fijiensis is a functional ortholog of C. fulvum Avr4 that protects fungal cell walls against hydrolysis by plant chitinases through binding to chitin and, despite the low overall sequence homology, triggers a Cf-4-mediated hypersensitive response (HR) in tomato. Furthermore, three homologs of C. fulvum Ecp2 are found in M. fijiensis, one of which induces different levels of necrosis or HR in tomato lines that lack or contain a putative cognate Cf-Ecp2 protein, respectively. In contrast to Avr4, which acts as a defensive virulence factor, M. fijiensis Ecp2 likely promotes virulence by interacting with a putative host target causing host cell necrosis, whereas Cf-Ecp2 could possibly guard the virulence target of Ecp2 and trigger a Cf-Ecp2-mediated HR. Overall our data suggest that Avr4 and Ecp2 represent core effectors that are collectively recognized by single cognate Cf-proteins. Transfer of these Cf genes to plant species that are attacked by fungi containing these cognate core effectors provides unique ways for breeding disease-resistant crops.

  4. Isolation and Cloning of cDNA Fragment of Gene Encoding for Multidrug Resistance Associated Protein from M. affine.

    Directory of Open Access Journals (Sweden)

    Utut Widyastuti Suharsono

    2008-11-01

    Full Text Available Isolation and Cloning of cDNA Fragment of Gene Encoding for Multidrug Resistance Associated Protein from M. affine. M. affine can grow well in acid soil with high level of soluble aluminum. One of the important proteins in the detoxifying xenobiotic stress including acid and Al stresses is a multidrug resistance associated protein (MRP encoded by mrp gene. The objective of this research is to isolate and clone the cDNA fragment of MaMrp encoding MRP from M. affine. By reverse transcription, total cDNA had been synthesized from the total RNA as template. The fragment of cDNA MaMrp had been successfully isolated by PCR by using total cDNA as template and mrp primer designed from A. thaliana, yeast, and human. This fragment was successfully inserted into pGEM-T Easy and the recombinant plasmid was successfully introduced into E. coli DH5α. Nucleotide sequence analysis showed that the lenght of MaMrp fragment is 633 bp encoding 208 amino acids. Local alignment analysis based on nucleotide of mRNA showed that MaMrp fragment is 69% identical to AtMrp1 and 63% to AtMrp from A. thaliana. Based on deduced amino acid sequence, MaMRP is 84% identical to part of AtMRP13, 77% to AtMRP12, and 73% to AtMRP1 from A. thaliana respectively. Alignment analysis with AtMRP1 showed that MaMRP fragment is located in TM1 and NBF1 domains and has a specific amino acid sequence QCKAQLQNMEEE.

  5. Acute post-exercise myofibrillar protein synthesis is not correlated with resistance training-induced muscle hypertrophy in young men.

    Directory of Open Access Journals (Sweden)

    Cameron J Mitchell

    Full Text Available Muscle hypertrophy following resistance training (RT involves activation of myofibrillar protein synthesis (MPS to expand the myofibrillar protein pool. The degree of hypertrophy following RT is, however, highly variable and thus we sought to determine the relationship between the acute activation of MPS and RT-induced hypertrophy. We measured MPS and signalling protein activation after the first session of resistance exercise (RE in untrained men (n = 23 and then examined the relation between MPS with magnetic resonance image determined hypertrophy. To measure MPS, young men (24±1 yr; body mass index  = 26.4±0.9 kg•m² underwent a primed constant infusion of L-[ring-¹³C₆] phenylalanine to measure MPS at rest, and acutely following their first bout of RE prior to 16 wk of RT. Rates of MPS were increased 235±38% (P<0.001 above rest 60-180 min post-exercise and 184±28% (P = 0.037 180-360 min post exercise. Quadriceps volume increased 7.9±1.6% (-1.9-24.7% (P<0.001 after training. There was no correlation between changes in quadriceps muscle volume and acute rates of MPS measured over 1-3 h (r = 0.02, 3-6 h (r = 0.16 or the aggregate 1-6 h post-exercise period (r = 0.10. Hypertrophy after chronic RT was correlated (r = 0.42, P = 0.05 with phosphorylation of 4E-BP1(Thr37/46 at 1 hour post RE. We conclude that acute measures of MPS following an initial exposure to RE in novices are not correlated with muscle hypertrophy following chronic RT.

  6. Dietary sardine protein lowers insulin resistance, leptin and TNF-α and beneficially affects adipose tissue oxidative stress in rats with fructose-induced metabolic syndrome.

    Science.gov (United States)

    Madani, Zohra; Louchami, Karim; Sener, Abdullah; Malaisse, Willy J; Ait Yahia, Dalila

    2012-02-01

    The present study aims at exploring the effects of sardine protein on insulin resistance, plasma lipid profile, as well as oxidative and inflammatory status in rats with fructose-induced metabolic syndrome. Rats were fed sardine protein (S) or casein (C) diets supplemented or not with high-fructose (HF) for 2 months. Rats fed the HF diets had greater body weight and adiposity and lower food intake as compared to control rats. Increased plasma glucose, insulin, HbA1C, triacylglycerols, free fatty acids and impaired glucose tolerance and insulin resistance was observed in HF-fed rats. Moreover, a decline in adipose tissues antioxidant status and a rise in lipid peroxidation and plasma TNF-α and fibrinogen were noted. Rats fed sardine protein diets exhibited lower food intake and fat mass than those fed casein diets. Sardine protein diets diminished plasma insulin and insulin resistance. Plasma triacylglycerol and free fatty acids were also lower, while those of α-tocopherol, taurine and calcium were enhanced as compared to casein diets. Moreover, S-HF diet significantly decreased plasma glucose and HbA1C. Sardine protein consumption lowered hydroperoxide levels in perirenal and brown adipose tissues. The S-HF diet, as compared to C-HF diet decreased epididymal hydroperoxides. Feeding sardine protein diets decreased brown adipose tissue carbonyls and increased glutathione peroxidase activity. Perirenal and epididymal superoxide dismutase and catalase activities and brown catalase activity were significantly greater in S-HF group than in C-HF group. Sardine protein diets also prevented hyperleptinemia and reduced inflammatory status in comparison with rats fed casein diets. Taken together, these results support the beneficial effect of sardine protein in fructose-induced metabolic syndrome on such variables as hyperglycemia, insulin resistance, hyperlipidemia and oxidative and inflammatory status, suggesting the possible use of sardine protein as a protective

  7. Cross-Resistance between Cry1 Proteins in Fall Armyworm (Spodoptera frugiperda) May Affect the Durability of Current Pyramided Bt Maize Hybrids in Brazil.

    Science.gov (United States)

    Bernardi, Daniel; Salmeron, Eloisa; Horikoshi, Renato Jun; Bernardi, Oderlei; Dourado, Patrick Marques; Carvalho, Renato Assis; Martinelli, Samuel; Head, Graham P; Omoto, Celso

    2015-01-01

    Genetically modified plants expressing insecticidal proteins from Bacillus thuringiensis (Bt) offer valuable options for managing insect pests with considerable environmental and economic benefits. Despite the benefits provided by Bt crops, the continuous expression of these insecticidal proteins imposes strong selection for resistance in target pest populations. Bt maize (Zea mays) hybrids have been successful in controlling fall armyworm (Spodoptera frugiperda), the main maize pest in Brazil since 2008; however, field-evolved resistance to the protein Cry1F has recently been reported. Therefore it is important to assess the possibility of cross-resistance between Cry1F and other Cry proteins expressed in Bt maize hybrids. In this study, an F2 screen followed by subsequent selection on MON 89034 maize was used to select an S. frugiperda strain (RR) able to survive on the Bt maize event MON 89034, which expresses the Cry1A.105 and Cry2Ab2 proteins. Field-collected insects from maize expressing the Cry1F protein (event TC1507) represented most of the positive (resistance allele-containing) (iso)families found. The RR strain showed high levels of resistance to Cry1F, which apparently also conferred high levels of cross resistance to Cry1A.105 and Cry1Ab, but had only low-level (10-fold) resistance to Cry2Ab2. Life history studies to investigate fitness costs associated with the resistance in RR strain revealed only small reductions in reproductive rate when compared to susceptible and heterozygous strains, but the RR strain produced 32.2% and 28.4% fewer females from each female relative to the SS and RS (pooled) strains, respectively. Consistent with the lack of significant resistance to Cry2Ab2, MON 89034 maize in combination with appropriate management practices continues to provide effective control of S. frugiperda in Brazil. Nevertheless, the occurrence of Cry1F resistance in S. frugiperda across Brazil, and the cross-resistance to Cry1Ab and Cry1A.105

  8. Penicillin-Binding Protein Transpeptidase Signatures for Tracking and Predicting β-Lactam Resistance Levels in Streptococcus pneumoniae

    Directory of Open Access Journals (Sweden)

    Yuan Li

    2016-06-01

    Full Text Available β-Lactam antibiotics are the drugs of choice to treat pneumococcal infections. The spread of β-lactam-resistant pneumococci is a major concern in choosing an effective therapy for patients. Systematically tracking β-lactam resistance could benefit disease surveillance. Here we developed a classification system in which a pneumococcal isolate is assigned to a “PBP type” based on sequence signatures in the transpeptidase domains (TPDs of the three critical penicillin-binding proteins (PBPs, PBP1a, PBP2b, and PBP2x. We identified 307 unique PBP types from 2,528 invasive pneumococcal isolates, which had known MICs to six β-lactams based on broth microdilution. We found that increased β-lactam MICs strongly correlated with PBP types containing divergent TPD sequences. The PBP type explained 94 to 99% of variation in MICs both before and after accounting for genomic backgrounds defined by multilocus sequence typing, indicating that genomic backgrounds made little independent contribution to β-lactam MICs at the population level. We further developed and evaluated predictive models of MICs based on PBP type. Compared to microdilution MICs, MICs predicted by PBP type showed essential agreement (MICs agree within 1 dilution of >98%, category agreement (interpretive results agree of >94%, a major discrepancy (sensitive isolate predicted as resistant rate of <3%, and a very major discrepancy (resistant isolate predicted as sensitive rate of <2% for all six β-lactams. Thus, the PBP transpeptidase signatures are robust indicators of MICs to different β-lactam antibiotics in clinical pneumococcal isolates and serve as an accurate alternative to phenotypic susceptibility testing.

  9. Potential of Central, Eastern and Western Africa Medicinal Plants for Cancer Therapy: Spotlight on Resistant Cells and Molecular Targets

    Directory of Open Access Journals (Sweden)

    Armelle T. Mbaveng

    2017-06-01

    Full Text Available Cancer remains a major health hurdle worldwide and has moved from the third leading cause of death in the year 1990 to second place after cardiovascular disease since 2013. Chemotherapy is one of the most widely used treatment modes; however, its efficiency is limited due to the resistance of cancer cells to cytotoxic agents. The present overview deals with the potential of the flora of Central, Eastern and Western African (CEWA regions as resource for anticancer drug discovery. It also reviews the molecular targets of phytochemicals of these plants such as ABC transporters, namely P-glycoprotein (P-gp, multi drug-resistance-related proteins (MRPs, breast cancer resistance protein (BCRP, ABCG2 as well as the epidermal growth factor receptor (EGFR/ErbB-1/HER1, human tumor suppressor protein p53, caspases, mitochondria, angiogenesis, and components of MAP kinase signaling pathways. Plants with the ability to preferentially kills resistant cancer cells were also reported. Data compiled in the present document were retrieved from scientific websites such as PubMed, Scopus, Sciencedirect, Web-of-Science, and Scholar Google. In summary, plant extracts from CEWA and isolated compounds thereof exert cytotoxic effects by several modes of action including caspases activation, alteration of mitochondrial membrane potential (MMP, induction of reactive oxygen species (ROS in cancer cells and inhibition of angiogenesis. Ten strongest cytotoxic plants from CEWA recorded following in vitro screening assays are: Beilschmiedia acuta Kosterm, Echinops giganteus var. lelyi (C. D. Adams A. Rich., Erythrina sigmoidea Hua (Fabaceae, Imperata cylindrical Beauv. var. koenigii Durand et Schinz, Nauclea pobeguinii (Pobég. ex Pellegr. Merr. ex E.M.A., Piper capense L.f., Polyscias fulva (Hiern Harms., Uapaca togoensis Pax., Vepris soyauxii Engl. and Xylopia aethiopica (Dunal A. Rich. Prominent antiproliferative compounds include: isoquinoline alkaloid isotetrandrine (51

  10. Potential of Central, Eastern and Western Africa Medicinal Plants for Cancer Therapy: Spotlight on Resistant Cells and Molecular Targets

    Science.gov (United States)

    Mbaveng, Armelle T.; Kuete, Victor; Efferth, Thomas

    2017-01-01

    Cancer remains a major health hurdle worldwide and has moved from the third leading cause of death in the year 1990 to second place after cardiovascular disease since 2013. Chemotherapy is one of the most widely used treatment modes; however, its efficiency is limited due to the resistance of cancer cells to cytotoxic agents. The present overview deals with the potential of the flora of Central, Eastern and Western African (CEWA) regions as resource for anticancer drug discovery. It also reviews the molecular targets of phytochemicals of these plants such as ABC transporters, namely P-glycoprotein (P-gp), multi drug-resistance-related proteins (MRPs), breast cancer resistance protein (BCRP, ABCG2) as well as the epidermal growth factor receptor (EGFR/ErbB-1/HER1), human tumor suppressor protein p53, caspases, mitochondria, angiogenesis, and components of MAP kinase signaling pathways. Plants with the ability to preferentially kills resistant cancer cells were also reported. Data compiled in the present document were retrieved from scientific websites such as PubMed, Scopus, Sciencedirect, Web-of-Science, and Scholar Google. In summary, plant extracts from CEWA and isolated compounds thereof exert cytotoxic effects by several modes of action including caspases activation, alteration of mitochondrial membrane potential (MMP), induction of reactive oxygen species (ROS) in cancer cells and inhibition of angiogenesis. Ten strongest cytotoxic plants from CEWA recorded following in vitro screening assays are: Beilschmiedia acuta Kosterm, Echinops giganteus var. lelyi (C. D. Adams) A. Rich., Erythrina sigmoidea Hua (Fabaceae), Imperata cylindrical Beauv. var. koenigii Durand et Schinz, Nauclea pobeguinii (Pobég. ex Pellegr.) Merr. ex E.M.A., Piper capense L.f., Polyscias fulva (Hiern) Harms., Uapaca togoensis Pax., Vepris soyauxii Engl. and Xylopia aethiopica (Dunal) A. Rich. Prominent antiproliferative compounds include: isoquinoline alkaloid isotetrandrine (51), two

  11. Resistance: a threat to the insecticidal crystal proteins of Bacillus thuringiensis

    Science.gov (United States)

    Leah S. Bauer

    1995-01-01

    Insecticidal crystal proteins (also known as d-endotoxins) synthesized by the bacterium Bacillus thuringiensis Berliner (Bt) are the active ingredient of various environmentally friendly insecticides that are 1) highly compatible with natural enemies and other nontarget organisms due to narrow host specificity, 2) harmless to vertebrates, 3) biodegradable in the...

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

    DEFF Research Database (Denmark)

    Agergaard, Jakob; Bülow, Jacob; Jensen, Jacob K

    2017-01-01

    to 13 h of supine rest. After 2.5 h of rest, unilateral LL-RE, consisting of leg extensions (10 sets, 36 repetitions) at 16% of 1 repetition maximum (RM), was conducted. Subsequently, the subjects were randomized to oral intake of 4 g of whey protein per hour (PULSE, n = 10), 28 g of whey protein at 0 h...... and 12 g of whey protein at 7 h postexercise (BOLUS, n = 10), or 4 g of maltodextrin per hour (placebo, n = 10). Quadriceps muscle biopsies were taken at 0, 3, 7, and 10 h postexercise from the resting and the exercised leg of each subject. Myofibrillar FSR and activity of select targets from...... persisted in the placebo group only. Levels of phosphorylated (T37/46) eukaryotic translation initiation factor 4E-binding protein 1 increased throughout the postexercise period in the exercised leg in the placebo and BOLUS groups and peaked at 7 h. In all three groups, phosphorylated (T56) eukaryotic...

  13. Functional activity of Gi alpha protein in detergent resistant membrane domains from rat brain cortex

    Czech Academy of Sciences Publication Activity Database

    Stöhr, Jiří; Rudajev, Vladimír; Bouřová, Lenka; Lisý, Václav; Novotný, Jiří; Svoboda, Petr

    2007-01-01

    Roč. 101, Suppl.1 (2007), s. 52-52 ISSN 0022-3042. [European Society for Neurochemistry Meeting /17./. 19.05.2007-22.05.2007, Salamanca] Institutional research plan: CEZ:AV0Z50110509 Keywords : cpo1 * GABAB receptor * Gi protein * membrane domains Subject RIV: ED - Physiology

  14. The relationship of platinum resistance and ERCC1 protein expression in epithelial ovarian cancer

    DEFF Research Database (Denmark)

    Steffensen, Karina Dahl; Waldstrøm, Marianne; Jakobsen, Anders

    2009-01-01

    : Formalin-fixed, paraffin-embedded tissue sections from 101 patients with newly diagnosed ovarian cancer were used for immunohistochemical staining for the ERCC1 protein. All patients received carboplatin-paclitaxel combination chemotherapy. RESULTS: Excision repair cross-complementation group 1 enzyme...

  15. Systematic mutagenesis of genes encoding predicted autotransported proteins of Burkholderia pseudomallei identifies factors mediating virulence in mice, net intracellular replication and a novel protein conferring serum resistance.

    Directory of Open Access Journals (Sweden)

    Natalie R Lazar Adler

    Full Text Available Burkholderia pseudomallei is the causative agent of the severe tropical disease melioidosis, which commonly presents as sepsis. The B. pseudomallei K96243 genome encodes eleven predicted autotransporters, a diverse family of secreted and outer membrane proteins often associated with virulence. In a systematic study of these autotransporters, we constructed insertion mutants in each gene predicted to encode an autotransporter and assessed them for three pathogenesis-associated phenotypes: virulence in the BALB/c intra-peritoneal mouse melioidosis model, net intracellular replication in J774.2 murine macrophage-like cells and survival in 45% (v/v normal human serum. From the complete repertoire of eleven autotransporter mutants, we identified eight mutants which exhibited an increase in median lethal dose of 1 to 2-log10 compared to the isogenic parent strain (bcaA, boaA, boaB, bpaA, bpaC, bpaE, bpaF and bimA. Four mutants, all demonstrating attenuation for virulence, exhibited reduced net intracellular replication in J774.2 macrophage-like cells (bimA, boaB, bpaC and bpaE. A single mutant (bpaC was identified that exhibited significantly reduced serum survival compared to wild-type. The bpaC mutant, which demonstrated attenuation for virulence and net intracellular replication, was sensitive to complement-mediated killing via the classical and/or lectin pathway. Serum resistance was rescued by in trans complementation. Subsequently, we expressed recombinant proteins of the passenger domain of four predicted autotransporters representing each of the phenotypic groups identified: those attenuated for virulence (BcaA, those attenuated for virulence and net intracellular replication (BpaE, the BpaC mutant with defects in virulence, net intracellular replication and serum resistance and those displaying wild-type phenotypes (BatA. Only BcaA and BpaE elicited a strong IFN-γ response in a restimulation assay using whole blood from seropositive donors

  16. Bioinformatics analysis to assess potential risks of allergenicity and toxicity of HRAP and PFLP proteins in genetically modified bananas resistant to Xanthomonas wilt disease.

    Science.gov (United States)

    Jin, Yuan; Goodman, Richard E; Tetteh, Afua O; Lu, Mei; Tripathi, Leena

    2017-11-01

    Banana Xanthomonas wilt (BXW) disease threatens banana production and food security throughout East Africa. Natural resistance is lacking among common cultivars. Genetically modified (GM) bananas resistant to BXW disease were developed by inserting the hypersensitive response-assisting protein (Hrap) or/and the plant ferredoxin-like protein (Pflp) gene(s) from sweet pepper (Capsicum annuum). Several of these GM banana events showed 100% resistance to BXW disease under field conditions in Uganda. The current study evaluated the potential allergenicity and toxicity of the expressed proteins HRAP and PFLP based on evaluation of published information on the history of safe use of the natural source of the proteins as well as established bioinformatics sequence comparison methods to known allergens (www.AllergenOnline.org and NCBI Protein) and toxins (NCBI Protein). The results did not identify potential risks of allergy and toxicity to either HRAP or PFLP proteins expressed in the GM bananas that might suggest potential health risks to humans. We recognize that additional tests including stability of these proteins in pepsin assay, nutrient analysis and possibly an acute rodent toxicity assay may be required by national regulatory authorities. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

  17. Ion Transport in Confined Geometries below the Nanoscale: Access Resistance Dominates Protein Channel Conductance in Diluted Solutions.

    Science.gov (United States)

    Alcaraz, Antonio; López, M Lidón; Queralt-Martín, María; Aguilella, Vicente M

    2017-10-24

    Synthetic nanopores and mesoscopic protein channels have common traits like the importance of electrostatic interactions between the permeating ions and the nanochannel. Ion transport at the nanoscale occurs under confinement conditions so that the usual assumptions made in microfluidics are challenged, among others, by interfacial effects such as access resistance (AR). Here, we show that a sound interpretation of electrophysiological measurements in terms of channel ion selective properties requires the consideration of interfacial effects, up to the point that they dominate protein channel conductance in diluted solutions. We measure AR in a large ion channel, the bacterial porin OmpF, by means of single-channel conductance measurements in electrolyte solutions containing varying concentrations of high molecular weight PEG, sterically excluded from the pore. Comparison of experiments performed in charged and neutral planar membranes shows that lipid surface charges modify the ion distribution and determine the value of AR, indicating that lipid molecules are more than passive scaffolds even in the case of large transmembrane proteins. We also found that AR may reach up to 80% of the total channel conductance in diluted solutions, where electrophysiological recordings register essentially the AR of the system and depend marginally on the pore characteristics. These findings may have implications for several low aspect ratio biological channels that perform their physiological function in a low ionic strength and macromolecule crowded environment, just the two conditions enhancing the AR contribution.

  18. Multilayer Choline Phosphate Molecule Modified Surface with Enhanced Cell Adhesion but Resistance to Protein Adsorption.

    Science.gov (United States)

    Chen, Xingyu; Yang, Ming; Liu, Botao; Li, Zhiqiang; Tan, Hong; Li, Jianshu

    2017-08-22

    Choline phosphate (CP), which is a new zwitterionic molecule, and has the reverse order of phosphate choline (PC) and could bind to the cell membrane though the unique CP-PC interaction. Here we modified a glass surface with multilayer CP molecules using surface-initiated atom-transfer radical polymerization (SI-ATRP) and the ring-opening method. Polymeric brushes of (dimethylamino)ethyl methacrylate (DMAEMA) were synthesized by SI-ATRP from the glass surface. Then the grafted PDMAEMA brushes were used to introduce CP groups to fabricate the multilayer CP molecule modified surface. The protein adsorption experiment and cell culture test were used to evaluate the biocompatibility of the modified surfaces by using human umbilical veinendothelial cells (HUVECs). The protein adsorption results demonstrated that the multilayer CP molecule decorated surface could prevent the adsorption of fibrinogen and serum protein. The adhesion and proliferation of cells were improved significantly on the multilayer CP molecule modified surface. Therefore, the biocompatibility of the material surface could be improved by the modified multilayer CP molecule, which exhibits great potential for biomedical applications, e.g., scaffolds in tissue engineering.

  19. Two Seven-Transmembrane Domain MILDEW RESISTANCE LOCUS O Proteins Cofunction in Arabidopsis Root Thigmomorphogenesis[C][W

    Science.gov (United States)

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

    2009-01-01

    Directional root expansion is governed by nutrient gradients, positive gravitropism and hydrotropism, negative phototropism and thigmotropism, as well as endogenous oscillations in the growth trajectory (circumnutation). Null mutations in phylogenetically related Arabidopsis thaliana genes MILDEW RESISTANCE LOCUS O 4 (MLO4) and MLO11, encoding heptahelical, plasma membrane–localized proteins predominantly expressed in the root tip, result in aberrant root thigmomorphogenesis. mlo4 and mlo11 mutant plants show anisotropic, chiral root expansion manifesting as tightly curled root patterns upon contact with solid surfaces. The defect in mlo4 and mlo11 mutants is nonadditive and dependent on light and nutrients. Genetic epistasis experiments demonstrate that the mutant phenotype is independently modulated by the Gβ subunit of the heterotrimeric G-protein complex. Analysis of expressed chimeric MLO4/MLO2 proteins revealed that the C-terminal domain of MLO4 is necessary but not sufficient for MLO4 action in root thigmomorphogenesis. The expression of the auxin efflux carrier fusion, PIN1-green fluorescent protein, the pattern of auxin-induced gene expression, and acropetal as well as basipetal auxin transport are altered at the root tip of mlo4 mutant seedlings. Moreover, addition of auxin transport inhibitors or the loss of EIR1/AGR1/PIN2 function abolishes root curling of mlo4, mlo11, and wild-type seedlings. These results demonstrate that the exaggerated root curling phenotypes of the mlo4 and mlo11 mutants depend on auxin gradients and suggest that MLO4 and MLO11 cofunction as modulators of touch-induced root tropism. PMID:19602625

  20. An OGA-Resistant Probe Allows Specific Visualization and Accurate Identification of O-GlcNAc-Modified Proteins in Cells.

    Science.gov (United States)

    Li, Jing; Wang, Jiajia; Wen, Liuqing; Zhu, He; Li, Shanshan; Huang, Kenneth; Jiang, Kuan; Li, Xu; Ma, Cheng; Qu, Jingyao; Parameswaran, Aishwarya; Song, Jing; Zhao, Wei; Wang, Peng George

    2016-11-18

    O-linked β-N-acetyl-glucosamine (O-GlcNAc) is an essential and ubiquitous post-translational modification present in nucleic and cytoplasmic proteins of multicellular eukaryotes. The metabolic chemical probes such as GlcNAc or GalNAc analogues bearing ketone or azide handles, in conjunction with bioorthogonal reactions, provide a powerful approach for detecting and identifying this modification. However, these chemical probes either enter multiple glycosylation pathways or have low labeling efficiency. Therefore, selective and potent probes are needed to assess this modification. We report here the development of a novel probe, 1,3,6-tri-O-acetyl-2-azidoacetamido-2,4-dideoxy-d-glucopyranose (Ac 3 4dGlcNAz), that can be processed by the GalNAc salvage pathway and transferred by O-GlcNAc transferase (OGT) to O-GlcNAc proteins. Due to the absence of a hydroxyl group at C4, this probe is less incorporated into α/β 4-GlcNAc or GalNAc containing glycoconjugates. Furthermore, the O-4dGlcNAz modification was resistant to the hydrolysis of O-GlcNAcase (OGA), which greatly enhanced the efficiency of incorporation for O-GlcNAcylation. Combined with a click reaction, Ac 3 4dGlcNAz allowed the selective visualization of O-GlcNAc in cells and accurate identification of O-GlcNAc-modified proteins with LC-MS/MS. This probe represents a more potent and selective tool in tracking, capturing, and identifying O-GlcNAc-modified proteins in cells and cell lysates.

  1. Proteome analyses of cellular proteins in methicillin-resistant Staphylococcus aureus treated with rhodomyrtone, a novel antibiotic candidate.

    Directory of Open Access Journals (Sweden)

    Wipawadee Sianglum

    Full Text Available The ethanolic extract from Rhodomyrtus tomentosa leaf exhibited good antibacterial activities against both methicillin-resistant Staphylococcus aureus (MRSA and S. aureus ATCC 29213. Its minimal inhibitory concentration (MIC values ranged from 31.25-62.5 µg/ml, and the minimal bactericidal concentration (MBC was 250 µg/ml. Rhodomyrtone, an acylphloroglucinol derivative, was 62.5-125 times more potent at inhibiting the bacteria than the ethanolic extract, the MIC and MBC values were 0.5 µg/ml and 2 µg/ml, respectively. To provide insights into antibacterial mechanisms involved, the effects of rhodomyrtone on cellular protein expression of MRSA have been investigated using proteomic approaches. Proteome analyses revealed that rhodomyrtone at subinhibitory concentration (0.174 µg/ml affected the expression of several major functional classes of whole cell proteins in MRSA. The identified proteins involve in cell wall biosynthesis and cell division, protein degradation, stress response and oxidative stress, cell surface antigen and virulence factor, and various metabolic pathways such as amino acid, carbohydrate, energy, lipid, and nucleotide metabolism. Transmission electron micrographs confirmed the effects of rhodomyrtone on morphological and ultrastructural alterations in the treated bacterial cells. Biological processes in cell wall biosynthesis and cell division were interrupted. Prominent changes including alterations in cell wall, abnormal septum formation, cellular disintegration, and cell lysis were observed. Unusual size and shape of staphylococcal cells were obviously noted in the treated MRSA. These pioneer findings on proteomic profiling and phenotypic features of rhodomyrtone-treated MRSA may resolve its antimicrobial mechanisms which could lead to the development of a new effective regimen for the treatment of MRSA infections.

  2. Crystal structure of tabtoxin resistance protein complexed with acetyl coenzyme A reveals the mechanism for beta-lactam acetylation.

    Science.gov (United States)

    He, Hongzhen; Ding, Yi; Bartlam, Mark; Sun, Fei; Le, Yi; Qin, Xincheng; Tang, Hong; Zhang, Rongguang; Joachimiak, Andrzej; Liu, Jinyuan; Zhao, Nanming; Rao, Zihe

    2003-01-31

    Tabtoxin resistance protein (TTR) is an enzyme that renders tabtoxin-producing pathogens, such as Pseudomonas syringae, tolerant to their own phytotoxins. Here, we report the crystal structure of TTR complexed with its natural cofactor, acetyl coenzyme A (AcCoA), to 1.55A resolution. The binary complex forms a characteristic "V" shape for substrate binding and contains the four motifs conserved in the GCN5-related N-acetyltransferase (GNAT) superfamily, which also includes the histone acetyltransferases (HATs). A single-step mechanism is proposed to explain the function of three conserved residues, Glu92, Asp130 and Tyr141, in catalyzing the acetyl group transfer to its substrate. We also report that TTR possesses HAT activity and suggest an evolutionary relationship between TTR and other GNAT members.

  3. Crystal structure of tabtoxin resistance protein complexed with acetyl coenzyme A reveals the mechanism for {beta}-lactam acetylation.

    Energy Technology Data Exchange (ETDEWEB)

    He, H.; Ding, Y.; Bartlam, M.; Sun, F.; Le, Y.; Qin, X.; Tang, H.; Zhang, R.; Joachimiak, A.; Liu, J.; Zhao, N.; Rao, Z.; Biosciences Division; Tsinghua Univ.; Chinese Academy of Science

    2003-01-31

    Tabtoxin resistance protein (TTR) is an enzyme that renders tabtoxin-producing pathogens, such as Pseudomonas syringae, tolerant to their own phytotoxins. Here, we report the crystal structure of TTR complexed with its natural cofactor, acetyl coenzyme A (AcCoA), to 1.55 {angstrom} resolution. The binary complex forms a characteristic 'V' shape for substrate binding and contains the four motifs conserved in the GCN5-related N-acetyltransferase (GNAT) superfamily, which also includes the histone acetyltransferases (HATs). A single-step mechanism is proposed to explain the function of three conserved residues, Glu92, Asp130 and Tyr141, in catalyzing the acetyl group transfer to its substrate. We also report that TTR possesses HAT activity and suggest an evolutionary relationship between TTR and other GNAT members.

  4. Circumvention of the multidrug-resistance protein (MRP-1) by an antitumor drug through specific inhibition of gene transcription in breast tumor cells.

    Science.gov (United States)

    Mansilla, Sylvia; Rojas, Marta; Bataller, Marc; Priebe, Waldemar; Portugal, José

    2007-04-01

    Multidrug-resistance protein 1 (MRP-1) confers resistance to a number of clinically important chemotherapeutic agents. The promoter of the mrp-1 gene contains an Sp1-binding site, which we targeted using the antitumor bis-anthracycline WP631. When MCF-7/VP breast cancer cells, which overexpress MRP-1 protein, were incubated with WP631 the expression of the multidrug-resistance protein gene decreased. Conversely, doxorubicin did not alter mrp-1 gene expression. The inhibition of gene expression was followed by a decrease in the activity of the MRP-1 protein. The IC(75) for WP631 (drug concentration required to inhibit cell growth by 75%) circumvented the drug-efflux pump, without addition of resistant modifiers. After treatment with WP631, MCF-7/VP cells were committed to die after entering mitosis (mitotic catastrophe), while treatment with doxorubicin did not affect cell growth. This is the first report on an antitumor drug molecule inhibiting the mrp-1 gene directly, rather than being simply a poor substrate for the transporter-mediated efflux. However, both situations appeared to coexist, thereby a superior cytotoxic effect was attained. Ours results suggest that WP631 offers great potential for the clinical treatment of tumors displaying a multidrug-resistance phenotype.

  5. Preparation and characterization of soy protein films with a durable water resistance-adjustable and antimicrobial surface.

    Science.gov (United States)

    Li, Shuzhao; Donner, Elizabeth; Xiao, Huining; Thompson, Michael; Zhang, Yachuan; Rempel, Curtis; Liu, Qiang

    2016-12-01

    A water resistant surface was first obtained by immobilizing hydrophobic copolymers, poly (styrene-co-glycidyl methacrylate) (PSG), with functional groups on soy protein isolate (SPI) films. XPS and AFM results showed that PSG copolymers were immobilized on the film by chemical bonding, and formed a rough surface with some bumps because of the segregation of two different phases on PSG copolymers. Water resistance of the modified films could be adjusted dramatically by further immobilizing different amounts of guanidine-based antimicrobial polymers, poly (hexamethylene guanidine hydrochloride) (PHMG) on the resulting hydrophobic surface. The introduction of hydrophilic PHMG on the resulting surface generated many micropores, which potentially increased the water uptake of the modified films. Furthermore, the modified SPI films showed higher thermostability compared to native SPI film and broad-spectrum antimicrobial activity by contact killing, attributed to the presence of PHMG on the surface. The modified SPI film with a multi-functional surface showed potential for applications in the packaging and medical fields. Crown Copyright © 2016. Published by Elsevier B.V. All rights reserved.

  6. The MAP kinase Pmk1 and protein kinase A are required for rotenone resistance in the fission yeast, Schizosaccharomyces pombe

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Yiwei; Gulis, Galina; Buckner, Scott; Johnson, P. Connor; Sullivan, Daniel [Department of Biological Sciences, The University of Alabama, Tuscaloosa, AL 35487 (United States); Busenlehner, Laura [Department of Chemistry, The University of Alabama, Tuscaloosa, AL 35487 (United States); Marcus, Stevan, E-mail: smarcus@bama.ua.edu [Department of Biological Sciences, The University of Alabama, Tuscaloosa, AL 35487 (United States)

    2010-08-20

    Research highlights: {yields} Rotenone induces generation of ROS and mitochondrial fragmentation in fission yeast. {yields} The MAPK Pmk1 and PKA are required for rotenone resistance in fission yeast. {yields} Pmk1 and PKA are required for ROS clearance in rotenone treated fission yeast cells. {yields} PKA plays a role in ROS clearance under normal growth conditions in fission yeast. -- Abstract: Rotenone is a widely used pesticide that induces Parkinson's disease-like symptoms in rats and death of dopaminergic neurons in culture. Although rotenone is a potent inhibitor of complex I of the mitochondrial electron transport chain, it can induce death of dopaminergic neurons independently of complex I inhibition. Here we describe effects of rotenone in the fission yeast, Schizosaccharomyces pombe, which lacks complex I and carries out rotenone-insensitive cellular respiration. We show that rotenone induces generation of reactive oxygen species (ROS) as well as fragmentation of mitochondrial networks in treated S. pombe cells. While rotenone is only modestly inhibitory to growth of wild type S. pombe cells, it is strongly inhibitory to growth of mutants lacking the ERK-type MAP kinase, Pmk1, or protein kinase A (PKA). In contrast, cells lacking the p38 MAP kinase, Spc1, exhibit modest resistance to rotenone. Consistent with these findings, we provide evidence that Pmk1 and PKA, but not Spc1, are required for clearance of ROS in rotenone treated S. pombe cells. Our results demonstrate the usefulness of S. pombe for elucidating complex I-independent molecular targets of rotenone as well as mechanisms conferring resistance to the toxin.

  7. Relation of Absolute or Relative Adiposity to Insulin Resistance, Retinol Binding Protein-4, Leptin, and Adiponectin in Type 2 Diabetes

    Directory of Open Access Journals (Sweden)

    You Lim Kim

    2012-12-01

    Full Text Available BackgroundCentral fat mass (CFM correlates with insulin resistance and increases the risk of type 2 diabetes and cardiovascular complications; however, peripheral fat mass (PFM is associated with insulin sensitivity. The aim of this study was to investigate the relation of absolute and relative regional adiposity to insulin resistance index and adipokines in type 2 diabetes.MethodsTotal of 83 overweighted-Korean women with type 2 diabetes were enrolled, and rate constants for plasma glucose disappearance (KITT and serum adipokines, such as retinol binding protein-4 (RBP4, leptin, and adiponectin, were measured. Using dual X-ray absorptiometry, trunk fat mass (in kilograms was defined as CFM, sum of fat mass on the lower extremities (in kilograms as PFM, and sum of CFM and PFM as total fat mass (TFM. PFM/TFM ratio, CFM/TFM ratio, and PFM/CFM ratio were defined as relative adiposity.ResultsMedian age was 55.9 years, mean body mass index 27.2 kg/m2, and mean HbA1c level 7.12±0.84%. KITT was positively associated with PMF/TFM ratio, PMF/CFM ratio, and negatively with CFM/TFM ratio, but was not associated with TFM, PFM, or CFM. RBP4 levels also had a significant relationship with PMF/TFM ratio and PMF/CFM ratio. Adiponectin, leptin, and apolipoprotein A levels were related to absolute adiposity, while only adiponectin to relative adiposity. In correlation analysis, KITT in type 2 diabetes was positively related with HbA1c, fasting glucose, RBP4, and free fatty acid.ConclusionThese results suggest that increased relative amount of peripheral fat mass may aggravate insulin resistance in type 2 diabetes.

  8. The MAP kinase Pmk1 and protein kinase A are required for rotenone resistance in the fission yeast, Schizosaccharomyces pombe

    International Nuclear Information System (INIS)

    Wang, Yiwei; Gulis, Galina; Buckner, Scott; Johnson, P. Connor; Sullivan, Daniel; Busenlehner, Laura; Marcus, Stevan

    2010-01-01

    Research highlights: → Rotenone induces generation of ROS and mitochondrial fragmentation in fission yeast. → The MAPK Pmk1 and PKA are required for rotenone resistance in fission yeast. → Pmk1 and PKA are required for ROS clearance in rotenone treated fission yeast cells. → PKA plays a role in ROS clearance under normal growth conditions in fission yeast. -- Abstract: Rotenone is a widely used pesticide that induces Parkinson's disease-like symptoms in rats and death of dopaminergic neurons in culture. Although rotenone is a potent inhibitor of complex I of the mitochondrial electron transport chain, it can induce death of dopaminergic neurons independently of complex I inhibition. Here we describe effects of rotenone in the fission yeast, Schizosaccharomyces pombe, which lacks complex I and carries out rotenone-insensitive cellular respiration. We show that rotenone induces generation of reactive oxygen species (ROS) as well as fragmentation of mitochondrial networks in treated S. pombe cells. While rotenone is only modestly inhibitory to growth of wild type S. pombe cells, it is strongly inhibitory to growth of mutants lacking the ERK-type MAP kinase, Pmk1, or protein kinase A (PKA). In contrast, cells lacking the p38 MAP kinase, Spc1, exhibit modest resistance to rotenone. Consistent with these findings, we provide evidence that Pmk1 and PKA, but not Spc1, are required for clearance of ROS in rotenone treated S. pombe cells. Our results demonstrate the usefulness of S. pombe for elucidating complex I-independent molecular targets of rotenone as well as mechanisms conferring resistance to the toxin.

  9. Resveratrol Inhibits Porcine Intestinal Glucose and Alanine Transport: Potential Roles of Na+/K+-ATPase Activity, Protein Kinase A, AMP-Activated Protein Kinase and the Association of Selected Nutrient Transport Proteins with Detergent Resistant Membranes

    Directory of Open Access Journals (Sweden)

    Stefanie Klinger

    2018-03-01

    Full Text Available Background: Beneficial effects of Resveratrol (RSV have been demonstrated, including effects on transporters and channels. However, little is known about how RSV influences intestinal transport. The aim of this study was to further characterize the effects of RSV on intestinal transport and the respective mechanisms. Methods: Porcine jejunum and ileum were incubated with RSV (300 µM, 30 min in Ussing chambers (functional studies and tissue bathes (detection of protein expression, phosphorylation, association with detergent resistant membranes (DRMs. Results: RSV reduced alanine and glucose-induced short circuit currents (ΔIsc and influenced forskolin-induced ΔIsc. The phosphorylation of sodium–glucose-linked transporter 1 (SGLT1, AMP-activated protein kinase (AMPK, protein kinase A substrates (PKA-S and liver kinase B1 (LKB1 increased but a causative relation to the inhibitory effects could not directly be established. The DRM association of SGLT1, peptide transporter 1 (PEPT1 and (phosphorylated Na+/H+-exchanger 3 (NHE3 did not change. Conclusion: RSV influences the intestinal transport of glucose, alanine and chloride and is likely to affect other transport processes. As the effects of protein kinase activation vary between the intestinal localizations, it would appear that increasing cyclic adenosine monophosphate (cAMP levels are part of the mechanism. Nonetheless, the physiological responses depend on cell type-specific structures.

  10. Preparation of protein- and cell-resistant surfaces by hyperthermal hydrogen induced cross-linking of poly(ethylene oxide).

    Science.gov (United States)

    Bonduelle, Colin V; Lau, Woon M; Gillies, Elizabeth R

    2011-05-01

    The functionalization of surfaces with poly(ethylene oxide) (PEO) is an effective means of imparting resistance to the adsorption of proteins and the attachment and growth of cells, properties that are critical for many biomedical applications. In this work, a new hyperthermal hydrogen induced cross-linking (HHIC) method was explored as a simple one-step approach for attaching PEO to surfaces through the selective cleavage of C-H bonds and subsequent cross-linking of the resulting carbon radicals. In order to study the effects of the process on the polymer, PEO-coated silicon wafers were prepared and the effects of different treatment times were investigated. Subsequently, using an optimized treatment time and a modified butyl polymer with increased affinity for PEO, the technique was applied to butyl rubber surfaces. All of the treated surfaces exhibited significantly reduced protein adsorption and cell growth relative to control surfaces and compared favorably with surfaces that were functionalized with PEO using conventional chemical methods. Thus HHIC is a simple and effective means of attaching PEO to non-functional polymer surfaces.

  11. [Relationship between phenomenon of acquired activated protein C resistance and antiphospholipid antibodies in patients with systemic lupus erythematosus].

    Science.gov (United States)

    Hu, Y Q; Chen, F P; Xie, Q Z

    2001-10-28

    To determine the occurrence of activated protein C resistance (APCR), to identify APCR is associated with thrombotic events (TEs), and acquired APCR is associated with the presence of antiphospholipid antibodies (APLAs) in 30 patients with systemic lupus erythematosus (SLE). Laboratory tests included dilute Russell's viper venom time assay for LA (dRVVT-LA), ELISA assay for ACL, APC sensitivity ratio, and factor V Leiden were detected by PCR-Mnl/I digestion. Acquired APCR was presented in 14(46.67%) of 30 patients. Factor V Leiden was not found in any patients. The incidence of TEs in the APCR-positive patients was significantly higher than that in the APCR-negative patients (42.85% vs 6.25%, P TEs in the LA-positive patients was also significantly higher than that in the LA-negative patients (50% vs 11.1%, P TEs (P TEs. Acquired APCR may not reflect the interference of LAs with the protein C pathway which may represent a mechanism of LA-associated TEs.

  12. Poly(l-glutamic acid)-g-poly(ethylene glycol) external layer in polyelectrolyte multilayer films: Characterization and resistance to serum protein adsorption.

    Science.gov (United States)

    Szczepanowicz, Krzysztof; Kruk, Tomasz; Świątek, Wiktoria; Bouzga, Aud M; Simon, Christian R; Warszyński, Piotr

    2018-06-01

    Formation of protein-resistant surfaces is a major challenge in the design of novel biomaterials and an important strategy to prevent protein adsorption is the formation of protein-resistant coatings. It can be achieved by proper modification of surfaces, e.g., by immobilization of hydrophilic polymers such as poly(ethylene glycol) (PEG). An appropriate method to immobilize PEG at charged surfaces is the adsorption of copolymers with PEG chains grafted onto polyelectrolyte backbone. The growing interest in the use of polyelectrolyte multilayer coatings in biomedical applications to improve biocompatibility and/or to prepare coating with antiadhesive properties has been the main reason for these studies. Therefore the aim was to produce protein resistant polyelectrolyte multilayer films. They were formed via the layer-by-layer approach, while their pegylation by the deposition of pegylated polyanion, PGA-g-PEG, as an external layer. The influence of PEG chain length and grafting density of PGA-g-PEG copolymers on the protein antiadhesive properties of pegylated polyelectrolyte multilayer films was investigated. To monitor the formation of pegylated and non-pegylated multilayer films, adsorption of the following proteins: HSA, Fibrinogen, and FBS were measured by quartz crystal microbalance (QCM - D). We found that protein adsorption onto all pegylated polyelectrolyte multilayers was significantly reduced in comparison to non-pegylated ones. Long-term performance tests confirmed the stability and the durability of the protein resistant properties of the pegylated multilayers. Antiadhesive properties of tested surfaces pegylated by PGA-g-PEG were compared to the available data for pegylated polycation PLL-g-PEG. Copyright © 2018 Elsevier B.V. All rights reserved.

  13. The effect of taurine and β-alanine supplementation on taurine transporter protein and fatigue resistance in skeletal muscle from mdx mice.

    Science.gov (United States)

    Horvath, Deanna M; Murphy, Robyn M; Mollica, Janelle P; Hayes, Alan; Goodman, Craig A

    2016-11-01

    This study investigated the effect of taurine and β-alanine supplementation on muscle function and muscle taurine transporter (TauT) protein expression in mdx mice. Wild-type (WT) and mdx mice (5 months) were supplemented with taurine or β-alanine for 4 weeks, after which in vitro contractile properties, fatigue resistance and force recovery, and the expression of the TauT protein and proteins involved in excitation-contraction (E-C) coupling were examined in fast-twitch muscle. There was no difference in basal TauT protein expression or basal taurine content between mdx than WT muscle. Supplementation with taurine and β-alanine increased and reduced taurine content, respectively, in muscle from WT and mdx mice but had no effect of TauT protein. Taurine supplementation reduced body and muscle mass, and enhanced fatigue resistance and force recovery in mdx muscle. β-Alanine supplementation enhanced fatigue resistance in WT and mdx muscle. There was no difference in the basal expression of key E-C coupling proteins [ryanodine receptor 1 (RyR1), dihydropyridine receptor (DHPR), sarco(endo)plasmic reticulum Ca 2+ -ATPase 1 (SERCA1) or calsequestrin 1 (CSQ1)] between WT and mdx mice, and the expression of these proteins was not altered by taurine or β-alanine supplementation. These findings suggest that TauT protein expression is relatively insensitive to changes in muscle taurine content in WT and mdx mice, and that taurine and β-alanine supplementation may be viable therapeutic strategies to improve fatigue resistance of dystrophic skeletal muscle.

  14. The stress granule protein Vgl1 and poly(A)-binding protein Pab1 are required for doxorubicin resistance in the fission yeast Schizosaccharomyces pombe

    Energy Technology Data Exchange (ETDEWEB)

    Morita, Takahiro [Laboratory of Molecular Pharmacogenomics, School of Pharmaceutical Sciences, Kinki University, Kowakae 3-4-1, Higashi-Osaka 577-8502 (Japan); Satoh, Ryosuke [Laboratory of Molecular Pharmacogenomics, School of Pharmaceutical Sciences, Kinki University, Kowakae 3-4-1, Higashi-Osaka 577-8502 (Japan); Japan Society for the Promotion of Science, 1-8 Chiyoda-ku, Tokyo 102-8472 (Japan); Umeda, Nanae; Kita, Ayako [Laboratory of Molecular Pharmacogenomics, School of Pharmaceutical Sciences, Kinki University, Kowakae 3-4-1, Higashi-Osaka 577-8502 (Japan); Sugiura, Reiko, E-mail: sugiurar@phar.kindai.ac.jp [Laboratory of Molecular Pharmacogenomics, School of Pharmaceutical Sciences, Kinki University, Kowakae 3-4-1, Higashi-Osaka 577-8502 (Japan)

    2012-01-06

    Highlights: Black-Right-Pointing-Pointer Stress granules (SGs) as a mechanism of doxorubicin tolerance. Black-Right-Pointing-Pointer We characterize the role of stress granules in doxorubicin tolerance. Black-Right-Pointing-Pointer Deletion of components of SGs enhances doxorubicin sensitivity in fission yeast. Black-Right-Pointing-Pointer Doxorubicin promotes SG formation when combined with heat shock. Black-Right-Pointing-Pointer Doxorubicin regulates stress granule assembly independent of eIF2{alpha} phosphorylation. -- Abstract: Doxorubicin is an anthracycline antibiotic widely used for chemotherapy. Although doxorubicin is effective in the treatment of several cancers, including solid tumors and leukemias, the basis of its mechanism of action is not completely understood. Here, we describe the effects of doxorubicin and its relationship with stress granules formation in the fission yeast, Schizosaccharomyces pombe. We show that disruption of genes encoding the components of stress granules, including vgl1{sup +}, which encodes a multi-KH type RNA-binding protein, and pab1{sup +}, which encodes a poly(A)-binding protein, resulted in greater sensitivity to doxorubicin than seen in wild-type cells. Disruption of the vgl1{sup +} and pab1{sup +} genes did not confer sensitivity to other anti-cancer drugs such as cisplatin, 5-fluorouracil, and paclitaxel. We also showed that doxorubicin treatment promoted stress granule formation when combined with heat shock. Notably, doxorubicin treatment did not induce hyperphosphorylation of eIF2{alpha}, suggesting that doxorubicin is involved in stress granule assembly independent of eIF2{alpha} phosphorylation. Our results demonstrate the usefulness of fission yeast for elucidating the molecular targets of doxorubicin toxicity and suggest a novel drug-resistance mechanism involving stress granule assembly.

  15. The stress granule protein Vgl1 and poly(A)-binding protein Pab1 are required for doxorubicin resistance in the fission yeast Schizosaccharomyces pombe

    International Nuclear Information System (INIS)

    Morita, Takahiro; Satoh, Ryosuke; Umeda, Nanae; Kita, Ayako; Sugiura, Reiko

    2012-01-01

    Highlights: ► Stress granules (SGs) as a mechanism of doxorubicin tolerance. ► We characterize the role of stress granules in doxorubicin tolerance. ► Deletion of components of SGs enhances doxorubicin sensitivity in fission yeast. ► Doxorubicin promotes SG formation when combined with heat shock. ► Doxorubicin regulates stress granule assembly independent of eIF2α phosphorylation. -- Abstract: Doxorubicin is an anthracycline antibiotic widely used for chemotherapy. Although doxorubicin is effective in the treatment of several cancers, including solid tumors and leukemias, the basis of its mechanism of action is not completely understood. Here, we describe the effects of doxorubicin and its relationship with stress granules formation in the fission yeast, Schizosaccharomyces pombe. We show that disruption of genes encoding the components of stress granules, including vgl1 + , which encodes a multi-KH type RNA-binding protein, and pab1 + , which encodes a poly(A)-binding protein, resulted in greater sensitivity to doxorubicin than seen in wild-type cells. Disruption of the vgl1 + and pab1 + genes did not confer sensitivity to other anti-cancer drugs such as cisplatin, 5-fluorouracil, and paclitaxel. We also showed that doxorubicin treatment promoted stress granule formation when combined with heat shock. Notably, doxorubicin treatment did not induce hyperphosphorylation of eIF2α, suggesting that doxorubicin is involved in stress granule assembly independent of eIF2α phosphorylation. Our results demonstrate the usefulness of fission yeast for elucidating the molecular targets of doxorubicin toxicity and suggest a novel drug-resistance mechanism involving stress granule assembly.

  16. Eukaryotic resistance to fluoride toxicity mediated by a widespread family of fluoride export proteins.

    Science.gov (United States)

    Li, Sanshu; Smith, Kathryn D; Davis, Jared H; Gordon, Patricia B; Breaker, Ronald R; Strobel, Scott A

    2013-11-19

    Fluorine is an abundant element and is toxic to organisms from bacteria to humans, but the mechanisms by which eukaryotes resist fluoride toxicity are unknown. The Escherichia coli gene crcB was recently shown to be regulated by a fluoride-responsive riboswitch, implicating it in fluoride response. There are >8,000 crcB homologs across all domains of life, indicating that it has an important role in biology. Here we demonstrate that eukaryotic homologs [renamed FEX (fluoride exporter)] function in fluoride export. FEX KOs in three eukaryotic model organisms, Neurospora crassa, Saccharomyces cerevisiae, and Candida albicans, are highly sensitized to fluoride (>200-fold) but not to other halides. Some of these KO strains are unable to grow in fluoride concentrations found in tap water. Using the radioactive isotope of fluoride, (18)F, we developed an assay to measure the intracellular fluoride concentration and show that the FEX deletion strains accumulate fluoride in excess of the external concentration, providing direct evidence of FEX function in fluoride efflux. In addition, they are more sensitive to lower pH in the presence of fluoride. These results demonstrate that eukaryotic FEX genes encode a previously unrecognized class of fluoride exporter necessary for survival in standard environmental conditions.

  17. Changes in Lean Mass and Serum Myostatin with Habitual Protein Intake and High-Velocity Resistance Training.

    Science.gov (United States)

    Binns, A; Gray, M; Henson, A C; Fort, I L

    2017-01-01

    Examine the associations between dietary protein intake, lean mass (LM), and serum myostatin (Mstn) levels among community-dwelling older adults participating in a 20-week high-velocity resistance training (HVRT) program. This longitudinal study consisted of 33 community-dwelling, older adults (mean age 77.0 years, SD = 6.4); all of which obtained physician clearance prior to study participation. Twenty-five females and eight males were randomized to a control (CON) or HVRT group. Anthropometric measures were obtained via dual energy x-ray absorptiometry (DXA) and peripheral venous blood draw used for serum myostatin analysis. Exercise was performed twice per week for 20 consecutive weeks. Food intake estimation with a diet history questionnaire (DHQ) was used for protein intake comparison to the recommended dietary allowance (RDA). All measures were recorded both prior to and following study participation. Altogether, protein was consumed in amounts more generous (1.01 ± 0.47 g·kg-1·d-1) than that of the RDA (0.8 g·kg-1·d-1). As a result of significant LM differences among men and women (p myostatin was greater among females (6681.8 ± 3155.0 pg·mL-1) than males (5560.0 ± 2946.1 pg·mL-1); however, these values were not significantly different (p = 0.39). Combined, protein consumption and serum myostatin did not significantly influence LM among males (p = 0.09) or females (p = 0.71). Irrespective of training group, significant changes were not exhibited in dietary intake patterns, LM, or serum myostatin. Contrary to the proposed hypothesis, results suggest protein consumption and circulating serum myostatin levels did not significantly influence LM among older adults. Although HVRT positively impacts LM, neither exercise group displayed significant changes in LM. Therefore, further research is needed examining dietary intake, exercise modality, and myostatin downregulation as non-pharmacological approaches to combating sarcopenia.

  18. Protein adsorption resistant surface on polymer composite based on 2D- and 3D-controlled grafting of phospholipid moieties

    International Nuclear Information System (INIS)

    Hoshi, Toru; Matsuno, Ryosuke; Sawaguchi, Takashi; Konno, Tomohiro; Takai, Madoka; Ishihara, Kazuhiko

    2008-01-01

    To prepare the biocompatible surface, a phosphorylcholine (PC) group was introduced on this hydroxyl group generated by surface hydrolysis on the polymer composite composed of polyethylene (PE) and poly (vinyl acetate) (PVAc) prepared by supercritical carbon dioxide. Two different procedures such as two-dimensional (2D) modification and three-dimensional (3D) modification were applied to obtain the steady biocompatible surface. 2D modification was that PC groups were directly anchored on the surface of the polymer composite. 3D modification was that phospholipid polymer was grafted from the surface of the polymer composite by surface-initiated atom transfer radical polymerization (SI-ATRP) of 2-methacryloyloxyethyl phosphorylcholine (MPC). The surfaces were characterized by X-ray photoelectron spectroscopy, dynamic water contact angle measurements, and atomic force microscope. The effects of the poly(MPC) chain length on the protein adsorption resistivity were investigated. The protein adsorption on the polymer composite surface with PC groups modified by 2D or 3D modification was significantly reduced as compared with that on the unmodified PE. Further, the amount of protein adsorbed on the 3D modified surface that is poly(MPC)-grafted surface decreased with an increase in the chain length of the poly(MPC). The surface with an arbitrary structure and the characteristic can be constructed by using 2D and 3D modification. We conclude that the polymer composites of PE/PVAc with PC groups on the surface are useful for fabricating biomedical devices due to their good mechanical and surface properties

  19. Transcription and activity of antioxidant proteins after ionization irradiation of radiation-resistant and radiation-sensitive mice

    International Nuclear Information System (INIS)

    Hardmeier, R.

    1998-03-01

    The involvernent of antioxidant proteins catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GSH px), and thioredoxin (TRX) in radiobiological processes has been described at the enzyme activity level. We were interested in examining the transcription of these proteins in a mammalian system following ionizing irradiation. In order to answer the question whether radiation effects in sensitive mice (Balb/c) (RS) showed differences at the transcriptional level from radiation effects in resistant mice (C3H) (RR). We exposed the whole body of these strains to X/rays doses of 2, 4, and 6 Gy and sacrificed the animals 5, 15, and 30 minutes after irradiation. The mRNA was isolated from liver and hybrized with probes for antioxidant enzymes and thioredoxin, β-actin was used as a housekeeping gene control. Antioxidant enzyme activities were determined by standard assays. Parameters for aromatic hydroxylation (o-Tyr) and lipid peroxidation (MDA) were determined by HPLC methods. Antioxidant transcription was unchanged in contrast to antioxidant activities. SOD and CAT activities were elevated within 15 minutes in RR animals but not in RS at all radiation doses. Glutathione peroxidase activity was not different between RR and RS mice, and was only moderately elevated after irradiation. No significant differences were found between RR and RS animals at the oxidation level, although a radiation dose-dependent increase of oxidation products was detected in both groups. Quantification of thioredoxin mRNA revealed that RR mice transcribed this protein at a significantly higher level at an earlier time point (5 minutes) than did RS mice. This delay may well be responsible for the radioresistance although no quantitative differences were found. As unchanged transcription of antioxidant enzymes could not have been responsible for the increased antioxidant enzyme activities, preformed antioxidant enzymes may have been released by irradiation. This would be in agreement

  20. Evaluation of iTRAQ and SWATH-MS for the Quantification of Proteins Associated with Insulin Resistance in Human Duodenal Biopsy Samples.

    Directory of Open Access Journals (Sweden)

    Sylvie Bourassa

    Full Text Available Insulin resistance (IR is associated with increased production of triglyceride-rich lipoproteins of intestinal origin. In order to assess whether insulin resistance affects the proteins involved in lipid metabolism, we used two mass spectrometry based quantitative proteomics techniques to compare the intestinal proteome of 14 IR patients to that of 15 insulin sensitive (IS control patients matched for age and waist circumference. A total of 3886 proteins were identified by the iTRAQ (Isobaric Tags for Relative and Absolute Quantitation mass spectrometry approach and 2290 by the SWATH-MS strategy (Serial Window Acquisition of Theoretical Spectra. Using these two methods, 208 common proteins were identified with a confidence corresponding to FDR < 1%, and quantified with p-value < 0.05. The quantification of those 208 proteins has a Pearson correlation coefficient (r2 of 0.728 across the two techniques. Gene Ontology analyses of the differentially expressed proteins revealed that annotations related to lipid metabolic process and oxidation reduction process are overly represented in the set of under-expressed proteins in IR subjects. Furthermore, both methods quantified proteins of relevance to IR. These data also showed that SWATH-MS is a promising and compelling alternative to iTRAQ for protein quantitation of complex mixtures.

  1. Middle East Respiratory Syndrome Coronavirus Nonstructural Protein 16 Is Necessary for Interferon Resistance and Viral Pathogenesis

    Energy Technology Data Exchange (ETDEWEB)

    Menachery, Vineet D.; Gralinski, Lisa E.; Mitchell, Hugh D.; Dinnon, Kenneth H.; Leist, Sarah R.; Yount, Boyd L.; Graham, Rachel L.; McAnarney, Eileen T.; Stratton, Kelly G.; Cockrell, Adam S.; Debbink, Kari; Sims, Amy C.; Waters, Katrina M.; Baric, Ralph S.; Fernandez-Sesma, Ana

    2017-11-15

    ABSTRACT

    Coronaviruses (CoVs) encode a mixture of highly conserved and novel genes, as well as genetic elements necessary for infection and pathogenesis, raising the possibility of common targets for attenuation and therapeutic design. In this study, we focused on highly conserved nonstructural protein 16 (NSP16), a viral 2'O-methyltransferase (2'O-MTase) that encodes critical functions in immune modulation and infection. Using reverse genetics, we disrupted a key motif in the conserved KDKE motif of Middle East respiratory syndrome CoV (MERS-CoV) NSP16 (D130A) and evaluated the effect on viral infection and pathogenesis. While the absence of 2'O-MTase activity had only a marginal impact on propagation and replication in Vero cells, dNSP16 mutant MERS-CoV demonstrated significant attenuation relative to the control both in primary human airway cell cultures andin vivo. Further examination indicated that dNSP16 mutant MERS-CoV had a type I interferon (IFN)-based attenuation and was partially restored in the absence of molecules of IFN-induced proteins with tetratricopeptide repeats. Importantly, the robust attenuation permitted the use of dNSP16 mutant MERS-CoV as a live attenuated vaccine platform protecting from a challenge with a mouse-adapted MERS-CoV strain. These studies demonstrate the importance of the conserved 2'O-MTase activity for CoV pathogenesis and highlight NSP16 as a conserved universal target for rapid live attenuated vaccine design in an expanding CoV outbreak setting.

    IMPORTANCECoronavirus (CoV) emergence in both humans and livestock represents a significant threat to global public health, as evidenced by the sudden emergence of severe acute respiratory syndrome CoV (SARS-CoV), MERS-CoV, porcine epidemic diarrhea virus, and swine delta CoV in the 21st century. These studies describe an approach that

  2. Application of resistivity measurements to the control of protein elutions using cellulose ion-exchangers; Application de la mesure de resistivite au controle d'une elution de proteines a partir de celluloses echangeuses d'ions

    Energy Technology Data Exchange (ETDEWEB)

    Duplaa, A M; Brandolin, G [Commissariat a l' Energie Atomique, Grenoble (France). Centre d' Etudes Nucleaires

    1969-07-01

    The eluate obtained by chromatography of a mixture of proteins on columns of cellulose ion-exchangers (DEAE-cellulose and CM-cellulose) sometimes have very low proteins concentrations. The resistivity measurement gives more information than the UV control which is often inadequate. The modifications undergone by elution buffers are recorded and the best conditions for the extraction of an enzymatic protein are determined. The tests are performed without proteins on the columns; they consist in a double control of resistivity and ph of elution buffers after they pass on the exchangers columns. (author) [French] Apres chromatographie d'un melange de proteine; sur colonnes d'echangeurs d'ions tels que DEAE-cellulose et CM-cellulose, les eluats obtenus ont quelquefois des concentrations en proteines tres faibles. Au controle en UV souvent insuffisant, on a ajoute la mesure de resistivite apportant des donnees complementaires. Des essais, realises en l'absence de proteines et consistant a effectuer un double controle de resistivite et de pH des tampons d'elution apres leur passage sur colonnes d'echangeurs, ont permis d'enregistrer les modifications subies par ces tampons et de determiner leurs meilleures conditions d'utilisation pour l'extraction d'une proteine enzymatique. (auteur)

  3. R-Flurbiprofen Traps Prostaglandins within Cells by Inhibition of Multidrug Resistance-Associated Protein-4

    Directory of Open Access Journals (Sweden)

    Ivonne Wobst

    2016-12-01

    Full Text Available R-flurbiprofen is the non-COX-inhibiting enantiomer of flurbiprofen and is not converted to S-flurbiprofen in human cells. Nevertheless, it reduces extracellular prostaglandin E2 (PGE2 in cancer or immune cell cultures and human extracellular fluid. Here, we show that R-flurbiprofen acts through a dual mechanism: (i it inhibits the translocation of cPLA2α to the plasma membrane and thereby curtails the availability of arachidonic acid and (ii R-flurbiprofen traps PGE2 inside of the cells by inhibiting multidrug resistance–associated protein 4 (MRP4, ABCC4, which acts as an outward transporter for prostaglandins. Consequently, the effects of R-flurbiprofen were mimicked by RNAi-mediated knockdown of MRP4. Our data show a novel mechanism by which R-flurbiprofen reduces extracellular PGs at physiological concentrations, particularly in cancers with high levels of MRP4, but the mechanism may also contribute to its anti-inflammatory and immune-modulating properties and suggests that it reduces PGs in a site- and context-dependent manner.

  4. High Expression of Antiviral Proteins in Mucosa from Individuals Exhibiting Resistance to Human Immunodeficiency Virus.

    Directory of Open Access Journals (Sweden)

    Sandra Milena Gonzalez

    Full Text Available Several soluble factors have been reported to have the capacity of inhibiting HIV replication at different steps of the virus life cycle, without eliminating infected cells and through enhancement of specific cellular mechanisms. Yet, it is unclear if these antiviral factors play a role in the protection from HIV infection or in the control of viral replication. Here we evaluated two cohorts: i one of 58 HIV-exposed seronegative individuals (HESNs who were compared with 59 healthy controls (HCs, and ii another of 13 HIV-controllers who were compared with 20 HIV-progressors. Peripheral blood, oral and genital mucosa and gut-associated lymphoid tissue (GALT samples were obtained to analyze the mRNA expression of ELAFIN, APOBEC3G, SAMHD1, TRIM5α, RNase 7 and SerpinA1 using real-time PCR.HESNs exhibited higher expression of all antiviral factors in peripheral blood mononuclear cells (PBMCs, oral or genital mucosa when compared with HCs. Furthermore, HIV-controllers exhibited higher levels of SerpinA1 in GALT.These findings suggest that the activity of these factors is compartmentalized and that these proteins have a predominant role depending on the tissue to avoid the infection, reduce the viral load and modulate the susceptibility to HIV infection.

  5. Bph32, a novel gene encoding an unknown SCR domain-containing protein, confers resistance against the brown planthopper in rice

    Science.gov (United States)

    Ren, Juansheng; Gao, Fangyuan; Wu, Xianting; Lu, Xianjun; Zeng, Lihua; Lv, Jianqun; Su, Xiangwen; Luo, Hong; Ren, Guangjun

    2016-01-01

    An urgent need exists to identify more brown planthopper (Nilaparvata lugens Stål, BPH) resistance genes, which will allow the development of rice varieties with resistance to BPH to counteract the increased incidence of this pest species. Here, using bioinformatics and DNA sequencing approaches, we identified a novel BPH resistance gene, LOC_Os06g03240 (MSU LOCUS ID), from the rice variety Ptb33 in the interval between the markers RM19291 and RM8072 on the short arm of chromosome 6, where a gene for resistance to BPH was mapped by Jirapong Jairin et al. and renamed as “Bph32”. This gene encodes a unique short consensus repeat (SCR) domain protein. Sequence comparison revealed that the Bph32 gene shares 100% sequence identity with its allele in Oryza latifolia. The transgenic introgression of Bph32 into a susceptible rice variety significantly improved resistance to BPH. Expression analysis revealed that Bph32 was highly expressed in the leaf sheaths, where BPH primarily settles and feeds, at 2 and 24 h after BPH infestation, suggesting that Bph32 may inhibit feeding in BPH. Western blotting revealed the presence of Pph (Ptb33) and Tph (TN1) proteins using a Penta-His antibody, and both proteins were insoluble. This study provides information regarding a valuable gene for rice defence against insect pests. PMID:27876888

  6. Bph32, a novel gene encoding an unknown SCR domain-containing protein, confers resistance against the brown planthopper in rice.

    Science.gov (United States)

    Ren, Juansheng; Gao, Fangyuan; Wu, Xianting; Lu, Xianjun; Zeng, Lihua; Lv, Jianqun; Su, Xiangwen; Luo, Hong; Ren, Guangjun

    2016-11-23

    An urgent need exists to identify more brown planthopper (Nilaparvata lugens Stål, BPH) resistance genes, which will allow the development of rice varieties with resistance to BPH to counteract the increased incidence of this pest species. Here, using bioinformatics and DNA sequencing approaches, we identified a novel BPH resistance gene, LOC_Os06g03240 (MSU LOCUS ID), from the rice variety Ptb33 in the interval between the markers RM19291 and RM8072 on the short arm of chromosome 6, where a gene for resistance to BPH was mapped by Jirapong Jairin et al. and renamed as "Bph32". This gene encodes a unique short consensus repeat (SCR) domain protein. Sequence comparison revealed that the Bph32 gene shares 100% sequence identity with its allele in Oryza latifolia. The transgenic introgression of Bph32 into a susceptible rice variety significantly improved resistance to BPH. Expression analysis revealed that Bph32 was highly expressed in the leaf sheaths, where BPH primarily settles and feeds, at 2 and 24 h after BPH infestation, suggesting that Bph32 may inhibit feeding in BPH. Western blotting revealed the presence of Pph (Ptb33) and Tph (TN1) proteins using a Penta-His antibody, and both proteins were insoluble. This study provides information regarding a valuable gene for rice defence against insect pests.

  7. FNDC5 Gene Expression and Irisin Protein Level of Visceral Fat Tissue after Eight Weeks of Resistance Training in Type 2 Diabetic Rats

    Directory of Open Access Journals (Sweden)

    Ali Abdi

    2018-04-01

    Conclusion: It seems that FNDC5 gene and Irisin protein have an important role in metabolic diseases and can be affected by resistance training. Perhaps the changes in the levels of these metabolic indicators is a potential new target for the treatment of metabolic disorders, such as T2DM (type 2 diabetes.

  8. Leptin receptor 170 kDa (OB-R170) protein expression is reduced in obese human skeletal muscle: a potential mechanism of leptin resistance

    DEFF Research Database (Denmark)

    Fuentes, T; Ara, I; Guadalupe-Grau, A

    2010-01-01

    To examine whether obesity-associated leptin resistance could be due to down-regulation of leptin receptors (OB-Rs) and/or up-regulation of suppressor of cytokine signalling 3 (SOCS3) and protein tyrosine phosphatase 1B (PTP1B) in skeletal muscle, which blunt janus kinase 2-dependent leptin...

  9. Effects of preoperative feeding with a whey protein plus carbohydrate drink on the acute phase response and insulin resistance. A randomized trial

    Directory of Open Access Journals (Sweden)

    Dock-Nascimento Diana B

    2011-06-01

    Full Text Available Abstract Background Prolonged preoperative fasting increases insulin resistance and current evidence recommends carbohydrate (CHO drinks 2 hours before surgery. Our hypothesis is that the addition of whey protein to a CHO-based drink not only reduces the inflammatory response but also diminish insulin resistance. Methods Seventeen patients scheduled to cholecystectomy or inguinal herniorraphy were randomized and given 474 ml and 237 ml of water (CO group or a drink containing CHO and milk whey protein (CHO-P group respectively, 6 and 3 hours before operation. Blood samples were collected before surgery and 24 hours afterwards for biochemical assays. The endpoints of the study were the insulin resistance (IR, the prognostic inflammatory and nutritional index (PINI and the C-reactive protein (CRP/albumin ratio. A 5% level for significance was established. Results There were no anesthetic or postoperative complications. The post-operative IR was lower in the CHO-P group when compared with the CO group (2.75 ± 0.72 vs 5.74 ± 1.16; p = 0.03. There was no difference between the two groups in relation to the PINI. The CHO-P group showed a decrease in the both CRP elevation and CRP/albumin ratio (p Conclusions Shortening the pre-operative fasting using CHO and whey protein is safe and reduces insulin resistance and postoperative acute phase response in elective moderate operations. Trial registration ClinicalTrail.gov NCT01354249

  10. Direct detection of methicillin resistance in Staphylococcus aureus in blood culture broth by use of a penicillin binding protein 2a latex agglutination test.

    Science.gov (United States)

    Qian, Qinfang; Venkataraman, Lata; Kirby, James E; Gold, Howard S; Yamazumi, Toshiaki

    2010-04-01

    We studied the utility of performing a penicillin binding protein 2a latex agglutination (PBP-LA) assay directly on Bactec blood culture broth samples containing Staphylococcus aureus to rapidly detect methicillin resistance. The sensitivity, specificity, positive predictive value, and negative predictive value of this method were 94.1%, 97.5%, 98%, and 92.9%, respectively.

  11. Direct Detection of Methicillin Resistance in Staphylococcus aureus in Blood Culture Broth by Use of a Penicillin Binding Protein 2a Latex Agglutination Test▿

    OpenAIRE

    Qian, Qinfang; Venkataraman, Lata; Kirby, James E.; Gold, Howard S.; Yamazumi, Toshiaki

    2010-01-01

    We studied the utility of performing a penicillin binding protein 2a latex agglutination (PBP-LA) assay directly on Bactec blood culture broth samples containing Staphylococcus aureus to rapidly detect methicillin resistance. The sensitivity, specificity, positive predictive value, and negative predictive value of this method were 94.1%, 97.5%, 98%, and 92.9%, respectively.